Method for verifying CRISPR (clustered regularly interspaced short palindromic repeats) or Cas9 carrier activity and gene function in melon hairy roots

Abstract

The invention discloses a method for verifying CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) or Cas9 carrier activity and gene functions in muskmelon hairy roots, which comprises the following steps: by using cotyledons of muskmelon seeds germinating for two days as explants, infecting by using agrobacterium rhizogenes K599, co-culturing, inducing the hairy roots, and taking green fluorescent protein eGFP as a selection marker of transgenic hairy roots, so as to verify CRISPR or Cas9 carrier activity and gene functions in muskmelon hairy roots. And hairy roots for converting the CRISPR / Cas9 vector can be obtained in about one month. By utilizing the transformation material, availability of gene editing target sites of multiple genes is analyzed, it is found that increase of the copy number of sgRNA expression units has a promoting effect on gene editing efficiency, and it is found that after CmRHL1 is edited, muskmelon root hair cannot be formed. Therefore, the invention provides a convenient and fast way for verifying the activity of the CRISPR / Cas9 vector and researching the function of the gene in the root development process, and is a beneficial supplement for a stable genetic transformation method of the muskmelon.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and particularly relates to a method for verifying CRISPR or Cas9 vector activity and gene function in melon hairy roots. Background Art

[0002] Melon (Cucumis melo L) is a fruit crop with important nutritional and economic value and is widely planted throughout the world. CRISPR or Cas9 gene editing technology can accurately modify the target gene, create excellent germplasm resources, and promote crop genetic improvement. CRISPR or Cas9 technology has been used in melon scientific research, but the current melon gene editing technology relies on the genetic transformation of Agrobacterium tumefaciens and plant tissue culture to obtain regenerated transformed plants. The entire process is long and requires large human and financial resources. CRISPR or Cas9 technology often fails due to the unavailability of gene editing target sites. At the same time, the editing efficiency in melon is low. Most of the gene-edited seedlings obtained in contemporary times are heterozygous and chimeric editing events. Therefore, it is necessary to screen for efficient gene editing targets before formal genetic transformation, and to further improve the gene editing efficiency of melons in certain ways.

[0003] Unlike Agrobacterium tumefaciens, Agrobacterium rhizogenes can quickly induce the production of hairy roots after infecting plants. The hairy roots grow rapidly and are easy to transform. The present invention combines the transformation of melon hairy roots with CRISPR or Cas9 gene editing technology to quickly obtain transgenic melon hairy roots transformed with CRISPR or Cas9 vectors. Using the transformed material, the activity of CRISPR or Cas9 vectors can be quickly verified, the availability of gene editing target sites can be analyzed, the effects of different vector construction methods on the editing efficiency of the CRISPR or Cas9 system can be compared, and the function of genes in the root development process can be analyzed. Summary of the invention

[0004] The present invention aims to provide a method for verifying the activity of CRISPR or Cas9 vectors and gene functions in melon hairy roots.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is as follows:

[0006] The method for verifying the activity of CRISPR or Cas9 vector and gene function in melon hairy roots comprises the following steps:

[0007] S1. Construction of gene editing vector:

[0008] Design the gene editing target, assemble it into the pBSE402 vector, obtain the gene editing vector of the target gene, transform Escherichia coli, and extract and construct the correct plasmid.

[0009] S2. Preparation of engineered Agrobacterium rhizogenes:

[0010] Prepare Agrobacterium rhizogenes K599 containing the pBSE402 gene editing vector and preserve the bacterial liquid.

[0011] S3. Preparation of cotyledon explants:

[0012] Select melon seeds with full and intact seeds, soak them at 55℃ for 30 minutes, and remove the seed shells. Disinfect them with 75% alcohol for 1 minute, 3% sodium hypochlorite for 15 minutes, and rinse them with sterile water three times in a clean bench. After the seeds are disinfected, place them in a 28℃ incubator for germination for 48 hours.

[0013] On sterile filter paper, use sterilized tweezers to cut off the proximal part of the cotyledons of the seeds below 2 mm after germination, separate the two cotyledons, and brush the cotyledon wound with a 3nm nanobrush about 10 times to obtain the processed explants.

[0014] S4. Infection of cotyledon explants

[0015] The above-preserved Agrobacterium rhizogenes culture was added to 40 mL TY liquid culture medium (containing 50 mg / L streptomycin and 50 mg / L kanamycin) at a ratio of 1:100, and cultured at 28°C and 220 rpm for 6 to 8 hours. 600 When the OD reaches 0.6-0.8, centrifuge at 5000 rpm for 10 min to collect the bacteria. Dilute the bacterial solution with the infection solution to OD 600 The concentration of the cotyledon explants was 0.1-0.2, and the cotyledon explants were mixed with Agrobacterium rhizogenes and shaken in a conical flask for infection for 15 minutes.

