A method for constructing a transgenic plasmid for extending the healthy lifespan of Caenorhabditis elegans and its application

Abstract

The present invention provides a method for constructing a transgenic plasmid for extending the healthy life span of Caenorhabditis elegans. The method comprises constructing pMD18-Tsimple-pU6::shRNA(RPS-30 UbL ) plasmid, construction of pMD18‑Tsimple‑pRPS‑30::GFP::RPS‑30 S30 Plasmids and constructions pPD95.77‑pRPS‑30::GFP::RPS‑30 S30 Plasmids and construction pPD95.77‑pU6::shRNA(RPS‑30 UbL )‑pRPS‑30::GFP::RPS‑30 S30 The plasmid is a transgenic plasmid that prolongs the healthy lifespan of Caenorhabditis elegans, and an application is also provided for prolonging the healthy lifespan of Caenorhabditis elegans. The transgenic plasmid prepared by the present invention that prolongs the healthy lifespan of Caenorhabditis elegans is transfected into the wild-type standard worm strain N2 of Caenorhabditis elegans by microinjection, and the worm lifespan is significantly prolonged, and no significant difference is observed in the worm development, egg laying, and movement ability.

Description

Technical Field

[0001] The invention belongs to the field of biological science technology, and specifically relates to a method for constructing a transgenic plasmid for extending the healthy lifespan of Caenorhabditis elegans and its application. Background Art

[0002] Healthy longevity has always been a goal pursued by mankind, and the regulatory mechanism of healthy longevity has always been a research hotspot in the fields of medicine and life sciences.

[0003] Caenorhabditis elegans has been widely used as a model organism for the study of lifespan regulation mechanisms due to its advantages such as short life cycle, easy cultivation and observation, complete cell lineage, powerful genetic analysis methods and highly conserved genome. Many signaling pathways that regulate lifespan were first discovered and elucidated in Caenorhabditis elegans. The lifespan of Caenorhabditis elegans is affected by many factors: genetic factors, environmental factors and a variety of conservative signal transduction pathways, such as IIS, TGF-β, TOR, AMPK, etc. At present, the methods for extending the lifespan of Caenorhabditis elegans mainly include: low temperature culture, feeding restriction, genetic manipulation, etc. Among them, genetic manipulation is a method of affecting the lifespan of Caenorhabditis elegans through genetic factors, mainly knocking out or silencing specific genes, such as daf-2, hcf-1, let-363, daf-15, hif-1, etc. However, these genes are all essential genes for the worm, such as the insulin signaling pathway receptor gene daf-2. The deletion mutation of this gene can lead to death in the embryonic stage or early larval development, and the daf-2 mutants used for lifespan and development research are mostly point mutations with partial functional loss, which are completed through random induction of chemical mutagens and high-throughput screening. This makes it impossible to promote this lifespan extension technology in other species. Although the knockout or silencing of other genes can lead to longevity of the worm, it also causes the worm to develop slowly, fail to lay eggs, or fail to develop to the adult stage, or have a significantly reduced ability to move, and other unhealthy longevity states. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for constructing a transgenic plasmid for extending the healthy lifespan of Caenorhabditis elegans and its application in view of the deficiencies of the above-mentioned prior art. The transgenic plasmid for extending the healthy lifespan of Caenorhabditis elegans is used to extend the healthy lifespan of Caenorhabditis elegans.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for constructing a transgenic plasmid for extending the healthy lifespan of Caenorhabditis elegans, the method comprising:

[0006] S1. Construction of pMD18-T simple-pU6::shRNA(RPS-30 UbL )Plasmid:

[0007] S101, using pLKO.1puro plasmid as a template, amplifying the U6 promoter sequence by PCR, and cloning the U6 promoter PCR product into the pMD18-T simple plasmid by TA cloning to construct the pMD18-T simple-pU6 plasmid, and then performing double digestion of the obtained pMD18-T simple-pU6 plasmid with AgeI / EcoRI restriction endonucleases to obtain AgeI / EcoRI double digestion products, and performing gel recovery on the AgeI / EcoRI double digestion products, the specific steps include:

[0008] S1011. Using pLKO.1puro plasmid as a template, performing PCR reaction with upstream primer U6P1 and downstream primer U6P2 to amplify U6 promoter, and obtaining U6 promoter PCR product; the nucleotide sequence of the upstream primer U6P1 is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer U6P2 is shown in SEQ ID NO: 2;

[0009] The PCR reaction system was: pLKO.1puro plasmid 0.2 μL, upstream primer U6P1 1 μL, downstream primer U6P2 1 μL, LATaq 0.25 μL, 10×LATaq buffer 2.5 μL, ddH2O to 25 μL;

[0010] The PCR reaction program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, 25 cycles; extension at 72°C for 5 min; storage at 4°C;

[0011] S1012. At 16°C, the U6 promoter PCR product obtained in S1011 was ligated into the pMD18-T simple plasmid overnight by TA cloning to obtain the TA cloning ligation product pMD18-T simple-pU6.

[0012] Ligation reaction system: 4 μL of U6 promoter PCR product, 0.5 μL of pMD18-T simple plasmid, 4.5 μL of DNA ligation buffer Solution I, and ddH2O to 10 μL;

[0013] S1013, conversion coated plate:

[0014] Take out the competent bacteria of Escherichia coli TOP10 from the storage temperature of -80°C, add the TA cloning ligation product pMD18-T simple-pU6 obtained in S1012, mix well, ice bath for 30 minutes, then heat stress at 42°C for 90 seconds, ice bath for 2 minutes, add 1 mL of LB liquid culture medium, culture at 37°C and a shaking speed of 250 rpm / min for 1 hour, obtain the residual bacterial liquid after transformation by centrifugation, add the residual bacterial liquid after transformation to a solid LB culture plate containing ampicillin (AMP) resistance, spread evenly, and culture overnight in a bacterial incubator at 37°C;

[0015] S1014, Sequencing Identification:

[0016] Monoclonal colonies were picked from the solid LB culture plate cultured overnight in S1013, and placed in 3 mL of LB liquid culture medium containing ampicillin (AMP) resistance, respectively, and cultured for 10 h at 37°C and a shaking speed of 250 rpm / min. The obtained bacterial liquids were respectively taken for sequencing identification, and the monoclonal colonies with correct sequencing results were screened;

[0017] S1015. Extract pMD18-T simple-pU6 plasmid:

[0018] The correctly sequenced monoclonal colony obtained in S1014 was extracted using the AxyPrep Plasmid MiniprepKit plasmid extraction kit to obtain the pMD18-T simple-pU6 plasmid;

[0019] Double digestion with S1016, AgeI / EcoRI restriction enzymes;

[0020] At 37°C, the pMD18-T simple-pU6 plasmid obtained in S1015 was subjected to a 5hAgeI / EcoRI restriction endonuclease double digestion reaction to obtain an AgeI / EcoRI double digestion product;

[0021] Reaction system: pMD18-T simple-pU6 plasmid 1.5 μg, 10×FastDigestGreen buffer 2 μL, AgeI restriction endonuclease 1 μL, EcoRI restriction endonuclease 1 μL, ddH2O to 20 μL;

[0022] S1017, gel recovery of AgeI / EcoRI double digestion products:

[0023] The AgeI / EcoRI double digestion product obtained in S1016 was subjected to 1% agarose gel electrophoresis, and the target DNA band of 270 bp was cut out and recovered using the AxyPrep DNA Gel Extraction Kit gel recovery kit to obtain the AgeI / EcoRI double digestion gel recovery product;

[0024] S102, annealing primer UbLshR1 and primer UbLshR2 to obtain an annealing product, i.e., targeting RPS-30 UbL Short hairpin RNA of the region: shRNA (RPS-30 UbL ); the nucleotide sequence of the primer UbLshR1 is shown in SEQ ID NO:3, and the nucleotide sequence of the primer UbLshR2 is shown in SEQ ID NO:4;

[0025] Annealing system: primer UbLshR1 1.5 μL, primer UbLshR2 1.5 μL, 10×NEBbuffer II 5 μL, ddH2O to 50 μL;

[0026] Annealing procedure: pre-denaturation at 95°C for 5 min; denaturation at 70°C for 15 min; decrease the temperature to 22°C at a rate of 0.1°C per second; store at -40°C;

[0027] S103, the gel recovery product obtained in S101 after the AgeI / EcoRI double digestion was connected with the annealing product obtained in S102 to construct pMD18-T simple-pU6::shRNA (RPS-30 UbL ) plasmid, the specific steps include:

[0028] S1031. At 16°C, the gel-recovered product obtained in S101 after double digestion with EcoRI was ligated with the annealed product obtained in S102 overnight to obtain the ligation product pMD18-T simple-pU6::shRNA (RPS-30 UbL );

[0029] Ligation reaction system: 100 ng of gel recovery product after AgeI / EcoRI double digestion, 500 ng of annealing product, 8 μL of LigationMix, and 25 μL of ddH2O;

[0030] S1032, conversion coated plate:

[0031] Take out the E. coli Stbl3 competent bacteria from the storage temperature of -80°C, add the ligation product pMD18-T simple-pU6::shRNA (RPS-30 UbL), mix well, ice bath for 30 minutes, then heat stress at 42°C for 90 seconds, ice bath for 2 minutes, add 1 mL of LB liquid culture medium, culture at 37°C and a shaking speed of 250 rpm / min for 1 hour, centrifuge to obtain the residual bacterial liquid after transformation, drop the residual bacterial liquid after transformation into a solid LB culture plate containing ampicillin (Amp) resistance, spread evenly, and place in a bacterial culture incubator at 37°C for overnight culture;

[0032] S1033, sequencing identification:

[0033] After overnight culture in S1032, single clones were picked from the solid LB culture plate transformed and placed in 3 mL of LB liquid culture medium containing ampicillin (AMP) resistance, respectively, and cultured for 10 h at 37° C. and 250 rpm / min on a shaker. The obtained bacterial liquids were taken for sequencing and identification, and the single clones with correct sequencing results were screened out;

[0034] S1034, extract pMD18-T simple-pU6::shRNA (RPS-30 UbL ) plasmid;

[0035] The correctly sequenced monoclonal colony obtained in S1033 was extracted using the AxyPrep Plasmid Miniprep Kit plasmid extraction kit to obtain pMD18-T simple-pU6::shRNA (RPS-30 UbL ) plasmid;

[0036] S2. Construction of pMD18-T simple-pRPS-30::GFP::RPS-30 S30 Plasmids:

[0037] S201, using the genomic DNA sequence of Caenorhabditis elegans as a template, amplifying the rps-30 promoter sequence by PCR to obtain the rps-30 promoter PCR product; using the pEGFP-c2 plasmid as a template, amplifying the GFP nucleic acid sequence by PCR to obtain the GFP PCR product; using the cDNA obtained by reverse transcription of the Caenorhabditis elegans RNA sequence as a template, amplifying the RPS-30 promoter by PCR S30 Nucleic acid sequence, RPS-30 S30 PCR products, the specific steps include:

[0038] S2011. Extraction of Caenorhabditis elegans genomic DNA:

[0039] The genomic DNA of Caenorhabditis elegans was extracted using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0;

[0040] S2012. Using the C. elegans genomic DNA obtained in S2011 as a template, PCR amplified the rps-30 promoter sequence:

[0041] Using Caenorhabditis elegans genomic DNA as a template, an upstream primer rps30P1 and a downstream primer rps30P2 were used to perform a PCR reaction to amplify the rps-30 promoter sequence, thereby obtaining an rps-30 promoter PCR product; the nucleotide sequence of the upstream primer rps30P1 is shown in SEQ ID NO:5, and the nucleotide sequence of the downstream primer rps30P2 is shown in SEQ ID NO:6;

[0042] PCR reaction system: 2 μL of C. elegans genomic DNA, 1 μL of upstream primer rps30P1, 1 μL of downstream primer rps30P2, 0.25 μL of LA Taq, 2.5 μL of 10×LA Taq buffer, and ddH2O to 25 μL;

[0043] PCR reaction program: 95℃ pre-denaturation for 4 min; 94℃ denaturation for 1 min, 50℃ annealing for 1 min, 72℃ extension for 2 min, 28 cycles; 72℃ extension for 10 min; 4℃ storage;

[0044] S2013. Using pEGFP-c2 plasmid as template, PCR amplified GFP nucleic acid sequence:

[0045] Using pEGFP-c2 plasmid as a template, PCR reaction was performed with upstream primer GFP1 and downstream primer GFP2 to amplify the GFP nucleic acid sequence to obtain a GFP PCR product; the nucleotide sequence of the upstream primer GFP1 is shown in SEQ ID NO:7, and the nucleotide sequence of the downstream primer GFP2 is shown in SEQ ID NO:8;

[0046] PCR reaction system: pEGFP-c2 plasmid 0.2 μL, upstream primer GFP 11 μL, downstream primer GFP 21 μL, LATaq 0.25 μL, 10×LATaq buffer 2.5 μL, ddH2O to 25 μL;

[0047] PCR reaction program: 95℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 30 s, 25 cycles; 72℃ extension for 5 min; 4℃ storage;

[0048] S2014, extract total RNA from Caenorhabditis elegans;

[0049] S2015, C. elegans RNA sequence was reverse transcribed to obtain cDNA;

[0050] S2016, using the cDNA obtained in S2015 as a template, PCR amplified RPS-30 S30 Nucleic acid sequence, the specific method is:

[0051] The cDNA obtained in S2015 was used as a template and PCR was performed with upstream primer s30F and downstream primer s30R to amplify RPS-30. S30 Nucleic acid sequence, RPS-30 S30 PCR product; the nucleotide sequence of the upstream primer s30F is shown in SEQ ID NO:9, and the nucleotide sequence of the downstream primer s30R is shown in SEQ ID NO:10;

[0052] PCR reaction system: cDNA 2 μL, upstream primer s30F 1 μL, downstream primer s30R 1 μL, LATaq 0.25 μL, 10×LATaq buffer 2.5 μL, ddH2O to 25 μL;

[0053] PCR reaction program: 95℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 30 s, 25 cycles; 72℃ extension for 5 min; 4℃ storage;

[0054] S202, the rps-30 promoter PCR product, GFP PCR product and RPS-30 obtained in S2012 were cloned by TA cloning. S30 The PCR product was connected to the pMD18-T simple plasmid and sequenced to obtain the pMD18-Tsimple-pRPS-30 plasmid, pMD18-T simple-GFP plasmid and pMD18-T simple-RPS-30 S30 Plasmid, the specific steps include:

[0055] S2021. The rps-30 promoter PCR product obtained in S201 was ligated into the pMD18-T simple plasmid by TA cloning at 16°C overnight to obtain the ligation product pMD18-T simple-pRPS-30.

[0056] At 16°C, the GFP PCR product obtained in S2013 was ligated into the pMD18-T simple plasmid by TA cloning overnight to obtain the ligation product pMD18-Tsimple-GFP;

[0057] At 16°C, RPS-30 obtained from S2016 was cloned by TA cloning. S30 The PCR products were connected to the pMD18-T simple plasmid overnight to obtain the connection product pMD18-Tsimple-RPS-30 S30 ;

[0058] Ligation reaction system: PCR product 4 μL, pMD18-T simple plasmid 0.5 μL, DNA ligation buffer Solution I 4.5 μL, ddH2O to 10 μL; the PCR product is rps-30 promoter PCR product or GFP PCR product or RPS-30 S30 PCR products;

[0059] S2022, conversion coated board:

[0060] Three Escherichia coli TOP10 competent bacteria were taken out at a storage temperature of -80°C. The connection product pMD18-T simple-pRPS-30 obtained in S2021 was added to the first Escherichia coli TOP10 competent bacteria. The connection product pMD18-T simple-GFP obtained in S2021 was added to the second Escherichia coli TOP10 competent bacteria. The connection product pMD18-T simple-RPS-30 obtained in S2021 was added to the third E. coli TOP10 competent bacteria. S30 , after being mixed evenly, ice-bathed for 30 minutes, then heat-stressed at 42°C for 90 seconds, ice-bathed for 2 minutes, added with 1 mL of LB liquid culture medium, cultured at 37°C and a shaking speed of 250 rpm / min for 1 hour, centrifuged to obtain the residual bacterial liquid after transformation, added dropwise to a solid LB culture plate containing ampicillin (AMP) resistance, and spread evenly, and placed in a bacterial incubator at 37°C for overnight culture;

[0061] S2023, Sequencing Identification:

[0062] Monoclonal colonies were picked from the solid LB culture plates cultured overnight in S2022, and then placed in 3 mL of LB liquid culture medium containing ampicillin (AMP) resistance, and cultured for 10 hours at 37°C and a shaking speed of 250 rpm / min. The obtained bacterial liquids were taken for sequencing identification, and the monoclonal colonies with correct sequencing results were screened out;

[0063] S2024, extract pMD18-T simple-pRPS-30 plasmid, pMD18-T simple-GFP plasmid and pMD18-Tsimple-RPS-30 S30 Plasmids:

[0064] The correctly sequenced monoclonal colonies obtained in S2023 were extracted using the AxyPrep Plasmid Miniprep Kit to obtain pMD18-T simple-pRPS-30 plasmid, pMD18-T simple-GFP plasmid and pMD18-T simple-RPS-30 plasmid, respectively. S30 Plasmids;

[0065] S203, the pMD18-T simple-pRPS-30 plasmid obtained in S2024 and pMD18-T simple-RPS-30 S30 The plasmids were double-digested with NheI / EcoRI restriction endonucleases, and the gel-recovered products were obtained by gel recovery. The gel-recovered products of NheI / EcoRI double-digestion were ligated to construct pMD18-T simple-pRPS-30::RPS-30. S30 Plasmid, the specific steps include:

[0066] Double digestion with S2031, NheI / EcoRI restriction enzymes;

