Construction method and application of CRISPR-Cas9 silence system of cress PDS gene

Abstract

The invention discloses a construction method and application of a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) silence system of a cress PDS (protein dehydrogenase) gene, the method effectively realizes fixed-point editing of the cress gene OjPDS, and the sequence of the cress gene OjPDS is shown as SEQ ID NO.1 or SEQ ID NO.2. The invention further discloses a construction method of the cress PDS gene CRISPR-Cas9 silence system of the cress PDS gene. The invention lays a foundation for deep research on cress gene functions, provides a new way for accurately improving cress characters, provides a new idea for breeding new cress varieties, and has important significance for shortening the cress breeding period and accelerating the cress research progress.

Description

Technical Field

[0001] The invention relates to a construction method and application of a CRISPR-Cas9 silencing system of a water celery PDS gene, and belongs to the technical field of genetic engineering. Background Art

[0002] Genome editing technology is a biotechnology that targets and modifies specific DNA sequences in organisms. It can be divided into zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats and associated protein systems (CRISPR / Cas) according to different types of DNA enzymes. Among them, the CRISPR / Cas system is widely used in agronomy, biological gene editing, and genetic molecular breeding due to its advantages such as easy vector construction and high editing efficiency. CRISPR / Cas9 technology is the most widely used one among the CRISPR / Cas systems.

[0003] The CRISPR / Cas9 system uses Cas9 protein and sgRNA to precisely locate and cut the target site in the genome of the target organism. The cutting of the Cas9 protein causes double-strand breaks in the genomic DNA, and then introduces mutations through the main non-homologous end joining repair or the less efficient homologous recombination repair method. During the main non-homologous end joining repair, it usually causes the deletion, insertion or replacement of small fragments of nucleotides, which leads to changes in the expression products of the target gene, loss of function, and ultimately changes in the corresponding traits of the target organism. At present, crops including rice, corn, and wheat have used this technology to achieve the breeding of excellent trait germplasm and key gene editing, but the application of this gene editing technology in aquatic vegetables such as water celery is still rarely reported.

[0004] Water celery (Oenanthejavanica (Bl.) DC.) is a perennial herb of the Apiaceae family and the genus Oenanthe. It is deeply loved by consumers for its special aroma and crisp taste. It is mainly cultivated in the Yangtze River Basin and the areas to its south in my country. Water celery is a plant that is both medicinal and edible. It is not only rich in nutrients, but also has the functions of calming the liver and lowering blood pressure, anti-thrombosis, calming the nerves, and preventing and fighting cancer. However, water celery seeds are double-hanging fruits, and the problems of difficult seed germination, difficult harvesting, and low seed reproduction coefficient have greatly restricted the selection and breeding of new varieties of water celery, making the innovation and application of water celery breeding technology lag behind other crops. Therefore, it is particularly important to establish an efficient water celery gene editing system for the selection and breeding of high-quality trait germplasm of water celery, improving the quality of water celery, and assisting in the identification and verification of key gene functions of water celery. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a celery phytoene dehydrogenase gene (OjPDS), and a construction method and application of a CRISPR-Cas9 silencing system for silencing the gene.

[0006] Technical solution: The present invention provides a celery phytoene dehydrogenase gene, and the full-length sequence of the celery phytoene dehydrogenase gene is shown in SEQ ID NO.2.

[0007] Furthermore, the CDS sequence of the celery phytoene dehydrogenase gene is shown as SEQ ID NO.1.

[0008] The present invention also provides a method for constructing a CRISPR-Cas9 silencing system of a PDS gene of water celery, comprising the following steps:

[0009] (1) Designing a target site according to the full-length sequence of the celery phytoene dehydrogenase gene as shown in SEQ ID NO.2 or the CDS sequence of the celery phytoene dehydrogenase gene as shown in SEQ ID NO.1, synthesizing a vector expression cassette sequence and connecting it to the linearized basic vector to construct a celery phytoene dehydrogenase gene CRISPR / Cas9 expression vector;

[0010] (2) The CRISPR / Cas9 expression vector of the phytoene dehydrogenase gene of the water celery was transformed into Agrobacterium to obtain the CRISPR-Cas9 silencing system of the PDS gene of the water celery.

[0011] Furthermore, the target sites described in step (1) include any one or more combinations shown in SEQ ID NOs. 5 to 8.