[0016] S5. Co-cultivation of cotyledons with Agrobacterium

[0017] The infected explants were dried on sterile filter paper, and a layer of sterile filter paper was spread on the co-cultivation medium. The cotyledons were placed on the filter paper with the abaxial side facing downwards. After sealing, the explants were cultured in a 25°C incubator in the dark for 5 days.

[0018] S6. Hairy root induction

[0019] After co-cultivation, the cotyledons were transferred to hairy root induction medium and hairy roots were induced at 25°C, 16 h light / 8 h dark, and a light intensity of 22000 Lx for 2 to 3 weeks.

[0020] S7. Observation of positive hairy root phenotype

[0021] Irradiate the hairy roots with ultraviolet light, and judge whether the pBSE402 gene editing vector is successfully transformed by whether the hairy roots can emit green fluorescence. Use a stereo microscope to observe the positive hairy root phenotype and take pictures, and save the corresponding hairy roots for DNA extraction.

[0022] S8. Detection of gene editing

[0023] The DNA of the above-mentioned hairy roots was extracted using the CTAB method, and primers were designed upstream and downstream of the gene editing target site for PCR amplification and Sanger sequencing of the PCR products. The presence of gene editing was analyzed based on the peak graph, and the gene editing form was analyzed to calculate the editing efficiency.

[0024] Preferably, the gene editing vector described in step S1 is a pBSE402 vector, which contains an eGFP expression frame. The expression product can emit green fluorescence under blue light irradiation and can be used for screening positive transgenic individuals.

[0025] Preferably, the specific arrangement of seeds during germination in step S3 is as follows: add 1.5 mL of sterilized water into a sterile culture dish with a diameter of 90 mm, spread a sterilized filter paper, spread the sterilized seeds on the filter paper, and place about 15 seeds in each culture dish.

[0026] Preferably, in step S4, the infection solution is prepared by dissolving 4.43 g MS powder and 30 g sucrose in 1 L of ddH2O, adjusting the pH to 5.8, sterilizing at 121°C for 20 min, and adding 100 μmol of acetosyringone, 1.25 mmol of 2-morpholineethanesulfonic acid, and 200 μL of surfactant Silwet-L77 sterilized by suction filtration in a clean bench.

[0027] Preferably, in step S5, the co-culture medium is prepared by dissolving 4.43 g MS powder and 8 g agar in 1 L ddH2O, adjusting the pH to 5.8, and sterilizing at 121°C for 20 min. Add 100 μmol of acetosyringone, 1.25 mmol of 2-morpholineethanesulfonic acid, 250 μmol of lipoic acid, and 2 mg of silver nitrate sterilized by suction filtration in a clean bench.

[0028] Preferably, in step S6, the preparation method of the hairy root induction medium is: dissolve 4.43g MS powder, 30g sucrose, and 8g agar in 1L ddH2O, adjust the pH to 5.8, sterilize at 121°C for 20min, and add 200mg of timentin and 200mg of cephalosporin sterilized by suction filtration in a clean bench.

[0029] Preferably, in step S8, the sequences of the PCR primer pair are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0030] The present invention adopts the above-mentioned Agrobacterium rhizogenes-mediated melon hairy root editing method, and its technical effects are as follows:

[0031] The cotyledons of germinated melon seeds are used as explants to induce hairy roots and to transform CRISPR or Cas9 gene editing vectors, and transgenic hairy roots can be obtained in about a month. The transgenic hairy roots obtained can be used to analyze the availability of gene targets, compare the editing efficiency of different vectors, and analyze the function of genes in the root development process. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of dual-target gene editing vector;

[0033] Figure 2 2-day-germinated melon seeds for preparing cotyledon explants;

[0034] Figure 3 The cotyledon explants of muskmelon were plated on co-cultivation medium after infection;

[0035] Figure 4 These are bright field and GFP fluorescence images of melon cotyledon explants co-cultured for 5 days;

[0036] Figure 5 The induction and GFP fluorescence images of hairy roots;

[0037] Figure 6 Analysis of editing forms of gene editing targets of CmERECTA, CmTCP1, CmACS7, and CmBRC1;

[0038] Figure 7 Schematic diagram of gene editing vector construction for different sgRNA expression units;

[0039] Figure 8 To compare the gene editing efficiency of hairy roots transformed with different sgRNA expression units;

[0040] Fig. 9 Schematic diagram for designing gene editing targets for the root hair gene CmRHL1;

[0041] Fig.10 Phenotypic observation of positive hairy roots containing CmRHL1 gene editing vector;

[0042] Fig.11 Analysis of CmRHL1 gene editing type in positive hairy roots. DETAILED DESCRIPTION

[0043] The technical method of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be noted that the following embodiments are only illustrative and the present invention is not limited to these embodiments.

[0044] Unless otherwise defined, scientific and technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention pertains.

