Agrobacterium tumefaciens-mediated open cotton hairy root transformation method

Abstract

The invention relates to the technical field of biology, in particular to an agrobacterium tumefaciens-mediated open cotton hairy root transformation method which comprises the following steps: selecting eight-day-old cotton seedlings, transversely cutting a hypocotyledonary axis 0.5-1cm above a rhizome junction, and cutting off an underground part; then, agrobacterium rhizogenes liquid is injected into the notch of the hypocotyl, the notch of the hypocotyl is subjected to vacuum filtration treatment, then seedlings are cut into wet vermiculite and placed in a seedling raising tray, a proper amount of clear water is added into the seedling raising tray, the seedling raising tray is covered with a moisturizing cover, dark treatment is conducted for 24 h, and then light culture is conducted in the environment with the relative humidity being 65-75%; an efficient induction transformation system of the cotton hairy roots under the open condition can be established. The method for rapidly inducing the cotton transgenic hairy roots under the open condition is established, sterile operation is not needed, a culture medium does not need to be used, cotton genotype limitation is avoided, and a simple, convenient and efficient research method is provided for cotton gene function analysis and root system biology research.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an Agrobacterium-mediated open cotton hairy root transformation method. Background Art

[0002] Cotton, a plant of the genus Gossypium in the family Malvaceae, is an important economic crop. It is not only an important source of natural fiber in the world's textile industry, but also an important source of edible oil. Efficient and simple genetic transformation technology is a powerful tool for analyzing cotton gene functions, metabolic pathways and molecular breeding. However, due to the long growth cycle of cotton and the low efficiency of genetic transformation, cotton genomics research has been greatly limited.

[0003] Hairy roots mediated by Agrobacterium rhizogenes have the characteristics of rapid growth, genetic stability, and strong ability to produce secondary metabolites, and have been used for research on gene function and root biology. At present, cotton hairy roots have been verified to be applicable to the study of the mechanism of action of cotton nematodes and the production of secondary metabolites such as gossypol. A transformation system based on perforation of cotyledons under sterile conditions and injection of cotton apical buds under open conditions has been established to induce hairy roots. However, the induction of cotyledon hairy roots under sterile conditions can only be subcultured on culture media, and the operation is relatively complicated and not conducive to the study of gene function. For the hairy roots induced by injection of cotton apical buds under open conditions, the non-transgenic roots in the underground part still exist, and the transgenic roots in the aboveground part cannot play the role of roots. Such complex plants are not suitable for cotton root biology research. Summary of the invention

[0004] To solve the above problems, the present invention provides an Agrobacterium-mediated open cotton hairy root transformation method, establishes an efficient induction system for transgenic cotton hairy roots under open conditions, does not require sterile operation, and has no cotton genotype restrictions, providing a simple and efficient research method for cotton gene function analysis and root biology research.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] An Agrobacterium-mediated open cotton hairy root transformation method comprises the following steps:

[0007] S1. Construct the vector and complete the transformation of Agrobacterium;

[0008] S2. Select eight-day-old cotton seedlings, cut the hypocotyl 0.5 to 1 cm above the junction of the rhizome, and remove the underground part;

[0009] S3, injecting Agrobacterium rhizogenes solution into the hypocotyl incision, vacuum filtering the hypocotyl incision, and then inserting the seedlings into moist vermiculite, keeping them moist and dark for 24 hours, and then culturing them under light for 35 days in an environment with a relative humidity of 65-75%;

[0010] S4. The seedlings with induced hairy roots are cultured hydroponically. During the hydroponic process, the wild-type roots are gradually removed by continuous pruning, so that the transgenic hairy roots are enriched in large quantities.

[0011] Furthermore, in the step S1, the vector is an overexpression vector of the RUBY gene, and the Agrobacterium is Agrobacterium rhizogenes K599.

[0012] Furthermore, when constructing the overexpression vector of the RUBY gene, a primary module of p35S:RUBY was first constructed through a modular cloning method and named pHL001; pHL001 was then connected to a secondary backbone vector, the correctly sequenced plasmid was saved and named pHL002, and pHL002 was transferred into C58C1, Ar 1193, K599, and MSU440 competent cells for subsequent infection.

[0013] Furthermore, in step S1, the vector is a p35S:RolABC-AmCyan vector, and the Agrobacterium is Agrobacterium tumefaciens EHA105 and GV3101.

