Preparation method based on application of OsLEC1 gene in rice callus differentiation
By using CRISPR-Cas9 technology to knock out the OsLEC1 gene in rice, the problem of low callus differentiation rate in the prior art was solved, and the effect of significantly improving the differentiation rate and transgenic seedling efficiency was achieved.
Patent Information
- Application Number
- CN202411103031.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-13
- Publication Date
- 2025-03-18
- Estimated Expiration
- 2044-08-13
AI Technical Summary
The prior art has a variety and chaotic method for improving the differentiation rate of callus in rice, and these methods are effective rather than universal for certain plant varieties, making it difficult to meet the needs of rice.
Using CRISPR-Cas9 technology based on the OsLEC1 gene, specific gRNA target sites were designed and introduced, and the OsLEC1 gene was knocked out through Agrobacterium-mediated transformation, thereby promoting the differentiation of rice callus.
Through knockout of OsLEC1 gene, the differentiation rate of rice callus is significantly improved, the emergence efficiency of transgenic plants is improved, and an important target gene is provided to improve the conversion efficiency of grass family crops.
Smart Images
Figure CN118620950B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of rice callus differentiation, and in particular to a method for OsLEC1 The invention discloses a method for applying gene in rice callus differentiation. Background Art
[0002] Plant tissue culture technology was developed in the early 20th century. It refers to the process of culturing isolated plant organs, tissues, cells, protoplasts, etc. under appropriate conditions under sterile conditions to induce the production of callus tissue, adventitious buds, etc., and further form complete plants.
[0003] In order to improve the differentiation rate of rice callus, researchers have adopted a series of innovations, such as drying, radiation, different hormone ratios, adding different carbon sources, adding copper, silver and other elements, adding proline, adding paclobutrazol (MET) and adding activated carbon. Drying and radiation treatments are time-consuming, and radiation treatments require specific instruments; adding copper, silver and other elements will cause additional heavy metal pollution. In short, the above methods are numerous and messy, and some methods only have significant effects on certain specific plant varieties, not applicable to rice, and it is difficult to meet the needs of staff. Summary of the invention
[0004] In order to solve the above technical problems, a OsLEC1 The preparation method of applying genes in rice callus differentiation solves the problem proposed in the above background technology that in order to improve the differentiation rate of rice callus, researchers have adopted a series of innovations, such as drying treatment, radiation treatment, different hormone ratios, adding different carbon sources, adding copper, silver and other elements, adding proline, adding paclobutrazol (MET) and adding activated carbon. Drying treatment and radiation treatment are time-consuming, and the radiation treatment method also requires specific instruments; adding copper, silver and other elements will cause additional heavy metal pollution, etc. In short, the above methods are numerous and messy, and some methods only have significant effects on certain specific plant varieties and are not applicable to rice.
[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is:
[0006] based on OsLEC1 The preparation method of applying gene in rice callus differentiation comprises:
[0007] S1. Select the gRNA target site. The sequence of the target site is located at OsLEC1 Genetically, OsLEC1 Gene is the sequence one recorded in the text;
[0008] S2, clone the tandem fragment containing gRNA;
[0009] S3, connect each gRNA fragment;
[0010] S4, performing PCR amplification on the ligation product;
[0011] S5, purification of product and digestion of target vector;
[0012] S6, transformation;
[0013] S7, Agrobacterium-mediated rice transgenic;
[0014] S8. Screening and identification of transgenic plants.
[0015] Preferably, the selection of the gRNA target site specifically comprises the following steps:
[0016] According to the target site design principles of CRISPR-Cas9 technology;
[0017] In close OsLEC1 Two gRNA target sites were designed on the gene sequence at the 5' end of the gene.
[0018] Preferably, the cloning of the tandem fragment containing gRNA specifically comprises the following steps:
[0019] Using plasmid pGTR as a template, three fragments L1, L2, and L3 containing gRNA sequences were amplified by PCR;
[0020] Take 5-10 μL of the product, detect it by 1% agarose gel electrophoresis, purify and recover the target fragment, and measure the three PCR products L1, L2, and L3.
[0021] Preferably, the connecting of the gRNA fragments specifically comprises the following steps:
[0022] According to the determined PCR product concentration, the three fragments were mixed in equal amounts;
[0023] T7 ligase and BsaI enzyme were added at the same time, and the reaction was carried out in a PCR instrument.
[0024] Preferably, the reaction is carried out at 37° C. for 5 min and at 20° C. for 10 min in the PCR instrument, and the reaction is cycled 30-50 times.
