An efficient Agrobacterium-mediated genetic transformation method for ophiopogon aviculare

Abstract

The present invention discloses an efficient Agrobacterium-mediated genetic transformation method for lycopersicum, which belongs to the technical field of plant genetic engineering. The method mainly includes: 1) activation culture of Agrobacterium; 2) seed disinfection and material selection; 3) induction of callus tissue; 4) preparation of infection solution: Agrobacterium is cultured in YEB liquid culture medium, added to MS liquid culture medium, and the bacterial solution is adjusted to OD 600 =0.3-0.9, then add 100μmol / L acetosyringone and 100μmol / L MgCl2, shake well; 5) infection; 6) co-cultivation; 7) washing and recovery culture; 8) screening differentiation and rooting induction; 9) plant regeneration and identification. Through the above steps, the genetic transformation system of ophiopogon can be successfully constructed, and the transformation rate of positive plants obtained by transformation is as high as 76.19%. The present invention has important significance and value for the rapid creation of transgenic ophiopogon germplasm.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, in particular to an efficient Agrobacterium-mediated genetic transformation method of sphaerocarpus ovali. Background Art

[0002] Flying grass is widely distributed in tropical and subtropical regions. It contains rich chemical components such as terpenes, flavonoids and alkaloids. It has pharmacological effects such as anti-allergy, anti-anxiety, anti-inflammatory, sedative and analgesic, anti-malice, antioxidant and anti-cancer. It is an important medicinal plant and was included in the "Pharmacopoeia of the People's Republic of China" in 2010. At present, the research related to Flying Grass mainly includes the identification of basic biological characteristics, extraction of chemical components and analysis of pharmacological activity. Cultivating high-yield, high-quality and resistant Flying Grass varieties is an urgent problem to be solved in the current Flying Grass research. However, the germplasm resources of Flying Grass are scarce and the breeding process is slow. At present, the use of genetic engineering technology to obtain new plant varieties is one of the most efficient breeding methods. However, there are no reports on the transgenic technology of Flying Grass at home and abroad, and no articles on the transgenic research of Flying Grass have been published.

[0003] Plant genetic transformation methods can be divided into three categories according to different implementation schemes. The first category is the vector-mediated transformation system, which is to connect the target gene to the vector plasmid and integrate the target gene into the plant genome with the help of the movement of the plasmid. Agrobacterium is commonly used as a medium. Agrobacterium is a type of Gram-negative bacteria commonly found in the soil. It mainly attaches to the surface of plant roots and relies on the nutrients of plant roots to survive. A plasmid in its body is called Ti plasmid. The T-DNA on it can be integrated into the plant genome and expressed. Due to this feature, it is widely used as a genetic transformation medium. The second method does not use any vector, but uses physical or chemical methods to directly introduce exogenous genes into recipient cells to complete gene transfer, mainly including gene gun method and electroporation transformation method. The gene gun method is a method that uses the power generated by compressed air to deliver gold powder with exogenous genes into the cell nucleus to achieve the transfer of exogenous genes. The advantage of this method is that it has a wide range of receptors. Materials such as callus tissue, suspension cells, and immature embryos can be transformed using this method. However, due to the particularity of the method, the cost is high and the transformation efficiency is low. The third category is to use the plant's own reproductive cells as a medium for genetic modification, including reproductive cell immersion method, embryo sac ovary injection, pollen tube channel method, etc. The most common of these is the pollen tube channel method, which uses the pollen tube channel produced when the plant is pollinated to inject exogenous DNA into the receptor cell and complete the gene transformation with the help of the development of the fertilized egg. Its advantages are simple operation and no reliance on tissue culture technology. The disadvantages are large workload and low transformation efficiency. Different genetic transformation methods have different advantages and disadvantages. To achieve the ideal transformation effect, it is necessary to choose the appropriate method according to the purpose of the experiment and the differences in materials. At present, Agrobacterium-mediated genetic transformation has the advantages of not affecting the expression of foreign genes by the integration position, not interfering with the stability of the host genome, stable transformation efficiency, low cost, and simple operation. As people's understanding of the mechanism of Agrobacterium-mediated transformation deepens, the transformation method is constantly being improved. However, for the important Chinese medicinal material, a complete and efficient Agrobacterium-mediated transgenic method has not yet been established. Summary of the invention

