PeLAP1 gene knockout system of Nanlin 895 poplar and its application

Abstract

The present invention discloses a 'Nanlin 895 Poplar' PeLAP1 gene knockout system and its application, and belongs to the technical field of plant genetic engineering. The present invention uses 'Nanlin 895 Poplar' tissue culture seedlings as materials, obtains the PeLAP1 gene by cloning, uses sgRNA target sites Target1, Target2, and Target3 to accurately edit the PeLAP1 gene, and on this basis constructs a knockout vector pYLCRISPR / Cas935S‑PeLAP1, which is transferred into 'Nanlin 895 Poplar' leaves, and cultivates and screens to obtain 'Nanlin 895 Poplar' PeLAP1 mutant plants with reduced branch numbers. The PeLAP1 mutant has almost no or very few branches, and the plant height of the mutant plants has increased to varying degrees. The average number of internodes of the mutant main stem is 17.25, which is 26.19% higher than the average number of internodes of CK in the same period, 13.67.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and more specifically relates to a Nanlin 895 poplar PeLAP1 gene knockout system and application thereof. Background Art

[0002] Poplar species represented by American black poplar and its hybrids have the advantages of fast growth, high timber yield, short rotation period and wide adaptability in the Huanghuai, Jianghuai and middle and lower reaches of the Yangtze River plains in my country. They are not only the preferred species for cultivating short-cycle industrial raw material forests, but also the pioneer species for urban and rural greening and afforestation. They play an extremely important role in alleviating the contradiction between wood supply and demand in my country, maintaining ecological balance, and achieving agricultural efficiency and farmers' income. As a model tree species for basic forest biology research, the functional genomics research of poplar has made great progress in recent years. Based on the utilization of hybrid vigor, combined with modern molecular biotechnology, the genetic regulation mechanism of poplar growth and development and adversity adaptation traits is comprehensively analyzed, and an efficient and precise gene editing breeding technology system is established, which will greatly promote the genetic improvement process of poplar and improve breeding efficiency.

[0003] Compared with traditional gene editing technologies ZFN (zinc finger nuclease) and TALEN (transcription activator-like effector nuclease), CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) gene editing technology has become a powerful tool for plant functional genomics research and molecular design breeding due to its advantages of simplicity, speed, efficiency and strong scalability. At present, CRISPR / Cas9 (or Cas12) gene editing technology has been preliminarily applied in the field of poplar functional genomics research. These studies focus on the analysis of the genetic basis of poplar growth and development traits. The genetic transformation and gene editing receptor materials used are limited to a few genotype materials, such as silver gland poplar 84K, European and American mountain poplar T89, silver gray poplar 717, white poplar 741 and Xinjiang poplar. There is no doubt that these receptor materials are good model systems for forest functional genomics research, but their field cultivation performance is poor and their adaptability is narrow, so they are not suitable for creating excellent germplasm with broad cultivation application prospects through gene editing technology.

[0004] Branching is an important component of the ideal plant type of poplars, which directly affects the survival rate and yield rate of afforestation. Using this as the target trait for precise improvement, the establishment of a genetic transformation and gene editing system for black poplar varieties can not only accelerate the creation of new and superior poplar germplasm with ideal plant types, but also lead the process of forest tree variety improvement. Nanlin 895 poplar has the advantages of fast growth, high quality, high yield, and high resistance. It is suitable for industrial timber afforestation and greening afforestation in plain areas such as Jianghuai and the middle and lower reaches of the Yangtze River in my country. It is a national-level fine variety obtained by Nanjing Forestry University (National S-SC-PE-002-2002). Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a Nanlin 895 poplar PeLAP1 gene knockout system; another technical problem to be solved by the present invention is to provide an application of the Nanlin 895 poplar PeLAP1 gene knockout system for genetic improvement breeding.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] The Nanlin 895 poplar PeLAP1 gene knockout system, the nucleotide sequence of the Nanlin 895 poplar PeLAP1 gene is shown as SEQ ID NO.1; the amino acid sequence of its expressed protein is shown as SEQ ID NO.2.