[0025] Preferably, the PCR amplification of the ligation product specifically comprises the following steps:
[0026] After the ligation reaction was completed, 1 μL of the ligation product was taken and diluted with 19 μL of water;
[0027] The diluted product was used as a template to perform PCR amplification with primers at both ends;
[0028] After the PCR was completed, 5 μL of the product was taken for electrophoresis detection. The product size was 500 bp, and the product was purified.
[0029] Preferably, the enzymatic digestion of the purified product and the target vector specifically comprises the following steps:
[0030] The purified PCR product was digested with FokI, while the target vector pRGEB32 was digested with BsaI;
[0031] After the digestion products were recovered, the purified PCR products and the target vector recovered after digestion were mixed in equal amounts and ligated with T4 ligase at 4°C overnight.
[0032] Preferably, the conversion specifically comprises the following steps:
[0033] The ligated vector was transformed into competent E. coli cells, plated, and incubated at 37°C overnight;
[0034] Pick a single colony and shake it, and use PCR to identify whether the target fragment is connected to the vector;
[0035] The correct extracted plasmid was identified and sequenced, and the plasmid with the correct sequencing result was electro-transformed into Agrobacterium EHA105.
[0036] Preferably, the Agrobacterium-mediated rice genetic modification specifically comprises the following steps:
[0037] The mature embryo callus of Nipponbare wild-type rice was used as explant;
[0038] The transformation was carried out by infection with Agrobacterium EHA105;
[0039] Transgenic plants were obtained through resistance screening, tissue differentiation, rooting and seedling hardening.
[0040] Preferably, the screening and identification of transgenic plants specifically includes the following steps:
[0041] Extract genomic DNA from transgenic plants and design primers on both sides of the two gRNA sequences;
[0042] The target fragment was amplified by PCR, and the mutants were detected by agarose gel electrophoresis and PAGE gel electrophoresis;
[0043] The PCR products of the mutant lines were purified and recovered, connected to the T vector for sequencing, and the mutant materials with the rice OsAFB4 gene knocked out were obtained.
[0044] Compared with the prior art, the present invention provides a OsLEC1 The preparation method of applying genes in rice callus differentiation has the following beneficial effects:
[0045] In the present invention, callus differentiation directly affects the efficiency of transgenic emergence. OsLEC1 Knockout of can promote the differentiation of rice callus, suggesting that it may be an important target gene for improving the transformation efficiency of rice and even Gramineae crops. It can be used as a starting point to construct various molecular tools to improve transformation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0046] Figure 1 For the present invention OsLEC1 Schematic diagram of the genome sequence editing and protein coding of the mutant;
[0047] Figure 2 For the present invention OsLEC1 Schematic diagram comparing the budding rates of callus differentiation between mutants and wild type. DETAILED DESCRIPTION
[0048] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.
[0049] Please refer to Figure 1-Figure 2 As shown, based on OsLEC1 The preparation method of applying gene in rice callus differentiation comprises:
[0050] S1. Select the gRNA target site. The sequence of the target site is located at OsLEC1 Genetically, OsLEC1 Gene is the sequence one recorded in the text;
[0051] S2, clone the tandem fragment containing gRNA;
[0052] S3, connect each gRNA fragment;
[0053] S4, performing PCR amplification on the ligation product;
[0054] S5, purification of product and digestion of target vector;
[0055] S6, transformation;
[0056] S7, Agrobacterium-mediated rice transgenic;
[0057] S8. Screening and identification of transgenic plants.
[0058] Selecting the gRNA target site specifically includes the following steps:
[0059] According to the target site design principles of CRISPR-Cas9 technology;
[0060] In close OsLEC1Two gRNA target sites were designed on the gene sequence at the 5' end of the gene.
[0061] It can be understood by those skilled in the art that OsLEC1 The gene is located on chromosome 2 of the rice genome. According to the target site design principle of CRISPR-Cas9 technology, the gRNA target site is designed in the exon region and is designed in OsLEC1 The 5' end of the gene. The designed primer sequences are as follows:
[0062]
[0063] Cloning the tandem fragment containing gRNA specifically includes the following steps:
[0064] Using plasmid pGTR as a template, three fragments L1, L2, and L3 containing gRNA sequences were amplified by PCR;
[0065] Take 5-10 μL of the product, detect it by 1% agarose gel electrophoresis, purify and recover the target fragment, and measure the three PCR products L1, L2, and L3.