[0067] At 37°C, the pMD18-Tsimple-pRPS-30 plasmid obtained in S2024 was double-digested with NheI / EcoRI restriction enzymes for 5 h to obtain the pMD18-T simple-pRPS-30 plasmid NheI / EcoRI double-digested product;

[0068] At 37°C, the pMD18-Tsimple-RPS-30 obtained in S2024 was cleaved with NheI / EcoRI restriction endonucleases. S30 The plasmid was subjected to double restriction digestion for 5 h to obtain pMD18-T simple-RPS-30 S30Plasmid NheI / EcoRI double digestion product;

[0069] Reaction system: pMD18-T simple-pRPS-30 plasmid or pMD18-T simple-RPS-30 S30 Plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, NheI restriction enzyme 1 μL, EcoRI restriction enzyme 1 μL, ddH2O to 20 μL;

[0070] S2032, gel recovery of NheI / EcoRI double digestion products:

[0071] The pMD18-T simple-pRPS-30 plasmid NheI / EcoRI double digestion product obtained in S2031 was subjected to 1% agarose gel electrophoresis, and the target DNA band of 4400 bp was cut out and recovered using the AxyPrep DNA Gel Extraction Kit gel recovery kit to obtain the pMD18-T simple-pRPS-30 plasmid NheI / EcoRI double digestion gel recovery product;

[0072] The pMD18-T simple-RPS-30 obtained in S2031 was S30 The plasmid NheI / EcoRI double digestion product was subjected to 1% agarose gel electrophoresis, and the 180 bp target DNA band was cut out and recovered using the AxyPrep DNA Gel Extraction Kit gel recovery kit to obtain pMD18-T simple-RPS-30 S30 The plasmid was double digested with NheI / EcoRI and the product was recovered from the gel;

[0073] S2033. At 16°C, the pMD18-T simple-pRPS-30 plasmid obtained in S2032 was digested with NheI / EcoRI and the recovered product was mixed with pMD18-T simple-RPS-30. S30 The plasmid NheI / EcoRI double digestion gel recovery product was connected overnight to obtain the connection product of the NheI / EcoRI double digestion gel recovery product:

[0074] Ligation reaction system: pMD18-T simple-pRPS-30 plasmid NheI / EcoRI double digestion gel recovery product 100ng, pMD18-T simple-RPS-30 S30After plasmid NheI / EcoRI double digestion, 500 ng of gel recovery product, 8 μL of LigationMix, and ddH2O were added to 25 μL;

[0075] S2034, conversion coated plate:

[0076] Take out the competent bacteria of Escherichia coli TOP10 from the storage temperature of -80°C, add the ligation product of the gel recovery product obtained in S2033 after NheI / EcoRI double digestion, mix evenly, ice bath for 30 minutes, then heat stress at 42°C for 90 seconds, ice bath for 2 minutes, add 1 mL of LB liquid culture medium for culture, culture at 37°C and a shaking speed of 250 rpm / min for 1 hour, obtain the residual bacterial liquid after transformation by centrifugation, drop the residual bacterial liquid after transformation into a solid LB culture plate containing ampicillin (AMP) resistance, spread evenly, and culture overnight in a bacterial incubator at 37°C;

[0077] S2035, Sequencing Identification:

[0078] Monoclonal colonies were picked from the solid LB culture plates cultured overnight in S2034, and then placed in 3 mL of LB liquid culture medium containing ampicillin (AMP) resistance, and cultured for 10 hours at 37°C and a shaking speed of 250 rpm / min. The obtained bacterial liquids were taken for sequencing and identification, and the monoclonal colonies with correct sequencing results were screened out;

[0079] S2036, extract pMD18-T simple-pRPS-30::RPS-30 S30 Plasmids;

[0080] The correctly sequenced monoclonal colony obtained in S2035 was extracted using the AxyPrep Plasmid MiniprepKit plasmid extraction kit to obtain pMD18-T simple-pRPS-30::RPS-30 S30 Plasmids;

[0081] S204, the pMD18-T simple-GFP plasmid obtained in S2024 and the pMD18-Tsimple-pRPS-30::RPS-30 obtained in S2036 were combined. S30 The plasmid was double digested with BglII / NheI restriction endonucleases, and the gel-recovered products after BglII / NheI double digestion were recovered separately. The gel-recovered products after BglII / NheI double digestion were ligated to construct pMD18-Tsimple-pRPS-30::GFP::RPS-30 S30Plasmid, the specific steps include:

[0082] Double digestion with S2041, BglII / NheI restriction endonuclease:

[0083] At 37°C, the pMD18-Tsimple-GFP plasmid obtained in S2024 was double-digested with BglII / NheI restriction enzymes for 5 h to obtain the pMD18-T simple-GFP plasmid BglII / NheI double-digested product;

[0084] At 37°C, the BglII / NheI restriction endonuclease was used to enzymatically cleave the pMD18-Tsimple-pRPS-30::RPS-30 obtained in S2036. S30 The plasmid was subjected to double restriction digestion for 5 h to obtain pMD18-T simple-pRPS-30::RPS-30 S30 Plasmid BglII / NheI double digestion product;

[0085] Reaction system: pMD18-T simple-GFP plasmid or pMD18-T simple-pRPS-30::RPS-30 S30 Plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, BglII restriction enzyme 1 μL, NheI restriction enzyme 1 μL, ddH2O to 20 μL;

[0086] S2042, gel recovery of BglII / NheI double digestion products:

[0087] The pMD18-T simple-GFP plasmid BglII / NheI double digestion product obtained in S2041 was subjected to 1% agarose gel electrophoresis, and the target DNA band of 720 bp was cut out and recovered using the AxyPrep DNA Gel Extraction Kit to obtain the pMD18-T simple-GFP plasmid BglII / NheI double digestion gel recovery product;

[0088] The pMD18-T simple-pRPS-30::RPS-30 obtained in S2041 was S30 The plasmid BglII / NheI double digestion product was subjected to 1% agarose gel electrophoresis, and the target DNA band of 4580 bp was cut out and recovered using the AxyPrep DNA Gel Extraction Kit gel recovery kit to obtain pMD18-T simple-pRPS-30::RPS-30 S30The plasmid was double digested with BglII / NheI and the product was recovered from the gel;

[0089] S2043. At 16°C, the pMD18-T simple-GFP plasmid obtained in S2042 was digested with BglII / NheI and the recovered products were mixed with pMD18-T simple-pRPS-30::RPS-30 S30 The plasmid BglII / NheI double digestion gel recovery product was ligated overnight to obtain the ligation product of the BglII / NheI double digestion gel recovery product;

[0090] Ligation reaction system: pMD18-T simple-GFP plasmid BglII / NheI double digestion gel recovery product 500ng, pMD18-T simple-pRPS-30::RPS-30 S30 After the plasmid BglII / NheI double digestion, the gel recovery product 100ng, Ligation Mix 8μL, ddH2O supplemented to 25μL;

[0091] S2044, conversion coated plate:

[0092] Take out the competent bacteria of Escherichia coli TOP10 from the storage temperature of -80°C, add the ligation product of the gel recovery product obtained after the BglII / NheI double digestion obtained in S2043, ice bath for 30 minutes, then heat stress at 42°C for 90 seconds, ice bath for 2 minutes, add 1 mL of LB liquid culture medium for culture, culture at 37°C and a shaking speed of 250 rpm / min for 1 hour, obtain the residual bacterial liquid after transformation by centrifugation, drop the residual bacterial liquid after transformation into a solid LB culture plate containing ampicillin (AMP) resistance, spread it evenly, and culture it in a bacterial incubator at 37°C overnight;

[0093] S2045, sequencing identification:

[0094] Monoclonal colonies were picked from the solid LB culture plates cultured overnight in S2044, and then placed in 3 mL of LB liquid culture medium containing ampicillin (AMP) resistance, and cultured for 10 hours at a temperature of 37° C. and a shaking speed of 250 rpm / min. The obtained bacterial liquids were taken for sequencing identification, and the monoclonal colonies with correct sequencing results were screened out;

[0095] S2046, extract pMD18-T simple-pRPS-30::GFP::RPS-30 S30 Plasmids;

[0096] The correctly sequenced monoclonal colony obtained in S2045 was extracted using the AxyPrep Plasmid MiniprepKit plasmid extraction kit to obtain pMD18-T simple-pRPS-30::GFP::RPS-30 S30 Plasmids;

[0097] S3. Construction of pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmid: pPD95.77 plasmid and pMD18-T simple-pRPS-30::GFP::RPS-30 obtained in S2046 S30 The plasmids were double digested with KpnI / EcoRI restriction endonucleases, and the gel-recovered products after KpnI / EcoRI double digestion were obtained. The gel-recovered products after KpnI / EcoRI double digestion were ligated to construct pPD95.77-pRPS-30::GFP::RPS-30. S30 Plasmid, the specific steps include:

[0098] Double digestion with S301, KpnI / EcoRI restriction endonuclease:

[0099] At 37°C, the pPD95.77 plasmid was double-digested with KpnI / EcoRI restriction enzymes for 5 hours to obtain the pPD95.77 plasmid KpnI / EcoRI double-digested product;

[0100] At 37°C, KpnI / EcoRI restriction endonucleases were used to enzymatically cleave the pMD18-Tsimple-pRPS-30::GFP::RPS-30 obtained from S2046. S30 The plasmid was subjected to double restriction digestion for 5 h to obtain pMD18-T simple-pRPS-30::GFP::RPS-30 S30 Plasmid KpnI / EcoRI double digestion product;

[0101] Double restriction enzyme digestion reaction system: pPD95.77 plasmid or pMD18-T simple-pRPS-30::GFP::RPS-30 S30 Plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, KpnI restriction enzyme 1 μL, EcoRI restriction enzyme 1 μL, ddH2O to 20 μL;

[0102] S302, gel recovery of KpnI / EcoRI double digestion products:

[0103] The pPD95.77 plasmid KpnI / EcoRI double digestion product obtained in S301 was subjected to 1% agarose gel electrophoresis, and the target DNA band of 3562 bp was cut out and recovered using the AxyPrep DNA Gel Extraction Kit to obtain the pPD95.77 plasmid KpnI / EcoRI double digestion gel recovery product;

[0104] The pMD18-T simple-pRPS-30::GFP::RPS-30 obtained in S301 was S30 The plasmid KpnI / EcoRI double digestion product was subjected to 1% agarose gel electrophoresis, and the target DNA band of 2900 bp was cut out and recovered using the AxyPrep DNA Gel Extraction Kit to obtain pMD18-T simple-pRPS-30::GFP::RPS-30 S30 The plasmid was double digested with KpnI / EcoRI and the product was recovered from the gel;

[0105] S303. At 16°C, the pPD95.77 plasmid obtained in S302 was digested with KpnI / EcoRI and the recovered product and pMD18-T simple-pRPS-30::GFP::RPS-30 were separated and then the gel was cleaned and ... S30 The plasmid KpnI / EcoRI double digestion gel recovery product was ligated overnight to obtain the KpnI / EcoRI double digestion gel recovery product ligation product;

[0106] Ligation reaction system: pPD95.77 plasmid KpnI / EcoRI double digestion gel recovery product 100ng, pMD18-Tsimple-pRPS-30::GFP::RPS-30 S30 After plasmid BglII / NheI double digestion, 500 ng of gel recovery product, 8 μL of LigationMix, and ddH2O were added to 25 μL;

[0107] S304, conversion coated plate:

[0108] Take out the competent bacteria of Escherichia coli TOP10 from the storage temperature of -80°C, add the ligation product of the gel recovery product obtained in S303 after KpnI / EcoRI double digestion, ice bath for 30 minutes, then heat stress at 42°C for 90 seconds, ice bath for 2 minutes, add 1 mL of LB liquid culture medium, culture at 37°C and a shaking speed of 250 rpm / min for 1 hour, obtain the residual bacterial liquid after transformation by centrifugation, drop the residual bacterial liquid after transformation into a solid LB culture plate containing ampicillin (AMP) resistance, spread it evenly, and culture it in a bacterial incubator at 37°C overnight;

[0109] S305, sequencing and identification;

[0110] Monoclonal colonies were picked from the solid LB culture plates cultured overnight in S304, and then placed in 3 mL of LB liquid culture medium containing ampicillin (AMP) resistance, and cultured for 10 hours at a temperature of 37° C. and a shaking speed of 250 rpm / min. The obtained bacterial liquids were taken for sequencing identification, and the monoclonal colonies with correct sequencing results were screened out;

[0111] S306, extract pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmids;

[0112] The correctly sequenced monoclonal colony obtained in S305 was extracted using the AxyPrep Plasmid MiniprepKit plasmid extraction kit to obtain pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmids;

[0113] S4. Construction of pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Plasmids:

[0114] pPD95.77-pRPS-30::GFP::RPS-30 obtained in S306 S30 The plasmid was the same as the pMD18-T simple-pU6::shRNA (RPS-30 UbL ) plasmid was double digested with SmaI / KpnI restriction endonucleases, and the SmaI / KpnI double digested gel-recovered products were obtained by gel ligation to obtain pPD95.77-pU6::shRNA(RPS-30 UbL)-pRPS-30::GFP::RPS-30 S30 Plasmid, i.e., transgenic plasmid that extends the healthy life span of Caenorhabditis elegans, the specific steps include:

[0115] Double digestion with S401, SmaI / KpnI restriction endonuclease:

[0116] At 37°C, the pPD95.77-pRPS-30::GFP::RPS-30 obtained in S306 was cleaved by SmaI / KpnI restriction endonucleases. S30 The plasmid was subjected to double restriction digestion for 5 h to obtain pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmid SmaI / KpnI double digestion product;

[0117] At 37°C, the pMD18-Tsimple-pU6::shRNA (RPS-30 UbL ) plasmid was subjected to double restriction digestion for 5 h to obtain pMD18-T simple-pU6::shRNA(RPS-30 UbL ) Plasmid SmaI / KpnI double digestion product;

[0118] Reaction system: pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmid or pMD18-T simple-pU6::shRNA(RPS-30 UbL ) plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, SmaI restriction enzyme 1 μL, KpnI restriction enzyme 1 μL, ddH2O to 20 μL;

[0119] S402, gel recovery SmaI / KpnI double digestion product:

[0120] The pPD95.77-pRPS-30::GFP::RPS-30 obtained in S401 was S30 Plasmid SmaI / KpnI double digestion product SmaI / KpnI double digestion product was subjected to 1% agarose gel electrophoresis, and the target DNA band of 6462 bp was cut out and recovered using AxyPrep DNA Gel Extraction Kit to obtain pPD95.77-pRPS-30::GFP::RPS-30 S30 The plasmid was double digested with SmaI / KpnI and the product was recovered from the gel;

[0121] The pMD18-T simple-pU6::shRNA (RPS-30 UbL ) plasmid SmaI / KpnI double digestion product was subjected to 1% agarose gel electrophoresis, and the target DNA band of 326 bp was cut out and recovered using AxyPrep DNA Gel Extraction Kit to obtain pMD18-T simple-pU6::shRNA(RPS-30 UbL ) Plasmid SmaI / KpnI double digestion and gel recovery product;

[0122] S403. At 16°C, pPD95.77-pRPS-30::GFP::RPS-30 obtained in S402 was added. S30 Plasmid SmaI / KpnI double digestion gel recovery products and pMD18-T simple-pU6::shRNA (RPS-30 UbL ) The plasmid SmaI / KpnI double digestion gel recovery product was ligated overnight to obtain a ligation product of the SmaI / KpnI double digestion gel recovery product;

[0123] Ligation reaction system: pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmid SmaI / KpnI double digestion gel recovery product 100ng, pMD18-T simple-pU6::shRNA(RPS-30 UbL ) After double digestion of plasmid SmaI / KpnI, 500 ng of gel recovery product, 8 μL of Ligation Mix, and ddH2O to 25 μL;

[0124] S404, conversion coated plate:

[0125] Take out the competent bacteria of Escherichia coli TOP10 from the storage temperature of -80°C, add the ligation product of the gel recovery product obtained in S403 after SmaI / KpnI double digestion, ice bath for 30 minutes, then heat stress at 42°C for 90 seconds, ice bath for 2 minutes, add 1 mL of LB liquid culture medium, culture at 37°C and a shaking speed of 250 rpm / min for 1 hour, obtain the residual bacterial liquid after transformation by centrifugation, drop the residual bacterial liquid after transformation into a solid LB culture plate containing ampicillin (AMP) resistance, spread it evenly, and culture it in a bacterial incubator at 37°C overnight;

[0126] S405, sequencing identification:

[0127] Monoclonal colonies were picked from the solid LB culture plates cultured overnight in S404, and then placed in 3 mL of LB liquid culture medium containing ampicillin (AMP) resistance, and cultured for 10 hours at 37°C and a shaking speed of 250 rpm / min. The obtained bacterial liquids were taken for sequencing identification, and the monoclonal colonies with correct sequencing results were screened out;

[0128] S406, extract pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Plasmids;

[0129] The correctly sequenced monoclonal colony obtained in S405 was extracted using the AxyPrep Plasmid Miniprep Kit plasmid extraction kit to obtain pPD95.77-pU6::shRNA (RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Plasmid, that is, a transgenic plasmid for extending the healthy life span of Caenorhabditis elegans; the pPD95.77-pU6::shRNA (RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The nucleotide sequence of the plasmid is shown in SEQ ID NO:11.

[0130] The present invention also provides the use of the above-mentioned transgenic plasmid for extending the healthy lifespan of Caenorhabditis elegans. The transgenic plasmid for extending the healthy lifespan of Caenorhabditis elegans is used to extend the healthy lifespan of Caenorhabditis elegans.