[0008] The target sequence of the Nanlin 895 poplar PeLAP1 gene knockout system is:

[0009] Target1: 5'-CTCAGTCCTATGTGATGCCG-3',

[0010] Target2: 5'-CTTCCCCCAAATAGTGCCTG-3',

[0011] Target3: 5'-TCTGCAGCTCAGAAATCGAC-3'.

[0012] Knockout vector containing the PeLAP1 gene of Nanlin 895 poplar.

[0013] The knockout vector is a plant expression vector.

[0014] The plant expression vector is pYLCRISPR / Cas935S-PeLAP1.

[0015] Application of Nanlin 895 Poplar PeLAP1 gene knockout system in knocking out Nanlin 895 Poplar PeLAP1 gene.

[0016] The application of the Nanlin 895 poplar PeLAP1 gene knockout system in reducing the number of plant branches includes the following steps:

[0017] (1) Construct a knockout system for the PeLAP1 gene in Nanlin 895 poplar;

[0018] (2) The PeLAP1 knockout system constructed in Nanlin 895 Populus was transformed into the leaves of Nanlin 895 Populus;

[0019] (3) Cultivate and screen Nanlin 895 poplar plants with reduced number of branches.

[0020] Application of the PeLAP1 gene knockout system in Nanlin 895 poplar to promote plant height increase.

[0021] Application of the PeLAP1 gene knockout system in Nanlin 895 poplar to increase the average internode number of plants.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention uses the tissue culture seedlings of Nanlin 895 poplar as materials, obtains the PeLAP1 gene of Nanlin 895 poplar by cloning, uses sgRNA target sites Target1, Target2, and Target3, accurately edits the PeLAP1 gene of Nanlin 895 poplar, and constructs a knockout vector pYLCRISPR / Cas935S-PeLAP1 on this basis, which is transferred into the leaves of Nanlin 895 poplar, and cultivates and screens to obtain Nanlin 895 poplar PeLAP1 mutant plants with reduced branch numbers. The PeLAP1 mutant has almost no or very few branches, while the average number of branches of the control group CK in the same period is significantly higher than that of the mutant. In addition, the plant height of the mutant plants has increased to varying degrees, with the highest strain being 66 (cm) and the shortest strain being 44 (cm), which is 35.37% higher than the average plant height (40.17 cm) of the CK in the same period; the average number of internodes of the mutant main stem is 17.25, which is 26.19% higher than the average number of internodes of the CK in the same period, 13.67. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the gRNA target information diagram of the PeLAP1 gene (T1, 2, and 3 represent three target sites respectively);

[0025] Figure 2 It is a map of CRISPR / Cas9 related vectors (a is the map of pYLsgRNA-AtU3b, pYLsgRNA-AtU3d, pYLsgRNA-AtU6-1, and pYLsgRNA-AtU6-29 intermediate vectors; b is the map of CRISPR / Cas9 binary vectors);

[0026] Figure 3 This is a graph showing the detection results of the sgRNA expression cassette in the first round of PCR reaction 1 (M is Marker III);

[0027] Figure 4 This is a graph showing the detection results of the sgRNA expression cassette in the first round of PCR reaction 2 (M is Marker III);

[0028] Figure 5 This is the result of the sgRNA expression cassette detection in the second round of PCR reaction (M is Marker III);

[0029] Figure 6This is a diagram of the construction of the CRISPR / Cas9-PeLAP1 vector (a is a detection diagram of the Escherichia coli culture solution of the knockout vector, in which lanes 1, 2, 3, 4, 5, 6, 8, 9, 11, and 12 are positive culture solutions; bd are sequencing peak diagrams of the knockout vector);

[0030] Figure 7 This is a picture of transgenic positive plants identified by PCR (M is Marker III; WT is blank control; lanes 1-11 are mutant lines, and lanes 2, 3, 5, 8, 9, and 10 are positive seedlings);