[0066] It is understood by those skilled in the art that the sizes of L1, L2 and L3 products are approximately 130 bp, 200 bp and 150 bp, and the PCR reaction system and PCR procedure for amplifying the L1-3 fragment are as follows:
[0067]
[0068]
[0069] Connecting each gRNA fragment specifically includes the following steps:
[0070] According to the determined PCR product concentration, the three fragments were mixed in equal amounts;
[0071] T7 ligase and BsaI enzyme were added at the same time, and the reaction was carried out in a PCR instrument.
[0072] The reaction was carried out in a PCR instrument at 37°C for 5 min and at 20°C for 10 min, and the reaction was repeated 30-50 times.
[0073]
[0074]
[0075] The PCR amplification of the ligation product specifically includes the following steps:
[0076] After the ligation reaction was completed, 1 μL of the ligation product was taken and diluted with 19 μL of water;
[0077] The diluted product was used as a template to perform PCR amplification with primers at both ends;
[0078] After the PCR was completed, 5 μL of the product was taken for electrophoresis detection. The product size was 500 bp, and the product was purified.
[0079] S1-F: CG GGTCTC A GGCA GGATG GGCAGTCTG GGCA
[0080] S1-R:TA GGTCTC C AAAC GGATG AGCGACAGC AAAC
[0081]
[0082]
[0083] The purification product and the target vector digestion specifically include the following steps:
[0084] The purified PCR product was digested with FokI, while the target vector pRGEB32 was digested with BsaI;
[0085] After the enzyme digestion products were recovered respectively, the purified PCR product and the target vector recovered after enzyme digestion were mixed in equal amounts and ligated with T4 ligase at 4°C overnight.
[0086]
[0087]
[0088] The conversion process includes the following steps:
[0089] The ligated vector was transformed into competent E. coli cells, plated, and incubated at 37°C overnight;
[0090] Pick a single colony and shake it, and use PCR to identify whether the target fragment is connected to the vector;
[0091] The correct extracted plasmid was identified and sequenced, and the plasmid with the correct sequencing result was electro-transformed into Agrobacterium EHA105.
[0092] The PCR identification and sequencing primer sequences are as follows:
[0093] U3-F: AGTACCACCTCGGCTATCCACA
[0094] UGW-gRNA-R: CGCGCTAAAAACGGACTAGC
[0095] Agrobacterium-mediated rice genetic modification specifically includes the following steps:
[0096] The mature embryo callus of wild-type rice (Oryza sativa L. ssp. japonica 'Nipponbare') was used as explant;
[0097] The transformation was carried out by infection with Agrobacterium EHA105;
[0098] Transgenic plants were obtained through resistance screening, tissue differentiation, rooting and seedling hardening.
[0099] The screening and identification of transgenic plants specifically include the following steps: extracting genomic DNA from transgenic plants and designing primers on both sides of the two gRNA sequences;
[0100] The target fragment was amplified by PCR, and the mutants were detected by agarose gel electrophoresis and PAGE gel electrophoresis;
[0101] The PCR products of the mutant lines were purified and recovered, connected to the T vector for sequencing, and the mutant materials with the rice OsAFB4 gene knocked out were obtained.
[0102] The primer sequences are as follows:
[0103] OsLEC1 CF:TGTGGTCAACACCTCGTAGC
[0104] OsLEC1 CR:GACTTGAGCATCCCACCGAC
[0105]
[0106]
[0107] The PCR products of the above mutant strains were purified and recovered, and sequenced. The sequencing results are shown in Figure 1 The sequencing results showed that there were 46bp and 25bp deletions and 2bp deletion and 1bp insertion at the two target sites gRNA3 and gRNA4 in strains 3# and 4#, respectively. This sequence change will cause the termination codon of the protein to be advanced, and the translated protein will be shortened to 76 and 35 amino acids, respectively. So far, we have obtained rice OsLEC1 Gene knockout materials. The gRNA1 and gRNA2 in the figure are marked with two reported strains OsLEC1 -1 and -2 knockout sites, the previous knockout sites were OsLEC1 Behind the key domain, its knockout does not affect the transcription of the key domain part, so a new mutant with the knockout site at the key domain was constructed.
[0108] like Figure 2 As shown, wild type and OsLEC130 fresh seeds of each mutant were shelled, disinfected with 70% alcohol for 3 minutes, 30% sodium hypochlorite solution for 10 minutes, then rinsed with sterile water 6 times, spread on callus induction medium, and cultured in a 28°C light incubator for about 1 month. When granular callus appeared, it was subcultured to a new callus induction medium for about 20 days to expand and propagate. After that, callus particles with good growth status and similar size were selected and placed on differentiation medium. Photos were taken and the germination rate was counted after about 30 days. The results showed that OsLEC1 The budding rate of callus differentiation in the mutant was significantly increased compared with the wild type. OsLEC1 Knockout of can promote the differentiation of rice callus.