[0012] Furthermore, the vector expression cassette sequence also includes a promoter and sgRNA.

[0013] Furthermore, the promoter includes AtU3b, AtU3d, AtU6-1 and AtU6-29.

[0014] Furthermore, the vector expression cassette sequence is shown as SEQ ID NO.9.

[0015] Furthermore, the basic vector is pYLCRISPR / Cas9P 35S -N.

[0016] The present invention also provides a method for cultivating water celery plants using the CRISPR-Cas9 silencing system of the water celery PDS gene obtained by the above-mentioned construction method, wherein the water celery callus is infected with Agrobacterium using the CRISPR-Cas9 silencing system of the water celery PDS gene, and then adventitious bud regeneration culture, cell embryo regeneration culture, subculture culture and rooting culture are carried out to obtain regenerated plants.

[0017] Furthermore, the regenerated plants are plants with kanamycin resistance.

[0018] Furthermore, the culture medium for the adventitious bud regeneration culture is CIM+50mg / L Km+300mg / L Cb.

[0019] Furthermore, the culture medium for somatic embryo regeneration culture is B5+50 mg / L Km+300 mg / L Cb+3% Sugar+0.8% Agar (pH=5.8).

[0020] Furthermore, the culture medium for the subculture is MS+50 mg / L Km+300 mg / L Cb+3% Sugar+0.8% Agar (pH=5.8).

[0021] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: The present invention establishes a CRISPR-Cas9 silencing system for the PDS gene of water celery, and effectively realizes the site-specific editing of the water celery gene OjPDS. The invention lays the foundation for in-depth research on the function of water celery genes, provides a new way to accurately improve the traits of water celery, and provides a new idea for the selection and breeding of new varieties of water celery. It is of great significance to shorten the breeding cycle of water celery and accelerate the research progress of water celery. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the construction of the water celery pYLCRISPR-OjPDS vector in Example 1 of the present invention.

[0023] Figure 2 This is the Oenanthe genetic transformation system established in Example 3 of the present invention.

[0024] Figure 3 The mutation types and corresponding peak diagrams of the PDS target site cloning and sequencing of Line 1-5 of the water celery in Example 3 of the present invention are shown.

[0025] Figure 4 The mutation types and corresponding peak diagrams of the Line 6-10 water celery PDS target site cloning and sequencing in Example 3 of the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0027] Example 1 Construction of CRISPR / Cas9 expression vector for water celery

[0028]

[0029] Table 1

[0030]

[0031] The reaction system is shown in Table 2:

[0032] Table 2

[0033]

[0034] Amplification conditions: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 60°C for 20 s, extension at 72°C for 90 s, for a total of 35 cycles; and final extension at 72°C for 5 min.

[0035] After the target gene was amplified, the target gene was analyzed using the CRISPR-GE online website (http:skl.scau.edu.cn / ), and the base sequences with high ranking and GC%>40% were selected as editing targets in the exon region of the OjPDS coding region. To ensure accurate knockout of the target gene, four target sites were selected, and the AtU3b, AtU3d, AtU6-1 and AtU6-29 promoters were used to drive the four target sites T1, T2, T3 and T4, respectively. The nucleotide sequences of the target sites are as follows:

[0036] T1 (SEQ ID NO.5): 5'-CTGGTGGAAAGGGGGGGAGTAGG-3';

[0037] T2 (SEQ ID NO.6): 5'-AACATCGTCATTGCTGGCCAGG-3';

[0038] T3 (SEQ ID NO.7): 5'-GAAGCAAGAGATGTTCTTGGTGG-3';

[0039] T4 (SEQ ID NO.8): 5'-GCTGCTTGGAAAGATGACGATGG-3',

[0040] The underlined nucleotide sequence is the PAM site.

[0041] (2) Construction of pYLCRISPR-OjPDS knockout vector.

[0042]

[0043] ② The published pYLCRISPR / Cas9P 35S -N plasmid (Ma et al., A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants, 2015, Molecular Plant) was cut into a linearized plasmid using Smal and Sbfl enzymes, and the reaction conditions were 25°C for 4 h.