[0131] RPS-30 is a chimeric protein that is cleaved in the cytoplasm to produce the ubiquitin-like protein RPS-30 UbL and ribosomal protein RPS-30 S30 .RPS30 S30 Enters the cell nucleus to form the small ribosome subunit; RPS30 UbL The protein modifies the transcription factor ELT-2 through ubiquitination, thereby inhibiting the expression of the heterotypic forkhead comb transcription factor DAF-16 / FOXO, thereby inhibiting the lifespan of the worm. After RPS-30 knockdown or knockout, the worm body shows an unhealthy biological phenotype of extended lifespan, failure to lay eggs, and inability to develop into an adult. UbL Knockdown or knockout related; unhealthy phenotype of the worm is related to RPS30 S30 Therefore, in the present invention, we constructed a plasmid that has the function of regulating healthy life span. The principle is that while knocking down RPS-30, RPS-30 is restored in the worm body.S30 Protein ensures the integrity of ribosome function, allowing the insect to extend its lifespan while presenting a healthy phenotype.

[0132] In the face of the problem of longevity but unhealthy life in the era of longevity, the present invention successfully established a transgenic plasmid that extends the healthy lifespan of Caenorhabditis elegans, which is expected to provide a theoretical basis for the research on the regulation of human healthy lifespan.

[0133] The rps-30 gene involved in the present invention encodes ribosomal protein small subunit 30 (RPS-30). RPS-30 is a chimeric protein that is cleaved in the cytoplasm to produce ubiquitin-like protein (RPS-30 UbL ) and ribosomal protein S30 (RPS-30 S30 ), RPS-30 UbL The two glycine residues at the C-terminus of the protein are the cleavage sites of RPS-30. S30 Enters the cell nucleus and participates in the formation of the small ribosome subunit; while RPS-30 UbL The protein has biological functions such as regulating cell apoptosis and regulating immune cell activity through ubiquitination modification. UbL The life span of the worm can be inhibited by inhibiting the expression level of the transcription factor DAF-16. Therefore, RPS-30 can be knocked out or knocked down. UbL Prolong the life of the insect body.

[0134] RPS-30 UbL As part of the chimeric protein, RPS-30 UbL Both with RPS-30 S30 The nucleic acid sequence of the worm is fused to form the rps-30 gene. The rps-30 gene is an essential gene for the worm. After knocking it out or knocking it down, the worm shows an unhealthy lifespan extension: the worm cannot develop into an adult and cannot lay eggs. The generation of unhealthy phenotypes is related to RPS-30. S30 Therefore, we further investigated the RPS-30 knockout strains. S30 The results showed that the insects showed a healthy and long-lived phenotype. Therefore, in the present invention, we constructed a transgenic plasmid that can knock down the rps-30 gene and simultaneously regulate RPS-30. S30 The plasmid was transfected into the wild-type standard strain N2 of C. elegans by microinjection, and the results showed that the life span of the worm body was significantly prolonged, and there was no significant difference in the worm body development, egg laying, and movement ability. Therefore, the present invention successfully constructed a transgenic plasmid that extends the healthy life span of C. elegans, and the plasmid can be applied to the research field of healthy longevity of C. elegans.

[0135] Compared with the prior art, the present invention has the following advantages:

[0136] The present invention successfully constructed a transgenic plasmid pPD95.77-pU6::shRNA (RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 , used to extend the healthy lifespan of C. elegans. The plasmid was transfected into the wild-type standard strain N2 of C. elegans by microinjection, which significantly extended the lifespan of the worm, and there was no significant difference in the worm's development, egg-laying, and motility.

[0137] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] Figure 1 The transgenic plasmid pPD95.77-pU6::shRNA (RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The physical map of .

[0139] Figure 2 is pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Actual image of the transgenic insect taken under a fluorescence microscope.

[0140] Figure 3 This is a relative quantitative analysis of gene transcription levels in transgenic insects in Example 2 of the present invention.

[0141] Figure 4 This is a graph showing the life span of transgenic insects in Example 2 of the present invention.

[0142] Figure 5 Detection diagram of the egg-laying amount of transgenic insects in Example 2 of the present invention.

[0143] Figure 6 Detection diagram of transgenic insect body length in Example 2 of the present invention.

[0144] Figure 7 A diagram showing the ability of transgenic insects to resist heat stress in Example 2 of the present invention.

[0145] Figure 8 Detection diagram of oxidative stress in transgenic insects according to Example 2 of the present invention. DETAILED DESCRIPTION

[0146] Example 1

[0147] This example is a transgenic plasmid pPD95.77-pU6::shRNA (RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Methods for constructing plasmids.

[0148] 1. Main reagents

[0149] Escherichia coli TOP10 competent strain, pPD95.77 Caenorhabditis elegans expression vector pLKO.1puro plasmid (Addgene#: 8453), pEGFP-c2 plasmid (GenBank Accession#: U57606) were stored in our laboratory, cloning vector pMD18-TVector (Simple); restriction endonucleases AgeI, SmaI, KpnI, BglII, NheI, EcoRI; DNA ligase; LATaq DNA polymerase; DNA Marker (3427Q); DNA Ligation Kit (DNA Ligation Kit<Mighty Mix> ) were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. (TaKaRa). Agarose and Tris were purchased from Promega; peptone and yeast extract were purchased from OXOID; mouse reverse transcriptase (M-MLV) and TRIzol were purchased from Invitrogen. AxyPrep Plasmid Miniprep Kit and AxyPrep DNA Gel Extraction Kit were purchased from Axygen.

[0150] 2. Main instruments and equipment

[0151] PCR instrument, gel imaging system, desktop centrifuge (Japan), constant temperature water bath, biochemical incubator.

[0152] 3. Main solution

[0153] Preparation of lysogeny broth (LB) medium: Use an analytical balance to accurately weigh 10 g of tryptone, 5 g of yeast extract powder and 5 g of NaCl. To prepare LB solid medium, add 15 g of agar, make up to 1 L with ddH2O and mix well. Use 1 M NaOH to adjust the pH to 7.0 (liquid culture) or 7.5 (solid culture medium).

[0154] 4. Primer design

[0155] Referring to the U6 promoter sequence and its downstream nucleotide sequence in the pLKO.1 puro plasmid (including AgeI and EcoRI restriction endonuclease sites), the upstream primer U6P1 and the downstream primer U6P2 were designed for cloning the U6 promoter region, and the restriction sites SmaI and KpnI were added at both ends, respectively; referring to the rps-30 gene sequence in the NCBI database (NM_072606.7), the rps-30 ubL The interference sequence primers UbLshR1 and UbLshR2 in the sequence region were annealed to form AgeI and EcoRI restriction sites at both ends; referring to the genome sequence of Caenorhabditis elegans, the upstream primer rps30P1 and the downstream primer rps30P2 were designed, and the restriction sites KpnI and EcoRI were added at both ends to clone the 2000bp sequence of the rps-30 promoter region (2000bp upstream of the start codon ATG of the rps-30 gene), and the BglII and NheI restriction sites were added to the downstream primer to insert the expression sequence to be promoted; referring to the GFP (green The upstream primer GFP1 and the downstream primer GFP2 were designed based on the nucleic acid sequence of GFP, and the restriction sites BglII and NheI were added at both ends respectively for cloning the GFP gene sequence. At the same time, the Kozak sequence (ggtagaaaaa) was added to the upstream primer GFP1 to enhance gene expression, and the GFP gene termination codon was removed from the downstream primer to facilitate the fusion expression of GFP and the subsequent protein; referring to the rps-30 gene sequence in the NCBI database, the upstream primer s30F and the downstream primer s30R were designed, and the restriction sites NheI and EcoRI were added at both ends respectively for cloning the rps-30 gene sequence. S30 Gene sequence, and a transcription termination signal sequence (tacgaatg) was added to the downstream primer.

[0156] The nucleotide sequence of the upstream primer U6P1 is shown in SEQ ID NO: 1;

[0157] The nucleotide sequence of the downstream primer U6P2 is shown in SEQ ID NO: 2;

[0158] The nucleotide sequence of the primer UbLshR1 is shown in SEQ ID NO: 3;

[0159] The nucleotide sequence of the primer UbLshR2 is shown in SEQ ID NO:4;

[0160] The nucleotide sequence of the upstream primer rps30P1 is shown in SEQ ID NO: 5;

[0161] The nucleotide sequence of the downstream primer rps30P2 is shown in SEQ ID NO:6;

[0162] The nucleotide sequence of the upstream primer GFP1 is shown in SEQ ID NO:7;

[0163] The nucleotide sequence of the downstream primer GFP2 is shown in SEQ ID NO: 8;

[0164] The nucleotide sequence of the upstream primer s30F is shown in SEQ ID NO:9;

[0165] The nucleotide sequence of the downstream primer s30R is shown in SEQ ID NO:10.

[0166] Construction of transgenic plasmid pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The physical map of Figure 1 shown.

[0167] 5. Plasmid construction:

[0168] S1. Construction of pMD18-T simple-pU6::shRNA(RPS-30 UbL )Plasmid:

[0169] S101, using pLKO.1puro plasmid (Addgene#: 8453) as a template, amplifying the U6 promoter sequence by PCR, and cloning the U6 promoter PCR product into the pMD18-T simple plasmid by TA cloning to construct the pMD18-T simple-pU6 plasmid, and then performing AgeI / EcoRI restriction endonuclease double digestion on the obtained pMD18-T simple-pU6 plasmid to obtain AgeI / EcoRI double digestion product, and performing gel recovery on the AgeI / EcoRI double digestion product, the specific steps include:

[0170] S1011. Using pLKO.1puro plasmid as a template, performing PCR reaction with upstream primer U6P1 and downstream primer U6P2 to amplify U6 promoter, and obtaining U6 promoter PCR product; the nucleotide sequence of the upstream primer U6P1 is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer U6P2 is shown in SEQ ID NO: 2;

[0171] The PCR reaction system was: pLKO.1puro plasmid 0.2 μL, upstream primer U6P1 1 μL, downstream primer U6P2 1 μL, LATaq 0.25 μL, 10×LATaq buffer 2.5 μL, ddH2O to 25 μL;

[0172] The PCR reaction program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, 25 cycles; extension at 72°C for 5 min; storage at 4°C;

[0173] S1012. At 16°C, the U6 promoter PCR product obtained in S1011 was ligated into the pMD18-T simple plasmid overnight by TA cloning to obtain the TA cloning ligation product pMD18-T simple-pU6.

[0174] Ligation reaction system: 4 μL of U6 promoter PCR product, 0.5 μL of pMD18-T simple plasmid, 4.5 μL of DNA ligation buffer Solution I, and ddH2O to 10 μL;

[0175] S1013, conversion coated plate;

[0176] Take out the competent bacteria of Escherichia coli TOP10 from the -80℃ storage temperature, put them on ice to melt, add the TA cloning ligation product pMD18-T simple-pU6 obtained in S1012, gently blow and mix, ice bath for 30min, heat stress at 42℃ for 90s, then ice bath for 2min, add 1mL The culture was cultured in LB liquid medium (1 g / 100 mL sodium chloride; 1 g / 100 mL tryptone; 0.5 g / 100 mL yeast extract), shaken and cultured at 37°C and 250 rpm / min for 1 h, centrifuged at 4000 g for 4 min, the supernatant was discarded, and about 30 μL of residual bacterial solution after transformation was left. The precipitate was gently beaten until the residual bacterial solution after transformation was uniform, and the residual bacterial solution after transformation was added dropwise to a solid LB culture plate containing ampicillin resistance (AMP) (1 g / 100 mL sodium chloride; 1 g / 100 mL tryptone; 0.5 g / 100 mL yeast extract; 1.5 g / 100 mL agar; 50 mM AMP), and evenly spread, and placed in a bacterial incubator at 37°C for overnight culture;

[0177] S1014, Sequencing Identification:

[0178] Monoclonal colonies were picked from the solid LB culture plate cultured overnight in S1013, and placed in 3 mL of LB liquid culture medium containing ampicillin resistance, and cultured for 10 h at 37°C and 250 rpm / min on a shaking table; 1 mL of bacterial solution was taken and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and monoclonal colonies with correct sequencing results were screened out;

[0179] S1015. Extract pMD18-T simple-pU6 plasmid:

[0180] AxyPrep Plasmid Miniprep Kit was used according to the plasmid extraction instructions of the kit: according to the sequencing results in S1014, the monoclonal colonies with correct sequencing were selected, 1 mL of bacterial solution was placed in a 1.5 mL EP tube (centrifuge tube), centrifuged at 12000 g for 1 min, the supernatant was discarded, 250 μL of S1 solution (suspension solution) was added, and the mixture was mixed by pipetting, 250 μL of S2 solution (lysis solution) was added, and the mixture was mixed by gentle inversion for 6-8 times, 350 μL of S3 solution (neutralization solution) was added, and the mixture was mixed by gentle inversion for 6-8 times, the supernatant was added to the adsorption column, and the mixture was centrifuged at 12000 g for 10 min, the supernatant was added to the adsorption column, and the mixture was centrifuged at 12000 g for 1 min, the filtrate was discarded, 500 μL of washing solution W1 buffer was added, the mixture was centrifuged at 12000 g for 1 min, the filtrate was discarded, 700 μL of washing solution W2 buffer was added to wash twice, the mixture was centrifuged at 12000 g for 1 min, and the filtrate was discarded; the adsorption column was placed in a new 1.5 mL On the EP tube, open the lid and dry for 3 min, add 50 μL ddH2O, let stand at room temperature for 5 min, centrifuge at 12000g for 1 min, obtain the pMD18-T simple-pU6 plasmid, and measure the plasmid concentration, which is 265 ng / μL;

[0181] Double digestion with S1016, AgeI / EcoRI restriction endonuclease:

[0182] The pMD18-T simple-pU6 plasmid obtained in S1015 was subjected to AgeI / EcoRI double digestion reaction at 37°C for 5 h to obtain AgeI / EcoRI double digestion product;

[0183] Reaction system: pMD18-T simple-pU6 plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, AgeI restriction endonuclease 1 μL, EcoRI restriction endonuclease 1 μL, ddH2O to 20 μL;

[0184] S1017, gel recovery of AgeI / EcoRI double digestion products:

[0185] Use AxyPrep DNA Gel Extraction Kit according to the kit gel recovery instructions: 20 μL of the AgeI / EcoRI double digestion product obtained in S1016 was subjected to 1% agarose gel electrophoresis, and the target DNA band (270 bp) was cut out. The gel block was about 100-150 mg and placed in 1.5 mL Add 300-450 μL DE-A to an EP tube, place the EP tube in a water bath at 75°C for 10 min (mix once every 2-3 min), then add DE-B and shake for 1 min; transfer the liquid to a recovery column placed in a 2 mL centrifuge tube, centrifuge at 12000 g for 1 min, discard the liquid in the centrifuge tube, wash once with 500 μL buffer W1, centrifuge at 12000 g for 1 min, discard the liquid in the centrifuge tube; wash twice with 700 μL buffer W2, centrifuge at 12000 g for 1 min, discard the liquid in the centrifuge tube; empty at 12000 g for 2 min, discard the centrifuge tube, place the recovery column in a new 1.5 mL EP tube, add 25 μL ddH2O, incubate at room temperature for 2 min, then centrifuge at 12000 g for 1 min, obtain a DNA recovery product in the 1.5 mL EP tube, and store the DNA recovery product at -40°C for later use; the volume ratio of DE-B to DE-A is 1:2;

[0186] S102, annealing primer UbLshR1 and primer UbLshR2 to obtain an annealing product, i.e., targeting RPS-30 UbL Short hairpin RNA of the region: shRNA (RPS-30 UbL ); the nucleotide sequence of the primer UbLshR1 is shown in SEQ ID NO:3, and the nucleotide sequence of the primer UbLshR2 is shown in SEQ ID NO:4;

[0187] Annealing system: primer UbLshR1 1.5 μL, primer UbLshR2 1.5 μL, 10×NEBbuffer II 5 μL, ddH2O to 50 μL;

[0188] Annealing procedure: pre-denaturation at 95°C for 5 min; denaturation at 70°C for 15 min; decrease the temperature to 22°C at a rate of 0.1°C per second; store at -40°C;

[0189] S103, the gel recovery product obtained in S101 after the AgeI / EcoRI double digestion was connected with the annealing product obtained in S102 to construct pMD18-T simple-pU6::shRNA (RPS-30 UbL ) plasmid, the specific steps include:

[0190] S1031, at 16°C, using DNA Ligation Kit<Mighty Mix> The AgeI / EcoRI double digestion gel recovery product obtained in S101 was ligated overnight with the annealing product obtained in S102 to obtain the ligation product pMD18-Tsimple-pU6::shRNA(RPS-30 UbL );

[0191] Ligation reaction system: 100 ng of gel recovery product after AgeI / EcoRI double digestion, 500 ng of annealing product, 8 μL of LigationMix, and 25 μL of ddH2O;

[0192] S1032, conversion coated plate:

[0193] Take out the competent bacteria of E. coli Stbl3 at -80°C, put them on ice to thaw, and then add 25 μL of the ligation product pMD18-T simple-pU6::shRNA (RPS-30 UbL ) was added thereto, gently beaten to mix, and placed in an ice bath for 30 minutes; heat stressed at 42°C for 90 seconds, then placed in an ice bath for 2 minutes, and 1 mL of LB liquid culture medium was added for culture, and the culture was carried out at 37°C and 250 rpm / min on a shaking table for 1 hour; centrifuged at 4000 g for 4 minutes, the supernatant was discarded, and about 30 μL of residual bacterial solution was left, and the precipitate was gently beaten until the bacterial solution was uniform, and the bacterial solution was added dropwise to a solid LB culture plate containing AMP resistance, and spread evenly, and placed in a 37°C bacterial incubator for overnight culture;

[0194] S1033, sequencing identification:

[0195] Monoclonal colonies were picked from the solid LB culture plate cultured overnight in S1032, and placed in 3 mL of liquid LB culture medium containing ampicillin resistance, and cultured for 10 h at 37°C and 250 rpm / min on a shaking table; 1 mL of bacterial solution was taken and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and monoclonal colonies with correct sequencing results were screened out;

[0196] S1034, extract pMD18-T simple-pU6::shRNA (RPS-30 UbL )Plasmid:

[0197] Use AxyPrep Plasmid MiniprepKit according to the plasmid extraction instructions of the kit: According to the sequencing results of S1033, select the monoclonal colony with correct sequencing, take 1mL of bacterial solution in a 1.5mL EP tube, centrifuge at 12000g for 1min, discard the supernatant, add 250μL S1 solution, beat to mix, add 250μL S2 solution, gently invert and mix 6-8 times, add 350μL S3 solution, gently invert and mix 6-8 times, centrifuge at 12000g for 10min, take the supernatant to the adsorption column, centrifuge at 12000g for 1min, discard the filtrate, add 500μL W1 buffer, centrifuge at 12000g for 1min, discard the filtrate, add 700μL W2 buffer to wash twice, centrifuge at 12000g for 1min, discard the filtrate; place the adsorption column in a new 1.5mL EP tube, open the lid and dry for 3 min, add 50 μL ddH2O, let stand at room temperature for 5 min, centrifuge at 12000 g for 1 min, and obtain pMD18-T simple-pU6::shRNA (RPS-30 UbL ) plasmid, and the plasmid concentration was measured to be 280 ng / μL;

[0198] S2. Construction of pMD18-T simple-pRPS-30::GFP::RPS-30 S30 Plasmids:

[0199] S201, using the genomic DNA sequence of Caenorhabditis elegans as a template, amplifying the rps-30 promoter sequence by PCR to obtain the rps-30 promoter PCR product; using the pEGFP-c2 plasmid (GenBank Accession#: U57606) as a template, amplifying the GFP nucleic acid sequence by PCR to obtain the GFP PCR product; using the cDNA obtained by reverse transcription of the RNA sequence of Caenorhabditis elegans as a template, amplifying the RPS-30 promoter by PCR S30 Nucleic acid sequence, RPS-30 S30 PCR products, the specific steps include:

[0200] S2011. Extraction of Caenorhabditis elegans genomic DNA:

[0201] Use the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (code: 9765) to extract genomic DNA. Follow the instructions of the kit as follows:

[0202] a. Take 20 mg of Caenorhabditis elegans and place it in a 1.5 mL EP tube. Grind it into a homogenate using a grinding rod on ice.