[0109]
[0110]
[0111] In summary, in the present invention, callus differentiation directly affects the efficiency of transgenic emergence. OsLEC1 Knockout of can promote the differentiation of rice callus, suggesting that it may be an important target gene for improving the transformation efficiency of rice and even Gramineae crops. It can be used as a starting point to construct various molecular tools to improve transformation efficiency.
[0112] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.
[0113] Sequence one
[0114] >O.sativa v7.0|LOC_Os02g49370|Chr2:30164272..30165441 reverse
[0115]
[0116] Sequence Two
[0117] >O.sativa v7.0|LOC_Os02g49370.1 CDS
[0118] ATGGAGGCCGGCTACCCGGGCACGGCGGCGAACGGCGCTGCCGCCGACGGGAACGGTGGCGCGCAGCAGGCGGCGGCCGCGCCGGCTATACGTGAGCAGGACCGGCTGATGCCGATCGCGAACGTGATCCGCATCATGCGCCGCGTGCTCCCGGCGCACGCCAAGATCTCGGACGACGCCAAGGAGACGATCCAGGAGTGCGTGTCGGAGTACATCAGCTTCATCACCGGGGAGGCCAACGAGCGGTGCCAGCGCGAGCAGCGCAAGACCATCACCGCCGAGGACGTGCTCTGGGCCATGAGCCGCCTCGGCTTCGACGACTACGTCGAGCCCCTCGGCGTCTACCTCCACCGCTACCGCGAGTTCGAGGGGGAGTCCCGCGGCGTCGGCGTCGGCGTCGGCGCCGCGCGCGGCGACCACCACCATGGTCACGTCGGTGGGATGCTCAAGTCCCGCGCGCAGGGCTCCATGGTGACGCACCACGACATGCAGATGCACGCCGCCATGTACGGTGGCGGCGCGGTGCCGCCGCCGCCGCATCCTCCTCCGCACCACCACGCGTTCCACCAGCTCATGCCGCCGCACCACGGCCAGTACGCGCCGCCGTACGACATGTACGGCGGCGAGCACGGGATGGCGGCGTACTACGGCGGGATGTACGCGCCCGGCAGCGGCGGCGACGGGAGCGGCAGCAGCGGCAGCGGTGGCGCCGGCACGCCGCAGACCGTCAACTTCGAGCACCAGCATCCGTTCGGATACAAGTAG
[0119] Sequence Three
[0120] >O.sativa v7.0|LOC_Os02g49370.1
[0121] MEAGYPGTAANGAAADGNGGAQQAAAAPAIREQDRLMPIANVIRIMRRVLPAHAKISDDAKETIQECVSEYISFITGEANERCQREQRKTITAEDVLWAMSRLGFDDYVEPLGVYLHRYREFEGESRGVGVGVGAARGDHHHGHVGGMLKSRAQGSMVTHHDMQMHAAMYGGGAVPPPPHPPPHHHAFHQLMPPHHGQYAPPYDMYGGEHGMAAYYGGMYAPGSGGDGSGSSGSGGAGTPQTVNFEHQHPFGYK。
Claims
1. Knockout OsLEC1 The application of the gene in promoting the differentiation of rice callus is characterized in that: include: S1. Select gRNA target site; S2, clone the tandem fragments containing gRNA; S3, connect each gRNA fragment; S4, performing PCR amplification on the ligation product; S5, purification of product and digestion of target vector; S6, transformation; S7, Agrobacterium-mediated rice transgenic; S8. Screening and identification of transgenic plants; Said OsLEC1 The gene is shown in sequence 1; The test method for promoting rice callus differentiation is as follows: A1, wild type and knockout OsLEC1 Thirty fresh seeds of each mutant were shelled, sterilized with 70% alcohol for 3 minutes and 30% sodium hypochlorite solution for 10 minutes; A2, after rinsing with sterile water for 6 times, spread on callus induction medium and culture in a 28℃ light incubator for 1 month; A3. When granular callus appears, it is subcultured to a new callus induction medium for 20 days to expand and propagate. Then, callus particles with good growth status and similar size are selected and placed on differentiation medium. After 30 days, photos are taken and the budding rate is calculated. The formula of callus induction medium is 3.859 g N6 medium powder, 2 mL 1 mg / ml 2,4-D, 2.8 g L-proline, 0.5 g tyrosinase, 30 g sucrose and 4.5 g phytagel, and the pH value of callus induction medium is 5.8; The formula of callus differentiation medium is 4.47 g MS powder, 3 mL 1 mg / ml 6-BA, 0.5 mL 1 mg / ml NAA, 20 g sorbitol, 30 g sucrose and 4.5 g phytagel, and the pH value of callus differentiation medium is 5.