[0014] Furthermore, when constructing the p35S:RolABC-AmCyan vector, firstly, lox71 and lox66 primers are annealed to synthesize double strands to construct a primary modular plasmid containing 35S promoter and lox71, and the correctly sequenced recombinant vector is named pHL015; RolABC and its promoter PCR products are connected to the primary vector backbone according to the Modular cloning reaction system, and the correctly sequenced recombinant vector is named pHL016; double-stranded lox66, RUBY 0-level modular plasmid, and 35S terminator 0-level modular plasmid are connected to the primary vector backbone according to the Modular cloning reaction system, and the correctly sequenced recombinant vector is named pHL017; then the primary vector modules pHL015, pHL016, pHL017 and pHL018 (CmYLCV:AmCyan:Ter) are connected to the primary vector backbone according to the Modular cloning reaction system. The cloning reaction system was connected to the secondary receptor backbone, and then the ligation product was transferred into DH5α competent cells, and single clones were picked for sequencing; the correctly sequenced plasmid was named pHL019; pHL019 was transferred into EHA105, GV3101 and K599 competent cells, and pTRV1 and pTRV2-Cre were transferred into GV3101 competent cells for subsequent infection.

[0015] Furthermore, in the step S2, when cross-cutting, the hypocotyl is inclined at an angle of 50 to 60 degrees.

[0016] Furthermore, in step S3, the concentration of Agrobacterium rhizogenes solution is OD 600 Between 0.3 and 1.5.

[0017] Furthermore, in the step S3, vacuum filtration is performed at a pressure of 0.1 MPa for 2 minutes.

[0018] Furthermore, in step S3, the lighting conditions are 16 h of light, 26 degrees Celsius; 8 h of darkness, 24 degrees Celsius.

[0019] Furthermore, in the step S3, first fill 3 / 4 of vermiculite into a 500 mL plastic cup, water it with clean water until the vermiculite is completely moistened, then place the seedlings after the hypocotyl incision has been injected with the Agrobacterium rhizogenes solution and vacuum treated into the pre-moistened vermiculite, and compact it with the vermiculite on one side.

[0020] The invention establishes a method for rapid induction of transgenic hairy roots of cotton under open conditions, which does not require aseptic operation, does not require the use of culture medium, and has no cotton genotype restrictions. After the cotton hypocotyls are infected with Agrobacterium rhizogenes, they are inserted into vermiculite, and transgenic hairy roots grow from the infected parts, thereby avoiding contamination caused by incomplete sterile culture and disinfection and cumbersome aseptic operation procedures, and can also achieve the removal of non-transgenic target elements and the study of root metabolic secretions. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0022] Figure 1 Flowchart for construction of overexpression p35S:RUBY vector;

[0023] In the figure: B is BsaI; P is PaqcI; Spec R Spectinomycin hydrochloride resistance; Carb R For carbenicillin resistance; Kan R Resistant to kanamycin.

[0024] Figure 2 Procedure for inoculating cotton seedling hypocotyls with Agrobacterium rhizogenes;

[0025] In the figure: (a) Cotton seedlings eight days after sowing; (b) A scalpel is used to make a transverse cut 0.5 to 1 cm above the junction of the root and stem; (c) The Agrobacterium infection solution is injected along the hypocotyl incision using a syringe; (d) The infected cotton seedlings are inserted into moist vermiculite; (e) A moisturizing cover is used to keep the cotton seedlings in the seedling pot moist.

[0026] Figure 3 To identify the phenotype and hairy roots of cotton seedlings infected with K599 and K599-RUBY by PCR;

[0027] In the figure: (a) Phenotype of cotton seedling hypocotyls 35 days after infection with Agrobacterium rhizogenes; (b) PCR detection of hairy roots induced by K599 and K599-RUBY. M: DL5000 DNA Marker, P: pRUBY plasmid

[0028] Figure 4 The induction rates of transgenic hairy roots infecting fluids with different concentrations of Agrobacterium (n=30, different letters indicate significant differences using One-way ANOVA statistical analysis, P<0.05).

[0029] Figure 5 The induction rates of transgenic hairy roots in different explants (n=30, different letters indicate significant differences by One-way ANOVA statistical analysis, P<0.05).

[0030] Figure 6 The induction rates of transgenic hairy roots at different vacuum filtration times (n=30, different letters indicate significant differences using One-way ANOVA statistical analysis, P<0.05).

[0031] Figure 7 The necrotic phenotype of the infected part of the plant was obtained by vacuum filtration for 4 minutes.