[0031] Figure 8 This is a comparison of the peak maps of 3 clone sequencing of the target sites of 4 PeLAP1 mutant strains (ad represents KO#10, KO#58, KO#66, and KO#78 strains, respectively, the green underline represents the target sequence, and the red frame represents the PAM sequence "NGG");

[0032] Fig. 9 This is a comparison of the peak maps of 3 clone sequencing of the target sites of 4 PeLAP1 mutant strains (ad represents KO#86, KO#87, KO#88, and KO#264 strains, respectively, the green underline represents the target sequence, and the red frame represents the PAM sequence "NGG");

[0033] Fig.10 Analysis diagram of 3 mutation sites at the target site of 8 PeLAP1 mutant strains (green font and underline represent target sequence and PAM, respectively; "-" represents base deletion; "+" represents inserted base; blue font represents inserted base; red font represents replaced base);

[0034] Fig.11 It is the amino acid sequence alignment of 8 PeLAP1 mutant strains;

[0035] Fig.12 The phenotypes of eight PeLAP1 mutant strains are shown;

[0036] Fig.13 The phenotypic statistics of 8 PeLAP1 mutant strains. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0038] The plant material used in the invention is Nanlin 895 poplar tissue culture seedlings, which are grown on WPM rooting medium under the culture conditions of 16 hours of light, 8 hours of darkness, and humidity set to 70%.

[0039] Trans1-T1 E. coli competent cells, -BluntZeroCloningKit cloning vector was purchased from Quanshijin (Beijing) Biotechnology Co., Ltd.

[0040] The EHA105 Agrobacterium strain was purchased from Qingke (Beijing) Biological Co., Ltd.

[0041] The overexpression vector pH35GS was stored in the laboratory of Nanjing Forestry University.

[0042] RNAprepPure polysaccharide and polyphenol plant total RNA extraction kit and polysaccharide and polyphenol plant genomic DNA extraction kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; AxyPrep TM DNA Gel Extraction Kit was purchased from AXYGEN (USA); PrimeSTAR Max DNA Polymerase high-fidelity enzyme, PrimeScript TM RTMasterMix reverse transcription reagent, PowerUP TM SYBRGreenMastermix quantitative reagent was purchased from ThermoFisher (USA); TIANpure Midi PlasmidKit high-purity plasmid mini-preparation and midi kit (centrifugal column type) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; One Step Cloning Kit was purchased from Novozymes (Nanjing) Biotechnology Co., Ltd.; T4DNA ligase and BsaI-HF enzyme were purchased from TaKaRa Biotechnology (Beijing) Co., Ltd.; other commonly used reagents for molecular experiments.

[0043] Example 1

[0044] 1. Extraction of Total RNA

[0045] The RNA from the roots, stems, and leaves of 30-day-old Nanlin 895 poplar tissue culture seedlings was extracted using a polysaccharide and polyphenol plant total RNA extraction kit and mixed in equal amounts. The experimental steps are as follows:

[0046] Add 475 μL of lysis buffer SL and 25 μL of β-mercaptoethanol to a 1.5 mL RNase-Free centrifuge tube; add the plant tissue to a high-temperature sterilized mortar and grind it immediately and quickly, add the ground plant tissue into a 1.5 mL centrifuge tube containing the lysis buffer, and then shake vigorously for 1 minute; centrifuge at 12000 rpm for 2 minutes at room temperature; transfer the supernatant to the filter column CS, centrifuge at 12000 rpm for 2 minutes at room temperature, and then carefully pipette the supernatant into a new 1.5 mL RNase-Free centrifuge tube; add 300 μL of anhydrous ethanol to the centrifuge tube, mix it with a pipette, and then transfer it to the adsorption column CR3, centrifuge it at 12000 rpm for 15 seconds, discard the waste liquid, and replace CR3. In the collection tube; add 350μL deproteinization solution RW1, centrifuge at 12000rpm for 15s, and discard the waste liquid; take out 10μL DNaseI from the -20℃ refrigerator, add 70μL RDD, and mix gently; add the DNaseI preparation solution to the center of the adsorption membrane of the CR3 adsorption column, and let it stand at room temperature for 15min; add 350μL deproteinization solution RW1, centrifuge at 12000rpm for 15s, and discard the waste liquid; add 500μL rinse solution PW, centrifuge at 12000rpm for 15s, discard the waste liquid, and put CR3 back into the collection tube and wash twice; centrifuge at 12000rpm for 2min, and put CR3 into a new centrifuge tube; add 35μL RNase-Free ddH2O to the center of the adsorption membrane in the CR3 adsorption column, let it stand on ice for 2min, and centrifuge at 12000rpm at 4℃ for 1min; repeat elution, and finally obtain RNA solution, which is stored in a -80℃ ultra-low temperature refrigerator.