[0045] Sources:

[0046] The gene editing vector pBSE402 comes from Li Zheng's research group at Northwest Agriculture and Forestry University. The vector contains eGFP, Cas9 and sgRNA expression frames. When the vector is transferred into plant cells, the eGFP sequence will express green fluorescent protein, which will emit green fluorescence under blue light and is used to screen positive transgenic individuals.

[0047] Vectors pDT1T2 and pDT2T3 ​​are supporting vectors for pBSE402. They come from Li Zheng's research group at Northwest Agriculture and Forestry University and are used as PCR amplification templates for assembling gene targets.

[0048] The melon material was XZM seeds preserved in our laboratory, which had been self-pollinated and purified for more than 10 generations.

[0049] Agrobacterium rhizogenes strain K599 was purchased from Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd.

[0050] Escherichia coli strain DH5α was purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.

[0051] Example 1

[0052] In this example, hairy root transformation was used to test the availability of two gene editing targets for each of the four genes, CmERECTA, CmTCP1, CmACS7, and CmBRC1, in melon.

[0053] 1. Construction of gene editing vector

[0054] DNA sequences were obtained from the Cucurbitaceae genome website according to gene IDs MELO3C016916 (CmERECTA), MELO3C022091 (CmTCP1), MELO 3C015444 (CmACS7), and MELO3C017286 (CmBRC1). The DNA sequences were submitted to the online website CRISPR-P v2.0 (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR2 / ) to design gene editing targets. Two high-scoring targets were selected for each gene to construct the corresponding sgRNA expression units, namely ERECTA-sgRNA1, ERECTA-sgRNA2, TCP1-sgRNA1, TCP1-sgRNA2, ACS7-sgRNA1, ACS7-sgRNA2, BRC1-sgRNA1, and BRC1-sgRNA2. The corresponding primers were designed to assemble the two target sites of the same gene into the same pBSE402 vector. The vector is shown in the figure below. Figure 1 The primer names and sequences used are as follows:

[0055] Primers for constructing CmERECTA dual-target gene editing vector: ERECTA-T1-F: ATATATGGTCTCGATT GTTCTGTGCTACCGTTGGTGGTTTTAGAGCTAGAAATAGC (SEQ ID NO.1); ERECT A-T2-R: ATTATTGGTCTCGAAACTCCTTTTTTTCACTGCCTTCAATCTCTTAGTCGACT CTAC (SEQ ID NO.2)

[0056] Primers for constructing CmTCP1 dual-target gene editing vector: TCP1-T1-F: ATATATGGTCTCGATTGAGA GAACCGGATCTTGCGGGTTTTAGAGCTAGAAATAGC (SEQ ID NO.3); TCP1-T2-R: ATTATTGGTCTCGAAACGCGTTGGCATCGACGGTTGCAATCTCTTAGTCGACTCTAC (SEQ ID NO.4).

[0057] Primers for constructing CmACS7 dual-target gene editing vector: ACS7-T1-F: ATATATGGTCTCGATTGCTTT CCAGCCAGCAAAATAGTTTTAGAGCTAGAAATAGC (SEQ ID NO.5); ACS7-T2-R: ATTATTGGTCTCGAAACCCTCTAATCTCTTCCATAACAATCTCTTAGTCGACTCTAC (SEQ ID NO.6).

[0058] Primers for constructing CmBRC1 dual-target gene editing vector: BRC1-T1-F: ATATATGGTCTCGATTGGAT TGTTGTTGAAGAAGAAGTTTTAGAGCTAGAAATAGC (SEQ ID NO.7); BRC1-T2-R: ATTATTGGTCTCGAAACCGTCTGAAACCGATGATGGCAATCTCTTAGTCGACTCTAC (SEQ ID NO.8).

[0059] The designed primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and PCR amplification was performed using the designed primers and the vector pDT1T2 as a template. The PCR amplification reaction system is shown in Table 1. The reaction procedure is: 95°C, 3 min; (95°C, 15 s; 56°C, 15 s; 72°C, 30 s) 30 cycles; 72°C, 5 min, and the PCR product was temporarily stored at 4°C.

[0060] Table 1 PCR reaction system

[0061]

[0062] The above PCR products were connected to the pBSE402 vector using the Golden gate method, and the target vectors were named pBSE 402-ERECTA-T1 / T2, pBSE402-TCP1-T1 / T2, pBSE402-ACS7-T1 / T2, and pBSE402-BRC1-T1 / T2. The Golden gate reaction system is shown in Table 2, and the reaction procedure is: 37°C, 5h; 50°C, 5min; 80°C, 10min.