[0032] Figure 8 The statistical results of cotton hairy root phenotype and hairy root transformation rate after 35 days of infection with different Agrobacterium rhizogenes strains;

[0033] In the figure: (a) phenotype of cotton hairy roots 35 days after infection with different rhizobacterium; (b) induction rate of transgenic hairy roots with different rhizobacterium (n=30, different letters indicate significant differences by One-way ANOVA statistical analysis, P<0.05).

[0034] Fig. 9 The statistical results of hairy root plant phenotype and hairy root conversion rate induced by different cotton varieties are shown in Figure 2.

[0035] In the figure: (a) Phenotypes of different cotton varieties at 35 days of infection; (b) Induction rate of transgenic hairy roots of different cotton varieties (n=30, different letters indicate significant differences by One-way ANOVA statistical analysis, P<0.05).

[0036] Fig.10 Flowchart for constructing the p35S:RolABC vector;

[0037] In the figure: B is BsaI; P is PaqcI; Spec R Spectinomycin hydrochloride resistance; Carb R For carbenicillin resistance; Kan R Resistant to kanamycin.

[0038] Fig.11 This is the fluorescent phenotype of cotton hairy roots 35 days after Agrobacterium infection.

[0039] Fig.12 For PCR identification of hairy roots and statistics of induction rate;

[0040] In the figure: (a) PCR identification of RolB gene in hairy roots; M: DL5000 DNA Marker, P: Plasmid; (b) Statistical results of hairy root induction rate (n=30, *P<0.05, t-test).

[0041] Fig.13 Enriching transgenic hairy roots of cotton for hydroponics;

[0042] In the figure: (a) Schematic diagram of hydroponic enrichment of transgenic hairy roots; (b) Phenotypic diagram of hydroponic cotton roots 35 days after pruning.

[0043] Fig.14 This is a diagram of the TRV-delivered Cre-mediated deletion pattern of the Rol gene in hairy roots.

[0044] Fig.15 Phenotype of cotton 30 days after inoculation with pTRV-Cre.

[0045] Fig.16 To test the efficiency of RolABC deletion in the hairy root genome;

[0046] In the figure: (a) PCR identification of the deletion efficiency of RolABC in hairy roots. M: DL5000 DNA Marker, P: Plasmid, 1: RolABC-transfected hairy roots, 2-4: recombinant hairy roots; (b) RT-PCR detection of the expression levels of TRV and RUBY in hairy roots.

[0047] Fig.17 This is the sequencing result after RolABC was deleted in hairy roots.

[0048] Fig.18 It is the overall flow chart of the present invention. DETAILED DESCRIPTION

[0049] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0050] Example 1

[0051] Establishment of a method for inducing transgenic hairy roots in cotton using Agrobacterium rhizogenes

[0052] (1) Culture medium preparation

[0053] 1L TY liquid medium: 5g peptone, 3g yeast powder, adjust pH to 7.0-7.4. When using, add 10mL sterilized 1M calcium chloride solution.

[0054] 1L of TY solid medium: 5g peptone, 3g yeast powder, 15g agar powder, adjust the pH to 7.0-7.4.

[0055] (2) Construction of RUBY overexpression vector

[0056] The present invention constructs a 35S promoter-driven RUBY gene overexpression vector based on the modular cloning system. Figure 1 As shown: the 35S promoter level 0 module pJC556, the RUBY level 0 module pCO920, and the 35S terminator level 0 module pCO576 in the modular cloning library were connected to the level 1 vector pJC267 according to the modular cloning reaction system. The plasmid with correct sequencing was saved, and the first-level module of 35S:RUBY:35Ster was obtained, which was named pHL001. Next, pHL001 was connected to the secondary backbone vector pJC337, and the plasmid with correct sequencing was saved and named pHL002.

[0057] (3) Transformation of Agrobacterium competent cells

[0058] Take the competent Agrobacterium stored at -80℃ and melt it on ice. Add 1μg pHL001 plasmid to 100μL K599 competent cells and mix them by gently pipetting. Then, place the mixture on ice for 5 minutes, freeze it in liquid nitrogen for 5 minutes, heat it in a 37℃ water bath for 5 minutes, and finally put it back on ice for 5 minutes. Add 700μL of antibiotic-free TY liquid culture medium and shake and culture it at 200rpm in a 28℃ shaker for 2h. Collect Agrobacterium by centrifugation at 5000rpm for 1 minute, and retain about 100μL of supernatant. Then gently blow and resuspend the bacterial block, spread it on TY medium containing 50mg / L kanamycin and streptomycin, and place it in an incubator at 28℃ for inverted culture for 2 to 3 days.