[0047] RNA quality detection: The quality of total RNA was determined by spectrophotometer, where OD260 / 280 should be in the range of 1.8-2.1; the integrity of RNA was detected by 1% agarose gel electrophoresis.

[0048] 2. cDNA Synthesis

[0049] The total RNA of plants was reverse transcribed into cDNA using the PrimeScript RT Master Mix kit.

[0050] The reaction system was: Prime Script RT Master Mix (5×) 4 μL, Total RNA 1000 ng X μL, RNase-Free ddH2O supplemented to 20 μL.

[0051] The reaction procedure is: 37°C, 15 min; 85°C, 5 s. After the reaction is completed, the cDNA product is diluted according to specific needs and stored at 4°C.

[0052] 3. DNA extraction

[0053] Use the polysaccharide and polyphenol DNA extraction kit to extract poplar genomic DNA. The specific steps are shown in the instruction manual.

[0054] 4. Primer design

[0055] Download the Populus trichocarpa v4 genome data from the JGI (https: / / phytozome-next.jgi.doe.gov / ) website, and design specific amplification primers based on the LAP1 sequence provided in the database. Use Oligo6 to design primers with a length of 23-28 nt, a GC content of 40%-70%, and an annealing temperature of Tm ≥ 60°C. The primer sequences are as follows:

[0056] PeLAP1-F: 5'-ATGGGAAGAGGTAGGGTTCAGTT-3',

[0057] PeLAP1-R: 5'-TCATGTTCCAAAGCATCCAAGGT-3'.

[0058] 5. Amplification of target fragment

[0059] Using Nanlin 895 poplar cDNA and DNA as templates, the target fragment was amplified using the high-fidelity enzyme PrimeSTARMaxDNAPolymerase in a 50μL system.

[0060] The reaction system is:

[0061]

[0062]

[0063] The reaction procedure is:

[0064]

[0065] The PCR amplification product was detected by 2% agarose gel electrophoresis, and a single and clear gel band was recovered for recovery and purification.

[0066] 6. Purification and ligation of target fragments

[0067] Using the AxyPrep Gel Extraction Kit TM DNA Gel Extraction Kit is used to recover and purify the target fragment. The experimental steps are as follows:

[0068] Place the separated agarose gel block in a 2mL centrifuge tube, peel and weigh it, add DE-A solution to the centrifuge tube at a ratio of gel block weight: DE-A = 1:3, and place it in a 75℃ water bath to melt; add 1 / 2 volume of DE-A DE-B solution, mix thoroughly to form a yellow solution, pipette it into the DNA preparation tube, centrifuge at 12000rpm for 1min, and pour out the waste liquid; add 500μLW1, centrifuge at 12000rpm for 30s, pour out the waste liquid; add 700μLW2, centrifuge at 12000rpm for 30s, pour out the waste liquid; add 700μLW2, centrifuge at 12000rpm for 1min; place the DNA preparation tube in a new 1.5mL centrifuge tube, vertically add 20μLddH2O to the center of the adsorption membrane and open the lid and let it stand for 2min. After centrifugation at 12000rpm for 2min, the purified solution of the target fragment can be obtained.

