Agrobacterium rhizogenes-mediated bottle gourd visual hairy root induction method

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to an agrobacterium rhizogenes-mediated visualized genetic transformation method for cucurbitaceae plants, which comprises the step of transfecting the cucurbitaceae plants by using agrobacterium rhizogenes containing recombinant plasmids, and the recombinant plasmids are expression cassettes containing Ruby reporter genes. A visual agrobacterium rhizogenes-mediated transformation system of the cucurbitaceae plants is established by utilizing the RUBY gene, professional equipment or chemical treatment is not needed, red hairy roots can be visually screened to identify and induce the positive plants, and the method is simple, rapid and efficient and can be used for identifying and inducing the positive plants of the cucurbitaceae plants such as bottle gourds and the like. And the gene is transferred into an exogenous gene or is subjected to gene editing on the cucurbitaceae plant, so that the gene has important significance.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for visualized genetic transformation of gourd mediated by Agrobacterium rhizogenes. Background Art

[0002] Gourd [Lagenaria siceraria (Molina) Standl.], also known as bottle gourd, is an important rootstock for grafted cultivation of watermelon, a Cucurbitaceae crop. Gourd has a well-developed root system and is significantly better than watermelon in resisting soil-borne diseases, which can effectively solve the problem of continuous cropping of watermelon. At present, biological research on bottle gourd is mostly limited to rootstock variety screening and gene localization of important agronomic traits. There are no reports on the genetic transformation system of bottle gourd. The genetic transformation efficiency of bottle gourd is limited by genotype and variety. The research on its genetic system is slow, and there is no efficient and stable genetic transformation system method, which seriously limits the progress of molecular precision breeding and gene function research of bottle gourd.

[0003] Plant genetic transformation is mainly achieved through Agrobacterium tumefaciens transformation and Agrobacterium rhizogenes transformation. Among them, Agrobacterium rhizogenes belongs to the genus Agrobacterium of the Rhizobium family and can effectively induce plants to produce hairy roots. It has the advantages of high transformation efficiency and simple operation steps. The positive transformed hairy roots induced by Agrobacterium rhizogenes can be used to quickly screen and verify the functions of genes related to root traits. For gourd rootstocks, molecular biology research on root traits is particularly important. Therefore, establishing a simple, rapid and efficient gourd rootstock-mediated hairy root transformation system is of great significance for the study of soil pests or root-related traits. Agrobacterium rhizogenes has been used to induce hairy roots in cucurbit crops such as watermelon and pumpkin, and reporter genes such as GFP and GUS have been successfully identified and used in tolerance studies of pumpkin rootstocks. However, reporter genes such as GFP and GUS require professional equipment or chemical treatment to identify the induction situation, and positive tissues cannot be directly screened. Summary of the invention

[0004] A technical problem to be solved by the present invention is how to establish a visualized Cucurbitaceae genetic transformation system.

[0005] RUBY is a new reporter gene that converts tyrosine into bright red betalain. Betalain is a natural plant product synthesized by tyrosine as a substrate and catalyzed by three enzymes (CYP76AD1, DODA, GT). Studies have shown that the RUBY system has been used in species such as Arabidopsis, rice, tobacco, tomato, wheat, corn, petunia and radish. The system does not require chemical treatment or special equipment, and the success of hairy roots can be identified by direct naked eye observation. In order to overcome the shortcomings of the prior art of the transformation method of gourd hairy roots, the present invention provides a transformation technology for quickly obtaining visualized hairy roots of gourd with the help of the RUBY reporter system. The RUBY reporter system is applied to the genetic transformation of gourd hairy roots mediated by Agrobacterium rhizogenes, which can achieve efficient and visual identification of transformed positive hairy roots. At the same time, this patent can provide the necessary hairy root gene editing method for studying the function of genes related to gourd root traits. The present invention can also provide a reference for the visualization operation of hairy root genetic transformation of other crops in the Cucurbitaceae family (watermelon, pumpkin, melon and bitter melon, etc.).

[0006] In order to solve the above technical problems, the present invention provides a method for visualized genetic transformation of gourd hairy roots mediated by Agrobacterium rhizogenes, comprising the step of transfecting cucurbitaceae plants with Agrobacterium rhizogenes containing a recombinant plasmid, wherein the recombinant plasmid is a vector containing a Ruby reporter gene expression cassette.

[0007] In the above method, the Ruby reporter gene encodes a protein having an amino acid sequence as shown in SEQ ID No.2.

[0008] In the above method, the sequence encoded by the Ruby reporter gene is shown as SEQ ID No. 1, 1-3933.

[0009] The recombinant plasmid further comprises a vector of fragment A, and the fragment A is T1 or T2 as follows:

[0010] T1, the fragment A may be the gene to be transferred A1, and the recombinant plasmid is an expression vector for expressing the Ruby reporter gene and the gene to be transferred A1;

[0011] T2. The fragment A may be an expression cassette of the sgRNA and Cas9 gene of the gene A2 to be edited, and the recombinant plasmid is a gene editing vector that expresses the Ruby reporter gene and edits the gene A2 to be edited.

[0012] In the above method, the expression of the Ruby reporter gene can preferably be driven by the CAMV 35S promoter.

[0013] In the above method, the Agrobacterium rhizogenes containing the recombinant plasmid is obtained by transferring the recombinant plasmid into Agrobacterium rhizogenes K599.

[0014] In the above method, the steps of transfecting Cucurbitaceae plants with Agrobacterium rhizogenes containing the recombinant plasmid are as follows:

[0015] S1, preparing the Agrobacterium rhizogenes containing the recombinant plasmid, resuspending the Agrobacterium rhizogenes containing the recombinant plasmid in a liquid culture medium to obtain an infection liquid, and culturing the Agrobacterium rhizogenes containing the recombinant plasmid in a solid culture medium to obtain a solid infection strain;

[0016] S2, pre-cultivating Cucurbitaceae plants and cutting off the stem tips as explants to be infected;

[0017] S3, after using the infection liquid to infect the incision of the explant to be infected, applying the solid infection strain to the incision;

[0018] S4, co-culturing the explants treated in step S3 under dark conditions, then transferring to normal light conditions for cultivation for 7-10 days, cutting off the roots, and continuing to cultivate until hairy roots grow, selecting plants with red hairy roots, and obtaining positively transformed Cucurbitaceae plants.

[0019] In the above method, the liquid culture medium in step S2 is a MS liquid culture medium with a sucrose concentration of 10 g / L, and the resuspending is resuspended to OD 600 Between 0.8-1.2.

[0020] In the above method, the solid culture medium in step S2 is a YEB solid culture medium containing 50 mg / L streptomycin and 50 mg / L kanamycin. The solid culture medium is cultured at 28° C. for 24-48 hours.

[0021] In the above method, the pre-culture step in step S2 is: culturing the seeds until the cotyledons unfold, cutting off the stem tip at a 45-60° angle 2-3 cm from the top of the cotyledons as the explants of the Cucurbitaceae plant to be infected.

[0022] In the above method, the normal lighting condition is a daily light period of 16 hours and a dark period of 8 hours.

[0023] In the above method, the co-cultivation in step S4 lasts for 4 days, and the duration of continuing to cultivate until hairy roots grow is 18-22 days.