[0059] (4) Preparation of Agrobacterium infection solution

[0060] Pick a single clone of Agrobacterium rhizogenes K599 transformed with pHL002, inoculate it into TY liquid medium (about 5 mL of liquid medium in the test tube) containing 50 mg / L kanamycin and streptomycin, and then culture it in a shaker at 28°C and 200 rpm for 24 hours. Add 50 mL of TY medium to a 250 mL sterilized bacterial conical flask, draw 1 mL of the above Agrobacterium bacterial solution (according to a ratio of 1:50) and add it to the conical flask, place the conical flask in a shaker at 28°C and 200 rpm for 8 to 10 hours, until OD600 = 1.2 to 1.6. Pipette the Agrobacterium bacterial solution into a sterilized 50 mL centrifuge tube, centrifuge at 5000 rpm for 10 minutes at room temperature to collect the bacteria. Discard the supernatant, add 20 mL of Agrobacterium buffer, vortex mix, and centrifuge at 5000 rpm for 10 minutes at room temperature to collect the bacteria. Repeat twice. Add 1 mL of Agrobacterium buffer and vortex mix. Pipette an appropriate amount of the resuspension, prepare 50 mL of a resuspension with an OD600 of 0.7, and add 50 μL of 100 μmol / L acetosyringone. Finally, place the resuspension in a 28°C incubator for 2 hours away from light. After standing, the resuspension is used for subsequent transformation or infection experiments.

[0061] (5) Explant preparation

[0062] After removing the surface fluff from cotton seeds with concentrated sulfuric acid, rinse with running water for 20 minutes, transfer to a glass culture dish, add clean water until the seeds are submerged, and culture in a 37°C incubator for one day until they turn white. Fill 3 / 4 of vermiculite into a 500mL plastic cup, water it with clean water until the vermiculite is completely wet, sow the white cotton seeds in the moist vermiculite, and grow them in a light culture room (16h light, 8h dark) at a temperature of 26°C and a relative air humidity of 65-75%.

[0063] (6) Infection of explants

[0064] like Figure 2 As shown, eight-day-old cotton seedlings were selected, cut 0.5-1 cm above the junction of the rhizome, and the underground part was removed. The Agrobacterium resuspension was sucked with a syringe and injected from the phloem of the hypocotyl incision. 3 / 4 moist vermiculite was filled into a 500mL plastic cup, and the injected seedlings were inserted into the moist vermiculite and placed in a seedling tray. An appropriate amount of clean water was added to the seedling tray, and a moisturizing cover was covered. After 24 hours of dark treatment, it was placed on a culture rack for light culture (26°C, 16 hours of light, 8 hours of darkness).

[0065] (7) Statistical analysis of transgenic hairy root induction rate

[0066] The number of transgenic hairy root explants under different treatments was counted, and the induction rate of transgenic hairy roots was analyzed. Excel was used for data processing, and SPSS and GraphPad Prism were used for difference significance analysis and statistical graph preparation.

[0067] Transgenic hairy root induction rate (%) = number of explants producing transgenic hairy roots / total number of infected explants × 100.

[0068] (8) Identification of transgenic hairy roots

[0069] Observe the root RUBY signal 35 days after infection. Figure 3 As shown in a, cotton plants infected with K599-RUBY for 35 days have red roots, while cotton plants infected with K599 do not produce red roots. To further verify the transgenic event, the DNA of white and red roots infected with K599 and K599-RUBY was extracted using the CTAB method. Primers RUBY-F1 and RUBY-R1 were designed based on the RUBY gene sequence (primer sequences are shown in Appendix A), and the amplified length was 1435 bp. Figure 3 As shown in b, a band of 1435 bp was amplified in the genomic DNA of the red hairy roots of three randomly selected cotton plants, and no band was amplified in the genomic DNA of the hairy roots of three randomly selected cotton plants infected with K599. The above results show that the RUBY gene has been integrated into the genome of cotton hairy roots and can be stably expressed to promote the accumulation of betalain and produce red hairy roots.