[0203] b. Add 180 μL Buffer GL, 20 μL Proteinase K, and 10 μL 10 mg / mL RNase A, mix gently, and incubate in a 56°C water bath for 2 h until the tissue is completely lysed;

[0204] c. The lysed solution was centrifuged at 12000g for 2 min to remove impurities, and the supernatant was transferred to a new 1.5 mL EP tube to obtain the lysate;

[0205] d. Add 200 μL Buffer GB and 200 μL anhydrous ethanol to the lysate and mix thoroughly by pipetting;

[0206] e. Place the Spin column on the collection tube, transfer the solution to the Spin column, centrifuge at 12000g for 2 minutes, and discard the filtrate;

[0207] f. Add 500 μL of Buffer WA to the spin column, centrifuge at 12000 g for 1 min, and discard the filtrate;

[0208] g. Add 700 μL of Buffer WB to the spin column, centrifuge at 12000 g for 1 min, discard the filtrate, and wash again with Buffer WB;

[0209] h. Place the spin column on the collection tube and spin at 12000g for 2 min;

[0210] i. Place the Spin column on a new 1.5 mL EP tube, add 100 μL Elution Buffer to the center of the Spin column membrane, and let stand at room temperature for 5 minutes;

[0211] j. Centrifuge at 12000g for 2 min to elute the genomic DNA of Caenorhabditis elegans;

[0212] S2012. Using the C. elegans genomic DNA obtained in S2011 as a template, PCR amplified the rps-30 promoter sequence:

[0213] Using Caenorhabditis elegans genomic DNA as a template, an upstream primer rps30P1 and a downstream primer rps30P2 were used to perform a PCR reaction to amplify the rps-30 promoter sequence, thereby obtaining an rps-30 promoter PCR product; the nucleotide sequence of the upstream primer rps30P1 is shown in SEQ ID NO:5, and the nucleotide sequence of the downstream primer rps30P2 is shown in SEQ ID NO:6;

[0214] PCR reaction system: 2 μL of C. elegans genomic DNA, 1 μL of upstream primer rps30P1, 1 μL of downstream primer rps30P2, 0.25 μL of LATaq, 2.5 μL of 10×LATaq buffer, and ddH2O to 25 μL;

[0215] PCR reaction program: 95℃ pre-denaturation for 4 min; 94℃ denaturation for 1 min, 50℃ annealing for 1 min, 72℃ extension for 2 min, 28 cycles; 72℃ extension for 10 min; 4℃ storage;

[0216] S2013, using pEGFP-c2 plasmid as template, PCR amplified GFP nucleic acid sequence;

[0217] Using pEGFP-c2 plasmid as a template, PCR reaction was performed with upstream primer GFP1 and downstream primer GFP2 to amplify the GFP nucleic acid sequence to obtain a GFP PCR product; the nucleotide sequence of the upstream primer GFP1 is shown in SEQ ID NO:7, and the nucleotide sequence of the downstream primer GFP2 is shown in SEQ ID NO:8;

[0218] PCR reaction system: pEGFP-c2 plasmid 2 μL, upstream primer GFP 11 μL, downstream primer GFP 21 μL, LATaq 0.25 μL, 10×LATaq buffer 2.5 μL, ddH2O to 25 μL;

[0219] PCR reaction program: 95℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 30 s, 25 cycles; 72℃ extension for 5 min; 4℃ storage;

[0220] S2014. Extraction of total RNA from Caenorhabditis elegans:

[0221] a. Take 20-40 mg of C. elegans and place it in a 1.5 mL EP tube. Grind it into a homogenate using a grinding rod on ice.

[0222] b. Add 500 μL TRIzol reagent and shake vigorously for 1 min, then let stand at room temperature for 10 min;

[0223] c. Add 400 μL of chloroform, shake vigorously for 1 min, and let stand at room temperature for 10 min;

[0224] d. Centrifuge at 12000g for 15 min at 4°C, and transfer the supernatant into a new 1.5 mL EP tube;

[0225] e. Add isopropanol until the EP tube is full, and let it stand at -20℃ for 30 minutes;

[0226] f. Centrifuge at 13000g for 15 min at 4°C. The precipitate at the bottom of the EP tube is the extracted total RNA of Caenorhabditis elegans.

[0227] g. Discard the liquid, let the RNA dry naturally for about 5 minutes, add 20 μL RNase Free ddH2O, blow up and dissolve the precipitate, and store at -80℃ for later use:

[0228] S2015, C. elegans RNA sequence was reverse transcribed to obtain cDNA;

[0229] a. Take out the total RNA of C. elegans at -80℃, melt it on ice, and pipette 19μL into a 0.5mL EP tube;

[0230] b. Add 4 μL dNTP and 4 μL random primers to the EP tube, mix well and place in a water bath at 70°C for 5 min;

[0231] c. Take out the EP tube and place it on ice to cool for 1-2 minutes, then add 4 μL DDT and 8 μL 5× First-Strand Buffer respectively, mix well and place in a water bath at 37°C for 1 minute;

[0232] d. Add 1 μL of M-MLV reverse transcriptase to the EP tube, mix gently, incubate at 37°C for 3 h, and perform reverse transcription;

[0233] e. After reverse transcription, place the EP tube in a water bath at 70°C for 10 minutes, and then cool it on ice for 2 minutes to obtain cDNA. Store the product cDNA at -20°C for later use;

[0234] S2016, using the cDNA obtained in S2015 as a template, PCR amplified RPS-30 S30 Nucleic acid sequence, the specific method is:

[0235] The cDNA obtained in S2015 was used as a template and PCR was performed with upstream primer s30F and downstream primer s30R to amplify RPS-30. S30 Nucleic acid sequence, RPS-30 S30 PCR product; the nucleotide sequence of the upstream primer s30F is shown in SEQ ID NO:9, and the nucleotide sequence of the downstream primer s30R is shown in SEQ ID NO:10;

[0236] PCR reaction system: cDNA 2 μL, upstream primer s30F 1 μL, downstream primer s30R 1 μL, LA Taq 0.25 μL, 10×LA Taq buffer 2.5 μL, ddH2O to 25 μL;

[0237] PCR reaction program: 95℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 30 s, 25 cycles; 72℃ extension for 5 min; 4℃ storage;

[0238] S202, the rps-30 promoter PCR product, GFP PCR product and RPS-30 S30 The PCR product was connected to the pMD18-T simple plasmid and sequenced to obtain the pMD18-Tsimple-pRPS-30 plasmid, pMD18-T simple-GFP plasmid and pMD18-T simple-RPS-30 S30 Plasmid, the specific steps include:

[0239] S2021. The rps-30 promoter PCR product obtained in S201 was ligated into the pMD18-T simple plasmid by TA cloning at 16°C overnight to obtain the ligation product pMD18-T simple-pRPS-30.

[0240] At 16°C, the GFP PCR product obtained in S2013 was ligated into the pMD18-T simple plasmid (TaKaRa Code: D103A) by TA cloning overnight to obtain the ligation product pMD18-T simple-GFP.

[0241] At 16°C, RPS-30 obtained from S2016 was cloned by TA cloning. S30 The PCR products were connected to the pMD18-T simple plasmid overnight to obtain the connection product pMD18-Tsimple-RPS-30 S30 ;

[0242] Ligation reaction system: PCR product 4 μL, pMD18-T simple plasmid 0.5 μL, DNA ligation buffer Solution I 4.5 μL, ddH2O to 10 μL; the PCR product is rps-30 promoter PCR product or GFP PCR product or RPS-30 S30 PCR products;

[0243] S2022, conversion coated board:

[0244] Three Escherichia coli TOP10 competent bacteria were taken out at a storage temperature of -80°C. The ligation product pMD18-T simple-pRPS-30 obtained in S2021 was added to the first Escherichia coli TOP10 competent bacteria. The ligation product pMD18-T simple-GFP obtained in S2021 was added to the second Escherichia coli TOP10 competent bacteria. The ligation product pMD18-T simple-RPS-30 obtained in S2021 was added to the third Escherichia coli TOP10 competent bacteria. S30 , gently blow and mix them evenly, and then put them in an ice bath for 30 minutes; heat stress at 42°C for 90 seconds, then put them in an ice bath for 2 minutes, add 1 mL of LB liquid culture medium and culture them at 37°C and 250 rpm / min on a shaking table for 1 hour; centrifuge at 4000g for 4 minutes, discard the supernatant, and obtain the residual bacterial liquid after transformation by centrifugation. Gently beat the precipitate until the bacterial liquid is uniform, and add the residual bacterial liquid after transformation to the solid LB culture plate containing ampicillin resistance, spread it evenly, and place it in a bacterial incubator at 37°C for overnight culture;

[0245] S2023, Sequencing Identification:

[0246] Monoclonal colonies were picked from the solid LB culture plates cultured overnight in S2022, respectively, and placed in 3 mL of liquid LB culture medium containing AMP resistance, and cultured for 10 hours at 37°C and 250 rpm / min on a shaking table; 1 mL of bacterial solution was taken and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and monoclonal colonies with correct sequencing results were screened out;

[0247] S2024, extract pMD18-T simple-pRPS-30 plasmid, pMD18-T simple-GFP plasmid and pMD18-Tsimple-RPS-30 S30 Plasmids:

[0248] Use AxyPrep Plasmid Miniprep Kit according to the instructions for plasmid extraction: According to the S2023 sequencing results, select the correctly sequenced monoclonal colonies, take 1 mL of bacterial solution in 1.5 mL EP tubes, centrifuge at 12000g for 1 min, discard the supernatant, add 250 μL S1 solution, mix by pipetting, add 250 μL S2 solution, mix by gentle inversion 6-8 times, add 350 μL S3 solution, mix by gentle inversion 6-8 times, centrifuge at 12000g for 10 min, take the supernatant to the adsorption column, centrifuge at 12000g for 1 min, discard the filtrate, add 500 μL W1 buffer, centrifuge at 12000g for 1 min, discard the filtrate, add 700 μL W2 buffer, wash twice, centrifuge at 12000g for 1 min, discard the filtrate, and place the adsorption column in a new 1.5 mL EP tube, open the lid and dry for 3 minutes, add 50μL ddH2O, stand at room temperature for 5 minutes, centrifuge at 12000g for 1 minute, and obtain pMD18-T simple-pRPS-30 plasmid, pMD18-T simple-GFP plasmid, pMD18-T simple-RPS-30 S30 plasmid, and the plasmid concentration was measured to be 273 ng / μL;

[0249] S203, the pMD18-T simple-pRPS-30 plasmid obtained in S202 and pMD18-T simple-RPS-30 S30 The plasmid was double digested with NheI / EcoRI restriction endonucleases, and the gel-recovered product obtained by NheI / EcoRI double digestion was ligated to construct pMD18-T simple-pRPS-30::RPS-30. S30 Plasmid, the specific steps include:

[0250] Double digestion with S2031, NheI / EcoRI restriction endonuclease:

[0251] At 37°C, the pMD18-Tsimple-pRPS-30 plasmid obtained in S2024 was double-digested with NheI / EcoRI restriction enzymes for 5 h to obtain the pMD18-T simple-pRPS-30 plasmid NheI / EcoRI double-digested product;

[0252] At 37°C, the pMD18-Tsimple-RPS-30 obtained in S2024 was cleaved with NheI / EcoRI restriction enzymes. S30 The plasmid was subjected to double restriction digestion for 5 h to obtain pMD18-T simple-RPS-30S30 Plasmid NheI / EcoRI double digestion product;

[0253] Reaction system: pMD18-T simple-pRPS-30 plasmid or pMD18-T simple-RPS-30 S30 Plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, NheI restriction enzyme 1 μL, EcoRI restriction enzyme 1 μL, ddH2O to 20 μL;

[0254] S2032, gel recovery of NheI / EcoRI double digestion products:

[0255] AxyPrep DNA Gel Extraction Kit was used according to the kit gel recovery instructions: 20 μL of NheI / EcoRI double digestion product obtained in S2031 was subjected to 1% agarose gel electrophoresis to cut out the target DNA band (4400 bp); pMD18-T simple-RPS-30 S30 The plasmid NheI / EcoRI double digestion product was subjected to 1% agarose gel electrophoresis, and the target DNA band (180 bp) was cut out. The gel block is about 100-150 mg, which is placed in a 1.5 mL EP tube, and 300-450 μL DE-A is added. The EP tube is placed in a 75°C water bath for 10 min (mixed every 2-3 min), DE-B is added to the EP tube and shaken for 1 min; the liquid is transferred to a recovery column placed in a 2 mL centrifuge tube, centrifuged at 12000 g for 1 min, the liquid in the centrifuge tube is discarded, and the column is washed once with 500 μL buffer W1, centrifuged at 12000 g for 1 min, and the liquid in the centrifuge tube is discarded; the column is washed twice with 700 μL buffer W2, centrifuged at 12000 g for 1 min, and the liquid in the centrifuge tube is discarded; the column is emptied at 12000 g for 2 min, the centrifuge tube is discarded, and the recovery column is placed in a new 1.5 mL EP tube, 25 μL ddH2O is added, incubated at room temperature for 2 min, centrifuged at 12000 g for 1 min, and 1.5 mL The DNA recovery product was obtained in the EP tube; the DNA recovery product was stored at -40°C for later use; the volume ratio of DE-B to DE-A was 1:2;

[0256] After recovery, the pMD18-T simple-pRPS-30 plasmid NheI / EcoRI double digestion gel recovery product and pMD18-T simple-RPS-30 were obtained. S30 The plasmid was double digested with NheI / EcoRI and the product was recovered from the gel;

[0257] S2033, at 16°C, using DNA Ligation Kit<Mighty Mix> The pMD18-T simple-pRPS-30 plasmid obtained from S2032 was ligated overnight and the gel-recovered product after NheI / EcoRI double digestion was connected to pMD18-Tsimple-RPS-30 S30 The plasmid was double digested with NheI / EcoRI and the product was recovered from the gel to obtain the ligation product of the product recovered from the gel after double digestion with NheI / EcoRI:

[0258] Ligation reaction system: pMD18-T simple-pRPS-30 plasmid NheI / EcoRI double digestion gel recovery product 100ng, pMD18-T simple-RPS-30 S30 After plasmid NheI / EcoRI double digestion, 500 ng of gel recovery product, 8 μL of LigationMix, and ddH2O were added to 25 μL;

[0259] S2034, conversion coated plate:

[0260] Take out the competent bacteria of Escherichia coli TOP10 at -80°C, place them on ice to melt, add 25 μL of the ligation product of the gel recovery product obtained after NheI / EcoRI double digestion obtained in S2033, gently blow and mix, and place on ice for 30 minutes; heat stress at 42°C for 90 seconds, then place on ice for 2 minutes, add 1 mL of LB liquid culture medium, and culture at 37°C and 250 rpm / min on a shaking table for 1 hour; centrifuge at 4000g for 4 minutes, discard the supernatant, leave 30 μL of residual bacterial solution, gently beat the precipitate until the bacterial solution is uniform, add the bacterial solution dropwise to the solid LB culture medium containing ampicillin resistance, spread it evenly, and place it in a bacterial incubator at 37°C for overnight culture;

[0261] S2035, Sequencing Identification:

[0262] Monoclonal colonies were picked from the solid LB medium cultured overnight in S2034, and placed in 3 mL of liquid LB medium containing AMP resistance, respectively, and cultured for 10 h at 37°C and 250 rpm / min on a shaking table. 1 mL of bacterial solution was taken and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and monoclonal colonies with correct sequencing results were screened out;