[0063] Table 2 Golden gate reaction system

[0064]

[0065]

[0066] The ligation product was transformed into DH5α. For specific operations, refer to the instruction manual of E. coli DH5α. The PCR identification primers for positive clones are:

[0067] U6-26p-F: TGTCCCAGGATTAGAATGATTAGGC (SEQ ID NO.9)

[0068] U6-29p-R: AGCCCTCTTTCTTTCGATCCATCAAC (SEQ ID NO.10)

[0069] The PCR-positive strains were sent to a sequencing company (Sangon Biotech) for sequencing using the sequencing primer U6-26p-F. The strains with the correct sequencing were cultured and the plasmids were extracted.

[0070] 2. Preparation of engineered Agrobacterium rhizogenes

[0071] The pBSE402-ERECTA-T1 / T2, pBSE402-TCP1-T1 / T2, pBSE402-ACS7-T1 / T2, and pBSE402-BRC1-T1 / T2 plasmids were used to transform the competent Agrobacterium rhizogenes K599. The transformation method was the liquid nitrogen freeze-thaw method. For specific operations, refer to the K599 instruction manual to obtain K599 strains containing the corresponding gene editing vectors, and save the positive strains for later use.

[0072] 3. Preparation of Cotyledon Explants

[0073] Select melon seeds with full seeds and no damage, soak them in 55℃ warm water for 30 minutes, and peel off the seed shells. Put the seeds into a 15mL sterile centrifuge tube in a clean bench, disinfect them with 75% alcohol for 1 minute, wash them with sterile water once, disinfect them with 3% sodium hypochlorite solution (containing 0.1% soil temperature 20) for 15 minutes, and rinse them with sterile water 3 times. Add 1.5mL sterile water to a sterile plastic culture dish with a diameter of 90mm, spread a piece of sterilized filter paper, and spread the sterilized seeds on the filter paper. Each culture dish can hold about 15 seeds. Seal the culture dish and place it in a 28℃ incubator for germination for 2 days.

[0074] The morphology of melon seeds after germination for 2 days is as follows Figure 2 As shown, on sterile filter paper, the cotyledon was cut 2 mm from the proximal end using sterile forceps, the cotyledon part was retained, and the two cotyledons were separated. A 3 nm nanobrush was used to brush the cotyledon cut 10 times to obtain a melon cotyledon explant that can be used for transformation.

[0075] 4. Infection of Explants

[0076] Add the K599 Agrobacterium rhizogenes culture solution stored in step 2 to 40 mL TY liquid medium (containing 50 mg / L streptomycin and 50 mg / L kanamycin) at a ratio of 1:100, and culture at 28°C and 220 rpm for 6 to 8 hours. 600 When the OD reaches 0.6-0.8, place the bacterial solution in a 50 mL centrifuge tube and centrifuge at 5000 rpm for 10 min to collect the solution. Readjust the OD of the bacterial solution with the infection solution. 600 The concentration is 0.1-0.2, mixed with the explants, and shaken in a sterile conical flask for infection for 15 minutes.

[0077] The preparation method of TY liquid medium is as follows: 5g tryptone, 3g yeast extract, and 10mmol calcium chloride are dissolved in 1L ddH2O, and sterilized at 121℃ for 20min. The preparation method of infection solution is as follows: 4.43g MS powder and 30g sucrose are dissolved in 1L ddH2O, pH is adjusted to 5.8, and sterilized at 121℃ for 20min. 100μmol acetosyringone, 1.25mmol 2-morpholineethanesulfonic acid, and 200μL surfactant Silwet-L77 are added in the clean bench.

[0078] 5. Co-cultivation of Explants and Agrobacterium rhizogenes

[0079] The infected explants were dried on sterile filter paper, and a sterile filter paper was spread on the co-culture medium. The abaxial side of the cotyledons was placed against the filter paper on the co-culture medium. About 30 cotyledons ( Figure 3 ) and seal the culture dish with breathable medical tape. In the incubator, cotyledons of melon and Agrobacterium K599 were co-cultured for 5 days at 25°C in dark conditions. After the co-culture, obvious green fluorescence was visible at the cut of the cotyledons under blue light ( Figure 4 ).

[0080] The co-culture medium was prepared by dissolving 4.43 g MS powder and 8 g agar in 1 L ddH2O, adjusting the pH to 5.8, and sterilizing at 121°C for 20 min. 100 μmol of acetosyringone, 1.25 mmol of 2-morpholineethanesulfonic acid, 250 μmol of lipoic acid, and 2 mg of silver nitrate were added in a clean bench.

[0081] 6. Induction of Hairy Roots

[0082] The cotyledons after co-cultivation were placed on the hairy root induction medium, with about 10 cotyledons placed in each culture dish, and cultured for 3 weeks at 25°C, 16h light / 8h dark, and a light intensity of 22000Lx to induce hairy roots ( Figure 5 ).

[0083] The preparation method of hairy root induction medium is as follows: dissolve 4.43g MS powder, 30g sucrose, and 8g agar in 1L ddH2O, adjust the pH to 5.8, sterilize at 121°C for 20min, and add 200mg of filtration-sterilized timentin and 200mg of cephalosporin in a clean bench.