[0070] (9) Effect of Agrobacterium infection solution concentration on the induction rate of transgenic hairy roots

[0071] This example selected six different concentrations of Agrobacterium infection solutions, namely OD 600 0.3, 0.5, 0.7, 0.9, 1.2, 1.5. Figure 4As shown in the figure, the concentration of Agrobacterium infection solution has a significant effect on the induction rate of transgenic hairy roots. With the increase of the concentration of Agrobacterium infection solution, the induction rate shows an upward trend, and the highest induction rate is 23.3% when OD600=0.7. Then, with the increase of infection solution concentration, the induction rate shows a downward trend, and the lowest induction rate of transgenic hairy roots is 6.6% when OD600 is 1.5. The results show that the concentration of Agrobacterium infection solution OD 600 =0.7, the induction rate of transgenic hairy roots was the highest, and the optimal Agrobacterium infection solution concentration OD600 was 0.7.

[0072] (10) Effects of different explants on the induction rate of transgenic hairy roots

[0073] As the number of growing days increases, the degree of lignification of the hypocotyls of cotton seedlings will gradually deepen. The induction rate of transgenic hairy roots is significantly different when cotton seedling explants with different growing days are selected. In order to compare the effect of explants with different growing days on the induction rate of transgenic hairy roots, cotton seedling explants 6, 8, and 10 days after sowing were selected for infection comparison. Figure 5 As shown, the induction rates of transgenic hairy roots in the hypocotyls of 6-day-old, 8-day-old and 10-day-old cotton seedlings were 13.3%, 26.7% and 14.5%, respectively. The induction rate of 8-day-old cotton seedling hypocotyls as explants was significantly higher than that of 6-day-old and 10-day-old cotton seedlings. The results showed that more transgenic hairy roots could be obtained by selecting 8-day-old cotton seedling hypocotyls as explants.

[0074] (11) Effect of vacuum filtration time on the induction rate of transgenic hairy roots

[0075] In this embodiment, different gradients of vacuum treatment time are set at a pressure of 0.1 MPa, namely, vacuum treatment time of 0 min, 1 min, 2 min, 3 min and 4 min. When other conditions are kept the same, the induction rate of transgenic hairy roots is observed and counted with the vacuum treatment time as a variable.

[0076] like Figure 6 As shown in the figure, compared with not using vacuum filtration (vacuum filtration 0min), using vacuum filtration for 1min, 2min, and 3min can significantly increase the transgenic hairy root induction rate. When the vacuum filtration treatment time is 2min, the transgenic hairy root induction rate is as high as 33.5%. The transgenic hairy root induction rate of vacuum filtration for 4min is only 21.6%. If the filtration time is too long, the transformed cells will be severely damaged, and the infected parts of the plants will be necrotic, resulting in a decrease in the transgenic hairy root induction rate ( Figure 7 The above results show that the highest transgenic hairy root induction rate can be obtained when vacuum filtration is carried out for 2 minutes.

[0077] (12) Effects of different Agrobacterium rhizogenes strains on the induction rate of transgenic hairy roots

[0078] In this example, the Agrobacterium rhizogenes strains were optimized. The selected Agrobacterium rhizogenes strains were: C58C1, Ar 1193, K599, MSU440. The hairy root induction method established in this example was used for infection, and the results were as follows: Figure 8 As shown in a, four types of Agrobacterium rhizogenes can induce transgenic hairy roots to grow from the incision of cotton hypocotyls. Statistical analysis was performed on the induction rate of transgenic hairy roots of cotton hypocotyls infected with different Agrobacterium rhizogenes ( Figure 8 b), the results showed that K599 had the highest induction rate of transgenic hairy roots in cotton hypocotyl incisions, with an induction rate of 31.7%, significantly higher than that of several other rhizogenes. C58C1 had the lowest induction rate of transgenic hairy roots in cotton hypocotyl incisions, at 8.3%. The results of this example showed that K599 had the highest induction rate of transgenic hairy roots in cotton hypocotyl incisions of eight-day-old seedlings.

[0079] (13) Effects of different cotton varieties on the induction rate of transgenic hairy roots

[0080] In this example, five cotton varieties with different genotypes were selected to explore whether the cotton genotype affects the hairy root induction method. The selected cotton varieties are: R15, Zhongmian 24, TM-1, Jinkemian 11, and Hanza 1692. Fig. 9 As shown in a, all five cotton varieties induced transgenic hairy roots, and the results showed that the hairy root induction method established in this example is not limited by cotton genotype. Next, the induction rate of transgenic hairy roots of different varieties was statistically analyzed. The results are shown in Fig. 9 As shown in b, there was no significant difference in the induction rate of transgenic hairy roots among R15, Zhongmian 24, TM-1 and Handanza 1692. The induction rate of transgenic hairy roots of Jinkemian 11 was the lowest, at 25.5%, which was significantly lower than that of R15, Zhongmian 24 and Handanza 1692.