[0263] S2036, extract pMD18-T simple-pRPS-30::RPS-30 S30 Plasmids:

[0264] Use AxyPrep Plasmid Miniprep Kit according to the instructions for plasmid extraction: According to the sequencing results of S2035, select the monoclonal colony with the correct sequencing, take 1 mL of bacterial solution in a 1.5 mL EP tube, centrifuge at 12000g for 1 min, discard the supernatant, add 250 μL S1 solution, mix by pipetting, add 250 μL S2 solution, mix by gentle inversion 6-8 times, add 350 μL S3 solution, mix by gentle inversion 6-8 times, centrifuge at 12000g for 10 min, take the supernatant to the adsorption column, centrifuge at 12000g for 1 min, discard the filtrate, add 500 μL W1 buffer, centrifuge at 12000g for 1 min, discard the filtrate, add 700 μL W2 buffer to wash twice, centrifuge at 12000g for 1 min, discard the filtrate; place the adsorption column in a new 1.5 mL EP tube, open the lid and dry for 3 minutes, add 50 μL ddH2O, stand at room temperature for 5 minutes, centrifuge at 12000g for 1 minute, and obtain pMD18-T simple-pRPS-30::RPS-30 S30 plasmid, and the plasmid concentration was measured to be 268 ng / μL;

[0265] S204, the pMD18-T simple-GFP plasmid obtained in S202 and the pMD18-Tsimple-pRPS-30::RPS-30 obtained in S203 S30 The plasmid was double digested with BglII / NheI restriction endonucleases, and the gel-recovered products after BglII / NheI double digestion were recovered separately. The gel-recovered products after BglII / NheI double digestion were ligated to construct pMD18-Tsimple-pRPS-30::GFP::RPS-30 S30 Plasmid, the specific steps include:

[0266] Double digestion with S2041, BglII / NheI restriction endonuclease:

[0267] At 37°C, the pMD18-Tsimple-GFP plasmid obtained in S2024 was double-digested with BglII / NheI restriction enzymes for 5 h to obtain the pMD18-T simple-GFP plasmid BglII / NheI double-digested product;

[0268] At 37°C, the BglII / NheI restriction enzymes were used to enzymatically cleave the pMD18-Tsimple-pRPS-30::RPS-30 obtained in S2036. S30 The plasmid was subjected to double restriction digestion for 5 h to obtain pMD18-T simple-pRPS-30::RPS-30 S30Plasmid BglII / NheI double digestion product;

[0269] Reaction system: pMD18-T simple-GFP plasmid or pMD18-T simple-pRPS-30::RPS-30 S30 Plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, BglII restriction enzyme 1 μL, NheI restriction enzyme 1 μL, ddH2O to 20 μL;

[0270] S2042, gel recovery of BglII / NheI double digestion products:

[0271] AxyPrep DNA Gel Extraction Kit was used according to the kit gel extraction instructions: the pMD18-T simple-GFP plasmid obtained in S2041 was subjected to 1% agarose gel electrophoresis with BglII / NheI double digestion products, and the target DNA band of 720 bp was cut out; the pMD18-T simple-pRPS-30::RPS-30 obtained in S2041 was subjected to 1% agarose gel electrophoresis with BglII / NheI double digestion products, and the target DNA band of 720 bp was cut out; S30 The plasmid BglII / NheI double digestion product was subjected to 1% agarose gel electrophoresis, and the target DNA band of 4580bp was cut out; about 100-150mg of the gel block was placed in a 1.5mL EP tube, 300-450μL DE-A was added, and the EP tube was placed in a 75°C water bath for 10min (mixed once every 2-3min), DE-B was added to the EP tube and shaken for 1min; the liquid was transferred to a recovery column placed in a 2mL centrifuge tube, centrifuged at 12000g for 1min, the liquid in the centrifuge tube was discarded, and washed once with 500μL buffer W1, centrifuged at 12000g for 1min, and the liquid in the centrifuge tube was discarded; washed twice with 700μL buffer W2, centrifuged at 12000g for 1min, and the liquid in the centrifuge tube was discarded; 12000g was emptied for 2min, the centrifuge tube was discarded, and the recovery column was placed in a new 1.5mL EP tube, 25μL ddH2O, incubate at room temperature for 2 minutes, centrifuge at 12000g for 1 minute, obtain DNA recovery product in 1.5mL EP tube, store the DNA recovery product at -40°C for later use; the volume ratio of DE-B to DE-A is 1:2;

[0272] The AxyPrep DNA Gel Extraction Kit was used to recover the plasmid pMD18-T simple-GFP and pMD18-T simple-pRPS-30::RPS-30 respectively. S30The plasmid was double digested with BglII / NheI and the product was recovered from the gel;

[0273] S2043, at 16°C, using DNA Ligation Kit<Mighty Mix> The pMD18-T simple-GFP plasmid obtained in S2042 was digested with BglII / NheI and the recovered products were mixed with pMD18-T simple-pRPS-30::RPS-30 S30 The plasmid BglII / NheI double digestion gel recovery product was ligated overnight to obtain the ligation product of the BglII / NheI double digestion gel recovery product:

[0274] Ligation reaction system: pMD18-T simple-GFP plasmid BglII / NheI double digestion gel recovery product 500ng, pMD18-T simple-pRPS-30::RPS-30 S30 After plasmid BglII / NheI double digestion, gel recovery product 100ng, ligation mix 8μL, ddH2O to 25μL;

[0275] S2044, conversion coated plate:

[0276] Take out the competent bacteria of E.coli TOP10 at -80℃, put them on ice to melt, add 25μL of the ligation product of the gel recovery product obtained from the double digestion of BglII / NheI obtained in S2043, gently blow and mix, and place on ice for 30min; heat stress at 42℃ for 90s, then place on ice for 2min, add 1mL LB for culture, and culture at 37℃ and 250rpm / min on a shaking table for 1h; centrifuge at 4000g for 4min, discard the supernatant, leave 30μL of residual bacterial solution, gently beat the precipitate until the bacterial solution is uniform, add the bacterial solution dropwise to the solid LB medium containing AMP resistance, spread it evenly, and place it in a 37℃ bacterial incubator for overnight culture;

[0277] S2045, sequencing identification:

[0278] Monoclonal colonies were picked from the solid LB culture plates cultured overnight in S2044, respectively, and placed in 3 mL of liquid LB culture medium containing AMP resistance, and cultured for 10 hours at 37°C and 250 rpm / min on a shaking table; 1 mL of bacterial solution was taken and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and monoclonal colonies with correct sequencing results were screened out;

[0279] S2046, extract pMD18-T simple-pRPS-30::GFP::RPS-30 S30Plasmids:

[0280] Use AxyPrep Plasmid Miniprep Kit according to the plasmid extraction instructions of the kit: according to the sequencing results of S2045, select the monoclonal colony with correct sequencing, take 1mL of bacterial solution in a 1.5mL EP tube, centrifuge at 12000g for 1min, discard the supernatant, add 250μL S1 solution, mix by pipetting, add 250μL S2 solution, mix by gentle inversion 6-8 times, add 350μL S3 solution, mix by gentle inversion 6-8 times, centrifuge at 12000g for 10min, take the supernatant to the adsorption column, centrifuge at 12000g for 1min, discard the filtrate, add 500μL W1 buffer, centrifuge at 12000g for 1min, discard the filtrate, add 700μL W2 buffer to wash twice, centrifuge at 12000g for 1min, discard the filtrate; place the adsorption column in a new 1.5mL EP tube, open the lid and dry for 3 minutes, add 50 μL ddH2O, let stand at room temperature for 5 minutes, centrifuge at 12000g for 1 minute, and obtain pMD18-T simple-pRPS-30::GFP::RPS-30 S30 plasmid, and the plasmid concentration was measured to be 258 ng / μL;

[0281] S3. Construction of pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmid: pPD95.77 plasmid (Addgene #: 1495) was combined with pMD18-T simple-pRPS-30::GFP::RPS-30 obtained from S2046. S30 The plasmid was double digested with KpnI / EcoRI restriction endonucleases, and the gel-recovered product after KpnI / EcoRI double digestion was obtained. The gel-recovered product after KpnI / EcoRI double digestion was ligated to construct pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmid, the specific steps include:

[0282] Double digestion with S301, KpnI / EcoRI restriction enzymes;

[0283] At 37°C, the pPD95.77 plasmid was double-digested with KpnI / EcoRI restriction enzymes for 5 hours to obtain the pPD95.77 plasmid KpnI / EcoRI double-digested product;

[0284] At 37°C, KpnI / EcoRI restriction endonucleases were used to enzymatically cleave the pMD18-Tsimple-pRPS-30::GFP::RPS-30 obtained from S2046. S30The plasmid was subjected to double restriction digestion for 5 h to obtain pMD18-T simple-pRPS-30::GFP::RPS-30 S30 Plasmid KpnI / EcoRI double digestion product;

[0285] Reaction system: pPD95.77 plasmid or pMD18-T simple-pRPS-30::GFP::RPS-30 S30 Plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, KpnI restriction enzyme 1 μL, EcoRI restriction enzyme 1 μL, ddH2O to 20 μL;

[0286] S302, gel recovery of KpnI / EcoRI double digestion products:

[0287] AxyPrepDNA Gel Extraction Kit was used according to the kit gel extraction instructions: the KpnI / EcoRI double digestion product of pPD95.77 obtained in S301 was subjected to 1% agarose gel electrophoresis, and the 3562 bp target DNA band was cut out; the pMD18-T simple-pRPS-30::GFP::RPS-30 obtained in S301 was purified by PCR. S30 The plasmid KpnI / EcoRI double digestion product was subjected to 1% agarose gel electrophoresis, and the target DNA band of 2900bp was cut out; about 100-150mg of the gel block was placed in a 1.5mL EP tube, 300-450μL DE-A was added, and the EP tube was placed in a 75°C water bath for 10min (mixed once every 2-3min), DE-B was added to the EP tube and shaken for 1min; the liquid was transferred to a recovery column placed in a 2mL centrifuge tube, centrifuged at 12000g for 1min, the liquid in the centrifuge tube was discarded, and washed once with 500μL buffer W1, centrifuged at 12000g for 1min, and the liquid in the centrifuge tube was discarded; washed twice with 700μL buffer W2, centrifuged at 12000g for 1min, and the liquid in the centrifuge tube was discarded; 12000g was emptied for 2min, the centrifuge tube was discarded, the recovery column was placed in a new 1.5mL EP tube, 25μL ddH2O, incubate at room temperature for 2 minutes, centrifuge at 12000g for 1 minute, obtain DNA recovery product in 1.5mL EP tube, store the DNA recovery product at -40°C for later use; the volume ratio of DE-B to DE-A is 1:2;

[0288] The gel recovery products of pPD95.77 plasmid KpnI / EcoRI double digestion and pMD18-T simple-pRPS-30::GFP::RPS-30 were obtained by AxyPrep DNA Gel Extraction Kit gel recovery kit. S30 The plasmid was double digested with KpnI / EcoRI and the product was recovered from the gel;

[0289] S303, at 16°C, using DNA Ligation Kit<Mighty Mix> The pPD95.77 plasmid obtained in S302 was digested with KpnI / EcoRI and the recovered products were mixed with pMD18-T simple-pRPS-30::GFP::RPS-30 S30 The plasmid KpnI / EcoRI double digestion gel recovery product was connected overnight to obtain the KpnI / EcoRI double digestion gel recovery product connection product:

[0290] Ligation reaction system: pPD95.77 plasmid KpnI / EcoRI double digestion gel recovery product 100ng, pMD18-Tsimple-pRPS-30::GFP::RPS-30 S30 After plasmid BglII / NheI double digestion, 500 ng of gel recovery product, 8 μL of LigationMix, and ddH2O were added to 25 μL;

[0291] S304, conversion coated plate:

[0292] Take out the competent bacteria of E. coli TOP10 from the storage temperature of -80°C, add the ligation product of the gel recovery product obtained in S303 after KpnI / EcoRI double digestion, ice-bathe for 30 minutes, then heat-stress at 42°C for 90 seconds, ice-bathe for 2 minutes, add 1 mL of LB liquid culture medium, culture at 37°C and a shaking speed of 250 rpm / min for 1 hour, obtain the residual bacterial liquid after transformation by centrifugation, drop the residual bacterial liquid after transformation into a solid LB culture plate containing ampicillin resistance, spread it evenly, and culture it in a bacterial incubator at 37°C overnight;

[0293] S305, Sequencing Identification:

[0294] Monoclonal colonies were picked from the solid LB culture plates cultured overnight in S304, respectively, and placed in 3 mL of liquid LB culture medium containing AMP resistance, and cultured for 10 hours at 37°C and 250 rpm / min on a shaking table; 1 mL of bacterial solution was taken and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and monoclonal colonies with correct sequencing results were screened out;

[0295] S306, extract pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmids:

[0296] Use AxyPrep Plasmid Miniprep Kit according to the instructions for plasmid extraction: According to the sequencing results of S305, select the monoclonal colony with the correct sequencing, take 1 mL of bacterial solution in a 1.5 mL EP tube, centrifuge at 12000g for 1 min, discard the supernatant, add 250 μL S1 solution, mix by pipetting, add 250 μL S2 solution, mix by gentle inversion 6-8 times, add 350 μL S3 solution, mix by gentle inversion 6-8 times, centrifuge at 12000g for 10 min, take the supernatant to the adsorption column, centrifuge at 12000g for 1 min, discard the filtrate, add 500 μL W1 buffer, centrifuge at 12000g for 1 min, discard the filtrate, add 700 μL W2 buffer, wash twice, centrifuge at 12000g for 1 min, discard the filtrate; place the adsorption column in a new 1.5 mL EP tube, open the lid and dry for 3 minutes, add 50mL ddH2O, stand at room temperature for 5 minutes, centrifuge at 12000g for 1 minute, and obtain pPD95.77-pRPS-30::GFP::RPS-30 S30 plasmid, and the plasmid concentration was measured to be 205 ng / μL;

[0297] S4. Construction of pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Plasmid: pPD95.77-pRPS-30::GFP::RPS-30 obtained in S306 S30 The plasmid was the same as the pMD18-T simple-pU6::shRNA (RPS-30 UbL ) plasmid was double digested with SmaI / KpnI restriction endonucleases, and the SmaI / KpnI double digested gel-recovered products were obtained by gel ligation to obtain pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Plasmid, i.e., transgenic plasmid that extends the healthy life span of Caenorhabditis elegans, the specific steps include:

[0298] Double digestion with S401, SmaI / KpnI restriction enzymes;

[0299] At 37°C, the pPD95.77-pRPS-30::GFP::RPS-30 obtained in S306 was cleaved by SmaI / KpnI restriction endonucleases. S30 The plasmid was subjected to double restriction digestion for 5 h to obtain pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmid SmaI / KpnI double digestion product;

[0300] At 37°C, the pMD18-Tsimple-pU6::shRNA (RPS-30 UbL ) plasmid was subjected to double restriction digestion for 5 h to obtain pMD18-T simple-pU6::shRNA(RPS-30 UbL ) Plasmid SmaI / KpnI double digestion product;

[0301] Reaction system: pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmid or pMD18-T simple-pU6::shRNA(RPS-30 UbL ) plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, SmaI restriction enzyme 1 μL, KpnI restriction enzyme 1 μL, ddH2O to 20 μL;

[0302] S402, gel recovery SmaI / KpnI double digestion product:

[0303] Use AxyPrep DNA Gel Extraction Kit according to the kit gel recovery instructions: pPD95.77-pRPS-30::GFP::RPS-30 obtained in S401 was added to S30 Plasmid SmaI / KpnI double digestion product SmaI / KpnI double digestion product was subjected to 1% agarose gel electrophoresis, and the target DNA band of 6462 bp was cut out; pMD18-T simple-pU6::shRNA (RPS-30 UbL) Plasmid SmaI / KpnI double digestion product was subjected to 1% agarose gel electrophoresis, and the target DNA band of 326 bp was cut out; about 100-150 mg of gel blocks were placed in 1.5 mL EP tubes, 300-450 μL DE-A was added, and the EP tube was placed in a 75°C water bath for 10 min (mixed once every 2-3 min), DE-B was added to the EP tube and shaken for 1 min; the liquid was transferred to a recovery column placed in a 2 mL centrifuge tube, centrifuged at 12000g for 1 min, the liquid in the centrifuge tube was discarded, washed once with 500 μL buffer W1, centrifuged at 12000g for 1 min, and the liquid in the centrifuge tube was discarded; washed twice with 700 μL buffer W2, centrifuged at 12000g for 1 min, and the liquid in the centrifuge tube was discarded; 12000g was emptied for 2 min, the centrifuge tube was discarded, the recovery column was placed in a new 1.5 mL EP tube, 25 μL ddH2O, incubate at room temperature for 2 minutes, centrifuge at 12000g for 1 minute, obtain DNA recovery product in 1.5mL EP tube, store the DNA recovery product at -40°C for later use; the volume ratio of DE-B to DE-A is 1:2;

[0304] After recovery by AxyPrep DNA Gel Extraction Kit, pPD95.77-pRPS-30::GFP::RPS-30 was obtained. S30 Plasmid SmaI / KpnI double digestion and gel recovery products, pMD18-T simple-pU6::shRNA(RPS-30 UbL ) Plasmid SmaI / KpnI double digestion and gel recovery product;

[0305] S403, at 16°C, using DNA Ligation Kit<Mighty Mix> The pPD95.77-pRPS-30::GFP::RPS-30 obtained in S402 was S30 The plasmid SmaI / KpnI double digestion gel recovery product and pMD18-Tsimple-pU6::shRNA(RPS-30 UbL ) The plasmid SmaI / KpnI double digestion gel recovery product was connected overnight to obtain the SmaI / KpnI double digestion gel recovery product:

[0306] Ligation reaction system: pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmid SmaI / KpnI double digestion gel recovery product 100ng, pMD18-T simple-pU6::shRNA(RPS-30 UbL) After double digestion of plasmid SmaI / KpnI, 500 ng of gel recovery product, 8 μL of Ligation Mix, and ddH2O to 25 μL;

[0307] S404, conversion coated plate:

[0308] Take out the competent bacteria of Escherichia coli TOP10 at -80°C, put them on ice to melt, add 25 μL of the ligation product of the gel recovery product obtained after KpnI / EcoRI double digestion in S403, gently blow and mix, and place on ice for 30 minutes; heat stress at 42°C for 90 seconds, then place on ice for 2 minutes, add 1 mL of LB liquid culture medium, and culture at 37°C and 250 rpm / min on a shaking table for 1 hour; centrifuge at 4000g for 4 minutes, discard the supernatant, leave 30 μL of residual bacterial solution, gently beat the precipitate until the bacterial solution is uniform, add the bacterial solution dropwise to the solid LB culture plate containing AMP resistance, spread it evenly, and place it in a 37°C bacterial incubator for overnight culture;

[0309] S405, sequencing identification:

[0310] Monoclonal colonies were picked from the solid LB culture plates cultured overnight in S404, respectively, and placed in 3 mL of LB liquid culture medium containing ampicillin resistance, and cultured for 10 hours at 37°C and 250 rpm / min on a shaking table; 1 mL of bacterial solution was taken and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and monoclonal colonies with correct sequencing results were screened out;

[0311] S406, extract pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Plasmids;

[0312] Use AxyPrep Plasmid Miniprep Kit according to the instructions for plasmid extraction: According to the sequencing results of S405, select the monoclonal colony with correct sequencing, take 1 mL of bacterial solution in a 1.5 mL EP tube, centrifuge at 12000g for 1 min, discard the supernatant, add 250 μL S1 solution, mix by pipetting, add 250 μL S2 solution, mix by gentle inversion 6-8 times, add 350 μL S3 solution, mix by gentle inversion 6-8 times, centrifuge at 12000g for 10 min, take the supernatant to the adsorption column, centrifuge at 12000g for 1 min, discard the filtrate, add 500 μL W1 buffer, centrifuge at 12000g for 1 min, discard the filtrate, add 700 μL W2 buffer, wash twice, centrifuge at 12000g for 1 min, discard the filtrate; place the adsorption column in a new 1.5 mL EP tube, open the lid and dry for 3 min, add 50 μL ddH2O, let stand at room temperature for 5 min, centrifuge at 12000 g for 1 min, and obtain pPD95.77-pU6::shRNA (RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The plasmid was prepared and its concentration was measured to be 215 ng / μL. The nucleotide sequence was shown in SEQ ID NO: 11. The plasmid was named as the transgenic plasmid for extending the healthy lifespan of Caenorhabditis elegans.

[0313] Example 2

[0314] This example is the transgenic gene for extending the healthy life span of Caenorhabditis elegans in Example 1, i.e., the plasmid pPD95.77-pU6::shRNA (RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Application of plasmid, the transgenic plasmid for extending the healthy lifespan of Caenorhabditis elegans is used to extend the healthy lifespan of Caenorhabditis elegans.

[0315] 1. Caenorhabditis elegans used in the experiment;

[0316] The wild-type standard strain N2 of Caenorhabditis elegans was maintained and cultured in our laboratory.

[0317] 2. Main reagents;

[0318] The uracil mutant strain of Escherichia coli (OP50) was preserved in our laboratory; the oil used for microinjection, Halocarbon oil 700, was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Sigma).

[0319] 3. Instruments and equipment;

[0320] Table centrifuge (Japan), inverted fluorescence microscope (Olympus IX71), micro-manipulation syringe (IM300, Narishige), MOTIC body dissecting microscope (China), P-2000 horizontal programmable laser microelectrode pulling instrument (Sutter Company, USA), biochemical incubator.

[0321] 4. Main solution

[0322] (1) Preparation of M9 buffer: Use an analytical balance to accurately weigh 3 g KH2PO4, 6 g Na2HPO4, and 5 g NaCl, add 1 mL 1 M MgSO4, and make up to 1 L with ddH2O, mix well, and sterilize at high temperature.

[0323] (2) Preparation of Nematode Growth Medium (NGM): Use an analytical balance to accurately weigh 3g NaCl, 2.5g tryptone, and 17g agar, add 975mL ddH2O and mix well, and sterilize at high temperature. When the sterilized NGM cools to 55°C, add 1mL 1M CaCl2, 1mL 5mg / mL cholesterol solution (solvent is ethanol), 1mL 1M MgSO4 and 25mL 1M K2HPO4-KH2PO4 buffer in sequence on a sterile operating table, mix well, and pour onto a plate (10mLNGM / 35mm culture dish).

[0324] 5. Transfection plasmids into Caenorhabditis elegans;

[0325] The transgenic plasmid prepared in Example 1 for extending the healthy life span of Caenorhabditis elegans is pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Plasmid, concentration is 215ng / μL.

[0326] 6. Microinjection transfection of Caenorhabditis elegans;

[0327] ① Preparation for microinjection;

[0328] Preparation of Pad: Drop 2% agarose on a cover glass, use another cover glass to press it into a very thin agarose sheet, leave it to dry at room temperature overnight, and store it at 4℃ for later use;

[0329] Preparation of microinjection needles: Capillary glass tubes (FHC, Borosil 1.0 mm OD × 0.75 mm ID / Fiber) were made into microinjection needles using a P-200 horizontal programmable optical microelectrode puller (Sutter, USA);

[0330] Oil for microinjection: Halocarbon oil 700;

[0331] ② Preparation of C.elegans (N2) worms;

[0332] C.elegans (N2) was cultured in NGM medium at 20°C until the fourth larvae (L4), and then larvae close to L4 were selected and placed in new NGM medium for 12 to 14 h before microinjection;

[0333] ③ Preparation of plasmid for microinjection;

[0334] The pPD95.77-pU6::shRNA (RPS-30 UbL )-pRPS-30::GF P::RPS-30 S30 The plasmid was diluted with ultrapure water to a final concentration of 50 ng / μL, mixed and centrifuged at 8000 rpm / min for 10 min;

[0335] ④Microinjection procedure;

[0336] a. Turn on the microinjector, press the mode key, and adjust to the action interface;

[0337] b. Open the main valve of the N2 cylinder, and then open the pressure valve so that the pointer points to 0.4-0.5;

[0338] c. Press the balance button on the microinjector;

[0339] d. Use the foot pedal to check whether the air outlet of the needle carrier is smooth;

[0340] e. Install the needle. Gently loosen the end of the needle carrier, then gently insert the tail of the glass needle loaded with the injection plasmid into the needle carrier, and tighten the end to fix the needle;

[0341] f. To find the needle, first adjust the 10× objective lens to the top, adjust the needle tip to make it 30 to 45 degrees and close to the center of the objective lens vertically above, turn on the light source, adjust the objective lens in the field of view to slowly lower it until the needle tip appears in the field of view, and then adjust the needle tip to the best position in the 40× field of view;

[0342] g. Strike the needle. Place the prepared striker slide on the stage, adjust the 40× objective lens to clearly see the glass tube, and adjust the slide so that the glass tube is perpendicular to the injection needle. Adjust the needle carrier to slowly move the needle down until the needle tip appears in the field of view and is in the same plane as the glass tube. Gently strike the glass tube with the needle (it is best to press the pedal while striking), and then press the pedal to check whether the liquid flows out into droplets;

[0343] h. Pick insects. Drop a drop of Halocarbonoil700 on the prepared pad, observe the insect bodies (C.elegans) in the NGM medium, pick out the insect bodies with more than 5 to a single row of eggs, and fix them in the halocarbon oil on the pad (it is best not to fix the head);

[0344] i. Injection: There are two relatively clear transparent areas at the upper edge of the tail and the lower edge of the pharynx, which are the gonads of the worm. Insert the needle tip obliquely into the gonad syncytium area and step on the pedal to inject (it is best to step on the pedal when the needle tip enters the syncytium to avoid clogging the needle tip);

[0345] j. After injection, adjust the needle carrier to lift the needle, remove the pad and pick the insect body into NGM culture medium under a microscope; 7. Screen transgenic insect strains;

[0346] After microinjection and transfection, the worms (F0) were placed in NGM culture medium, 4 per dish. Three days later, the green fluorescent L4 worms were picked under a fluorescence microscope as the F1 generation and placed in new NGM culture medium, 4 per dish; four days later, the green fluorescent L4 worms were picked under a fluorescence microscope as the F2 generation. The F2 generation worms were used for the next step of ultraviolet irradiation to construct a stable transgenic strain.

[0347] 8. Construction of stable transgenic insect strains by ultraviolet irradiation (using Spectrolinker UV crosslinker XLE-1000)

[0348] Pick 60 green fluorescent L4 worms and put them into a new NGM culture plate (without OP50), 20 per plate. After culturing at 15℃ for 2h, open the lid of the dish and irradiate the worms with 120J / m2 UV irradiation in a UV crosslinker. The irradiated worms are F0 generation and cultured at 20℃. Pick 100 green fluorescent F1 generation L4 worms under a fluorescence microscope, 1 per plate, and culture at 20℃. Observe the F2 generation worms, select the F1 generation plate that produces a large number (about 70%) of green fluorescent F2 generation, pick 4 F2 generation L4 worms per plate, 1 per plate, and culture at 20℃. Observe the F3 generation worms, find the F2 generation plate with 100% green fluorescent F3 generation, pick 8 F3 generation L4 worms with green fluorescence per plate, 1 per plate, and culture at 20℃. Continue to propagate the insect body until the fluorescence intensity of the insect body remains unchanged, and the stable inheritance of pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Transgenic insect strains. Figure 2 As shown, Figure 2 For transgenic insects, Figure 2A, B, and C in the figure are pPD95.77-pRPS-30::GFP transgenic worms (control worms), where A is the bright field (DI C) image of the transgenic worms, B is the green fluorescence (GFP) image of the transgenic worms, and C is the fusion (Merge) image of the bright field image and the green fluorescence image of the transgenic worms; Figure 2 D, E, and F are pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The transgenic insects with the plasmid that extends the healthy lifespan, where D is the bright field (DIC) image of the transgenic insects, E is the green fluorescence (GFP) image of the transgenic insects, and F is the fusion (Merge) image of the bright field image and the green fluorescence image of the transgenic insects. Figure 2 In the pPD95.77-pRPS-30::GFP transgenic worms (control group), GFP was present throughout the body and diffused in the cells, while in the pPD95.77-pU6::shRNA (RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 In transgenic worms, GFP was expressed systemically and could enter the cell nucleus, indicating that RPS-30 S30 It can enter the nucleus and participate in ribosome assembly, and ribosome function is successfully rescued.

[0349] 9. Detection of various biological phenotypes of transgenic insects;

[0350] (1) Synchronization of insect bodies

[0351] In NGM medium, C. elegans was cultured to the adult stage, the worm body was washed with 3 mL M9 buffer, and transferred to a 5 mL centrifuge tube, the total volume of the solution was adjusted to 2.8 mL, and then 0.8 mL NaOCl solution and 0.4 mL 5 M NaOH solution were added in sequence, and the mixture was fully mixed. When about 2 / 3 of the worm body was lysed, centrifuged at 5500 rpm for 1 min, the supernatant was discarded, and the worm eggs in the adult body were washed twice with M9 buffer to obtain the eggs; the prepared eggs were placed in 4 mL M9 buffer for overnight incubation to obtain L1 larvae; the L1 larvae were inoculated in NGM medium and cultured at 20°C, and L2, L3, L4, and adult worm bodies were obtained at 12 h, 20 h, 30 h, and 48 h, respectively.

[0352] (2) shRNA (RPS-30 UbL ) Interference effect and RPS-30 S30 Expression level detection

[0353] ① Extract total RNA from insects and obtain cDNA through reverse transcription

[0354] The method is the same as S2014 and S2015.

[0355] ②In order to detect the ubiquitin-like protein RPS-30 UbL Short hairpin RNA region: shRNA (RPS-30 UbL ) on the rps-30 gene in the worm and the rps-30 gene complemented in the transgenic worm ribosomal protein RPS-30 S30 Area (RPS-30 S30 This is the RPS-30 cloned in S201 S30 The expression of PCR products in the worm body, that is, the expression level of the complemented ribosomal protein region, we used relative fluorescence quantitative PCR (qRT-PCR) method to detect the rps-30 gene, rps-30 in the worm body at different stages. S30 、rps-30 UbL According to the rps-30 gene sequence, qRT-PCR detection primers were designed. RTrps30P1 and RTrps30P2 were used to detect the transcription level of rps-30 gene; RT s30P1 and RT s30P2 were used to detect the transcription level of rps-30 gene. S30 Transcript levels; RTUbLP1 and RTUbLP2 were used to detect rps-30 UbL Transcription level. The primer sequences are as follows:

[0356] The nucleotide sequence of RTrps30P1 is shown in SEQ ID NO:12;

[0357] The nucleotide sequence of RTrps30P2 is shown in SEQ ID NO:13;

[0358] The nucleotide sequence of RT s30P1 is shown in SEQ ID NO:14;

[0359] The nucleotide sequence of RT s30P2 is shown in SEQ ID NO: 15;

[0360] The nucleotide sequence of RTUbLP1 is shown in SEQ ID NO:16;

[0361] The nucleotide sequence of RTUbLP2 is shown in SEQ ID NO: 17;

[0362] qRT-PCR uses a 25μl system, and the specific operation is carried out according to THUNDERBIRDSYBRq PCR Mix (TOYOBO). qRT-PCR reaction system: cDNA, 2μL; RTrps30P1 or RT s30P1 or RTUbLP1, 0.5μL; RTrps30P2 or RTs30P2 or RTUbLP2, 0.5μL; qPCRMix 5μL; H2O, 2μL. The qRT-PCR reaction conditions are: 50℃, 2min, 95℃, 1min one cycle, 95℃, 15s, 60℃, 15s, 72℃, 31s, 40 cycles, and the melting curve is generated in the last cycle. Each sample was repeated three times, and the average threshold (CT) was taken for data analysis. At the same time, the Caenorhabditis elegans act-1 gene was selected as an internal reference, and the primer sequences are as follows:

[0363] The nucleotide sequence of RTact-1P1 is shown in SEQ ID NO:18;

[0364] The nucleotide sequence of RTact-1P2 is shown in SEQ ID NO: 19;

[0365] The relative quantification was calculated by comparing CT values. The data were analyzed by one-way ANOVA and Student's t-tests, and p < 0.05 was considered significant.

[0366] The results show:

[0367] rps-30 and rps-30 in transgenic insects S30 、rps-30 UbL Relative quantitative analysis of gene transcription levels Figure 3 As shown, N2 is the wild-type standard strain of Caenorhabditis elegans (blank control group); N2; GPF is the pPD95.77-pRPS-30::GFP transgenic worm (experimental control group); N2; shRNA (RPS-30 UbL )-pRPS-30 S30 Plasmid pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Transgenic insects (experimental group).

[0368] pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The transcription level of rps-30 gene in transgenic insects was significantly reduced. Figure 3As shown in A; rps-30 S30 No significant changes were observed in the transcription level. Figure 3 As shown in B; rps-30 UbL The transcription level was significantly reduced. Figure 3 As shown in C.

[0369] The results showed that pU6::shRNA(RPS-30 UbL ) successfully interfered with the transcription level of rps-30 gene in Caenorhabditis elegans; while pRPS-30::GFP::RPS-30 S30 Successfully recovered rps-30 S30 After the transgenic plasmid was used, rps-30 in the transgenic insect strain was successfully knocked down. UbL Transcription level.

[0370] (3) Transgenic insect lifespan detection

[0371] Pick 10 L4 worms and place them in NGM medium and culture them at 20℃ for 15h. After the worms lay eggs, pick out the adults. After the eggs hatch, place the larvae in new NGM medium, 1 worm / dish, and culture at 20℃. Observe once a day until the worms die. During the egg-laying period, the worms should be replaced with new culture medium every two days. The criterion for judging the death of nematodes is that there is no reaction when the worm is touched with platinum wire, which means death. The survival time of each worm is recorded. This experiment was repeated 3 times, and 40 worms were tested each time. The data obtained were subjected to one-way ANOVA statistical analysis and Student's t-tests, and p<0.05 was considered significant.

[0372] The life test results show:

[0373] Compared with pPD95.77-pRPS-30::GFP transgenic worms and wild-type standard strain N2 of C. elegans, pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The life span of transgenic insects was significantly extended, N2; GFPvsN2; shRNA (RPS-30 UbL )-RPS-30 S30 =14±0.3(days)vs26±0.5(days), such as Figure 4 As shown. N2 is the wild-type standard strain of Caenorhabditis elegans (blank control group); N2; GPF is the pPD95.77-pRPS-30::GFP transgenic worm (experimental control group); N2; shRNA (RPS-30 UbL )-pRPS-30 S30Plasmid pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Transgenic insects (experimental group).

[0374] The results of life span detection showed that pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GF P::RPS-30 S30 Transfection of plasmids into Caenorhabditis elegans can extend the lifespan of Caenorhabditis elegans.

[0375] (4) Detection of transgenic insect egg production

[0376] 40 L4 stage worms were selected and placed in NGM medium, 1 worm / dish, and cultured at 20℃. When the worms were in the egg-laying period, the new medium was replaced every day; after 3 days of the old medium, when the eggs were hatched and developed into adults, the number of worms was counted, which was the daily egg-laying amount of the worms, until the egg-laying period stopped, and finally the total egg-laying amount of each worm was calculated. This experiment was repeated 3 times. The data obtained were subjected to one-way ANOVA statistical analysis and Student's t-tests, and p<0.05 was considered significant.