[0084] 7. Identification of Transgenic Hairy Roots

[0085] Use LUYOR-3105 fluorescent lamp to illuminate the hairy roots. If the hairy roots emit green fluorescence, it means that the pBSE402 vector has been successfully transferred ( Figure 5 ). The statistical results in Table 3 show that 76.67% of the cotyledon explants of the present invention can produce positive hairy roots, the proportion of positive hairy roots to the total hairy roots reaches 68.61%, and the average number of hairy roots produced by each explant is 2.61.

[0086] Table 3. Statistics of hairy root transformation efficiency

[0087]

[0088] 8. Gene Editing Analysis

[0089] The DNA of the hairy roots was extracted by CTAB method as a template for PCR amplification. A pair of specific primers were designed upstream and downstream of the target gene target site for PCR amplification. The PCR amplification product was sent to the company for sequencing. The sequencing results were analyzed and edited using the online software CRISPR-GE (http: / / skl.scau.edu.cn / home / ). The analysis results are as follows Figure 6 As shown: According to the test results, these eight gene editing target sites can be divided into three types. The first category: complete editing, including CmERECTA target site 1, CmERECTA target site 2, CmTCP1 target site 2, CmACS7 target site 1, CmBRC1 target site 1; the second category: partial editing, both edited sequences and wild-type sequences can be detected, including CmACS7 target site 2 and CmBRC1 target site 2; the third category, no editing, only including CmTCP1 target site 1.

[0090] The PCR primers used for each gene are as follows:

[0091] ERECTA-IF: GGTGTTGGTTTTTAAGGGGTTCTCAG (SEQ ID NO. 11);

[0092] ERECTA-IR: ACAGATATTGCAGCACTTCATTCCAG (SEQ ID NO. 12);

[0093] TCP1-IF: GCCCAAAGAGGGCTAAAGTTTACC (SEQ ID NO.13);

[0094] TCP1-IR: CAATTTGGAGTCAAATTGGAAGCTGG (SEQ ID NO. 14);

[0095] ACS7-IF:GAGGTGCCCATTGTAAGAGCAAC (SEQ ID NO. 15);

[0096] ACS7-IR: GGATAGTAAGGAGTGGGGACAAGC (SEQ ID NO. 16);

[0097] BRC1-IF: GGGGAAAAGAGATCAAAAGAGAGATATG (SEQ ID NO. 17);

[0098] BRC1-IR:GAGTTGGAGGGGCTCCACCT(SEQ ID NO.18)

[0099] Example 2

[0100] This example uses hairy root transformation to examine the effect of the number of sgRNA expression units on gene editing efficiency.

[0101] 1. Construction of gene editing vector

[0102] The CmACS7 target site 2 and CmBRC1 target site 2 in implementation case 1 were selected to construct the expression units of ACS7-sgRNA2 and BRC1-sgRNA2. For these two sgRNAs, primers were designed to construct a single expression unit, two expression units, and three expression units of pBSE402 gene editing vectors, respectively. The vector schematic diagram is shown in Figure 7 , the primer sequences used are:

[0103] Primers for constructing the gene editing vector of the ACS7-sgRNA2 single expression unit: ACS7-T2-single-F: ATTGTTTATGGAAGAGATTAGAGGG (SEQ ID NO.19); ACS7-T2-single-R: AAACCCTCTAATCT CTTCCATAAA (SEQ ID NO.20)

[0104] Primers for constructing the gene editing vector of the two expression units of ACS7-sgRNA2: ACS7-T2-double-F: TATATGGTCTCGATTGTTTATGGAAGAGATTAGAGGGTTTTAGAGCTAGAAATAGCAAG (SEQ ID NO.21); ACS7-T2-double-R: TTATTGGTCTCGAAACCCTCTAATCTCTTCCATAA ACAATCTCTTAGTCGACTCTAC (SEQ ID NO.22)

[0105] Primers for constructing the gene editing vector of the three expression units of ACS7-sgRNA2: ACS7-T2-triple-F1: TATATG GTCTCGATTGTTTATGGAAGAGATTAGAGGGTTTTAGAGCTAGAAATAGCAAG (SEQ ID NO.23); T0-R1: TTATTGGTCTCAATCTCTTAGTCGACTCTACCAAT (SEQ ID NO.24); ACS7-T2-triple-F2: TTATTGGTCTCAAGATTGTTTATGGAAGAGATTAGAGGG TTTTAGAGCTAGAAATAGC (SEQ ID NO.25); ACS7-T2-triple-R: TTATTGGTCTCGA AACCCTCTAATCTCTTCCATAAACAATCACTACTTCGTCTCTAACC (SEQID NO.26)

[0106] Primers for constructing gene editing vector of BRC1-sgRNA2 single expression unit: BRC1-T2-single-F: ATTGCCATCATCGGTTTCAGACG (SEQ ID NO.27); BRC1-T2-single-R: AAACCGTCTGAAAC CGATGATGG (SEQID NO.28)