[0004] The purpose of the present invention is to provide an efficient Agrobacterium-mediated genetic transformation method for sphagnum fasciatum to solve the problems existing in the above-mentioned prior art. By screening and optimizing conditions such as the concentration of the infection solution, the infection time, the co-cultivation time, and the resistance screening of the callus tissue after the transformation during the genetic transformation process, a genetic transformation system for sphagnum fasciatum was successfully constructed, which effectively solves the problem of the imperfect genetic transformation system of sphagnum mediated by sphagnum fasciatum in the prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an efficient Agrobacterium-mediated genetic transformation method of sphaerocarpus aviculare, comprising the following steps:

[0007] The leaves of sterile spatholobiaceae seedlings are used as explants and inoculated into a callus induction medium for callus induction; the callus induction medium comprises the following components: MS liquid medium + naphthaleneacetic acid + 6-benzylaminopurine + sucrose + agar;

[0008] The induced callus tissue is inoculated with Agrobacterium inoculation solution. After the inoculation is completed, the inoculation solution is discarded and the callus tissue is transferred to a co-culture medium for culture; the co-culture medium comprises the following components: MS liquid culture medium + sucrose + agar + naphthaleneacetic acid + 6-benzylaminopurine + acetosyringone + timentin;

[0009] After the co-culture, the callus tissue is transferred to a recovery culture medium for recovery culture, and then screened and differentiated through a differentiation culture medium, and rooting induced through a rooting culture medium, and transplanted after rooting to obtain flying grass seedlings; the recovery culture medium comprises the following components: MS liquid culture medium + sucrose + agar + naphthaleneacetic acid + 6-benzylaminopurine + timentin; the differentiation culture medium comprises the following components: MS liquid culture medium + sucrose + agar powder + naphthaleneacetic acid + 6-benzylaminopurine + acetosyringone + timentin + kanamycin; the rooting culture medium comprises the following components: MS liquid culture medium + indolebutyric acid + sucrose + agar + potato.

[0010] Preferably, the callus induction medium comprises the following components: MS liquid medium + 0.5 mg / L naphthaleneacetic acid + 1.5 mg / L 6-benzylaminopurine + 30 g / L sucrose + 6 g / L agar;

[0011] The callus induction conditions are: 28° C. in dark culture.

[0012] Preferably, the Agrobacterium infection solution is obtained by centrifuging the fermentation solution of the recombinant Agrobacterium and then resuspending the cells in MS liquid medium to an OD of 600 The pH value is 0.3-0.9, and 100 μmol / L acetosyringone and 100 μmol / L magnesium chloride are added to obtain the result.

[0013] Preferably, the recombinant Agrobacterium is obtained by transferring the HbCBF1 gene of rubber tree to an expression vector to construct a recombinant vector, and then transforming the recombinant vector into Agrobacterium; the nucleotide sequence of the HbCBF1 gene is shown in SEQ ID NO:5.

[0014] Preferably, the soaking time is 20 min.

[0015] Preferably, the co-culture medium comprises components of the following concentrations: MS liquid medium + 30 g / L sucrose + 8 g / L agar + 0.5 mg / L naphthaleneacetic acid + 2 mg / L 6-benzylaminopurine + 100 µM / L acetosyringone + 300 mg / L timentin;

[0016] The co-cultivation condition is dark cultivation at 22°C for 3 days.