[0069] The recovered PCR products were tested for concentration and quality by spectrophotometer and then connected to -BluntZeroCloningVector carrier. The reaction system is as follows:

[0070]

[0071] The reaction procedure is: 25°C, 15 min. The ligation product is stored in a refrigerator at 4°C for later use.

[0072] Add 5 μL of the ligation product to the thawed Trans1-T1 Escherichia coli competent strain, gently stir to mix, and place on ice for 30 minutes; place in a 42°C water bath, heat shock for 40 seconds, immediately return to the ice box, and place on ice for 4 minutes; add 250 μL LB liquid culture medium, set the shaker at 200 rpm, and shake at 37°C for 1 hour; remove the bacterial solution from the shaker and centrifuge at 4000 rpm for 5 minutes; remove 600 μL of supernatant in the clean bench, mix the remaining bacterial solution with a pipette, and then drop it into LB solid culture medium with ampicillin (Amp) resistance, and spread it evenly with a coating rod; invert in a 37°C constant temperature incubator and culture for 12 hours.

[0073] Pick a single colony of appropriate size from the plate, inoculate each colony into 800 μL LB liquid culture medium carrying Amp resistance, and shake the culture on a temperature-controlled shaker at 250 rpm and 37°C for 4-6 hours. Then use the bacterial solution as a template for PCR cloning detection.

[0074] The reaction system is as follows;

[0075] 2× Rapid Taq Master Mix 5μL M13F Forward Primer (10μM) 0.5μL M13R Reverse Primer (10μM) 0.5μL Bacterial liquid 1μL <![CDATA[ddH2O]]> up to 10 μL

[0076] The reaction procedure is:

[0077]

[0078] The fragment size of the PCR product was preliminarily detected by 1.5% agarose gel electrophoresis. The corresponding positive bacterial solution with the band position consistent with the target fragment was selected and sent to Sangon for sequencing. According to the sequencing results, the sequence of the target gene was determined to be 684 bp as shown in SEQ ID NO.1, and was named PeLAP1. The protein sequence expressed by it is shown in SEQ ID NO.2.

[0079] Example 2

[0080] 1. sgRNA target site design

[0081] Design the target site using the targetDesign subroutine on the CRISPR-GE website (http: / / skl.scau.edu.cn / ). First, select SpCas9fromSterptococcuspyogenes:5'-NGG-3' for PAMtype, select P.trichocarpa (JGI2.0) for the reference genome, paste the PeLAP1 genome sequence in the "Sequence submission" box, and click "Submit" to start the run. Select the optimal target with a target GC content between 45% and 70%, a low off-target rate, and located in the CDS region from the results provided by the software.

[0082] Target 1 is located in the positive strand of the first exon, with a sequence of 5'-CTCAGTCCTATGTGATGCCG-3' and a PAM site of AGG; target 2 is located in the negative strand of the third exon, with a sequence of 5'-CTTCCCCCAAATAGTGCCTG-3' and a PAM site of TGG; target 3 is located in the negative strand of the fifth exon, with a sequence of 5'-TCTGCAGCTCAGAAATCGAC-3' and a PAM site of TGG. For more information on target sequences, see Figure 1 And Table 1.

[0083] Table 1 Target details

[0084] Target PAM Positive and negative chains GC content scope Off-target estimation Base pairing with sgRNA Target1 AGG + 55％ cds1 0.386 None Target2 TGG - 55％ cds3 0.156 None Target3 TGG - 50％ cds5 0.289 None

[0085] 2. Primer design

[0086] The CRISPR / sgRNA intermediate vectors used were pYLsgRNA-AtU3d, pYLsgRNA-AtU6-1, pYLsgRNA-AtU6-29 ( Figure 2 a); The CRISPR / Cas9 binary vector used is pYLCRISPR / Cas935S-DN (N = NTPII, kanamycin resistance) mainly used in dicotyledons ( Figure 2b), the backbone is pCAMBIA-1300 (ACCESSION: AF234296). After selecting the target sequence, click "primerDesign-V" in the CRISPR-GE software, select the Arabidopsis small nuclear RNA promoter (AtU3d, AtU6-1, AtU6-29) and method2 to automatically generate the required sgRNA amplification primers.