[0024] In the above method, the Cucurbitaceae plant can be gourd, watermelon, pumpkin, melon and bitter melon, etc., preferably gourd.

[0025] The present invention also protects the application of the above method in Y1 or Y2:

[0026] Y1. Application in the transfer of exogenous protein-encoding genes into Cucurbitaceae plants;

[0027] Y2. Application in gene editing of genes in the genome of Cucurbitaceae plants.

[0028] The present invention uses the RUBY gene to establish a visualized Cucurbitaceae plant transformation system mediated by Agrobacterium rhizogenes, which does not require professional equipment or chemical treatment and can visually screen red hairy roots to identify induced positive plants. The system is simple, rapid and efficient, and has important significance for the study of soil pests and diseases or root-related traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The transient transformation phenotype of the RUBY reporter system of the 35S promoter and the DR5 promoter in Example 1 of the present invention in tobacco. Figure 1 a is the pDR5:RUBY vector transiently transformed tobacco; Figure 1 b is the p35S:RUBY vector transiently transformed tobacco; Figure 1 c is a negative control of injection of sterile water.

[0030] Figure 2 This is the process of transformation of gourd hairy roots mediated by Agrobacterium rhizogenes in Example 2 of the present invention.

[0031] Figure 3 This is the result of amplification of the RUBY gene in the positively transformed hairy roots in Example 2 of the present invention.

[0032] Figure 4 The positive transformation and control hairy root phenotype of 'Suzhen No. 1' infected by Agrobacterium rhizogenes K599-p35S:RUBY carrying the p35S:RUBY vector in Example 2 of the present invention. Figure 4 a is the red positive transformation strain and root phenotype, Figure 4 b is the wild control strain and root phenotype of 'Suzhen 1'.

[0033] Figure 5 The positive transformation and control hairy root phenotype of 'Suzhen No. 3' infected by Agrobacterium rhizogenes K599-p35S:RUBY carrying p35S:RUBY vector in Example 2 of the present invention. Figure 5 a is the red positive transformation strain and root phenotype, Figure 5 b is the wild control strain and root phenotype of 'Suzhen 3'.

[0034] Figure 6 The hairy root phenotype of the LsNAC1 gene-edited strain of Gourd in Example 3 of the present invention. Figure 6 a is the editing status of LsNAC1 gene in gene-edited hairy roots. Figure 6b shows the hairy root phenotype of the negative control (CK) and gene-edited strain (T2-8) of K599-pKSE402:RUBY containing pKSE402:RUBY; Figure 6 c is the statistics of root hair length of the negative control (CK) and gene-edited strain (T2-8) of K599-pKSE402:RUBY strain containing pKSE402:RUBY. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0037] The gourd-type watermelon rootstock variety 'Suzhen No. 1' in the following embodiment passed the Jiangsu Province non-major crop variety certification in 2023, the non-major crop certification certificate number: 2023-1-011, certification number: Surenzhen 202201.

[0038] The gourd-type watermelon rootstock variety 'Suzhen No. 3' in the following embodiment passed the Jiangsu Province non-major crop variety certification on January 24, 2024, the non-major crop certification certificate number: 2023-3-021, certification number: Surenzhen 202301.

[0039] The plant expression vector pDR5:RUBY in the following examples was donated by Cheng Qinghui of China Agricultural University, and is the vector pDR5:RUBY in the non-patent document "He Y, Zhang T, Sun H, Zhan H, Zhao YA reporter for noninvasively monitoring gene expression and plant transformation. Hortic Res. 2020 Sep 19; 7(1): 152." The public can obtain it from the applicant to repeat the experiment of the present invention. The vector pDR5:RUBY contains the RUBY coding sequence, as shown in SEQ ID No. 1 (3939 bp, wherein CDS is from position 1 to 3933), and the amino acid sequence of the RUBY protein encoded by it is shown in SEQ ID No. 2:

[0040] SEQ ID No.1

[0041] ATGGATCATGCGACCCTCGCCATGATCCTCGCGATCTGGTTCATCAGCTTCCACTTCATCAAGCTGCTGTTCTCCCAGCA

[0042] GACCACCAAGCTGCTTCCGCCAGGACCAAAGCCGCTTCCGATCATCGGCAACATCCTTGAGGTGGGCAAGAAGCCGCATC

[0043] GGTCCTTCGCCAACCTCGCCAAGATTCACGGCCCACTCATTTCCCTCAGACTCGGCTCTGTGACCACCATCGTTGTGTCC

[0044] TCTGCCGACGTGGCCAAAGAGATGTTCCTCAAGAAGGATCACCCGCTCTCCAACCGCACGATCCCGAATAGTGTTACAGC

[0045] CGGCGACCACCACAAGCTCACCATGTCTTGGCTCCCGGTGTCTCCGAAGTGGCGCAACTTCCGCAAGATTACCGCCGTGC

[0046] ATCTGCTCTCCCCACAGAGACTCGATGCCTGCCAGACATTCAGGCACGCCAAGGTGCAGCAGCTCTACGAGTACGTTCAA

[0047] GAGTGCGCCCAGAAAGGCCAGGCCGTGGATATTGGCAAGGCCGCCTTTACGACCAGCCTCAACCTCCTCAGCAAGCTGTT

[0048] CTTCAGCGTCGAGCTGGCGCACCACAAGTCCCATACCAGCCAAGAGTTCAAAGAGCTGATCTGGAACATCATGGAAGATA

[0049] TAGGCAAGCCGAACTACGCCGACTACTTCCCGATTCTCGGCTGCGTTGACCCATCTGGCATTAGAAGAAGGCTCGCCTGC

[0050] TCCTTCGACAAGCTGATCGCCGTGTTCCAGGGCATCATCTGCGAGAGACTCGCCCCAGATTCCTCCACCACAACTACCAC

[0051] CACCACCGACGACGTGCTCGATGTGCTCCTCCAGCTGTTCAAGCAGAACGAGCTGACGATGGGCGAGATCAACCACCTCC

[0052] TCGTGGACATCTTCGACGCCGGCACCGATACCACATCCTCCACATTCGAGTGGGTGATGACCGAGCTGATCCGCAATCCA

[0053] GAGATGATGGAAAAGGCCCAAGAGGAAATCAAGCAGGTCCTCGGCAAGGACAAGCAGATCCAAGAGTCCGACATCATCAA

[0054] CCTGCCGTACCTCCAGGCGATCATCAAAGAGACACTCCGCCTCCATCCGCCGACCGTGTTCTTGCTCCCAAGAAAGGCCG

[0055] ACACCGATGTCGAGCTGTACGGCTACATCGTGCCGAAGGATGCCCAGATCCTCGTGAACCTCTGGGCCATTGGCAGGGAC

[0056] CCAAACGCCTGGCAGAACGCCGATATTTTCAGCCCAGAGCGCTTCATCGGCTGCGAGATCGATGTTAAGGGCCGCGATTT

[0057] CGGCCTCCTTCCATTTGGCGCTGGCCGCAGAATTTGCCCAGGCATGAATCTCGCCATCAGGATGCTCACCCTCATGCTCG

[0058] CCACACTCCTCCAGTTCTTCAACTGGAAGCTCGAAGGCGACATCTCCCCGAAGGACCTCGACATGGACGAGAAGTTCGGC

[0059] ATTGCGCTCCAAAAGACCAAGCCGCTCAAGCTCATCCCGATTCCGCGCTACCAATTGTTGAATTTTGATTTGTTGAAGTT

[0060] GGCTGGAGATGTTGAATCTAATCCTGGACCTAAGATGATGAACGGCGAGGACGCCAACGACCAGATGATCAAAGAGTCCT

[0061] TCTTCATCACCCACGGCAACCCGATCCTCACCGTCGAGGATACACATCCGCTCAGGCCGTTCTTCGAGACATGGCGCGAG

[0062] AAGATTTTCTCCAAGAAGCCGAAGGCCATCCTCATCATCTCCGGCCACTGGGAGACAGTGAAGCCAACCGTGAACGCCGT

[0063] GCACATCAACGACACCATCCACGACTTCGACGACTACCCAGCCGCCATGTACCAGTTCAAGTACCCAGCTCCAGGCGAGC

[0064] CAGAGCTTGCGAGAAAGGTGGAAGAGATCCTCAAGAAGTCCGGGTTCGAGACAGCCGAGACAGACCAAAAGAGGGGCCTT

[0065] GATCACGGCGCCTGGGTTCCACTCATGCTCATGTATCCAGAGGCGGACATCCCGGTGTGCCAGCTCTCAGTTCAGCCACA

[0066] TCTCGACGGCACCTACCACTACAATCTCGGCAGAGCCCTCGCGCCGCTCAAGAATGATGGCGTGCTCATTATTGGCTCCG

[0067] GCAGCGCCACACATCCACTCGATGAGACACCGCACTACTTCGATGGTGTTGCCCCTTGGGCCGCTGCCTTCGATTCTTGG

[0068] CTTAGGAAGGCCCTCATCAACGGCCGCTTCGAGGAAGTGAACATCTACGAGAGCAAGGCCCCGAACTGGAAGCTCGCCCA

[0069] TCCATTTCCAGAGCACTTCTACCCGCTCCACGTTGTGCTCGGCGCTGCTGGTGAAAAGTGGAAGGCCGAGCTGATCCACT

[0070] CCTCCTGGGATCATGGCACACTTTGCCACGGCTCCTACAAGTTCACCTCCGCCCAATTGTTGAATTTTGATTTGTTGAAG

[0071] TTGGCTGGAGATGTTGAATCTAATCCTGGACCTACCGCCATCAAGATGAACACCAACGGCGAGGGCGAGACACAGCACAT

[0072] CCTCATGATCCCGTTCATGGCGCAGGGCCACCTCAGGCCATTTCTCGAACTCGCCATGTTCCTCTACAAGCGCTCCCACG

[0073] TGATCATCACCCTGCTCACAACTCCGCTCAACGCCGGCTTCCTCAGGCACCTCCTTCACCACCATTCCTACTCCTCCAGC

[0074] GGCATCAGGATCGTCGAGCTGCCATTCAACTCCACCAACCACGGACTCCCACCGGGCATCGAGAACACCGATAAGCTCAC

[0075] ACTCCCGCTCGTGGTGTCCCTCTTCCATTCCACCATCAGCCTCGATCCGCACCTCCGCGATTACATCTCCAGGCATTTCA

[0076] GCCCAGCCAGGCCACCACTCTGCGTGATCCATGATGTGTTCCTCGGCTGGGTTGACCAGGTGGCCAAGGATGTGGGCTCT

[0077] ACAGGCGTGGTGTTCACAACAGGCGGCGCTTATGGCACATCCGCCTACGTGTCCATCTGGAACGATCTCCCGCACCAGAA

[0078] CTACTCCGACGACCAAGAGTTCCCGCTGCCAGGCTTCCCAGAGAACCATAAGTTCCGCAGGTCCCAGCTCCATCGGTTCC

[0079] TCAGATATGCCGACGGCTCCGACGATTGGTCCAAGTATTTCCAGCCGCAGCTCCGCCAGTCCATGAAGTCTTTTGGCTGG

[0080] CTCTGCAACTCCGTGGAAGAGATCGAGACACTCGGCTTCTCCATCCTCCGCAACTACACCAAGCTGCCGATCTGGGGCAT

[0081] CGGCCCACTTATTGCTTCCCCAGTGCAGCACTCCTCCTCCGACAACAATTCAACAGGCGCCGAGTTCGTGCAGTGGCTCA

[0082] GCCTCAAAGAGCCGGACTCCGTCCTCTACATCTCCTTCGGCTCCCAGAACACGATCAGCCCGACGCAGATGATGGAACTC

[0083] GCTGCTGGCCTTGAGTCCTCCGAGAAGCCATTCCTCTGGGTGATCAGAGCCCCGTTCGGCTTCGACATCAACGAAGAGAT

[0084] GCGCCCAGAGTGGCTGCCAGAGGGCTTTGAGGAACGCATGAAGGTGAAGAAACAGGGCAAGCTCGTGTACAAGCTCGGCC

[0085] CGCAGCTTGAGATCCTCAACCATGAATCCATCGGCGGCTTTCTCACCCACTGCGGATGGAACAGCATCCTTGAGTCTCTT

[0086] CGCGAGGGCGTTCCGATGCTTGGATGGCCACTTGCTGCCGAGCAGGCCTACAACCTCAAGTACCTCGAAGATGAGATGGG

[0087] CGTCGCGGTTGAGCTTGCTAGAGGCCTCGAAGGCGAGATCTCCAAAGAGAAGGTCAAGCGCATCGTCGAGATGATCCTTG

[0088] AGCGCAACGAGGGCTCCAAAGGCTGGGAGATGAAGAATCGCGCCGTGGAAATGGGCAAAAAGCTCAAGGACGCCGTGAAC

[0089] GAGGAAAAAGAGCTGAAGGGCTCCTCCGTGAAGGCGATCGACGATTTCCTCGACGCCGTCATGCAGGCCAAACTTGAGCC

[0090] AAGCCTCCAGTGATAGTGA

[0091] SEQ ID No.2

[0092]

[0093] The gene editing vector pKSE402 in the following examples was kindly donated by Professor Huang Sanwen's team at the Chinese Academy of Agricultural Sciences and is described in the non-patent document "Xin T, Zhang Z, Li S, Zhang S, Li Q, Zhang ZH, Huang S, Yang X. Genetic Regulation of Ethylene Dosage for Cucumber Fruit Elongation. Plant Cell. 2019 May; 31(5): 1063-1076. doi: 10.1105 / tpc.18.00957. Epub 2019 Mar 26. PMID: 30914499; PMCID: PMC6533019." The public can obtain it from the applicant to repeat the experiments of the present invention.

[0094] The competent cells in the following examples were purchased from Shanghai Weidi Biotechnology Co., Ltd.: Escherichia coli DH5α-m, catalog number: DL1004; Agrobacterium tumefaciens GV3101, catalog number: AC1001; Agrobacterium rhizogenes K599, catalog number: AC1080; Agrobacterium rhizogenes Ar Qual, catalog number: AC1060.