[0044] ③ The expression cassette sequence of the vector is connected to pYLCRISPR / Cas9P by ligase 35S -N linearized vector was connected, and the connection conditions were metal bath 50℃, 20min; 5min on ice. Then, 10μL of the cooled recombinant product was taken in the clean bench and added to 100μL of DH5α competent cells, and the tube wall was flicked several times to mix, and incubated on ice for 30min, heat-shocked at 42℃ for 90s, and stood on ice for 2min. Then, 700μL of anti-LB liquid medium was added to the centrifuge tube, and cultured at 37℃, 200rpm for 1h. 100μL of bacterial solution was aspirated and gently spread on the LB solid medium plate containing kanamycin with a sterile coating stick. After the bacterial solution was absorbed, the plate was inverted in a 37℃ incubator for overnight culture.

[0045] ④Use Primer Premier 6.0 primer design software to design primers in pYLCRISPR / Cas9P 35S -N vector target site connection site about 300-400bp upstream and downstream primers for PCR amplification were designed, the primer sequences are shown in Table 3, and the primer synthesis was entrusted to Nanjing Qingke Biotechnology Co., Ltd.

[0046] Table 3

[0047]

[0048]

[0049] ⑤Use M13-F and pYLCRISPR-R primers to perform PCR identification on the colonies, screen the positive clones, and identify the correct recombinant vector and name it: pYLCRISPR-OjPDS.

[0050] The reaction system is shown in Table 4:

[0051] Table 4

[0052]

[0053] Reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s, annealing at 55°C for 20 s, extension at 72°C for 90 s, for a total of 35 cycles; and final extension at 72°C for 5 min.

[0054] Schematic diagram of pYLCRISPR-OjPDS vector construction is shown in Figure 1 As shown, A is a schematic diagram of four small guide RNAs (sgRNA, black boxes) targeting the OjPDS coding sequence. Boxes of different colors represent exons, and the black part in the middle of the exons represents introns. B is a schematic diagram of the method for constructing sgRNA expression cassettes. On the left, the backbone of the sgRNA shows that any specific targeting sequence can be inserted between the promoter and the unchanged part of the guide RNA; on the right, the four promoters AtU3b, AtU3d, AtU6-1 and AtU6-29 in Arabidopsis drive the four OjPDS target sites respectively. C is a schematic diagram of plant stable transformation mediated by Agrobacterium tumefaciens sgRNA and Cas9 expression cassettes assembled on a vector. The vector expression cassette is connected to the pYLCRISPR vector through the designed tail after cutting.

[0055] Example 2 Agrobacterium transformation of water celery pYLCRISPR-OjPDS expression vector plasmid

[0056] ① Take 5 μL of the recombinant product pYLCRISPR-OjPDS vector plasmid and add it to 100 μL of GV3101 Agrobacterium tumefaciens competent cells, gently stir the bottom of the tube to mix, and then stand on ice for 10 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes. Add 900 μL of antibiotic-free YEB liquid medium to the centrifuge tube and shake and culture for 3 hours at 28°C and 200 rpm. Centrifuge at 6000 rpm for 1 minute to collect the bacteria, keep 100 μL of the supernatant and gently blow the resuspended bacteria block to apply it on the YEB solid medium plate containing 50 μg / mL Kan (kanamycin) and 20 μg / mL Rif (rifampicin), and invert it in a 28°C incubator for 48 hours to grow a single colony of Agrobacterium tumefaciens.

[0057] ② Select a single clone of Agrobacterium tumefaciens, add 700 μL of YEB liquid culture medium containing kanamycin and rifampicin, and culture at 28°C and 200 rpm for 24 hours before performing bacterial testing. Use the primers shown in Table 5 to perform bacterial testing under the PCR reaction system.

[0058] Table 5

[0059]

[0060] The reaction system is shown in Table 6:

[0061] Table 6

[0062]

[0063] Reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s, annealing at 55°C for 20 s, extension at 72°C for 90 s, for a total of 35 cycles; and final extension at 72°C for 5 min.

[0064] ③After the bacterial test is completed, select the bacterial solution with brighter bands for bacterial preservation. One tube of bacteria is placed in one conical flask, and 10mL of YEB liquid medium containing kanamycin and rifampicin is added. Oscillate at 28℃ and 200rpm for 12h. One conical flask corresponds to one 1.5mL centrifuge tube. Pipette 1300μL of bacterial solution, centrifuge at 12000rpm and 4℃ for 1min, pour out the supernatant, continue to add 1300μL of bacterial solution for centrifugation, repeat this operation until 650μL of bacterial solution is left in the centrifuge tube. At this time, add 40% equal volume of glycerol, mix well by pipetting, and store in a -40℃ refrigerator for subsequent Agrobacterium infection of explants.