[0017] Preferably, the recovery medium comprises components of the following concentrations: MS liquid medium + 30 g / L sucrose + 8 g / L agar + 0.5 mg / L naphthaleneacetic acid + 2 mg / L 6-benzylaminopurine + 300 mg / L timentin;

[0018] The differentiation medium includes components of the following concentrations: MS liquid medium + 30 g / L sucrose + 0.8 g / L agar powder + 0.5 mg / L naphthaleneacetic acid + 2 mg / L 6-benzylaminopurine + 100 µM / L acetosyringone + 300 mg / L timentin + 150 mg / L kanamycin;

[0019] The rooting medium includes components with the following concentrations: MS liquid medium+1.5 mg / L indolebutyric acid+30 g / L sucrose+3.5 g / L agar+20 g / L potato.

[0020] Preferably, after culturing on the recovery medium under a weak light of 5000 lux for 7 days, the cells are transferred to the differentiation medium for culturing, and after 14 days, they are transferred to the rooting medium for rooting induction.

[0021] Preferably, when the rooting is induced, the seedlings grow to 5-10 cm, are moved into vermiculite for indoor culture for 7 days, and then are moved outdoors to be cultured into adult flying grass seedlings; the vermiculite is soaked with 1 / 2 MS solution before use, and the vermiculite is irrigated with tap water during the culture process, and the 1 / 2 MS solution includes the following concentration components: 1 / 2 MS liquid culture medium + 30 g / L sucrose.

[0022] Preferably, the sterile spatholobi seedlings are obtained by aseptically inoculating large spatholobi seeds into MS solid culture medium.

[0023] The present invention discloses the following technical effects:

[0024] The present invention uses the callus of the herbaceous plant as a transformation receptor, and successfully constructs an efficient Agrobacterium-mediated genetic transformation method of the herbaceous plant by screening and optimizing the key factors affecting the transformation efficiency, such as the infection concentration of the bacterial solution, the infection time, and the co-cultivation time, and screening the resistance concentration of the transformed callus, and successfully obtains the transformed herbaceous plant seedlings, which changes the current situation of obtaining herbaceous plant without genetic engineering technology, and achieves a breakthrough from zero to one. The present invention provides sufficient experimental basis for the establishment of a genetic transformation system of herbaceous plants of the genus Euphorbia of the Euphorbiaceae family, and has important significance and value for the rapid creation of transgenic herbaceous plant germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 It is the electrophoresis diagram of PCR amplification of HbCBF1 gene; M is the standard DNA molecule, 1-4 are samples;

[0027] Figure 2 This is the map of HbCBF1 recombinant plasmid vector;

[0028] Figure 3 This is a graph showing the effect of different concentrations of antibiotics (kanamycin) on the browning rate of callus tissue;

[0029] Figure 4 This is a graph showing the effect of different concentrations of Agrobacterium GV3101 on callus transformation efficiency;

[0030] Figure 5 This is a graph showing the effect of different infection times of Agrobacterium GV3101 on callus transformation efficiency;

[0031] Figure 6 The figure shows the effect of different co-cultivation time on callus transformation efficiency;

[0032] Figure 7 These are the GUS staining identification pictures of some transgenic T0 plants of HbCBF1; the first row is GUS staining negative, and the second row is GUS staining positive;

[0033] Figure 8 This is the electrophoresis diagram of PCR identification of some transgenic T0 plants of HbCBF1; lanes 1-3 are 2k Marker, wild-type Sphagnum sphagnum samples and negative control (ddH2O), and lanes 4-18 are 1-15 Sphagnum sphagnum T0 plant samples. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Example 1

[0040] An efficient Agrobacterium-mediated genetic transformation method for sphaerocarp, comprising the following steps:

[0041] 1) Activation culture of Agrobacterium

[0042] RNA extracted from rubber trees was reverse transcribed into cDNA, which was used as a template to PCR amplify HbCBF1 in rubber trees using specific primers (shown in SEQ ID NO.1 and SEQ ID NO.2). The results are shown in Figure 1As shown in FIG. 5 , the amplified fragment size of HbCBF1 is 696 bp (SEQ ID NO. 5). The pCAMBIA2301-HbCBF1 vector was constructed by homologous recombination. Figure 2 As shown, the size of the recombinant plasmid vector is about 13664 bp; the correctly sequenced recombinant was transformed into Agrobacterium GV3101 (pSoup-p19), inoculated on YEB solid medium, and cultured at 28°C for three days. On the transformation plate, a single clone was selected for PCR identification and inoculated into 2 mL YEB liquid medium, and activated at 28°C and 200 r / min to OD 600 =0.7, aspirate the culture medium and place it in new YEB liquid medium, dilute it according to the ratio of bacterial liquid to YEB liquid = 1:20, place it in a 200mL conical flask, and expand the culture under the above conditions until OD 600 =0.8, introduced into a 50 mL centrifuge tube, centrifuged at 5000 g / min, and the bacteria were collected; wherein the liquid YEB medium included: 0.5% beef extract, 0.1% yeast extract powder, 0.5% peptone, 0.5% sucrose, 0.4% magnesium sulfate, a final concentration of 20 mg / L rifampicin, and 10 mg / L, 30 mg / L, 50 mg / L, 100 mg / L, 150 mg / L or 300 mg / L kanamycin.

[0043] PCR identification system: Primer-F 0.5 μL, Primer-R 0.5 μL, cDNA 1 μL (200 ng / μL), 2× Rapid Taq Master Mix 25 μL, ddH2O 23 μL. Program: 98 ℃ / 5 min, 98 ℃ / 30 s, 55 ℃ / 10 s, 72 ℃ / 1 min (To 2 Steps, 35 cycles in total), 72 ℃ / 1 min, stored at 4 ℃.

[0044] 2) Seed disinfection and material selection

[0045] Take 30 to 50 wild-type large flying grass seeds that have been dried and reserved, remove surface impurities, place them in a 1.5 mL centrifuge tube, add 50% chlorine-containing disinfectant, and disinfect for 5 min. Then, pour out the disinfectant in the clean bench, wash the disinfected seeds with sterile water 5 to 7 times until the water is clean and transparent. Finally, use a micropipette to sow the seeds on MS culture medium, place them in a constant temperature and light incubator at 28°C and a light intensity of 10,000 lx (16 h light / 8 h dark), and the seeds in a healthy state will germinate within 3 to 7 days. After the sterile seedlings grow for a period of time, take their leaves and cut them into about 0.5 cm × 0.5 cm in size with a sterilized scalpel in the clean bench for subsequent infection.

[0046] 3) Callus induction

[0047] The leaves cut in step 2) were inoculated into callus induction medium (MS liquid medium + 0.5 mg / L naphthaleneacetic acid + 1.5 mg / L 6-benzylaminopurine (6-BA) + 30 g / L sucrose + 6 g / L agar) for callus induction. Ten explants were inoculated per dish and cultured in a constant temperature and light incubator under dark conditions at 28°C. During the culture period, the callus induction was observed every 7 days, and subculture was performed every 14 days. The kanamycin concentration was set to 10, 30, 50, 100, 150 and 300 mg / L, respectively. In the clean bench, the vigorously growing calli that had been cultured on the callus induction medium for about 14 days were inoculated into the MS medium containing kanamycin. The growth and development of the calli were observed every 7 days, and subculture was performed every 14 days. After 30 days of culture, the growth and development of the calli and the browning mortality rate were counted.

[0048] The results are as follows Figure 3 As shown in the figure: when the kanamycin concentration was 10 mg / L, the browning rate was only 7%. As the kanamycin concentration increased, the browning rate of callus also increased. When the concentration reached 150 mg / L, the browning rate reached 100%. In order to achieve the best screening effect and save costs, the critical value of the browning rate of 150 mg / L was selected as the subsequent screening concentration of transgenic callus.

[0049] 4) Preparation of infection solution

[0050] The cells obtained in step 1) were centrifuged at 5000 r / min and 4°C for 10 min, the supernatant was discarded, and the cells were resuspended in MS liquid medium to adjust the OD 600 To 0.3, 0.6, and 0.9, add 100 μmol / L acetosyringone and 100 μmol / L magnesium chloride, shake well, and let stand in the dark for 1 h to complete the preparation of the infection solution.