[0095] Example 1

[0096] 1p35S:RUBY vector construction

[0097] p35S:RUBY is a plant expression vector that uses pDR5:RUBY as a backbone and replaces the DR5 promoter on the DR5:RUBY vector with the CaMV 35S promoter (amplified from the gene editing vector pKSE402) by homologous recombination. The specific steps are as follows:

[0098] a, pDR5:RUBY vector double restriction enzyme digestion: pDR5:RUBY vector was double-digested with restriction endonucleases XmaI and PstI (NEB) to obtain pDR5:RUBY linearized vector.

[0099] Enzyme digestion and ligation system (50 μL): pDR5:RUBY vector 1 μg, rCutSmart 5 μL, XmaI and PstI 1 μL each, ddH 2 0 to make up the total volume to 50 μL.

[0100] Enzyme digestion reaction program: 37°C for 1h, 85°C for 15min.

[0101] b. PCR amplification of the full-length sequence of CAMV 35S promoter: The full-length sequence of CAMV 35S promoter was amplified using vector pKSE402 as a template. The primer pair consisted of 35S-782-F and 35S-782-R in Table 1. The length of the amplified product was 782 bp, which was the 35S promoter fragment.

[0102] Table 1p35S: RUBY vector construction primer sequences

[0103]

[0104]

[0105] c. Connection: Connection between target gene and expression vector: according to Novozymes Ultra OneStep Cloning Kit V2 Recombination Cloning Kit (Cat. No.: C116) Instructions for Use.

[0106] Optimal amount of cloning vector used = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol)

[0107] Optimal amount of insert fragment = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol)

[0108] Ligation reaction system (10 μL): pDR5:RUBY linearized vector 3 ng, 35S promoter fragment 1 ng, 2×CE Mix 5 μL, ddH 2 HO to make up the total volume to 10 μL.

[0109] Ligation reaction procedure: 50°C, 5 min; reduce to 4°C or immediately cool on ice.

[0110] D. The ligation product was transformed into Escherichia coli DH5α (Shanghai, Weidi). For specific operations, refer to the instructions. After culturing at 28°C for 16-24 hours, a single clone was picked for PCR identification of the colony. The detection primer pair consisted of JC-35S:RUBY-F and JC-35S:RUBY-Ru (see Table 1). The positive bacterial solution with a product length of 1018bp was sent to the company (Beijing, Qingke) for sequencing analysis. The strains with correct sequencing results were activated and the vector was extracted (Nanjing, Novizan). The construction of the gene editing vector p35S:RUBY was completed.

[0111] The gene editing vector p35S:RUBY is a recombinant plasmid obtained by replacing the DR5 promoter between the restriction endonuclease XmaI and PstI recognition sequences of the pDR5:RUBY vector with the CAMV 35S promoter, while keeping the other sequences of the pDR5:RUBY vector unchanged. P35S:RUBY contains the RUBY coding sequence (as shown in SEQ ID No. 1, where positions 3937-3939 are terminators).

[0112] 2 Transformation of Agrobacterium: Use heat stimulation to transform the vector p35S:RUBY into Agrobacterium tumefaciens GV3101, Agrobacterium rhizogenes K599 and Ar Qual competent cells respectively. Add 0.1-1μg of the vector to each 100μL competent cell, mix well with a pipette, and place in ice for 5min, liquid nitrogen for 5min, 37℃ water bath for 5min, and ice bath for 5min. Add 700μL of TY liquid medium without antibiotics, and culture at 28℃ and 200rpm for 2h. Centrifuge at 4000rpm for 1min, take 100μL of the supernatant, gently blow and resuspend the bacterial block, and spread it on the TY plate containing 50mg / L streptomycin and 50mg / L kanamycin with antibiotics, and culture it upside down at 28℃ for 2-3d. Single colonies were picked, identified by PCR and verified by sequencing, and cultured at 180 rpm for 1 day in TY liquid medium containing 50 mg / L streptomycin and 50 mg / L kanamycin for later use. Among them, p35S:RUBY was transformed into Agrobacterium tumefaciens GV3101 to obtain Agrobacterium tumefaciens GV3101-p35S:RUBY, p35S:RUBY was transformed into Agrobacterium rhizogenes K599 to obtain Agrobacterium rhizogenes K599-p35S:RUBY, and p35S:RUBY was transformed into Agrobacterium rhizogenes Ar Qual to obtain Agrobacterium rhizogenes Ar Qual-p35S:RUBY.

[0113] 3. Transient transformation of tobacco

[0114] (b) pDR5:RUBY was used to transform Agrobacterium tumefaciens GV3101 to obtain Agrobacterium tumefaciens GV3101-pDR5:RUBY bacterial solution.

[0115] p19 was transformed into Agrobacterium tumefaciens GV3101 to obtain Agrobacterium tumefaciens GV3101-p19 bacterial liquid.

[0116] Take 10 μL of Agrobacterium tumefaciens GV3101-p35S:RUBY bacterial solution, Agrobacterium tumefaciens GV3101-pDR5:RUBY bacterial solution and Agrobacterium tumefaciens GV3101-p19 bacterial solution, and put them into 10 mL of TY medium containing corresponding antibiotics (50 mg / L kanamycin and 20 mg / L rifampicin) for propagation. The TY medium contains 150 μM acetosyringone. Cultivate at 28°C and 200 rpm until the Agrobacterium OD reaches 0.600 =1.5.

[0117] (2) The cells were collected by centrifugation at 5000 rpm for 10 min at room temperature and then impregnated with MES-KOH solution (containing 10 mM MgCl 2 , 10 mM MES, 150 μM acetosyringone, pH = 5.6) and suspended the cells to OD 600 = 1.0. Mix Agrobacterium tumefaciens GV3101-p35S:RUBY: Agrobacterium tumefaciens GV3101-p19 at a volume ratio of 1:1 to obtain a mixed bacterial solution for p35S:RUBY transformation, and mix Agrobacterium tumefaciens GV3101-pDR5:RUBY: Agrobacterium tumefaciens GV3101-p19 at a volume ratio of 1:1 to obtain a mixed bacterial solution for pDR5:RUBY transformation. After fully mixing, stand at room temperature in the dark for 2-3 hours.

[0118] (3) After gently making a small incision on the back of a tobacco leaf with a 1 mL syringe, use a syringe without the needle to draw up the mixed bacterial solution for p35S:RUBY transformation and inject it into the leaf from the wound. Use a marker to mark the water-stained area of ​​the tobacco leaf, and use sterile water as a control. Inject all true leaves of a whole tobacco plant except for the 2-3 true leaves at the growth point.

[0119] As a positive control, the mixed bacterial solution used for pDR5:RUBY transformation was injected instead of the mixed bacterial solution used for p35S:RUBY transformation.

[0120] As a negative control, sterile water was used instead of the mixed bacterial solution used for p35S:RUBY transformation.

[0121] Three biological replicates were performed for each treatment.

[0122] After injection, the tobacco plants were cultured under weak light at 25°C for 12 hours, and then cultured normally with 16 hours of light / 8 hours of darkness. After 2-3 days, the color changes of the tobacco area injected with Agrobacterium were observed.

[0123] Transient transformation experiments of p35:RUBY vector and pDR5:RUBY vector in tobacco Figure 1 , using the RUBY system tobacco plants driven by the DR5 promoter as a reference ( Figure 1 a), the expression level of the RUBY gene driven by the 35S promoter in the p35S:RUBY vector used in the present invention is higher than that in the reference ( Figure 1 b), and the tobacco plants injected with sterile water showed no color change ( Figure 1c). The above results indicate that 35S in the vector p35S:RUBY constructed in the present invention can drive the expression of RUBY, and the expression effect is better than that of DR5 promoter driving RUBY in the vector pDR5:RUBY, and can be used for subsequent hairy root transformation experiments.