5.
2. The knockout according to claim 1 OsLEC1 The application of the gene in promoting the differentiation of rice callus is characterized in that: The selection of the gRNA target site specifically comprises the following steps: According to the target site design principles of CRISPR-Cas9 technology; In close OsLEC1 Two gRNA target sites were designed on the gene sequence at the 5' end of the gene.
3. Knockout according to claim 2 OsLEC1 The application of the gene in promoting the differentiation of rice callus is characterized in that: The cloning of the tandem fragment containing gRNA specifically comprises the following steps: Using plasmid pGTR as a template, three fragments L1, L2, and L3 containing gRNA sequences were amplified by PCR; Take 5-10 μL of the product, detect it by 1% agarose gel electrophoresis, purify and recover the target fragment, and measure the three PCR products L1, L2, and L3; L1-F:CGGGTCTCAGGCAGGATGGGCAGTCTGGGCAACAAAGCACCAGTGG L1-R:CGGGTCTCAGCGTGCGCCGGGTGCACCAGCCGGG L2-F:TAGGTCTCCACGCCAAGATCTGTTTTAGAGCTAGAA L2-R:CGGGTCTCATAGCCGGCCTCCTGCACCAGCCGGG L3-F:TAGGTCTCCGCTACCCGGGCAGTTTTAGAGCTAGAA L3-R:TAGGTCTCCAAACGGATGAGCGACAGCAAACAAAAAAAAAAGCACCGACTCG.
4. The knockout according to claim 3 OsLEC1 The application of the gene in promoting the differentiation of rice callus is characterized in that: The connecting of each gRNA fragment specifically comprises the following steps: According to the determined PCR product concentration, the three fragments were mixed in equal amounts; T7 ligase and BsaI enzyme were added at the same time, and the reaction was carried out in a PCR instrument.
5. The knockout according to claim 4 OsLEC1 The application of the gene in promoting the differentiation of rice callus is characterized in that: The reaction was carried out in the PCR instrument at 37°C for 5 minutes and at 20°C for 10 minutes, and the reaction was repeated 30-50 times.
6. The knockout according to claim 4 OsLEC1 The application of the gene in promoting the differentiation of rice callus is characterized in that: The PCR amplification of the ligation product specifically comprises the following steps: After the ligation reaction was completed, 1 μL of the ligation product was taken and diluted with 19 μL of water; The diluted product was used as a template to perform PCR amplification with primers at both ends; After the PCR was completed, 5 μL of the product was taken for electrophoresis detection. The product size was 500 bp, and the product was purified.
7. The knockout according to claim 6 OsLEC1 The application of the gene in promoting the differentiation of rice callus is characterized in that: The enzyme digestion of the purified product and the target vector specifically comprises the following steps: The purified PCR product was digested with FokI, while the target vector pRGEB32 was digested with BsaI; After the digestion products were recovered, the purified PCR products and the target vector recovered after digestion were mixed in equal amounts and ligated with T4 ligase at 4°C overnight.
8. The knockout according to claim 7 OsLEC1 The application of the gene in promoting the differentiation of rice callus is characterized in that: The conversion specifically comprises the following steps: The ligated vector was transformed into competent E. coli cells, plated, and incubated at 37°C overnight; Pick a single colony and shake it, and use PCR to identify whether the target fragment is connected to the vector; The correct extracted plasmid was identified and sequenced, and the plasmid with the correct sequencing result was electro-transformed into Agrobacterium EHA105.
9. The knockout according to claim 8 OsLEC1 The application of the gene in promoting the differentiation of rice callus is characterized in that: The Agrobacterium-mediated rice transgenic method specifically comprises the following steps: The mature embryo callus of Nipponbare wild-type rice was used as explant; Transformation was performed using Agrobacterium EHA105; Transgenic plants were obtained through resistance screening, tissue differentiation, rooting and seedling hardening.
10. The knockout according to claim 9 OsLEC1 The application of the gene in promoting the differentiation of rice callus is characterized in that: The screening and identification of the transgenic plants specifically includes the following steps: Extract genomic DNA from transgenic plants and design primers on both sides of the two gRNA sequences; The target fragment was amplified by PCR, and the mutants were detected by agarose gel electrophoresis and PAGE gel electrophoresis; The PCR products of the mutant lines were purified and recovered, connected to the T vector for sequencing, and the knockout rice was obtained. OsLEC1 Gene mutation material.