[0377] The egg laying test results show:

[0378] Compared with pPD95.77-pRPS-30::GFP transgenic worms and wild-type standard strain N2 of C. elegans, pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The egg laying amount of transgenic insects was not significantly reduced, which was N2; shRNA (RPS-30 UbL )-RPS-30 S30 vsN2; GFP = 289 ± 5 (pieces) vs 312 ± 8 (pieces), such as Figure 5 As shown, N2 is the wild-type standard strain of Caenorhabditis elegans (blank control group); N2; GPF is the pPD95.77-pRPS-30::GFP transgenic worm (experimental control group); N2; shRNA (RPS-30 UbL )-pRPS-30 S30 Plasmid pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Transgenic insects (experimental group).

[0379] The results of egg laying test showed that:

[0380] pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Transfection of the plasmid into C. elegans did not lead to a significant decrease in the amount of eggs laid by the nematodes, and the reproductive capacity of the nematodes was normal.

[0381] (5) Detection of transgenic insect body length

[0382] The insects with different treatments were picked up on a glass slide, photographed, and measured using a Java image processing program. The length of the insect body was from the nose to the tip of the tail. The data obtained were subjected to one-way ANOVA statistical analysis and Student's t-tests, and p < 0.05 was considered significant.

[0383] The body length test results show:

[0384] Compared with pPD95.77-pRPS-30::GFP transgenic worms and wild-type standard strain N2 of C. elegans, pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The length of the transgenic insects at each stage was not significantly shortened. Figure 6 As shown, N2 is the wild-type standard strain of Caenorhabditis elegans (blank control group); N2; GPF is the pPD95.77-pRPS-30::GFP transgenic worm (experimental control group); N2; shRNA (RPS-30 UbL )-pRPS-30 S30 Plasmid pP D95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Transgenic insects (experimental group).

[0385] The body length test results show that:

[0386] pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The plasmid transfected into Caenorhabditis elegans did not shorten the body length of Caenorhabditis elegans, and the worms grew normally.

[0387] (6) Detection of heat stress in transgenic insects

[0388] Pick L4 and put them into new culture medium, 40 per dish, culture at 20℃ for 18h, then raise the temperature to 37℃. Take out 3 culture mediums directly from 37℃ every hour and culture at 20℃ for 12h. Discard them directly after counting. Heat stress death is detected by touching the head of the insect with a platinum needle, and the insect that does not move is considered dead. The data obtained were subjected to one-way ANOVA statistical analysis and Student's t-tests, and p<0.05 was considered significant.

[0389] The heat stress test results showed:

[0390] Compared with pPD95.77-pRPS-30::GFP transgenic worms and wild-type standard strain N2 of C. elegans, pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The survival time of transgenic insects under heat stress was significantly prolonged, which was N2; GFPvsN2; shRNA(RPS-30 UbL )-RPS-30 S30 =4.3±0.2(h)vs8.2±0.4(h), such as Figure 7 As shown, N2 is the wild-type standard strain of Caenorhabditis elegans (blank control group); N2; GPF is the pPD95.77-pRPS-30::GFP transgenic worm (experimental control group); N2; shRNA (RPS-30 UbL )-pRPS-30 S30 Plasmid pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Transgenic insects (experimental group).

[0391] The results of heat stress test showed that:

[0392] pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The plasmid was transfected into Caenorhabditis elegans, which enhanced the worm's resistance to heat stress.

[0393] (7) Oxidative stress detection in transgenic insects

[0394] Take a 24-well culture plate and add 200 μL of M9 buffer containing 3 mM H2O2 to each well. Pick up the adult insects and place them in a 24-well culture plate, 50 per well, 3 wells per plate per group. Culture at 20°C, take out one culture plate every hour for observation and counting, and then discard it directly. The detection index of death is: touch the head of the insect with a platinum needle, and it is considered dead if it does not move. The data obtained were subjected to one-way ANOVA statistical analysis and Student's t-tests, and p<0.05 was considered significant.

[0395] Oxidative stress test results showed:

[0396] Compared with pPD95.77-pRPS-30::GFP transgenic worms and wild-type standard strain N2 of C. elegans, pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The survival rate of transgenic insects increased significantly under oxidative stress conditions: 8 hours under oxidative stress conditions N2; GFPvsN2; shRNA (RPS-30 UbL )-RPS-30 S30 =62.5±2.5vs100±0; 10 hours under oxidative stress, N2; GFPvsN2; shRNA(RPS-30 UbL )-RPS-30 S30 =26±2vs95±1.5, such as Figure 8 As shown, N2 is the wild-type standard strain of Caenorhabditis elegans (blank control group); N2; GPF is the pPD95.77-pRPS-30::GFP transgenic worm (experimental control group); N2; shRNA (RPS-30 UbL )-pRPS-30 S30 Plasmid pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Transgenic insects (experimental group).

[0397] The results of oxidative stress test showed that:

[0398] pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The plasmid was transfected into Caenorhabditis elegans, which enhanced the worm's resistance to oxidative stress.