[0124] Example 2

[0125] 1. Sowing and germination of gourd explants

[0126] Select full and uniform-sized 'Suzhen No. 1' and 'Suzhen No. 3' gourd seeds and place them in sterilized vermiculite (particle size 3-6mm, density 2.65g / cm 3 ) (seed: vermiculite = 1:5), sealed with plastic wrap, and placed in a 31℃ incubator for dark culture. After 3 days, the plastic wrap was opened for ventilation, and the culture was placed in a 26℃ light incubator for culture. After about 7 days, when the cotyledons of the gourd were fully expanded and the true leaves were just exposed, the subsequent infection could be carried out.

[0127] 2. Strain activation and preparation of infectious strains

[0128] Single clones of Agrobacterium rhizogenes K599-p35S:RUBY and ArQual-p35S:RUBY strains carrying the 35S:RUBY vector identified by PCR were inoculated into 1 mL of YEP liquid medium (50 mg / L streptomycin and 50 mg / L kanamycin) and cultured overnight at 28°C and 200 rpm until OD 600 =1.5.

[0129] (b) Liquid infection solution preparation:

[0130] Take 200 μL of Agrobacterium rhizogenes K599-p35S:RUBY bacterial solution in 50 mL of YEB liquid medium (50 mg / L streptomycin and 50 mg / L kanamycin), culture at 28°C, 200 rpm for 10-12 hours; centrifuge at 25°C, 4000 rpm for 10 minutes and discard the supernatant. Resuspend the cells with 5-10 mL (containing 1% sucrose) MS suspension and adjust the OD 600 The concentration is between 0.8 and 1.2, and the solution is allowed to stand in the dark at room temperature for 1 to 2 hours to obtain the infection solution of Agrobacterium rhizogenes K599-p35S:RUBY.

[0131] Take 200 μL of Agrobacterium rhizogenes Ar Qual-p35S: RUBY bacterial solution in 50 mL of YEB liquid medium (50 mg / L streptomycin and 50 mg / L kanamycin), culture at 28°C, 200 rpm for 10-12 hours; centrifuge at 25°C, 4000 rpm for 10 minutes and discard the supernatant. Resuspend the bacteria in 5-10 mL of MS liquid medium containing 1% (i.e. 10 g / L) sucrose and adjust the OD600 The concentration of Agrobacterium rhizogenes Ar Qual-p35S: RUBY infection solution is prepared after the concentration is between 0.8 and 1.2 and placed in the dark at room temperature for 1 to 2 hours.

[0132] (2) Preparation of solid infection strains:

[0133] Take 10 μL of Agrobacterium rhizogenes K599-p35S:RUBY bacterial liquid and spread it evenly in a YEB solid culture medium containing 50 mg / L streptomycin and 50 mg / L kanamycin. Then, culture it in a 28°C constant temperature incubator for 24-48 hours to obtain the Agrobacterium rhizogenes K599-p35S:RUBY solid infection strain to be used.

[0134] Take 10 μL of Agrobacterium rhizogenes Ar Qual-p35S: RUBY bacterial liquid and put it into a YEB solid culture dish (50 mg / L streptomycin and 50 mg / L kanamycin), spread it evenly, and culture it in a 28°C constant temperature incubator for 24-48 hours to obtain the Agrobacterium rhizogenes Ar Qual-p35S: RUBY solid infection strain to be used.

[0135] 3 Hairy root induction steps

[0136] The steps for inducing hairy roots of gourd are shown in Figure 2 , the specific method is as follows:

[0137] (1) Explant selection: Place the gourd seeds in vermiculite with a water content of 70%. After 6-8 days, wait for the cotyledons to unfold. Use a scalpel to cut the gourd stem at an angle of 45-60 degrees about 2-3 cm from the top of the cotyledons (leave the stem tip with cotyledons as the explant) to obtain the explant to be infected.

[0138] (2) Infection:

[0139] Infection with Agrobacterium rhizogenes K599-p35S:RUBY: place the explant incision in the infection solution of Agrobacterium rhizogenes K599-p35S:RUBY, infect for about 1 hour at room temperature, and gently wipe off the excess infection solution on the stem wound; continue to evenly apply the solid infection strain of Agrobacterium rhizogenes K599-p35S:RUBY in the culture dish to the wound of the gourd stem.

[0140] Infection with Agrobacterium rhizogenes Ar Qual-p35S:RUBY: Place the explant incision in the infection solution of Agrobacterium rhizogenes Ar Qual-p35S:RUBY and infect for about 1 hour at room temperature. Gently wipe off the excess infection solution from the stem wound; continue to evenly apply the solid infection strain of Agrobacterium rhizogenes Ar Qual-p35S:RUBY in the culture dish to the wound of the gourd stem.

[0141] (3) Co-cultivation: The seedlings infected with Agrobacterium rhizogenes K599-p35S:RUBY and the seedlings infected with Agrobacterium rhizogenes Ar Qual-p35S:RUBY were vertically inserted into plug trays containing vermiculite soaked with 1% MS culture solution, respectively. The trays were sealed with transparent plastic covers to maintain high humidity. The cells were co-cultivated at 25°C in the dark for 4 days.

[0142] (4) Plug tray culture: After the co-cultivation, the seedlings were placed in plug trays (peat soil, perlite, vermiculite at a ratio of 2:1:1) and cultured under normal light conditions with a photoperiod of 16 h (light period) / 8 h (dark period) and a temperature of 26°C.

[0143] (5) Root cutting: After 7-10 days of cultivation, remove all the growing hairy roots and place them in a plug tray (peat soil, perlite, vermiculite in a ratio of 2:1:1) to continue growing.

[0144] (6) Obtaining positive (red) transformed hairy roots: After about 20 days, observe the root growth and directly observe the red positive transformed hairy roots.

[0145] The infection test was carried out with three biological replicates, and 50 plants were infected in each replicate. The uninfected wild-type plants were used as wild control plants.

[0146] 4 Identification and statistical analysis of transformed hairy roots

[0147] (1) Phenotypic identification: After the transformed hairy roots are formed, take out the gourd seedlings in the plug tray to directly observe whether the gourd root system has bright red hairy roots. The transformed positive roots are visible to the naked eye due to the expression of the betalain gene (RUBY), while the wild-type hairy roots are milky white. Without damaging the root system, it is possible to preliminarily determine whether the hairy roots are successfully transformed, count and record them. At the same time, wash the positive transformed roots and further analyze the phenotypic traits of the transformed roots.

[0148] (2) Target gene identification: The CTAB method was used to extract DNA from positively transformed root samples, and the RUBY tag gene was detected by PCR. The samples with the target band were considered positive.

[0149] PCR reaction system (25 μL): 1 μL DNA working solution, 12.5 μL 2× Super Pfx Master Mix (Dye) (CW2965, Kangwei Century), 1 μL forward primer and 1 μL reverse primer (10 μM / L; 0.4 μM / L) (RUBY gene identification primers are shown in Table 2), and 9.5 μL ddHO. 2 0. Make up the total volume to 25 μL.