[0065] Example 3 Agrobacterium-mediated transformation of water celery with CRISPR / Cas9

[0066] 1. Explant pre-culture: Select healthy and strong aseptic water celery seedlings, cut 70-80 water celery petioles of about 0.8 cm in size on a clean bench, spread them on callus induction medium (CIM) of B5+0.5 mg / L 2,4-D+1.0 mg / L 6-BA+100 mg / L SER+3% Sugar+0.8% Agar (pH=5.8) and place them in a dark incubator at 25°C for pre-culture for 10 days.

[0067] 2. Agrobacterium activation: Take 100 μL of the Agrobacterium culture prepared in Example 2 and inoculate it into 10 mL YEB liquid culture medium supplemented with 100 mg / L R, 50 mg / L Km, and 50 mg / L Gen and shake overnight at 28°C and 200 rpm. At 10:00 a.m. the next day, transfer 1 mL of the culture solution to 40 mL YEB liquid culture medium and continue shaking and culturing. When OD 600 When the value was greater than 1.0, the bacterial solution was transferred into a 50 mL centrifuge tube and centrifuged at 4000 rpm for 8 min, and the supernatant was discarded; the solution was poured into B5 liquid culture medium at 4000 rpm, centrifuged again for 8 min and the supernatant was discarded, and the solution was resuspended and diluted to 50 mL with CIM liquid culture medium (pH = 5.8) (40 mg / L acetosyringone AS was added), and then shaken at 200 rpm at 28 ° C for 1 h before use for explant infection.

[0068] 3. Explant infection and co-cultivation: After 10 days of pre-cultivation, transfer the explants into 20 mL CIM + AS liquid culture medium and soak for 5 minutes, then pour the activated bacterial solution into the culture medium for infection for 20 minutes (shake every 5 minutes). After infection, filter the Agrobacterium from the explants and spread them on the solid explant co-culture medium (CIM + 40 mg / LAS), seal them with sealing film, and place them in a dark incubator at 25°C for co-cultivation for 2 days.

[0069] 4. Adventitious bud regeneration, screening and plant formation: After 2 days of co-cultivation, the explants were transferred to a 50 mL Erlenmeyer flask, rinsed twice with sterile water, then rinsed twice with sterile water + carboxybenzyl (300 mg / L), and finally placed on a shaker at 100 rpm and 25°C for 10 min. The washed explants were transferred to sterile filter paper to absorb the water, then inoculated on differentiation medium (CIM + 50 mg / L Km + 300 mg / L Cb), and placed in a dark incubator at 25°C for adventitious bud regeneration culture, and the medium was changed every 4 weeks. After 12 weeks of dark culture, the adventitious buds with kana resistance were transferred to somatic embryo regeneration medium (B5 + 50 mg / L Km + 300 mg / L Cb + 3% Sugar + 0.8% Agar (pH = 5.8)), and placed in a light incubator with 16 h light, 8 h dark, and a temperature of 25°C for screening culture. After 6 weeks, the seedlings with kana resistance were transferred to the subculture medium (MS + 50 mg / L Km + 300 mg / L Cb + 3% Sugar + 0.8% Agar (pH = 5.8)) for propagation, and then cultured for rooting until they grew into complete regenerated plants. Figure 2 As shown, a is the sterile seedling of water celery. b is the petiole of water celery inoculated on CIM medium for pre-culture. c is the infection of Agrobacterium after 10 days of pre-culture. d is the induced resistant bud of water celery. e is the resistant bud of water celery. f is the positive seedling of water celery.

[0070] 5. Identification of positive plants: In this experiment, 14 Oenanthe serrata seedlings that were positive for antibiotic screening were obtained, named Line 1-14. The leaves of the transgenic plants were taken to extract total DNA (DNA extraction kit: FastPure Plant DNA Isolation Mini Kit-BOX 2 (Nanjing Novezan Company) and Primer Premier 6.0 was used to design primers, the sequences are shown in Table 7, and the primer synthesis was entrusted to Nanjing Qingke Biotechnology Co., Ltd. PCR amplification was performed using the cDNA of the positive plants as a template, and the knockout results were accurately detected by Sanger sequencing.

[0071] Table 7

[0072]

[0073] The reaction system is shown in Table 8:

[0074] Table 8

[0075]

[0076] Reaction conditions: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 60°C for 20 s, extension at 72°C for 90 s, for a total of 35 cycles; and final extension at 72°C for 5 min.