[0081] In summary, using the cultivated upland cotton variety Zhongmian 24 as the experimental material and RUBY as the reporter gene, the K599 root-generating Agrobacterium liquid was injected to infect the hypocotyls of cotton seedlings, and transgenic hairy roots could be successfully induced. Through the optimization of bacterial liquid concentration and explant, it was found that the concentration OD600 = 0.7, the infection of 8-day-old cotton seedling hypocotyls was the optimal infection and transformation condition, and the transgenic hairy root induction rate was 26.7%. On this basis, adding vacuum filtration (0.1Mpa, 2min) can further increase the induction rate to 33.5%. The induction transformation efficiency of four root-generating Agrobacterium was compared and tested. The results showed that the induction rate of transgenic hairy roots of the K599 strain was stably higher than 30%, which was significantly better than the other three strains. Under the optimal infection conditions, transgenic hairy roots were induced in five genotype upland varieties, and transgenic hairy roots were efficiently induced, proving that this method is suitable for the induction and transformation of hairy roots of different genotypes of cotton.

[0082] Example 2

[0083] Establishment of a method for transgenic cotton hairy roots mediated by Agrobacterium tumefaciens

[0084] (1) Culture medium preparation

[0085] 1L LB liquid medium: 5.0g yeast powder, 10.0g tryptone and 10.0g NaCl, adjust the pH to 7.0-7.4.

[0086] 1L LB solid medium: 5.0g yeast powder, 10.0g tryptone, 10.0g NaCl, 15g agar powder, adjust the pH to 7.0-7.4.

[0087] (2) Construction of Cre / loxP system RolABC vector and pTRV2-Cre vector

[0088] Cre / loxP is one of the most widely used site-specific recombinase systems, which consists of a 38kD recombinase Cre and two 34bp recognition sequences loxP. On a DNA molecule chain, when the two loxP sites are in the same direction, the Cre recombinase can efficiently delete the sequence between them and generate a new loxP site. RolABC is the main gene for inducing hairy roots by Agrobacterium rhizogenes. First, download the RolABC gene and its promoter sequence and the Cre recombinase gene sequence from NCBI (https: / / www.ncbi.nlm.nih.gov / ), and design the corresponding primers to amplify the sequence according to the sequence information (primers are shown in Appendix A). In this example, the full length of the RolABC gene and its promoter sequence were obtained by PCR amplification.

[0089] like Fig.10The lox71 and lox66 primers were annealed to synthesize double strands, and then the 35S promoter 0-level module pJC556 and double-stranded lox71 were connected to the 1-level vector pJC268 according to the Modular cloning reaction system, and the recombinant vector with correct sequencing was named pHL015. The RolABC and its promoter PCR products were connected to the 1-level vector backbone pJC270 according to the Modular cloning reaction system, and the recombinant vector with correct sequencing was named pHL016. The double-stranded lox66, RUBY 0-level module pCO920, and 35S terminator 0-level module pCO576 were connected to the 1-level vector pJC272 according to the Modular cloning reaction system, and the recombinant vector with correct sequencing was named pHL017. Then the first-level vector modules pHL015, pHL016, pHL017 and pHL018 (CmYLCV:AmCyan:Ter) were connected to the secondary receptor backbone pJC337 according to the Modular cloning reaction system, and then the ligation products were transferred into DH5α competent cells, and single clones were selected for sequencing. The correctly sequenced plasmid was named pHL019. pHL019 was transferred into EHA105 and GV3101 competent cells.