[0150] PCR amplification reaction program: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 5 min.

[0151] Table 2 Primer sequences for RUBY gene identification

[0152] Primer name Sequence (5'-3') RUBY-595-F GGCAACATCCTTGAGGTGGG RUBY-595-R AGCAGGCGAGCCTTCTTCTA

[0153] The phenotypic statistical results were consistent with the PCR test results. Figure 3 As shown, the RUBY fragment band was amplified in the transformed roots containing betalain and showing red color visible to the naked eye, and the negative roots with the same color as the wild-type roots did not amplify the RUBY fragment band. The statistical results show that the RUBY marker was successfully transformed into the gourd hairy roots by Agrobacterium rhizogenes, and the accuracy of identifying positive hairy roots by red phenotype can reach 100% (see Table 3), indicating that the method of the present invention is suitable for the transformation of gourd roots and can efficiently and quickly identify positive hairy roots.

[0154] Table 3 Efficiency of RUBY marker in identifying positive hairy roots of Gourd

[0155] Gourd varieties (Agrobacterium type) Phenotypic identification Gene identification Accuracy (%) 'Suzhen No. 1' (K599) 33 33 100 'Suzhen No. 3' (K599) 23 23 100 'Suzhen No. 1' (Ar Qual) 17 17 100 'Suzhen No. 3' (Ar Qual) 6 6 100

[0156] The induction efficiency of the two rhizogenes Agrobacterium on different gourd varieties is not the same, as shown in Table 4. The rhizogenes K599-p35S:RUBY and Ar Qual-p35S:RUBY carrying the p35S:RUBY vector were used to infect gourds of 'Suzhen No. 1' and 'Suzhen No. 3', respectively. The induction efficiency of the two gourd varieties by the rhizogenes K599-p35S:RUBY was significantly higher than that of the Ar Qual-p35S:RUBY. Among them, the induction efficiency of the hairy roots of the 'Suzhen No. 1' infected by the rhizogenes K599-p35S:RUBY was the highest at 66%, and the induction efficiency of the hairy roots of the 'Suzhen No. 3' infected by the Ar Qual-p35S:RUBY was the lowest at 12%. The positive hairy roots of 'Suzhen 1' infected by Agrobacterium rhizogenes K599-p35S:RUBY and the control phenotypes are shown in Figure 4 , Agrobacterium rhizogenes K599-p35S:RUBY infected 'Suzhen 3' positive hairy roots and control phenotypes are shown in Figure 5 .

[0157] Table 4 Different gourd varieties and infection efficiency of Agrobacterium rhizogenes

[0158] Agrobacterium Type 'Suzhen No. 1' 'Suzhen No. 3' K599 66％ 46％ Ar Qual 33％ 12％

[0159] Example 3

[0160] The application of the visualized hairy root induction method of gourd in gene editing is as follows:

[0161] Plant material: Gourd variety 'Suzhen No. 1'.

[0162] 1. Vector Construction

[0163] (b) Construction of pKSE402:RUBY vector

[0164] Using pKSE402 as the backbone and pDR5:RUBY as the template for amplifying the full-length sequence of the RUBY gene, the specific steps are as follows:

[0165] b, Double restriction enzyme digestion of pKSE402 vector: pKSE402 vector was double-digested with restriction endonucleases BsrGI and SpeI (NEB) to obtain pKSE402 linearized vector.

[0166] Enzyme digestion and ligation system (50 μL): pKSE402 vector 1 μg, rCutSmart 5 μL, BsrGI and SpeI 1 μL each, ddH 2 0 to make up the total volume to 50 μL.

[0167] Enzyme digestion reaction program: 37°C for 1h, 85°C for 15min.

[0168] b. PCR amplification of RUBY gene: using vector pDR5:RUBY as template, the amplification primer pair consisted of RUBY-3939-F and RUBY-3939-R in Table 5, and the product length was 3939 bp, as shown in SEQ ID No. 1, referred to as pKSE402 fragment.

[0169] Table 5 pKSE402: RUBY vector construction primer sequences

[0170]

[0171]

[0172] c. Connection: Connection between target gene and expression vector: according to Novozymes Ultra OneStep Cloning Kit V2 Recombination Cloning Kit (Cat. No.: C116) Instructions for Use.

[0173] Optimal amount of cloning vector used = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol)

[0174] Optimal amount of insert fragment = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol)

[0175] Ligation reaction system (10 μL): pKSE402 linearized vector 3 ng, RUBY gene target fragment 1 ng, 2×CE Mix 5 μL, ddH 2 HO to make up the total volume to 10 μL.

[0176] Ligation reaction procedure: 50°C, 5 min; reduce to 4°C or immediately cool on ice.

[0177] d. The ligation product was transformed into E. coli DH5α (Shanghai, Weidi). For specific operations, refer to the instructions. After culturing at 28°C for 16-24 hours, a single clone was picked for PCR identification of the colony. The detection primer pair consisted of JC-402:RUBY-F and JC-402:RUBY-R (see Table 5). The product length was 761bp. The positive bacterial solution was sent to the company (Beijing, Qingke) for sequencing analysis. The strains with correct sequencing results were activated and the vector was extracted (Nanjing, Novizan). The construction of the gene editing vector pKSE402:RUBY was completed.

[0178] The vector pKSE402:RUBY is a recombinant plasmid obtained by replacing the small fragment between the restriction endonuclease BsrGI and SpeI recognition sequences of the pKSE402 vector (containing the promoter CAMV 35S) with the pKSE402 fragment (as shown in SEQ ID No. 1), while keeping the other sequences of the pKSE402 vector unchanged. The expression of the RUBY gene on the pKSE402:RUBY vector is driven by the promoter CAMV 35S.

[0179] (2) Construction of LsNAC1 gene editing vector

[0180] The bottle gourd HG10013844 (NAC1) gene was used as the significantly differentially expressed gene in bottle gourd root samples in response to low temperature stress in the root zone (recorded in the non-patent literature "Liu J, Zhang M, Xu J, Yao X, Lou L, Hou Q, Zhu L, Yang X, Liu G, Xu JA Transcriptomic Analysis of Bottle Gourd-Type Rootstock RootsIdentifies Novel Transcription Factors Responsive to Low Root ZoneTemperature Stress. Int J Mol Sci. 2024 Jul 29; 25 (15): 8288.")

[0181] a. The CDS sequence of LsNAC1 gene was downloaded from the Lagenaria siceraria cv. Hangzhou Gourd genome database (shown in SEQ ID No.3). The amino acid sequence of the LsNAC1 protein encoded by it is shown in SEQ ID No.4.