[0051] 5) Infection

[0052] The 0.5×0.5 cm small leaf pieces prepared in step 2) were placed in the Agrobacterium infection solution prepared in step 4) and soaked for 20 min at 30°C and 100 r / min to ensure uniform infection. In order to explore the effect of bacterial solution concentration on the transformation effect of large flying grass callus, the present invention set up three different concentrations of Agrobacterium for treatment experiments as in step 4). The results are shown in Figure 2. Figure 4 Shown: Agrobacterium concentration OD 600 = 0.6 treatment group had the highest transformation efficiency and GUS expression, and most of the positive calli were stained dark blue. 600 = 0.3 and OD 600 =0.9 treatment group had a lower transformation efficiency, OD 600 =0.3 treatment group had a smaller number of GUS-positive calli, OD 600 =0.9 During the culture process of the treatment group, Agrobacterium over-reproduced and contaminated the culture medium, and the callus turned brown and died, resulting in a small number of surviving calli. 600 =0.6 is the optimal concentration for infection;

[0053] At the same time, the infection time of Agrobacterium is also one of the key factors affecting the transformation effect. The infection time must be long enough to allow the callus tissue to fully contact with the bacterial solution. If the time is too short, the infection is not sufficient, and the transformation efficiency of the callus is low. If the infection time is too long, the callus tissue will die. The experiment on the effect of infection time on transformation efficiency was set up with three treatments (10 min, 20 min and 30 min). The results are as follows Figure 5 As shown, select OD 600 =0.6, the genetic transformation efficiencies of the three treatments were similar, all between 30% and 40%. After one-way ANOVA, the transformation efficiency was significantly highest when the infection time was 20 min, thus confirming that 20 min was the optimal Agrobacterium treatment time.

[0054] 6) Co-culture

[0055] After the infection is completed, pour out the infection liquid, absorb the excess bacterial liquid on the surface of the material with sterile filter paper, and place it on the co-culture medium for dark culture at 22°C; the co-culture medium is: MS liquid medium + 30 g / L sucrose + 8 g / L agar powder + 0.5 mg / L naphthaleneacetic acid + 2 mg / L 6-benzylaminopurine + 100 µM / L acetosyringone + 300 mg / L timentin, pH = 5.8.

[0056] Agrobacterium needs to be in contact with plant tissue for a period of time to achieve the introduction and integration of foreign genes, so the infected callus needs to be placed in dark conditions for co-culture with Agrobacterium. This time is generally not less than 16 hours, but it cannot last too long. Too long co-culture time will cause Agrobacterium to over-reproduce and cause plant tissue death. Therefore, choosing the right co-culture time is one of the important factors affecting transformation efficiency. The present invention sets three co-culture times (2d, 3d, 4d) for experiments. The results are as follows Figure 6 As shown in the figure, the transformation efficiency of the treatment groups of 2 days and 3 days of co-cultivation is similar, and the transformation efficiency of the treatment group of 3 days of co-cultivation is slightly higher than that of the treatment group of 2 days of co-cultivation. The GUS staining area of ​​their calli is also similar, while the transformation efficiency of the treatment group of 4 days of co-cultivation is low. Observation of the experimental materials found that the calli of the treatment group of 4 days of co-cultivation had a large-scale overflow of Agrobacterium, resulting in the death of most calli, indicating that the risk of Agrobacterium contamination increases with the extension of co-cultivation time. Therefore, 3 days of co-cultivation is the best time point.

[0057] 7) Cleaning and recovery culture

[0058] The co-cultured materials were first washed with sterilized ddH2O, and then soaked in distilled water containing Timentin for 8 min at 30°C and 100 r / min to inhibit excessive proliferation of Agrobacterium. The liquid on the surface of the materials was blotted with sterile filter paper and placed on a recovery medium under weak light (light intensity 5000 lux) for 7 days; the recovery medium was: MS liquid medium + 30 g / L sucrose + 8 g / L agar powder + 0.5 mg / L naphthaleneacetic acid + 2 mg / L 6-benzylaminopurine + 300 mg / L Timentin, pH = 5.8.