[0087] 3. Plasmid extraction

[0088] Use a high-purity mini-preparation kit to extract the plasmids of the editing vector pYLCRISPR / Cas935S-DN strain and the promoter vector pYLgRNA-U3 / U6 strain. The editing vector pYLCRISPR / Cas935S-DN is Kan-resistant, and the promoter vectors pYLsgRNA-AtU3d, pYLsgRNA-AtU6-1, and pYLsgRNA-AtU6-29 are Amp-resistant. Refer to the kit instructions for the experimental steps.

[0089] 4. Construction of sgRNA expression cassette

[0090] (1) First round of PCR reaction

[0091] a. Reaction 1

[0092] The corresponding promoters pYLsgRNA-AtU3d, pYLsgRNA-AtU6-1, and pYLsgRNA-AtU6-29 were amplified using the universal primer UF (UF: 5'-CTCCGTTTTACCTGTGGAATCG-3') and PeLAP1-AtU3dT1 (5'-CGGCATCACATAGGACTGAGTgaccaatggtgctttg-3'), PeLAP1-AtU6-1T2 (5'-CAGGCACTATTTGGGGGAAGCaatcactacttcgtct-3'), and PeLAP1-AtU6-29T3 (5'-GTCGATTTCTGAGCTGCAGACaatctcttagtcgact-3'), and the three target sequences were introduced into the corresponding promoter downstream. The reaction system is:

[0093] PrimeSTAR Max premix (2×) 10μL pYLgRNA-U6 / U32~5ng 1μL UF 0.5μL U#-T# 0.5μL <![CDATA[ddH2O]]> up to 20 μL

[0094] The reaction procedure is:

[0095]

[0096] In total, three separate PCR products were obtained in reaction 1: U3d-PeLAP1T1, U6-1-PeLAP1T2, and U6-29-PeLAP1T3 ( Figure 3 ).

[0097] b. Reaction 2

[0098] Using pYLsgRNA-AtU3d, pYLsgRNA-AtU6-1 and pYLsgRNA-AtU6-29 plasmids as templates, the universal primer gR-R (gR-R: 5'-CGGAGGAAAATTCCATCCAC-3') and PeLAP1-gRT1 (5'-CTCAGTCCTATGTGATGCCGgttttagagctagaaat-3'), PeLAP1-gRT2 (5'-CTTCCCCCAAATAGTGCCTGgttttagagctagaaat-3'), PeLAP1-gRT3 (5'-TCTGCAGCTCAGAAATCGACgttttagagctagaaat-3') were used to amplify the corresponding sgRNA, and the target sequence was introduced upstream of the sgRNA. The reaction system is:

[0099]

[0100]

[0101] The reaction procedure was similar to that of reaction 1 above. In reaction 2, a total of three separate PCR products were obtained: PeLAP1-T1-sgRNA, PeLAP1-T2-sgRNA, and PeLAP1-T3-sgRNA ( Figure 4 ).

[0102] Use 1.5% agarose gel electrophoresis to check whether the bands are correct, and dilute the three PCR stock solutions of reactions 1 and 2 10 times for later use.

[0103] (2) Second round of PCR reaction

[0104] Using the Overlapping PCR method, the gRNA expression cassette fragments were connected using U-B1' (5'-TTCAGAggtctcTctcgACTAGTGGAATCGGCAGCAAAGG-3'), U-B2' (5'-TTCAGAggtctcTctgaCACTGGAATCGGCAGCAAAGG-3'), U-B3' (5'-TTCAGAggtctcTaagaCACTGGAATCGGCAGCAAAGG-3') and the corresponding gR-B2 (5'-AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC-3'), gR-B3 (5'-AGCGTGggtctcGtcttGGTCCATCCACTCCAAGCTC-3'), and gR-BL (5'-AGCGTGggtctcGaccgACGCGTCCATCCACTCCAAGCTC-3') as primers to obtain three complete gRNA expression cassettes. The three PCR products were mixed in equal proportions and then recovered and purified for subsequent ligation reactions. The specific reaction system is as follows:

[0105] PrimeSTAR Max premix (2×) 10μL Mixed universal primers 0.5μL Dilutions of Reaction 1 and Reaction 2 1μL <![CDATA[ddH2O]]> up to 20 μL

[0106] The reaction procedure refers to the first round of PCR.