[0182] SEQ ID No.3

[0183] ATGGAGAATAATATAAGCATGGTTGAGGCTAAACTTCCTCCTGGTTTTAGGTTTCATCCAAGAGATGAAGAATTGGTGTGTGATTATTTGATGAAGAAAATTGGCTCTGTTTCTGATTCTTCTTCTCTCTTGATTGAAGTTGACCTCAACAAGTGTGAGCCTTGGGATATTCCAAGAGAGGCATGCGTTGGAGGAAAAGAGTGGTACTTCTTCAGCCAGCGGGACCGTAAGTATGCGACTGGGCTCAGAACAAACCGCGCCACAGCCTCAGGGTATTGGAAGGCCACTGGCAAGGACAGGCCCGTCTTTCATAAGGCCAATCAACTCGTTGGGATGAGAAAAACTCTTGTTTTCTACCAAGGAAGGGCCCCTAAAGGCCGAAAAACTGAGTGGGTTATGCATGAATTTCGTCTTGAGGGTCCATTTTCTCCTCTCAAAGACCCATCTCCAAAGGAGGACTGGGTTCTGTGCAGAGAT TGTTCTGTAAACAGAAGGAAGTTACCGCCCAACCGAGCACAGGAAGCAGCAGCTGCTACAACGACATCGTTGCCTCTTCGTCGTCTCTCCCAGCTTTAATGGACTCATACATCAGTTTTGACCAAAATCCAAGTAGCCATTTAAATGAGTATGAGCAAGTGCCCTGCTTCTCCATTTTCTCTCTCAACCAAACCATCCCATCTCTCTCAAACCTCATACAAATGGAGCCAAACACACAGC CAATAACATCAAGAACTTTAGCACCATGTTTGGAGGAATGCCAAATTCAAGCACTTGTTCTTCAAACATTGACCCTTTTGCTTGTGACTCAAAAGTGCTCAAAGTTGTTCTAAATAACATTACTAAAATGGAAACAAATGGCAACTCCTTCATAGGGCAACCTAGCATGGGAGAAGGCAGCTCTGACAGCTACTTATCTGAGGTTGGAGATGACATTGCCAGCTTATGGAACAGATGA

[0184] SEQ ID No.4

[0185] MENNISMVEAKLPPGFRFHPRDEELVCDYLMKKIGSVSDSSSLLIEVDLNKCEPWDIPREACVGGKEWYFFSQRDRKYATGLRTNRATASGYWKATGKDRPVFHKANQLVGMRKTLVFYQGRAPKGRKTEWVMHEFRLEGPFSPLKDPSPKEDWVLCR LFCKQKEVTAQPSTGSSSCYNDIVASSSSLPALMDSYISFDQNPSSHLNEYEQVPCFSIFSQNQTIPSLSNLIQMEPNTANNIKNFSTMFGGMPNSSTCSSNIDPFACDSKVLKVVLNNITKMETNGNSFIGQPSMGEGSSDSYLSEVGDDIASLWNR

[0186] The online website CRISPR-P 2.0 was used to search for the target sequence of the target gene and select the best sgRNA target. The target sequence was located at CDS sequence 371-390 (positions 371-390 of SEQ ID No. 3), and the PAM was located at CDS sequence 391-393 (positions 391-393 of SEQ ID No. 3). The adapter sequences ATTG and AAAC were added to the 5' ends of the upstream and downstream primers, respectively. The sgRNA target primers were sgRNA-LsNAC1-F and sgRNA-LsNAC1-R in Table 6, and sent to the company (Qingke, Beijing) for synthesis.

[0187] Table 6 sgRNA target sequences

[0188] Primer name Sequence (5'-3') sgRNA-LsNAC1-F ATTGCTAAAGGCCGAAAAACTGAGTGG sgRNA-LsNAC1-R AAACCCACTCAGTTTTTCGGCCTTTAG

[0189] b, The synthesized sgRNA primers were annealed and paired, purified, digested, and ligated to the vector pKSE402:RUBY.

[0190] sgRNA primer annealing reaction system (50 μL): sgRNA-F / R 1.5 μL each (100 μM), 10×NEB buffer 5 μL, 42 μL ddH 2 0. Make up the total volume to 50 μL.

[0191] sgRNA reaction conditions: 95°C for 10 min, cooling to 20°C at 0.1°C / s.

[0192] Enzyme digestion and ligation system (10 μL): pKSE402:RUBY (100 ng) 2 μL, 10×NEB T4 Buffer 1 μL, 10xBSA 1 μL, BsaI (NEB) 1 μL, T4 Ligase (NEB) 1 μL, 3 μL ddH 2 0. Make up the total volume to 10 μL.

[0193] Enzyme digestion reaction program: 37°C for 3 h, 50°C for 5 min, and 80°C for 10 min.

[0194] c. Each ligation product was transformed into Escherichia coli DH5α (Shanghai, Weidi), and the specific operation was referred to the instruction manual. After culturing at 28°C for 16-24h, a single clone was picked for PCR identification of the colony (the identification primer pair consisted of LsNAC1-F and LsNAC1-R in Table 7), and the positive bacterial solution was sent to the company (Beijing, Qingke) for sequencing analysis. The strain with the correct sequencing result was activated and the vector was extracted (Nanjing, Novizuan). The construction of the gene editing vector pKSE402:RUBY:LsNAC1 was completed. The vector pKSE402:RUBY:LsNAC1 expresses RUBY, sgRNA for the LsNAC1 gene, and Cas9. The promoter for expressing RUBY is CAMV 35S, and the terminator is NOS; the promoter for expressing sgRNA is U6 promoter, and the promoter for expressing Cas9 is CAMV 35S promoter.

[0195] Table 7 sgRNA target identification primer sequences

[0196] Primer name Sequence (5'-3') LsNAC1-F CAATCTTCAAAAGTCCCACATCG LsNAC1-R TCGGAACTGCAAAACTCAAC

[0197] 2. Gene editing vector used for transformation of hairy roots of 'Suzhen No. 1' gourd

[0198] Transform the pKSE402:RUBY:NAC1 vector into Agrobacterium rhizogenes K599 (Shanghai, Weidi), add 0.1-1μg of the vector per 100μL competent medium, mix well with a pipette, and place in ice for 5min, liquid nitrogen for 5min, 37℃ water bath for 5min, and ice bath for 5min. Add 700μL of TY liquid medium without antibiotics, and culture at 28℃ and 200rpm for 2h. Centrifuge at 4000rpm for 1min, take 100μL of the supernatant, gently blow and resuspend the bacteria, and spread on the TY plate containing 50mg / L streptomycin and 50mg / L kanamycin, and culture at 28℃ for 2-3d. A single colony was picked, identified by PCR and verified by sequencing, and cultured at 180 rpm for 1 day in TY liquid medium containing 50 mg / L streptomycin and 50 mg / L kanamycin, and then retained for use to obtain Agrobacterium rhizogenes K599-pKSE402:RUBY:NAC1 transformed into Agrobacterium rhizogenes K599.

[0199] The pKSE402:RUBY vector was used as a negative control. The pKSE402:RUBY vector was transformed into Agrobacterium rhizogenes K599 to obtain Agrobacterium rhizogenes K599-pKSE402:RUBY.

[0200] Using the test material "Suzhen No. 1", full and uniform-sized seeds were selected and placed in sterilized vermiculite (particle size 3-6mm, density 2.65g / cm 3 ) (seed: vermiculite = 1:5), sealed with plastic wrap, and placed in a 31℃ incubator for dark culture. After 3 days, the plastic wrap was opened for ventilation, and the culture was placed in a 26℃ light incubator for culture. After about 7 days, when the cotyledons of the gourd were fully expanded and the true leaves were just exposed, the subsequent infection could be carried out.

[0201] Single clones of the K599-pKSE402:RUBY:NAC1 strain (K599-pKSE402:RUBY was used as the negative control) were inoculated into 1 mL of YEP liquid medium (50 mg / L streptomycin and 50 mg / L kanamycin) and cultured overnight at 28°C and 200 rpm until OD 600 =1.5.