[0090] (3) Establishment of a method for inducing transgenic hairy roots using Agrobacterium tumefaciens

[0091] In order to explore whether the transformation of RolABC by Agrobacterium tumefaciens can induce the formation of transgenic hairy roots in cotton, pRolABC-AmCyan was transferred into EHA105 and GV3101, respectively, and the hypocotyls of 8-day-old cotton seedlings were infected by the transgenic hairy root induction method optimized in Example 1. Fig.11 As shown, 35 days after inoculation, a handheld fluorescence device was used to observe that both EHA105 and GV3101 induced fluorescent hairy roots after the introduction of RolABC. A pair of primers was designed based on the RolB sequence information (primers are shown in Appendix A). The length of the amplified fragment was 690 bp, and PCR was performed using the extracted root DNA as a template. Fig.12 As shown in a, a target band of 690 bp was amplified in the genomic DNA of hairy roots of EHA105 and GV3101 infected plants. The results showed that the RolABC gene was integrated into the hairy root genome, thereby inducing the transformed cells to differentiate into hairy roots. The induction rate of transgenic hairy roots was statistically analyzed. The results are shown in Fig.12As shown in Fig. 2a, the hairy root transformation rates of EHA105 and GV3101 were 13.7% and 8.3%, respectively. The induction rate of transgenic hairy roots of EHA105 strain was significantly higher than that of GV3101. The results showed that the hairy root major induction gene RolABC can make Agrobacterium tumefaciens have the ability to induce hairy roots. In this example, a method for inducing transgenic hairy roots by Agrobacterium tumefaciens was established by transferring RolABC into Agrobacterium tumefaciens.

[0092] Example 3

[0093] Hydroponic enrichment culture of transgenic hairy roots

[0094] The transgenic hairy root plants induced by Agrobacterium rhizogenes and Agrobacterium tumefaciens obtained by implementing Case 1 and Example 2 are not conducive to the observation and operation of hairy roots by soil culture, and cotton roots under soil culture conditions are prone to wilt or even die after sampling. Therefore, in this example, the plants with induced hairy roots are cultured in a hydroponic manner, and the transgenic hairy roots are transferred to 1 / 2 Hoagland nutrient solution for culture, leaving only 2 cm of roots above the water surface.

[0095] After one week of cultivation in 1 / 2 Hoagland nutrient solution, the non-transgenic hairy roots were gradually cut off by pruning every three days using scissors. The operation steps are as follows: Fig.13 a. Based on this method, the transgenic RUBY hairy roots were successfully enriched ( Fig.13 b) The results of this example show that pruning and removing non-transgenic roots in hydroponics can achieve a large amount of enrichment of transgenic hairy roots.

[0096] Example 4

[0097] Establishment of a virus-mediated recombinant gene elimination system

[0098] (1) The amplified Cre recombinase CDS sequence was ligated to the multiple cloning site of pTRV2 to obtain the pTRV2-Cre vector. pTRV1 and pTRV2-Cre were transformed into GV3101 competent cells for subsequent infection.

[0099] (2) TRV remote delivery of Cre recombinase to efficiently delete hairy root recombination genes

[0100] In the pHL016 vector constructed in this example, lox71 and lox66 are in the same direction. After Cre recombination, RolABC between lox71 and lox66 is deleted, and RolABC is inserted between the 35S promoter and RUBY. After RolABC is removed, RUBY is expressed and the hairy roots turn red. Fig.14 The method shown was to inoculate pTRV-Cre into the phloem of the RolABC-induced hairy root plants of Agrobacterium tumefaciens EHA105. Fig.14As shown, 30 days after pTRV-Cre inoculation, red hairy roots were observed. The red hairy roots had fluorescent signals observed using a handheld fluorescent device. The results showed that the Cre recombinase delivered by TRV deleted RolABC. After the deletion, RUBY was expressed, causing the hairy roots to turn red. The genomic DNA of the red hairy roots was extracted according to Fig.11 Construct the sequence of the vector, design a pair of primers that can amplify the full length of RolABC at the 35S promoter and RUBY position (primers are shown in Appendix A), and perform PCR on the extracted DNA. Fig.16 As shown in a, the RolABC gene was not amplified in the deleted hairy roots, and only the target band with a length of 481bp after deletion was amplified. The full length of the RolABC gene was amplified in the hairy roots of the plants without Cre delivery. The mobility of TRV virus to the roots and the expression of RUBY in the hairy roots were detected by RT-PCR. The results showed that 30 days after inoculation, TRV moved from the phloem to the hairy roots, and RUBY had a higher expression level in the hairy roots after RolABC was deleted ( Fig.16 b) The results showed that the Cre recombinase delivered by TRV successfully deleted the RolABC gene in the hairy roots induced by GV3101. Next, the recombinant segment after deletion was sequenced, and the results were as follows: Fig.17 As shown, only the recombined sequence was detected in the hairy roots of the three deleted strains. The results of the above examples show that TRV can systemically infect cotton, and TRV delivery of Cre recombinase can efficiently delete the Rol gene inserted in the hairy root genome, and a virus-mediated recombinant gene elimination system is established on cotton.