[0107] Primers for constructing the gene editing vector of the two expression units of BRC1-sgRNA2: BRC1-T2-double-F: TATATGGTCTCGATTGCCATCATCGGTTTCAGACGGTTTTAGAGCTAGAAATAGCAAG (SEQ ID NO.29); BRC1-T2-double-R: TTATTGGTCTCGAAACCGTCTGAAACCGATGATGGC AATCTCTTAGTCGACTCTAC (SEQ ID NO.30)

[0108] Primers for constructing the gene editing vector of the three expression units of BRC1-sgRNA2: BRC1-T2-triple-F1: TATATG GTCTCGATTGCCATCATCGGTTTCAGACGGTTTTAGAGCTAGAAATAGCAAG (SEQ ID NO.31); T0-R1: TTATTGGTCTCAATCTCTTAGTCGACTCTACCAAT (SEQ ID NO.24); BRC1-T2-triple-F2: TTATTGGTCTCAAGATTGCCATCATCGGTTTCAGACGGTTT TAGAGCTAGAAATAGC (SEQ ID NO.32); BRC1-T2-triple-R: TTATTGGTCTCGAAA CCGTCTGAAACCGATGATGGCAATCACTACTTCGTCTCTAACC (SEQ ID NO.33)

[0109] The designed primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0110] Design of gene editing vectors with single sgRNA expression units

[0111] When constructing a gene editing vector with a single sgRNA expression unit, the primers Gene-T-single-F and Gene-T-single-R were mixed and annealed. The annealing reaction system is shown in Table 4. The annealing reaction steps are 95℃ for 4min, 0.1℃ / second, and then reduced to 20℃. The annealing product was Golden gate connected with the pBSE402 vector. The connection reaction conditions were the same as xx. The resulting vectors were named pBSE402-ACS7-sgRNA2*1 and pBSE402-BRC1-sgRNA2*1, respectively.

[0112] Table 4 Primer annealing reaction system

[0113]

[0114]

[0115] The ligation product was transformed into DH5α. For specific operations, refer to the instruction manual of E. coli DH5α. The PCR identification primers for positive clones are:

[0116] U6-26p-F: TGTCCCAGGATTAGAATGATTAGGC (SEQ ID NO.9)

[0117] U6-26t-R:CCCCAGAAATTGAACGCCGAAGAAC(SEQ ID NO.34)

[0118] The PCR-positive strains were sent to a sequencing company (Sangon Biotech) for sequencing using the sequencing primer U6-26p-F. The strains with the correct sequencing were cultured and the plasmids were extracted.

[0119] Design of gene editing vector with dual-copy sgRNA expression unit

[0120] PCR amplification was performed using the designed primers Gene-T-double-F and Gene-T-double-R with the vector pDT1T2 as the template. The PCR amplification reaction system was the same as Table 1. The PCR product was connected to the pBSE402 vector using the Golden gate method. The target vectors were named pBSE402-ACS7-sgRNA2*2 and pBSE402-BRC1-sgRNA2*2. The Golden gate reaction system was the same as Table 2.

[0121] The ligation product was transformed into DH5α. For specific operations, refer to the instruction manual of E. coli DH5α. The PCR identification primers for positive clones are:

[0122] U6-26p-F: TGTCCCAGGATTAGAATGATTAGGC (SEQ ID NO.9)

[0123] U6-29p-R: AGCCCTCTTTCTTTCGATCCATCAAC (SEQ ID NO.10)

[0124] The positive strains were sent to a sequencing company (Sangon Biotech) for sequencing, and the sequencing primer was U6-26p-F. The strains with correct sequencing were cultured by shaking, and the pBSE402-ACS7-sgRNA2*2 and pBSE402-BRC1-sgRNA2*2 plasmids were extracted.

[0125] Vector of three sgRNA expression units

[0126] PCR product 1 was obtained by amplifying pCBC-DT1T2 with primers Gene-T-triple-F1 and T0-R1, and PCR product 2 was obtained by amplifying pCBC-DT2T3 ​​with primers Gene-T-triple-F2 and Gene-T2-triple-R. The PCR reaction conditions were the same as those in Table xx. PCR product 1 and PCR product 2 were simultaneously connected to pBSE402 by Golden gate. The Golden gate reaction system is shown in Table 5. The resulting vectors were named pBSE402-ACS7-sgRNA2*3 and pBSE402-BRC1-sgRNA2*3.