[0059] 8) Screening, differentiation and rooting induction

[0060] After the co-culture of the transformed callus in step 7), the callus was restored to culture on a medium containing 300 mg / L timentin for 7 days. The callus after restoration culture was completely transferred to a differentiation medium containing kanamycin in a clean bench and screened under a light intensity of 5000 lux and a temperature of 26°C. After 14 days, it was transferred to a rooting medium to induce rooting. During the above screening process, re-screening or subculture was selected according to the budding situation. If there are more buds, further screening can be carried out. If there are fewer buds, subculture can be directly carried out. The kanamycin concentration of the culture medium after budding was adjusted to a final concentration of 50 mg / L; if there are few buds, GA and timentin are removed, and if there are many buds, the above culture medium is continued, and only the kanamycin concentration is adjusted.

[0061] The differentiation medium is: MS liquid medium + 30 g / L sucrose + 0.8 g / L agar powder + 0.5 mg / L naphthaleneacetic acid + 2 mg / L 6-benzylaminopurine + 100 µM / L acetosyringone + 300 mg / L timentin + 150 mg / L kanamycin;

[0062] The rooting medium was MS liquid medium + indolebutyric acid (IBA) 1.5 mg / L + sucrose 30 g / L + agar 3.5 g / L + potato flour 20 g / L.

[0063] 9) Seedling formation and identification

[0064] When the differentiated buds grow to 2-3 cm, they are cut off and transferred to the rooting medium for rooting. When the seedlings grow to about 8 cm, the root medium is washed off and transferred to vermiculite drenched with 1 / 2 MS solution. Later, tap water can be poured. After 7 days of indoor cultivation, they are transferred outdoors and transgenic plants are identified by PCR. Among them, the 1 / 2 MS solution contains 1 / 2MS liquid medium + 30 g / L sucrose, and sterilized ddH2O is added to make the volume 1 L and then the pH is adjusted to 5.8. The leaves or callus tissues of transgenic plants are placed in 1.5 mL centrifuge tubes, with the wild type as the negative control and the positive transgenic tobacco leaves obtained in the early stage as the positive control. The prepared GUS staining solution is added to each centrifuge tube until the plant tissue is immersed. After light protection, it is placed in a 37°C constant temperature incubator. After 12 hours, the staining solution is taken out and poured out, 75% alcohol is added, and the color is decolorized by shaking. After complete decolorization, the staining is observed and counted. Since the growth rates of T0 generation lycopodiella individuals vary to a certain extent, the mature transformed plants of the present invention were obtained in two batches, totaling 42 plants. 20 plants were randomly selected for staining, and the results are as follows: Figure 7 As shown: 12 strains were GUS positive, with a positive rate of 60%.

[0065] At the same time, the present invention uses the wild type and water as negative and blank controls respectively, and uses the specific primers of the target gene (shown in SEQ ID NO.3 and SEQ ID NO.4) to perform PCR amplification on the genomic DNA of the transformed plants, and the size of the target fragment is 539 bp. Figure 8 As shown in the figure, there were no amplified bands in the wild type and negative control (ddH2O), while clear target bands of the correct size were amplified in plants 1-9 and 11-15. Therefore, it can be confirmed that the target gene HbCBF1 has been integrated into the genome of the spatholobus plant. Statistical analysis of the results showed that a total of 32 plants were able to amplify the target bands, with a positive rate of about 76.19%.

[0066] SEQ ID NO.1 sequence is:

[0067] 5'-taagctagaagcgtgtctagaATGGATTGTTTTCCCTCAATATTCTG-3';

[0068] SEQ ID NO.2 sequence is:

[0069] 5'-gtacttatattttttactagtTATAGAAAAACTCCATAGTGACACATCAG-3';

[0070] SEQ ID NO.3 sequence is:

[0071] HbCBF1-F: 5'-CGCAAGACCCTTCCTCTA-3';

[0072] SEQ ID NO.4 sequence is:

[0073] HbCBF1-R: 5'-GCTGCTGCCTTCCTAATG-3'.