[0077] 6. Sequencing of gene editing results: For homozygous mutations, the PCR amplicon of each target site was directly sequenced, and the web-based DSDecode was used to decode the superimposed sequence chromatogram. Since DSDecode cannot decode chimeric mutations, the PCR product was connected to the pMD19-T vector (TaKaRa, Dalian, China) and transformed into Escherichia coli DH5α, and then a single clone was picked for sequencing. The sequence differences between the transgenic strains and the wild-type plants at the target site were compared, and the editing efficiency was calculated. The results are shown in Figure 3-4 As shown (the purple sequence represents sequence replacement, the blue sequence represents sequence insertion, and the red horizontal line represents sequence deletion): Gene editing mutation types include base deletion, base insertion, base substitution, etc., among which base substitution is the majority. Analysis of mutation results: Among the 14 Oenanthe seedlings that were positive for antibiotic screening, 10 were gene-edited, and among the 10 transgenic plants, 2 had a mosaic phenotype (Line 4, Line 5) and 4 had an obvious albino phenotype (Line 1, Line 2, Line 6, Line 8). Specifically: the mutation rate at Target 1 in the 10 transgenic plants was 90%, including 2 homozygous mutations and 4 biallelic mutations, and the rest were heterozygous mutations; at Target 2, the mutation rate was 70%, including 3 homozygous mutations and 4 heterozygous mutations; the mutation rate of Target 3 was lower, only 20%, including 1 heterozygous mutation and 1 biallelic mutation; the mutation rate of Target 4 was the same as Target 2, both 70%, including 6 heterozygous mutations and 1 biallelic mutation.

Claims

1. A celery phytoene dehydrogenase gene, characterized in that: The full-length sequence of the water celery phytoene dehydrogenase gene is shown in SEQ ID NO.

2.

2. The celery phytoene dehydrogenase gene according to claim 1, characterized in that: The CDS sequence of the water celery phytoene dehydrogenase gene is shown in SEQ ID NO.

1.

3. A method for constructing a CRISPR-Cas9 silencing system for the PDS gene of water celery, characterized in that: The following steps are involved: (1) Designing a target site according to the full-length sequence of the celery phytoene dehydrogenase gene as shown in SEQ ID NO.2 or the CDS sequence of the celery phytoene dehydrogenase gene as shown in SEQ ID NO.1, synthesizing a vector expression cassette sequence and connecting it to the linearized basic vector to construct a celery phytoene dehydrogenase gene CRISPR / Cas9 expression vector; (2) The CRISPR / Cas9 expression vector of the phytoene dehydrogenase gene of the water celery was transformed into Agrobacterium to obtain the CRISPR-Cas9 silencing system of the PDS gene of the water celery.

4. The method for constructing the CRISPR-Cas9 silencing system of the water celery PDS gene according to claim 3, characterized in that: The target sites described in step (1) include any one or more combinations shown in SEQ ID NOs. 5 to 8.

5. The method for constructing the CRISPR-Cas9 silencing system of the water celery PDS gene according to claim 3, characterized in that: The vector expression cassette sequence also includes a promoter and sgRNA.

6. The method for constructing the CRISPR-Cas9 silencing system of the water celery PDS gene according to claim 5, characterized in that: The promoters include AtU3b, AtU3d, AtU6-1 and AtU6-29.

7. The method for constructing the CRISPR-Cas9 silencing system of the water celery PDS gene according to claim 3, characterized in that: The vector expression cassette sequence is shown in SEQ ID NO.

9.

8. The method for constructing the CRISPR-Cas9 silencing system of the water celery PDS gene according to claim 3, characterized in that: The basic vector is pYLCRISPR / Cas9P 35S -N.

9. A method for cultivating water celery plants using the CRISPR-Cas9 silencing system of the water celery PDS gene obtained by the construction method according to any one of claims 3 to 8, characterized in that: The CRISPR-Cas9 silencing system of the PDS gene of water celery was used to infect the callus tissue of the water celery with Agrobacterium, and then adventitious bud regeneration culture, cell embryo regeneration culture, subculture culture and rooting culture were carried out to obtain regenerated plants.

10. The method for cultivating water celery plants according to claim 9, characterized in that: The regenerated plants are plants with kanamycin resistance.

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