[0127] Table 5 Golden gate reaction system

[0128]

[0129] The ligation product was transformed into DH5α. For specific operations, refer to the instruction manual of E. coli DH5α. The PCR identification primers for positive clones are:

[0130] U6-29p-F:TTAATCCAAACTACTGCAGCCTGAC(SEQ ID NO.9)

[0131] U6-1p-R: TATGCAAGTCTCACTCACACTCACG (SEQ ID NO.35)

[0132] The positive strains were sent to a sequencing company (Sangon Biotech) for sequencing. The sequencing primers were:

[0133] U6-29p-F:TTAATCCAAACTACTGCAGCCTGAC(SEQ ID NO.9)

[0134] U6-29p-R: AGCCCTCTTTCTTTCGATCCATCAAC (SEQ ID NO.10)

[0135] U6-29t-F:CGTGTTTCAGCTCTCATGATCCTTG(SEQ ID NO.36)

[0136] The correctly sequenced strains were cultured by shaking and the pBSE402-ACS7-sgRNA2*3 and pBSE402-BRC1-sgRNA2*3 plasmids were extracted.

[0137] The engineered Agrobacterium rhizogenes was obtained by referring to the method of Example 1, and the transformed melon hairy roots were obtained. For each vector, three transformed hairy roots were selected to extract DNA for PCR amplification. The names and sequences of PCR primers are as follows:

[0138] ACS7-IF:GAGGTGCCCATTGTAAGAGCAAC (SEQ ID NO. 15);

[0139] ACS7-IR: GGATAGTAAGGAGTGGGGACAAGC (SEQ ID NO. 16);

[0140] BRC1-IF: GGGGAAAAGAGATCAAAAGAGAGATATG (SEQ ID NO. 17);

[0141] BRC1-IR:GAGTTGGAGGGGCTCCACCT(SEQ ID NO.18)

[0142] After the PCR product is connected to the T vector, it is transformed into E. coli. Ten positive clones are selected from each ligation product for Sanger sequencing. The sequencing primers are the same as the PCR primers. The sequence is analyzed based on the sequencing results to determine whether the sequence is edited. The editing efficiency is equal to the number of clones with the target gene edited divided by the total number of clones. Figure 8 As shown, for a single ACS7-sgRNA2 expression unit, the editing efficiency was 32%, and the editing efficiency of two ACS7-sgRNA2 expression units was 72% (t-test, p<0.05). Similarly, compared with a single BRC1-sgRNA2 expression unit, the editing efficiency of two BRC1-sgRNA2 expression units increased from 26% to 64%. Compared with two sgRNA expression units, the editing efficiency of three sgRNA expression units was not further improved statistically.

[0143] Example 3

[0144] In this example, CRISPR was used to knock out the CmRHL1 gene in melon hairy roots, thereby examining the function of CmRHL1 in the root hair development process.

[0145] 1. Construction of CmRHL1 gene editing vector

[0146] According to gene ID MELO3C010237, the DNA sequence of the key gene CmRHL1 for root hair development was obtained from the Cucurbitaceae genome website (http: / / cucurbitgenomics.org / v2 / ). The DNA sequence was submitted to the online website CRISPR-Pv2.0 (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR2 / ) to design gene editing targets. Two targets with high scores and close to the 5' end of the coding sequence were selected from the design results of the website ( Fig. 9), design target assembly primers, construct a gene editing vector, pBSE402-RHL1-T1 / T2, the specific steps are the same as in Example 1, and the primer names and sequences are as follows:

[0147] RHL1-T1-F: ATATATGGTCTCGATTGATTGCATTTCTCTGCTACAGTTTTAGAGCT AGAAATAGC (SEQ ID NO. 37);

[0148] RHL1-T2-R: ATTATTGGTCTCGAAACCCTGGAGTTTGATTCATCACAATCTCTTA GTCGACTCTAC (SEQ ID NO.38)

[0149] 2. Obtaining hairy roots edited by CmRHL1 gene

[0150] Hairy roots transformed with the pBSE402-RHL1-T1 / T2 vector were obtained through infection with Agrobacterium rhizogenes K599, co-cultivation, hairy root induction and the like. The specific steps were the same as those in Example 1.

[0151] 3. Identification and phenotypic observation of transgenic hairy root bands

[0152] Use LUYOR-3105 ultraviolet lamp to irradiate the hairy roots. If the hairy roots emit green fluorescence, it means that the pBSE402 vector has been successfully transferred. Use a stereo microscope to observe the root hair phenotype of the hairy roots containing the pBSE402-RHL1-T1 / T2 vector and take pictures. Fig.10 As shown, unlike the control hairy roots which formed abundant root hairs at the upper part of the root tip, the hairy roots containing the pBSE402-RHL1-T1 / T2 gene editing vector had almost no root hairs formed. After phenotypic observation, the hairy roots were preserved for DNA extraction.