[0399] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for constructing a transgenic plasmid for extending the healthy lifespan of Caenorhabditis elegans, characterized in that: The method is: S1. Construction of pMD18-T simple-pU6::shRNA(RPS-30 UbL )Plasmid: S101, using pLKO.1 puro plasmid as a template, amplifying the U6 promoter sequence by PCR, and cloning the U6 promoter PCR product into the pMD18-T simple plasmid by TA cloning to construct the pMD18-T simple-pU6 plasmid, and then performing double digestion of the obtained pMD18-T simple-pU6 plasmid with AgeI / EcoRI restriction endonucleases to obtain AgeI / EcoRI double digestion products, and performing gel recovery on the AgeI / EcoRI double digestion products, the specific steps include: S1011. Using pLKO.1 puro plasmid as a template, performing PCR reaction with upstream primer U6P1 and downstream primer U6P2 to amplify U6 promoter, and obtaining U6 promoter PCR product; the nucleotide sequence of the upstream primer U6P1 is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer U6P2 is shown in SEQ ID NO: 2; The PCR reaction system was: pLKO.1 puro plasmid 0.2 μL, upstream primer U6P1 1 μL, downstream primer U6P2 1 μL, LATaq 0.25 μL, 10×LA Taq buffer 2.5 μL, ddH2O to 25 μL; The PCR reaction program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, 25 cycles; extension at 72°C for 5 min; storage at 4°C; S1012. At 16°C, the U6 promoter PCR product obtained in S1011 was ligated into the pMD18-T simple plasmid overnight by TA cloning to obtain the TA cloning ligation product pMD18-T simple-pU6. Ligation reaction system: 4 μL of U6 promoter PCR product, 0.5 μL of pMD18-T simple plasmid, 4.5 μL of DNA ligation buffer Solution I, and ddH2O to 10 μL; S1013, conversion coated plate: Take out from the storage temperature of -80℃ Escherichia coli TOP10 competent bacteria, add the TA cloning ligation product pMD18-T simple-pU6 obtained in S1012, mix well, ice bath for 30 minutes, then heat stress at 42°C for 90 seconds, ice bath for 2 minutes, add 1 mL LB liquid culture medium, culture at 37°C and a shaker speed of 250 rpm / min for 1 hour, centrifuge to obtain the residual bacterial liquid after transformation, add the residual bacterial liquid after transformation to a solid LB culture plate containing ampicillin resistance, spread it evenly, and place it in a bacterial incubator at 37°C for overnight culture; S1014, Sequencing Identification: Monoclonal colonies were picked from the solid LB culture plate cultured overnight in S1013, and placed in 3 mL of LB liquid culture medium containing ampicillin resistance, respectively, and cultured for 10 h at 37°C and a shaking speed of 250 rpm / min. The obtained bacterial liquids were respectively taken for sequencing identification, and the monoclonal colonies with correct sequencing results were screened; S1015. Extract pMD18-T simple-pU6 plasmid: The correctly sequenced monoclonal colony obtained in S1014 was extracted using the AxyPrep Plasmid Miniprep Kit to obtain the pMD18-T simple-pU6 plasmid; Double digestion with S1016, AgeI / EcoRI restriction enzymes; At 37°C, the pMD18-T simple-pU6 plasmid obtained in S1015 was subjected to a double digestion reaction with AgeI / EcoRI restriction endonucleases for 5 h to obtain an AgeI / EcoRI double digestion product; Reaction system: pMD18-T simple-pU6 plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, AgeI restriction endonuclease 1 μL, EcoRI restriction endonuclease 1 μL, ddH2O to 20 μL; S1017, gel recovery of AgeI / EcoRI double digestion products: The AgeI / EcoRI double digestion product obtained in S1016 was subjected to 1% agarose gel electrophoresis, and the target DNA band of 270 bp was cut out and recovered using the AxyPrep DNA Gel Extraction Kit to obtain the AgeI / EcoRI double digestion gel recovery product; S102, annealing primer UbLshR1 and primer UbLshR2 to obtain an annealing product, i.e., targeting RPS-30 UbL Short hairpin RNA of the region: shRNA (RPS-30 UbL ); the nucleotide sequence of the primer UbLshR1 is shown in SEQ ID NO: 3, and the nucleotide sequence of the primer UbLshR2 is shown in SEQ ID NO: 4; Annealing system: primer UbLshR1 1.5μL, primer UbLshR2 1.5μL, 10×NEB buffer II 5μL, ddH2O to 50μL; Annealing procedure: pre-denaturation at 95°C for 5 min; denaturation at 70°C for 15 min; decrease the temperature to 22°C at a rate of 0.1°C per second; store at -40°C; S103, the gel recovery product obtained in S101 after the AgeI / EcoRI double digestion was connected with the annealing product obtained in S102 to construct pMD18-T simple-pU6::shRNA (RPS-30 UbL ) plasmid, the specific steps include: S1031. At 16°C, the gel-recovered product obtained in S101 after double digestion with EcoRI was ligated with the annealed product obtained in S102 overnight to obtain the ligation product pMD18-T simple-pU6::shRNA (RPS-30 UbL ); Ligation reaction system: 100 ng of gel recovery product after AgeI / EcoRI double digestion, 500 ng of annealing product, 8 μL of LigationMix, and 25 μL of ddH2O; S1032, conversion coated plate: Take out the E. coli Stbl3 competent bacteria from the storage temperature of -80°C, add the ligation product pMD18-T simple-pU6::shRNA (RPS-30 UbL ), mix well, ice bath for 30 minutes, then heat stress at 42°C for 90 seconds, ice bath for 2 minutes, add 1 mL LB liquid culture medium for culture, culture at 37°C and shaking speed of 250 rpm / min for 1 hour, centrifuge to obtain the residual bacterial liquid after transformation, add the residual bacterial liquid after transformation to the solid LB culture plate containing ampicillin resistance, spread it evenly, and place it in a bacterial culture incubator at 37°C for overnight culture; S1033, sequencing identification: After overnight culture in S1032, single clones were picked from the solid LB culture plate transformed, and placed in 3 mL of LB liquid culture medium containing ampicillin resistance, and cultured for 10 h at 37°C and 250 rpm / min. The obtained bacterial liquids were taken for sequencing and identification, and the single clones with correct sequencing results were screened out; S1034, extract pMD18-T simple-pU6::shRNA (RPS-30 UbL ) plasmid; The correctly sequenced monoclonal colony obtained in S1033 was extracted using the AxyPrep Plasmid Miniprep Kit plasmid extraction kit to obtain pMD18-T simple-pU6::shRNA (RPS-30 UbL ) plasmid; S2. Construction of pMD18-T simple-pRPS-30::GFP::RPS-30 S30 Plasmids: S201, using the genomic DNA sequence of Caenorhabditis elegans as a template, amplifying the rps-30 promoter sequence by PCR to obtain the rps-30 promoter PCR product; using the pEGFP-c2 plasmid as a template, amplifying the GFP nucleic acid sequence by PCR to obtain the GFP PCR product; using the cDNA obtained by reverse transcription of the Caenorhabditis elegans RNA sequence as a template, amplifying the RPS-30 promoter by PCR S30 Nucleic acid sequence, RPS-30 S30 PCR product, the specific steps include: S2011. Extraction of Caenorhabditis elegans genomic DNA: The genomic DNA of Caenorhabditis elegans was extracted using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0; S2012. Using the C. elegans genomic DNA obtained in S2011 as a template, PCR amplified the rps-30 promoter sequence: Using the genomic DNA of Caenorhabditis elegans as a template, the rps-30 promoter sequence was amplified by PCR reaction using the upstream primer rps30P1 and the downstream primer rps30P2 to obtain the rps-30 promoter PCR product; the nucleotide sequence of the upstream primer rps30P1 is shown in SEQ ID NO: 5, and the nucleotide sequence of the downstream primer rps30P2 is shown in SEQ ID NO: 6; PCR reaction system: 2 μL of C. elegans genomic DNA, 1 μL of upstream primer rps30P1, 1 μL of downstream primer rps30P2, 0.25 μL of LA Taq, 2.5 μL of 10×LA Taq buffer, and ddH2O to 25 μL; PCR reaction program: 95℃ pre-denaturation for 4 min; 94℃ denaturation for 1 min, 50℃ annealing for 1 min, 72℃ extension for 2 min, 28 cycles; 72℃ extension for 10 min; 4℃ storage; S2013. Using pEGFP-c2 plasmid as template, PCR amplified GFP nucleic acid sequence: Using pEGFP-c2 plasmid as a template, PCR reaction was performed with upstream primer GFP1 and downstream primer GFP2 to amplify the GFP nucleic acid sequence to obtain a GFP PCR product; the nucleotide sequence of the upstream primer GFP1 is shown in SEQ ID NO: 7, and the nucleotide sequence of the downstream primer GFP2 is shown in SEQ ID NO: 8; PCR reaction system: pEGFP-c2 plasmid 0.2 μL, upstream primer GFP1 1 μL, downstream primer GFP2 1 μL, LA Taq 0.25 μL, 10×LA Taq buffer 2.5 μL, ddH2O to 25 μL; PCR reaction program: 95℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 30 s, 25 cycles; 72℃ extension for 5 min; 4℃ storage; S2014, extract total RNA from Caenorhabditis elegans; S2015, C. elegans RNA sequence was reverse transcribed to obtain cDNA; S2016, using the cDNA obtained in S2015 as a template, PCR amplified RPS-30 S30 Nucleic acid sequence, the specific method is: The cDNA obtained in S2015 was used as a template and PCR was performed with upstream primer s30F and downstream primer s30R to amplify RPS-30 S30 Nucleic acid sequence, RPS-30 S30 PCR product; the nucleotide sequence of the upstream primer s30F is shown in SEQ ID NO: 9, and the nucleotide sequence of the downstream primer s30R is shown in SEQ ID NO: 10; PCR reaction system: cDNA 2 μL, upstream primer s30F 1 μL, downstream primer s30R 1 μL, LA Taq 0.25 μL, 10×LA Taq buffer 2.5 μL, ddH2O to 25 μL; PCR reaction program: 95℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 30 s, 25 cycles; 72℃ extension for 5 min; 4℃ storage; S202, the rps-30 promoter PCR product, GFP PCR product and RPS-30 S30 The PCR product was connected to the pMD18-T simple plasmid and sequenced to obtain the pMD18-T simple-pRPS-30 plasmid, pMD18-T simple- GFP plasmid and pMD18-T simple-RPS-30 S30 Plasmid, the specific steps include: S2021. The rps-30 promoter PCR product obtained in S201 was ligated into the pMD18-T simple plasmid by TA cloning at 16°C overnight to obtain the ligation product pMD18-T simple-pRPS-30. The GFP PCR product obtained in S2013 was ligated into the pMD18-Tsimple plasmid by TA cloning at 16°C overnight to obtain the ligation product pMD18-T simple-GFP; At 16°C, RPS-30 obtained from S2016 was cloned by TA cloning. S30 The PCR products were connected to the pMD18-T simple plasmid overnight to obtain the connection product pMD18-T simple-RPS-30 S30 ; Ligation reaction system: PCR product 4 μL, pMD18-T simple plasmid 0.5 μL, DNA ligation buffer Solution I 4.5 μL, ddH2O to 10 μL; the PCR product is rps-30 promoter PCR product or GFP PCR product or RPS-30 S30 PCR products; S2022, conversion coated board: Take out 3 pieces from the storage temperature of -80℃ Escherichia coli TOP10 competent bacteria, the first Escherichia coli The ligation product pMD18-T simple-pRPS-30 obtained in S2021 was added to the TOP10 competent bacteria. Escherichia coli The ligation product pMD18-T simple-GFP obtained in S2021 was added to the TOP10 competent bacteria, and the ligation product pMD18-T simple-RPS-30 obtained in S2021 was added to the third E. coli TOP10 competent bacteria. S30 , after being mixed evenly, ice-bathed for 30 minutes, then heat-stressed at 42°C for 90 seconds, ice-bathed for 2 minutes, added with 1 mL of LB liquid culture medium, cultured at 37°C and a shaking speed of 250 rpm / min for 1 hour, centrifuged to obtain the residual bacterial liquid after transformation, added dropwise to a solid LB culture plate containing ampicillin resistance, and spread evenly, and placed in a bacterial incubator at 37°C for overnight culture; S2023, Sequencing Identification: Monoclonal colonies were picked from the solid LB culture plates cultured overnight in S2022, and then placed in 3 mL of LB liquid culture medium containing ampicillin resistance, and cultured for 10 hours at 37°C and a shaking speed of 250 rpm / min. The obtained bacterial liquids were taken for sequencing and identification, and the monoclonal colonies with correct sequencing results were screened out; S2024, extract pMD18-T simple-pRPS-30 plasmid, pMD18-T simple-GFP plasmid and pMD18-Tsimple-RPS-30 S30 Plasmids: The correctly sequenced monoclonal colonies obtained in S2023 were extracted using the AxyPrep Plasmid Miniprep Kit to obtain pMD18-T simple-pRPS-30 plasmid, pMD18-T simple-GFP plasmid and pMD18-T simple-RPS-30 plasmid, respectively. S30 Plasmids; S203, the pMD18-T simple-pRPS-30 plasmid obtained in S2024 and pMD18-T simple-RPS-30 S30 The plasmids were double-digested with NheI / EcoRI restriction endonucleases, and the gel-recovered products were obtained by gel recovery. The gel-recovered products of NheI / EcoRI double-digestion were ligated to construct pMD18-T simple-pRPS-30::RPS-30. S30 Plasmid, the specific steps include: Double digestion with S2031, NheI / EcoRI restriction enzymes; At 37°C, the pMD18-T simple-pRPS-30 plasmid obtained in S2024 was subjected to double restriction digestion reaction with NheI / EcoRI restriction enzymes for 5 h to obtain the pMD18-T simple-pRPS-30 plasmid NheI / EcoRI double restriction digestion product; At 37°C, the pMD18-T simple-RPS-30 obtained from S2024 was cleaved with NheI / EcoRI restriction enzymes. S30 The plasmid was subjected to double restriction digestion for 5 h to obtain pMD18-T simple-RPS-30 S30 Plasmid NheI / EcoRI double digestion product; Reaction system: pMD18-T simple-pRPS-30 plasmid or pMD18-T simple-RPS- 30 S30 Plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, NheI restriction enzyme 1 μL, EcoRI restriction enzyme 1 μL, ddH2O to 20 μL; S2032, gel recovery of NheI / EcoRI double digestion products: The pMD18-T simple-pRPS-30 plasmid NheI / EcoRI double digestion product obtained in S2031 was subjected to 1% agarose gel electrophoresis, and the target DNA band of 4400 bp was cut out and recovered using the AxyPrep DNA Gel Extraction Kit to obtain the pMD18-T simple-pRPS-30 plasmid NheI / EcoRI double digestion gel recovery product; The pMD18-T simple-RPS-30 obtained in S2031 was S30 The plasmid NheI / EcoRI double digestion product was subjected to 1% agarose gel electrophoresis, and the 180 bp target DNA band was cut out and recovered using the AxyPrep DNA Gel Extraction Kit to obtain pMD18-T simple-RPS-30 S30 The plasmid was double digested with NheI / EcoRI and the product was recovered from the gel; S2033. At 16°C, the pMD18-T simple- pRPS-30 plasmid obtained in S2032 was digested with NheI / EcoRI and the recovered product was mixed with pMD18-T simple-RPS-30. S30 The plasmid NheI / EcoRI double digestion gel recovery product was connected overnight to obtain the connection product of the NheI / EcoRI double digestion gel recovery product: Ligation reaction system: pMD18-T simple-pRPS-30 plasmid NheI / EcoRI double digestion gel recovery product 100ng, pMD18-T simple-RPS-30 S30 After plasmid NheI / EcoRI double digestion, 500 ng of gel recovery product, 8 μL of LigationMix, and ddH2O were added to 25 μL; S2034, conversion coated plate: Take out from the storage temperature of -80℃ Escherichia coli TOP10 competent bacteria, add the ligation product of the gel recovery product obtained after NheI / EcoRI double digestion obtained in S2033, mix evenly, ice bath for 30 minutes, then heat stress at 42°C for 90 seconds, ice bath for 2 minutes, add 1 mL LB liquid culture medium for culture, culture at 37°C and a shaking speed of 250 rpm / min for 1 hour, centrifuge to obtain the residual bacterial liquid after transformation, add the residual bacterial liquid after transformation to a solid LB culture plate containing ampicillin resistance, spread it evenly, and place it in a bacterial incubator at 37°C for overnight culture; S2035, Sequencing Identification: From the solid LB culture plate cultured overnight in S2034, monoclonal colonies were picked respectively, and then placed in 3 mL of LB liquid culture medium containing ampicillin resistance, and cultured for 10 hours at a temperature of 37°C and a shaking speed of 250 rpm / min. The obtained bacterial liquids were respectively taken for sequencing identification, and the monoclonal colonies with correct sequencing results were screened out; S2036, extract pMD18-T simple-pRPS-30::RPS-30 S30 Plasmids; The correctly sequenced monoclonal colony obtained in S2035 was extracted using the AxyPrep Plasmid Miniprep Kit plasmid extraction kit to obtain pMD18-T simple-pRPS-30::RPS- 30 S30 Plasmids; S204, the pMD18-T simple-GFP plasmid obtained in S2024 and the pMD18-T simple-pRPS-30::RPS-30 obtained in S2036 were combined. S30 The plasmid was double digested with BglII / NheI restriction endonucleases, and the gel-recovered products after BglII / NheI double digestion were recovered separately. The gel-recovered products after BglII / NheI double digestion were ligated to construct pMD18-T simple-pRPS-30::GFP::RPS-30 S30 Plasmid, the specific steps include: Double digestion with S2041, BglII / NheI restriction endonuclease: At 37°C, the pMD18-T simple -GFP plasmid obtained in S2024 was double-digested with BglII / NheI restriction enzymes for 5 hours to obtain a pMD18-T simple-GFP plasmid BglII / NheI double-digested product; At 37°C, the BglII / NheI restriction enzymes were used to enzymatically cleave the pMD18-T simple-pRPS-30::RPS-30 obtained from S2036. S30 The plasmid was subjected to double restriction digestion for 5 h to obtain pMD18-T simple-pRPS-30::RPS-30 S30 Plasmid BglII / NheI double digestion product; Reaction system: pMD18-T simple-GFP plasmid or pMD18-T simple-pRPS-30::RPS-30 S30 Plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, BglII restriction enzyme 1 μL, NheI restriction enzyme 1 μL, ddH2O to 20 μL; S2042, gel recovery of BglII / NheI double digestion products: The pMD18-T simple-GFP plasmid BglII / NheI double digestion product obtained in S2041 was subjected to 1% agarose gel electrophoresis, and the target DNA band of 720 bp was cut out and recovered using the AxyPrep DNA Gel Extraction Kit to obtain the pMD18-T simple-GFP plasmid BglII / NheI double digestion gel recovery product; The pMD18-T simple-pRPS-30::RPS-30 obtained in S2041 was S30 The plasmid BglII / NheI double digestion product was subjected to 1% agarose gel electrophoresis, and the target DNA band of 4580 bp was cut out and recovered using the AxyPrep DNA Gel Extraction Kit gel recovery kit to obtain pMD18-T simple-pRPS-30::RPS-30 S30 The plasmid was double digested with BglII / NheI and the product was recovered from the gel; S2043. At 16°C, the pMD18-T simple-GFP plasmid obtained in S2042 was digested with BglII / NheI and the recovered products were mixed with pMD18-T simple-pRPS-30::RPS -30 S30 The plasmid BglII / NheI double digestion gel recovery product was ligated overnight to obtain the ligation product of the BglII / NheI double digestion gel recovery product; Ligation reaction system: pMD18-T simple-GFP plasmid BglII / NheI double digestion gel recovery product 500ng, pMD18-T simple-pRPS-30::RPS-30 S30 After plasmid BglII / NheI double digestion, gel recovery product 100ng, ligation mix 8μL, ddH2O to 25μL; S2044, conversion coated plate: Take out from the storage temperature of -80℃ Escherichia coli TOP10 competent bacteria, add the ligation product of the gel recovery product obtained after the BglII / NheI double digestion obtained in S2043, ice bath for 30 minutes, then heat stress at 42°C for 90 seconds, ice bath for 2 minutes, add 1 mL of LB liquid culture medium, culture at 37°C and a shaking speed of 250 rpm / min for 1 hour, centrifuge to obtain the residual bacterial liquid after transformation, add the residual bacterial liquid after transformation to a solid LB culture plate containing ampicillin resistance, spread it evenly, and place it in a bacterial culture incubator at 37°C for overnight culture; S2045, sequencing identification: Monoclonal colonies were picked from the solid LB culture plates cultured overnight in S2044, and then placed in 3 mL of LB liquid culture medium containing ampicillin resistance, and cultured for 10 hours at 37°C and a shaking speed of 250 rpm / min. The obtained bacterial liquids were taken for sequencing and identification, and the monoclonal colonies with correct sequencing results were screened out; S2046, extract pMD18-T simple- p RPS-30::GFP::RPS-30 S30 Plasmids; The correctly sequenced monoclonal colony obtained in S2045 was extracted using the AxyPrep Plasmid Miniprep Kit plasmid extraction kit to obtain pMD18-T simple-pRPS-30:: GFP::RPS-30 S30 Plasmids; S3. Construction of pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmids: The pPD95.77 plasmid was combined with the pMD18-T simple-pRPS-30::GFP::RPS-30 obtained in S2046. S30 The plasmid was double-digested with KpnI / EcoRI restriction endonucleases, and the gel-recovered products after KpnI / EcoRI double-digestion were obtained. The gel-recovered products after KpnI / EcoRI double-digestion were ligated to construct pPD95.77-pRPS-30:: GFP::RPS-30. S30 Plasmid, the specific steps include: Double digestion with S301, KpnI / EcoRI restriction endonuclease: At 37°C, the pPD95.77 plasmid was double-digested with KpnI / EcoRI restriction enzymes for 5 h to obtain the pPD95.77 plasmid KpnI / EcoRI double-digested product; At 37°C, the KpnI / EcoRI restriction endonuclease pair obtained in S2046 was pMD18-T simple-pRPS-30::GFP::RPS-30 S30 The plasmid was subjected to double restriction digestion for 5 h to obtain pMD18-T simple-pRPS-30::GFP::RPS-30 S30 Plasmid KpnI / EcoRI double digestion product; Double restriction enzyme digestion reaction system: pPD95.77 plasmid or pMD18-T simple-pRPS-30::GFP::RPS-30 S30 Plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, KpnI restriction enzyme 1 μL, EcoRI restriction enzyme 1 μL, ddH2O to 20 μL; S302, gel recovery of KpnI / EcoRI double digestion products: The pPD95.77 plasmid KpnI / EcoRI double digestion product obtained in S301 was subjected to 1% agarose gel electrophoresis, and the target DNA band of 3562 bp was cut out and recovered using the AxyPrep DNA Gel Extraction Kit to obtain the pPD95.77 plasmid KpnI / EcoRI double digestion gel recovery product; The pMD18-T simple-pRPS-30::GFP::RPS-30 obtained in S301 was S30 The plasmid KpnI / EcoRI double digestion product was subjected to 1% agarose gel electrophoresis, and the target DNA band of 2900 bp was cut out and recovered using the AxyPrep DNA Gel Extraction Kit to obtain pMD18-T simple-pRPS -30::GFP::RPS-30 S30 The plasmid was double digested with KpnI / EcoRI and the product was recovered from the gel; S303, at 16°C, the pPD95.77 plasmid obtained in S302 was digested with KpnI / EcoRI and the recovered product and pMD18-T simple-pRPS-30::GFP::RPS-30 were S30 The plasmid KpnI / EcoRI double digestion gel recovery product was ligated overnight to obtain the KpnI / EcoRI double digestion gel recovery product ligation product; Ligation reaction system: pPD95.77 plasmid KpnI / EcoRI double digestion gel recovery product 100ng, pMD18-Tsimple-pRPS-30::GFP::RPS-30 S30 After plasmid BglII / NheI double digestion, 500 ng of gel recovery product, 8 μL of LigationMix, and ddH2O were added to 25 μL; S304, conversion coated plate: Take out from the storage temperature of -80℃ Escherichia coli TOP10 competent bacteria, add the ligation product of the gel recovery product obtained after KpnI / EcoRI double digestion obtained in S303, ice bath for 30 minutes, then heat stress at 42°C for 90 seconds, ice bath for 2 minutes, add 1 mL LB liquid culture medium for culture, culture at 37°C and a shaking speed of 250 rpm / min for 1 hour, centrifuge to obtain the residual bacterial liquid after transformation, add the residual bacterial liquid after transformation to a solid LB culture plate containing ampicillin resistance, spread it evenly, and place it in a bacterial incubator at 37°C for overnight culture; S305, sequencing and identification; Monoclonal colonies were picked from the solid LB culture plates cultured overnight in S304, and then placed in 3 mL of LB liquid culture medium containing ampicillin resistance, and cultured for 10 hours at 37°C and a shaking speed of 250 rpm / min. The obtained bacterial liquids were taken for sequencing and identification, and the monoclonal colonies with correct sequencing results were screened out; S306, extract pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmids; The correctly sequenced monoclonal colony obtained in S305 was extracted using the AxyPrep Plasmid Miniprep Kit plasmid extraction kit to obtain pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmids; S4. Construction of pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Plasmids: pPD95.77-pRPS-30::GFP::RPS-30 obtained in S306 S30 The plasmid was the same as pMD18-Tsimple-pU6::shRNA(RPS-30 UbL ) plasmid was double digested with SmaI / KpnI restriction endonucleases, and the SmaI / KpnI double digested gel-recovered products were obtained by gel ligation to obtain pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Plasmid, i.e., transgenic plasmid that extends the healthy life span of Caenorhabditis elegans, the specific steps include: Double digestion with S401, SmaI / KpnI restriction endonuclease: At 37°C, the pPD95.77-pRPS-30::GFP::RPS-30 obtained in S306 was cleaved by SmaI / KpnI restriction endonucleases. S30 The plasmid was subjected to double restriction digestion for 5 h to obtain pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmid SmaI / KpnI double digestion product; At 37°C, the pMD18-T simple-pU6::shRNA (RPS-30 UbL ) plasmid was subjected to double restriction digestion for 5 h to obtain pMD18-T simple-pU6::shRNA(RPS-30 UbL ) Plasmid SmaI / KpnI double digestion product; Reaction system: pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmid or pMD18-T simple -pU6::shRNA(RPS-30 UbL ) plasmid 1.5 μg, 10× FastDigest Green buffer 2 μL, SmaI restriction enzyme 1 μL, KpnI restriction enzyme 1 μL, ddH2O to 20 μL; S402, gel recovery SmaI / KpnI double digestion product: The pPD95.77-pRPS-30::GFP::RPS-30 obtained in S401 was S30 Plasmid SmaI / KpnI double digestion product SmaI / KpnI double digestion product was subjected to 1% agarose gel electrophoresis, and the target DNA band of 6462 bp was cut out and recovered using AxyPrepDNA Gel Extraction Kit to obtain pPD95.77-pRPS-30::GFP::RPS-30 S30 The plasmid was double digested with SmaI / KpnI and the product was recovered from the gel; The pMD18-T simple-pU6::shRNA (RPS-30 UbL ) plasmid SmaI / KpnI double digestion product was subjected to 1% agarose gel electrophoresis, and the target DNA band of 326 bp was cut out and recovered using AxyPrep DNA Gel Extraction Kit to obtain pMD18-T simple-pU6::shRNA(RPS-30 UbL ) Plasmid SmaI / KpnI double enzyme digestion and gel recovery product; S403. At 16°C, pPD95.77-pRPS-30:: GFP::RPS-30 obtained in S402 was added. S30 The plasmid SmaI / KpnI double digestion gel recovery product and pMD18-T simple -pU6::shRNA (RPS-30 UbL ) The plasmid SmaI / KpnI double digestion gel recovery product was ligated overnight to obtain a ligation product of the SmaI / KpnI double digestion gel recovery product; Ligation reaction system: pPD95.77-pRPS-30::GFP::RPS-30 S30 Plasmid SmaI / KpnI double digestion gel recovery product 100ng, pMD18-T simple-pU6::shRNA (RPS-30 UbL ) After double digestion of plasmid SmaI / KpnI, 500 ng of gel recovery product, 8 μL of Ligation Mix, and ddH2O to 25 μL; S404, conversion coated plate: Take out from the storage temperature of -80℃ Escherichia coli TOP10 competent bacteria, add the ligation product of the gel recovery product obtained after SmaI / KpnI double digestion obtained in S403, ice bath for 30 minutes, then heat stress at 42°C for 90 seconds, ice bath for 2 minutes, add 1 mL LB liquid culture medium for culture, culture at 37°C and shaking speed of 250 rpm / min for 1 hour, centrifuge to obtain the residual bacterial liquid after transformation, add the residual bacterial liquid after transformation to the solid LB culture plate containing ampicillin resistance, spread it evenly, and place it in a bacterial culture incubator at 37°C for overnight culture; S405, sequencing identification: Monoclonal colonies were picked from the solid LB culture plates cultured overnight in S404, and then placed in 3 mL of LB liquid culture medium containing ampicillin resistance, and cultured for 10 hours at 37°C and a shaking speed of 250 rpm / min. The obtained bacterial liquids were taken for sequencing and identification, and the monoclonal colonies with correct sequencing results were screened out; S406, extract pPD95.77-pU6::shRNA(RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Plasmids; The monoclonal colony with correct sequencing obtained in S405 was extracted using the AxyPrep Plasmid Miniprep Kit plasmid extraction kit to obtain pPD95.77-pU6::shRNA (RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 Plasmid, that is, a transgenic plasmid for extending the healthy life span of Caenorhabditis elegans; the pPD95.77-pU6::shRNA (RPS-30 UbL )-pRPS-30::GFP::RPS-30 S30 The nucleotide sequence of the plasmid is shown in SEQ ID NO:

11.

2. A use of a transgenic plasmid for extending the healthy life span of Caenorhabditis elegans as claimed in claim 1, characterized in that: The transgenic plasmid for extending the healthy life span of Caenorhabditis elegans is used for extending the healthy life span of Caenorhabditis elegans.

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