[0202] (1) Preparation of liquid infection solution:

[0203] Take 200 μL of K599-pKSE402:RUBY:NAC1 strain (negative control uses K599-pKSE402:RUBY) in 50 mL of YEB liquid medium (50 mg / L streptomycin and 50 mg / L kanamycin), culture at 28°C, 200 rpm for 10-12 h; centrifuge at 25°C, 4000 rpm for 10 min and discard the supernatant. Resuspend the cells in 5-10 mL of MS liquid medium containing 1% (i.e. 10 g / L) sucrose and adjust the OD 600 The concentration is between 0.8 and 1.2, and the solution is allowed to stand in the dark at room temperature for 1 to 2 hours to obtain the infection solution of K599-pKSE402:RUBY:NAC1 strain.

[0204] (2) Preparation of solid infection strains:

[0205] Take 10 μL of K599-pKSE402:RUBY:NAC1 bacterial liquid and place it in a YEB solid culture dish (50 mg / L streptomycin and 50 mg / L kanamycin), spread it evenly, and culture it in a 28°C constant temperature incubator for 24-48 hours to obtain the K599-pKSE402:RUBY:NAC1 solid infection strain to be used.

[0206] 3 Hairy root induction steps

[0207] The steps for inducing hairy roots of gourd are as follows:

[0208] (1) Selection of explants: Place the gourd seeds in vermiculite with a water content of 70%. After 6-8 days, wait for the cotyledons to unfold. Use a scalpel to cut the gourd stem at an angle of 45-60 degrees about 2-3 cm from the top of the cotyledons to obtain the explants to be infected.

[0209] (2) Infection:

[0210] The explant incision was placed in K599-pKSE402:RUBY:NAC1 infection solution and infected for about 1 hour at room temperature, and the excess infection solution on the stem wound was gently wiped off; the K599-pKSE402:RUBY:NAC1 solid infection strain in the culture dish was then evenly applied to the wound on the gourd stem.

[0211] (3) Co-cultivation: The seedlings infected with K599-pKSE402:RUBY:NAC1 were vertically inserted into plug trays containing vermiculite soaked with 1% MS culture solution, sealed with a transparent plastic cover to maintain high humidity, and co-cultivated at 25°C for 4 days in the dark.

[0212] (4) Plug tray culture: After the co-cultivation, the seedlings were placed in plug trays (peat soil, perlite, vermiculite at a ratio of 2:1:1) and cultured under normal light conditions, with a photoperiod of 16 h / 8 h and a temperature of 26 °C.

[0213] (5) Root cutting: After 7-10 days of cultivation, remove all the growing hairy roots and place them in a plug tray (peat soil, perlite, vermiculite in a ratio of 2:1:1) to continue growing.

[0214] (6) Obtaining positive (red) transformed hairy roots: After about 20 days, observe the root growth and directly observe the red positive transformed hairy roots.

[0215] The infection test was performed with three biological replicates, each replicate infected 50 strains. The K599-pKSE402:RUBY strain containing pKSE402:RUBY was used as a negative control (CK).

[0216] The Cucurbita guajava LsNAC1 (HG10013844) gene editing vector pKSE402:RUBY:NAC1 was used to transform the visible hairy roots in the 'Suzhen No. 1' material. After induction, positive transformed red hairy roots visible to the naked eye were successfully produced, of which two were numbered T2-3 and T2-8, and the gene editing was analyzed by PCR detection and target sequencing. The results showed that sgRNA successfully mediated the editing of the root genome of Cucurbita guajava 'Suzhen No. 1' by Cas9 protein (such as Figure 6 a)

[0217] Phenotypic identification of LsNAC1 knockout lines after low temperature stress in the root zone

[0218] The successfully knocked out gourd transformation strain T2-8 was subjected to 15℃ root zone low temperature stress, and the root phenotype and hairy root length (such as Figure 6 b, 6 c), preliminarily verified that Gourd LsNAC1 has the function of regulating root growth in response to low temperature in the root zone.

[0219] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for visualized genetic transformation of Cucurbitaceae plants mediated by Agrobacterium rhizogenes, characterized in that: The method comprises the step of using Agrobacterium rhizogenes containing a recombinant plasmid to transfect a cucurbitaceous plant, wherein the recombinant plasmid is a vector containing a Ruby reporter gene expression cassette; the Ruby reporter gene encodes a protein whose amino acid sequence is shown in SEQ ID No.

2.

2. The method according to claim 1, characterized in that: The recombinant plasmid further comprises fragment A; the fragment A is T1 or T2 as follows: T1, the fragment A is the gene to be transferred, and the gene to be transferred is a gene encoding protein; T2. The fragment A is an expression cassette of the sgRNA gene and the Cas9 gene targeting the gene to be edited.

3. The method according to claim 1 or 2, characterized in that: The expression of the Ruby reporter gene was driven by the CAMV 35S promoter.

4. The method according to claim 3, characterized in that: The Agrobacterium rhizogenes containing the recombinant plasmid is obtained by transferring the recombinant plasmid into Agrobacterium rhizogenes K599.

5. The method according to claim 4, characterized in that: The steps of transfecting Cucurbitaceae plants with Agrobacterium rhizogenes containing the recombinant plasmid are as follows: S1, preparing the Agrobacterium rhizogenes containing the recombinant plasmid, resuspending the Agrobacterium rhizogenes containing the recombinant plasmid in a liquid culture medium to obtain an infection liquid, and culturing the Agrobacterium rhizogenes containing the recombinant plasmid in a solid culture medium to obtain a solid infection strain; S2, pre-cultivating Cucurbitaceae plants and cutting off the stem tips as explants to be infected; S3, after using the infection liquid to infect the incision of the explant to be infected, applying the solid infection strain to the incision; S4, co-culturing the explants treated in step S3 under dark conditions, then transferring to normal light conditions for cultivation for 7-10 days, cutting off the roots, and continuing to cultivate until hairy roots grow, selecting plants with red hairy roots, and obtaining positively transformed Cucurbitaceae plants.

6. The method according to claim 5, characterized in that: The liquid culture medium in step S2 is a MS liquid culture medium with a sucrose concentration of 10 g / L, and the resuspending is resuspending to OD 600 The solid culture medium is YEB solid culture medium containing 50 mg / L streptomycin and 50 mg / L kanamycin. The solid culture medium is cultured at 28°C for 24-48 hours.

7. The method according to claim 5, characterized in that: The pre-culture step in step S2 is: culturing the seeds until the cotyledons are unfolded, and cutting off the stem tip at a 45-60° angle 2-3 cm from the top of the cotyledons as the explants of the Cucurbitaceae plant to be infected.

8. The method according to claim 5, characterized in that: The normal light condition is a 16-hour light period and an 8-hour dark period per day.

9. The method according to claim 5, characterized in that: The cucurbitaceae plant is gourd, watermelon, pumpkin, melon or bitter melon.

10. Use of the method according to any one of claims 1 to 9 in Y1 or Y2: Y1. Application in the transfer of exogenous protein-encoding genes into Cucurbitaceae plants; Y2. Application in gene editing of genes in the genome of Cucurbitaceae plants.

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