[0101] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. An Agrobacterium-mediated open cotton hairy root transformation method, characterized in that: The steps include: S1. Construct the vector and complete the transformation of Agrobacterium; S2. Select eight-day-old cotton seedlings, cut the hypocotyl 0.5 to 1 cm above the junction of the rhizome, and remove the underground part; S3, injecting Agrobacterium rhizogenes solution into the hypocotyl incision, vacuum filtering the hypocotyl incision, and then inserting the seedlings into moist vermiculite, keeping them moist and dark for 24 hours, and then culturing them under light for 35 days in an environment with a relative humidity of 65-75%; S4. The seedlings with induced hairy roots are cultured hydroponically. During the hydroponic process, the wild-type roots are gradually removed by continuous pruning, so that the transgenic hairy roots are enriched in large quantities.

2. The Agrobacterium-mediated open cotton hairy root transformation method according to claim 1, characterized in that: In the step S1, the vector is an overexpression vector of the RUBY gene, and the Agrobacterium is Agrobacterium rhizogenes K599.

3. The Agrobacterium-mediated open cotton hairy root transformation method according to claim 2, characterized in that: When constructing the overexpression vector of the RUBY gene, a primary module of p35S:RUBY is first constructed by a modular cloning method and named pHL001; pHL001 is then connected to a secondary backbone vector, the correctly sequenced plasmid is saved and named pHL002, and pHL002 is transferred into Agrobacterium rhizogenes C58C1, Ar 1193, K599, and MSU440 competent cells for subsequent infection.

4. The Agrobacterium-mediated open cotton hairy root transformation method according to claim 1, characterized in that: In the step S1, the vector is a p35S:RolABC-AmCyan vector, and the Agrobacterium is Agrobacterium tumefaciens EHA105 and GV3101.

5. The Agrobacterium-mediated open cotton hairy root transformation method according to claim 4, characterized in that: When constructing the p35S:RolABC-AmCyan vector, firstly, lox71 and lox66 primers are annealed to synthesize double chains, a primary module plasmid containing 35S promoter and lox71 is constructed, and the correctly sequenced recombinant vector is named pHL015; RolABC and its promoter PCR products are connected to the first-level vector backbone according to the Modular cloning reaction system, and the correctly sequenced recombinant vector is named pHL016; double-stranded lox66, RUBY 0-level module plasmid, and 35S terminator 0-level module plasmid are connected to the first-level vector backbone according to the Modular cloning reaction system, and the correctly sequenced recombinant vector is named pHL017; then the first-level vector modules pHL015, pHL016, pHL017 and pHL018 (CmYLCV:AmCyan:Ter) are connected to the secondary receptor backbone according to the Modular cloning reaction system, and then the connection products are transferred into DH5α competent cells, and single clones are picked for sequencing; The correctly sequenced plasmid was named pHL019; pHL019 was transformed into EHA105, GV3101 and K599 competent cells, and pTRV1 and pTRV2-Cre were transformed into GV3101 competent cells for subsequent infection.

6. The Agrobacterium-mediated open cotton hairy root transformation method according to claim 1, characterized in that: In the step S2, when cross-cutting, the hypocotyl is inclined at an angle of 50 to 60 degrees.

7. The Agrobacterium-mediated open cotton hairy root transformation method according to claim 1, characterized in that: In step S3, the concentration of Agrobacterium rhizogenes solution is OD 600 Between 0.3 and 1.

5.

8. The Agrobacterium-mediated open cotton hairy root transformation method according to claim 1, characterized in that: In the step S3, vacuum filtration is performed at a pressure of 0.1 MPa for 2 minutes.

9. The Agrobacterium-mediated open cotton hairy root transformation method according to claim 1, characterized in that: In step S3, the lighting conditions are 16 h of light, 26 degrees Celsius; 8 h of darkness, 24 degrees Celsius.

10. The Agrobacterium-mediated open cotton hairy root transformation method according to claim 1, characterized in that: In the step S3, first fill 3 / 4 of vermiculite into a 500 mL plastic cup, water it with clean water until the vermiculite is completely moistened, then insert the seedlings after injecting the rhizogenes solution into the hypocotyl incision and vacuum treating it into the pre-moistened vermiculite, and compact it with the vermiculite on one side.

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