[0074] SEQ ID NO.5 sequence is:

[0075] .

[0076] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An efficient Agrobacterium-mediated genetic transformation method of sphaerocarpus truncatus, characterized in that: The following steps are involved: The leaves of sterile spatholobi seedlings were used as explants and inoculated into a callus induction medium for callus induction; the callus induction medium included the following concentration components: MS liquid medium + 0.5 mg / L naphthaleneacetic acid + 1.5 mg / L 6-benzylaminopurine + 30 g / L sucrose + 6 g / L agar; The induced callus tissue was inoculated with Agrobacterium inoculation solution. After the inoculation was completed, the inoculation solution was discarded and the callus tissue was transferred to a co-culture medium for culture; the co-culture medium included the following components in concentrations: MS liquid culture medium + 30 g / L sucrose + 8 g / L agar + 0.5 mg / L naphthaleneacetic acid + 2 mg / L 6-benzylaminopurine + 100 µM / L acetosyringone + 300 mg / L timentin; the co-culture condition was dark culture at 22°C for 3 days; After co-cultivation, the callus tissue is transferred to a recovery medium for recovery culture, and then screened and differentiated by a differentiation medium, and rooting induced by a rooting medium. After rooting, it is transplanted to obtain flying grass seedlings; the recovery medium includes the following concentrations of components: MS liquid medium + 30 g / L sucrose + 8 g / L agar + 0.5 mg / L naphthaleneacetic acid + 2 mg / L 6-benzylaminopurine + 300 mg / L timentin; the differentiation medium includes the following concentrations of components: MS liquid medium + 30g / L sucrose + 0.8 g / L agar powder + 0.5 mg / L naphthaleneacetic acid + 2 mg / L 6-benzylaminopurine + 100 µM / L acetosyringone + 300 mg / L timentin + 150 mg / L kanamycin; the rooting medium includes the following concentrations of components: MS liquid medium + 1.5 mg / L indolebutyric acid + 30 g / L sucrose + 3.5 g / L agar + 20g / L potato; The Agrobacterium infection solution is prepared by centrifuging the fermentation solution of the recombinant Agrobacterium to obtain the bacterial cells, and then resuspending the cells in MS liquid medium to an OD of 600 is 0.6, and 100 μmol / L acetosyringone and 100 μmol / L magnesium chloride are added; The soaking time is 20 min.

2. The method according to claim 1, characterized in that The callus induction conditions are: 28° C. in dark culture.

3. The method according to claim 1, characterized in that The Agrobacterium recombinant bacteria is obtained by transferring the HbCBF1 gene of rubber tree to an expression vector to construct a recombinant vector, and then transforming the recombinant vector into Agrobacterium; the nucleotide sequence of the HbCBF1 gene is shown in SEQ ID NO:

5.

4. The method according to claim 1, characterized in that After culturing on the recovery medium under a weak light of 5000 lux for 7 days, the cells were transferred to the differentiation medium for culturing, and after 14 days, they were transferred to the rooting medium for rooting induction.

5. The method according to claim 1, characterized in that The specific steps of transplanting after rooting are: when the seedlings grow to 5-10 cm, they are moved into vermiculite for indoor cultivation for 7 days, and then moved outdoors for cultivation into adult flying grass seedlings; the vermiculite is soaked in 1 / 2 MS solution before use, and the vermiculite is irrigated with tap water during the cultivation process, and the 1 / 2 MS solution includes the following concentration components: 1 / 2 MS liquid culture medium + 30 g / L sucrose.

6. The method according to claim 1, characterized in that The sterile spatholobus seedlings are obtained by aseptically inoculating spatholobus seeds into MS solid culture medium for cultivation.

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