[0107] The products U3d-PeLAP1T1-sgRNA, U6-1-PeLAP1T2-sgRNA, U6-29-PeLAP1T3-sgRNA ( Figure 5 ), three complete sgRNA expression cassettes will be used to construct the pYLCRISPR / Cas935S-DN vector.

[0108] 5. CRISPR / Cas9-PeLAP1 vector construction

[0109] The purified product of the sgRNA expression cassette in the previous step and the pYLCRISPR / Cas935S-DN vector were digested with Bsa I and connected with T4 DNA ligase. The three sgRNA expression cassettes were assembled into the pYLCRISPR / Cas935S-DN empty vector by cutting and connecting using the Golden Gate Cloning method (Engler C, Kandzia R, Marillonnet SA OnePot, One Step, Precision Cloning Method with High Throughput Capability [J]. PLOSONE, 2008, 3 (11): e3647.). The ligation product was transferred into the competent E. coli Trans-T1, plated and cultured overnight, and a single colony was picked for bacterial liquid detection PCR using SP-DL and SP-R as primers. 1% agarose gel electrophoresis was used to preliminarily screen positive colonies.

[0110] The results are as follows Figure 6 As shown in a, lanes 1 and 2 are positive bacterial solutions containing three successfully constructed targets, and the remaining lanes are non-positive bacterial solutions. The positive bacterial solutions screened using SP-DL (SP-DL: 5'-GTCGTGCTCCACATGTTGACCG-3') and SP-R (SP-R: 5'-CCCGACATAGATGCAATAACTTC-3') sequencing primers were sent to the company for Sanger sequencing. The results showed that the sequence inserted into the knockout vector was completely consistent with the three designed sgRNA sequences ( Figure 6 b. Figure 6 c. Figure 6 d), indicating that the recombinant plasmid was successfully constructed.

[0111] 6. Positive screening of transgenic plants

[0112] After the successfully constructed knockout vector pYLCRISPR / Cas935S-PeLAP1 was transferred into Agrobacterium using the liquid nitrogen quick freeze-thaw method, the knockout vector pYLCRISPR / Cas935S-PeLAP1 with the correct sequence and insertion direction was transferred into the leaves of Nanlin 895 poplar by the Agrobacterium-mediated leaf disc transformation method. The polysaccharide polyphenol DNA kit was used to extract the DNA of CK and transgenic poplar leaves. Using DNA as a template, PCR amplification was performed using Cas9 primers (Cas9-F: 5'-CTGACGCTAACCTCGACAAG-3', Cas9-R: 5'-CCGATCTAGTAACATAGATGACACC-3') to preliminarily screen positive plants. Screening was performed using 2% agarose gel electrophoresis. Plants with a band length of 570bp were successfully transformed positive seedlings, and those without bands were not successfully transgenic. A total of 70 transgenic seedlings were tested, 27 of which were positive plants, with a transformation rate of 38.6%. The electrophoresis results of some positive individual plants are as follows Figure 7 shown.