[0153] 4. CmRHL1 gene editing analysis

[0154] The DNA of the hairy roots was extracted by CTAB method and used as a PCR amplification template. PCR amplification was performed using specific primers upstream and downstream of the CmRHL1 target site. The PCR primers used were as follows:

[0155] RHL1-IF: GTGCCAGAGAAGCTCCAGC (SEQ ID NO.39)

[0156] RHL1-IR: TGGGAACGAGTTCTTGCAGAGC (SEQ ID NO.40)

[0157] The PCR amplification products were sent to the company for sequencing (Sangon Biotech), and the editing forms were analyzed using the online software CRISPR-GE (http: / / skl.scau.edu.cn / home / ). The analysis results are shown in Fig.11 As shown, gene editing occurred in the three transformed positive root hairs tested. Root hair #1 lost four bases at both target sites. Root hair #2 produced allelic editing with 4 bases missing and 6 bases missing at target site 1, and a mutation with 8 bases inserted and 19 bases deleted at target site 2. Root hair #3 lost 640 bases between the two target sites.

Claims

1. A method for verifying CRISPR or Cas9 vector activity and gene function in melon hairy roots, characterized in that: The following steps are involved: a. Construction of gene editing vector; b. Preparation of engineered Agrobacterium rhizogenes; c. Preparation of cotyledon explants; d. Infection of explants; e. Co-cultivation of explants and Agrobacterium; f. Hairy root induction; g. Identification and phenotypic observation of positive hairy roots; h. Gene editing detection.

2. The method for verifying CRISPR or Cas9 vector activity and gene function in melon hairy roots according to claim 1, characterized in that: The gene editing vector is pBSE402, which contains eGFP protein, Cas9 protein and sgRNA expression frame. When the vector is successfully transferred into plant cells, the eGFP sequence will be translated to produce green fluorescent protein, which will emit green fluorescence under blue light, thereby screening successfully transformed hairy root individuals.

3. The method for verifying CRISPR or Cas9 vector activity and gene function in melon hairy roots according to claim 1, characterized in that: The explants were cotyledons of melon seeds that had germinated for two days. The specific preparation method was as follows: soak the melon seeds in 55°C warm water for 30 min, peel off the seed shells with tweezers, disinfect with 75% alcohol for 1 min in a clean bench, wash once with sterile water, disinfect with 3% sodium hypochlorite solution for 15 min, and rinse three times with sterile water; add sterile water to a sterile culture dish, spread a piece of sterilized filter paper, spread the sterilized seeds on the filter paper, seal the culture dish, and place it in a 28°C incubator for germination for 48 h; two days after the seeds germinate, cut off the part below 2 mm proximal to the cotyledon on the sterile filter paper to separate the two cotyledons.

4. The method for verifying CRISPR or Cas9 vector activity and gene function in melon hairy roots according to claim 1, characterized in that: Agrobacterium infection operation: Use infection solution to adjust the OD of the bacterial solution 600 The concentration of 0.1 to 0.2 was fully mixed with the explants, and then the explants were infected by shaking in a sterile conical flask for 15 min. The infection solution was prepared as follows: 4.43 g MS powder and 30 g sucrose were dissolved in 1 L ddH2O, the pH was adjusted to 5.8, and sterilized at 121°C for 20 min. 100 μmol of acetosyringone, 1.25 mmol of 2-morpholineethanesulfonic acid, and 200 μL of surfactant Silwet-L77 were added in a clean bench.

5. The method for verifying CRISPR or Cas9 vector activity and gene function in melon hairy roots according to claim 1, characterized in that: Dry the infected explants on sterile filter paper, place a piece of sterile filter paper on the co-culture medium, place the dried cotyledons with the abaxial side facing downward on the filter paper, seal with breathable medical tape, and culture in an incubator at 25°C in the dark for 4 to 6 days; The preparation method of co-culture medium is as follows: dissolve 4.43 g MS powder and 8 g agar in 1 L ddH2O, adjust the pH to 5.8, and sterilize at 121℃ for 20 min; add 100 μmol of acetosyringone, 1.25 mmol of 2-morpholineethanesulfonic acid, 250 μmol of lipoic acid, and 2 mg of silver nitrate sterilized by suction filtration in a clean bench.

6. The method for verifying CRISPR or Cas9 vector activity and gene function in melon hairy roots according to claim 1, characterized in that: Hairy root induction: Place the co-cultivated cotyledons on hairy root induction medium and culture at 25°C, 16 h light / 8 h dark, and 22,000 Lx for 2 to 3 weeks; The preparation method of hairy root induction medium is as follows: dissolve 4.43 g MS powder, 30 g sucrose, and 8 g agar in 1 L of ddH2O, adjust the pH to 5.8, sterilize at 121°C for 20 min, and add 200 mg of filtration-sterilized timentin and 200 mg of cephalosporin in a clean bench.

7. The method for verifying CRISPR or Cas9 vector activity and gene function in melon hairy roots according to claim 1, characterized in that: Identification of transgenic hairy roots: Use ultraviolet light to irradiate the hairy roots. Positive transgenic hairy roots will emit green fluorescence under ultraviolet light, while negative hairy roots will not emit fluorescence.

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