[0113] 7. Knockout efficiency detection of multi-target CRISPR / Cas9-PeLAP1 vector

[0114] Target site specific detection primers were designed at a position 50-100 bp away from the target site using oligo6 software. Since the three target sites are far apart, they are divided into two sections for detection. PeLAP1Test1-F (PeLAP1Test1-F: 5'-ATGGGAAGAGGTAGGGTTCAGCTGAAG-3') and PeLAP1Test1-R (PeLAP1Test1-R: 5'-GAAAAACCTAATTAAGATGTAAGGGTA-3') are used to detect target site 1, and PeLAP1Test2-F (PeLAP1Test2-F: 5'-CGGGTTCCAATAATACCGAGTTTAACT-3') and PeLAP1Test2-R (PeLAP1Test2-R: 5'-ATGATTATAGCTTCTAAGGTTGTACAG-3') are used to detect target sites 2 and 3. Specific target site detection primers were used to amplify the genomic DNA of positive plants, and the PCR reaction system was:

[0115]

[0116]

[0117] The reaction procedure is:

[0118]

[0119] The amplified products were sequenced by Sanger sequencing, and the peak graph was decoded and analyzed using DSDecode (http: / / skl.scau.edu.cn / dsdecode / ) to determine the mutation of the target site. The protein sequences of the mutant and the control group were compared using BioXM software to determine the transcription and translation status after the mutation.

[0120] The results are as follows Figure 8 , Fig. 9 and Fig.10 As shown, among the 27 positive plants, 8 edited plants were screened, namely KO#10, KO#58, KO#66, KO#78, KO#86, KO#87, KO#88, and KO#264. The mutation sites all occurred at the target site 3, and the total editing efficiency was 29.6%. No homozygous mutations were found in all mutant plants. The specific editing conditions of the 8 mutant strains are as follows: KO#10 has a mutation type of 1-bp (G) insertion; KO#58 has a 34-bp deletion; KO#66 has a base mutation type of 1-bp (C) insertion; KO#78 has a mutation type of 3-bp (ATC) deletion; KO#86 has two mutation types; one is a 41-bp insertion and the other is a 6-bp deletion; KO#87 has a base mutation type of 1-bp (C) insertion; KO#88 has a 6-bp deletion; KO#264 has a 1-bp (T) insertion.

[0121] The deletion, mutation and insertion of bases in the mutants may cause changes in ORF and lead to frameshift mutations, thus leading to premature termination of PeLAP1 protein translation. The nucleotide sequences of the PeLAP1 gene in all mutants were translated into amino acid sequences and compared with the control group CK. The specific results are as follows: Fig.11 shown.

[0122] 8. Phenotypic observation of PeLAP1 mutants

[0123] The control group and the PeLAP1 mutant were transplanted into sterilized nutrient soil, and their phenotypes were observed and counted when they grew to 150 days old. The observations included plant height, number of branches, and number of internodes. SPSS26 software was used for data processing.

[0124] The results are as follows Fig.12 , Fig.13As shown in Table 2, the PeLAP1 mutant had almost no or very few branches, while the average number of branches of the control group CK at the same period was significantly higher than that of the mutant. In addition, the height of the mutant plants increased to varying degrees, with the tallest plant being 66 (cm) and the shortest plant being 44 (cm), an increase of 35.37% over the average plant height of CK at the same period (40.17 cm); the average number of internodes of the mutant main stem was 17.25, an increase of 26.19% over the average number of internodes of CK at the same period (13.67).

[0125] Table 2 Statistical data of relevant phenotypes of the control group CK strain and mutant strains

[0126] Characteristics Control group Mutant Number of branches 1.5±1.05 0.125±0.35** Number of internodes 13.67±1.97 17.25±3.33* Plant height (cm) 40.17±5.34 54.38±11.39*

[0127] Note: Statistical data are the mean ± standard deviation (SD) of three independent replicates; * indicates 0.01

Claims

1. Nanlin 895 Yang PeLAP1 Application of gene knockout system in promoting the increase of poplar plant height, the Nanlin 895 poplar PeLAP1 The target sequence of the gene knockout system is: Target3:5'-TCTGCAGCTCAGAAATCGAC-3'。 2. Yang from Nanjing Forestry University 895 PeLAP1 Application of gene knockout system in increasing the average internode number of poplar, the Nanlin 895 poplar PeLAP1 The target sequence of the gene knockout system is: Target3:5'-TCTGCAGCTCAGAAATCGAC-3'。

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