Genetic transformation method suitable for shiny-leaved yellowhorn hairy root induction

Abstract

The invention provides a genetic transformation method suitable for shiny-leaved yellowhorn hairy root induction. The genetic transformation method comprises the following steps: culturing shiny-leaved yellowhorn seedlings infected by agrobacterium rhizogenes, preparing agrobacterium rhizogenes infection liquid, infecting the agrobacterium rhizogenes, inducing transgenic hairy roots by the agrobacterium rhizogenes and culturing. According to the method, a receptor material, a red tag gene RUBY and a green fluorescent gene GFP which are rapidly cultured and are suitable for agrobacterium rhizogenes infection are used as reporter genes, induction and transformation of hairy roots are carried out through agrobacterium rhizogenes mediation by utilizing a non-tissue culture infection and induction culture method, and the hairy roots are obtained. The cultivation period and cost of traditional plant hairy root induction are reduced, and a hairy root genetic transformation method suitable for shiny-leaved yellowhorn is established.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic transformation, and particularly relates to a method for inducing genetic transformation of Xanthoceras sorbifolia hairy roots. Background Art

[0002] Xanthoceras sorbifolium is a plant of the genus Xanthoceras in the family Sapindaceae. It is mainly distributed in Northeast, Northwest, and North my country. It has the advantages of a large suitable habitat, easy reproduction, and drought and barrenness resistance. It can adapt to the characteristics of my country's small cultivated land and rich mountain resources. The oil content of Xanthoceras sorbifolium kernels is 50% to 67%. Its seed oil has a high content of unsaturated fatty acids, including oleic acid 52.8% to 53.3%, linoleic acid 37.8% to 39.4%, as well as eicosenoic acid, palmitic acid, linolenic acid, etc. It is rich in nutrients and is an ideal raw material for the production of high-grade edible oil and biodiesel. It has attracted much attention. Therefore, it can be seen that the selection of Xanthoceras sorbifolium superior plants and clones with excellent agronomic traits and high fertility, which can be used for different economic production types or environmental adaptation types, is of great significance to the industrial production and practical application of Xanthoceras sorbifolium.

[0003] Transgenic hairy roots are produced by inserting the T-DNA on the expression plasmid containing the target gene into plant tissues or organs through Agrobacterium rhizogenes. The induced culture of hairy roots can produce a large number of transgenic hairy roots, which can be directly used for the study of the function of the target gene and as a bioreactor to synthesize the target metabolites, and can also be regenerated to form transgenic plants. However, the hairy root transformation methods of each plant are significantly different. In different plants, the infection and transformation culture methods will significantly affect the final target gene transformation efficiency and the survival rate of the plant. The current common hairy root induction genetic transformation methods all rely on sterile environment and tissue culture system experiments to induce plant hairy roots and genetic transformation of Agrobacterium rhizogenes, which usually require a suitable sterile environment and tissue culture technology of technicians.

[0004] At present, many plants have used genetic engineering technology to deeply reveal the regulatory genes and mechanisms of their excellent traits, and have achieved innovation and improvement of germplasm through genetic transformation. In the study of genetic improvement of forestry plants, great achievements have been made in the fields of stress resistance, high yield, and disease resistance through the extensive application of genetic engineering technology. The genetic transformation of hairy roots has greatly reduced the technical complexity of traditional tissue culture genetic transformation. However, as an environmentally adaptable economic oil tree species, there has been no report on the application of this technology. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for inducing genetic transformation of hairy roots of Xanthoceras sorbifolia in view of the deficiencies of the above-mentioned prior art. The method utilizes a non-tissue culture infection and induction culture method to induce and transform hairy roots through the mediation of Agrobacterium rhizogenes, thereby reducing the cultivation cycle and cost of traditional plant hairy root induction, and establishing a method for genetic transformation of hairy roots suitable for Xanthoceras sorbifolia.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for inducing genetic transformation of Xanthoceras sorbifolia hairy roots, the method comprising:

[0007] S1. Cultivation of Xanthoceras sorbifolia seedlings suitable for infection with Agrobacterium rhizogenes:

[0008] S101. Select fresh seeds with full grains grown in the current year, soak them in a sulfuric acid solution with a mass fraction of 75% for 1 minute, then take out the seeds, soak them in a 50g / L sodium bicarbonate solution for 10 minutes, rinse them with clean water, put the seeds in water-soaked absorbent cotton overnight, and then spread the seeds on a seedling tray padded with wet gauze to obtain seeds to be germinated;

[0009] S102, culturing the seeds to be germinated obtained from S101 under light conditions with a light intensity of 1000-5000 lux and a temperature of 24° C. for 12 h, and then culturing them under dark conditions at a temperature of 18° C. for 12 h, and artificially cultivating and accelerating germination and hardening the seedlings by alternating light and dark conditions for 15 days;

[0010] S103, after the artificial cultivation of germination and seedling hardening in S102 is completed, 1 / 4MS salt solution containing 20 mg / L calcium chloride and 20 mg / L 6-BA is sprayed every day for moisturizing and germination acceleration for 15 days to obtain Xanthoceras sorbifolia seedlings;

[0011] S2. Preparation of Agrobacterium rhizogenes infection solution:

[0012] S201, the expression vector containing RUBY gene was transformed into Agrobacterium rhizogenes R1601 competent cells, cultured on TY solid medium containing kanamycin at 26°C for 3 days, and a single clone was cultured in kanamycin-resistant liquid TY until the Agrobacterium OD 600 The value is 0.8, and bacterial solution a is obtained;

[0013] S202, take 200 μL of the bacterial solution a obtained in S201 and spread it on a TY solid medium plate containing Rif antibiotics and continue to culture for 18 h, pick a single colony of Agrobacterium rhizogenes again and put it into 30 mL of YEB liquid medium containing 25 mg / mL Rif and culture it until OD 600 Reaching 0.7, bacterial solution b was obtained;

[0014] S203, add 20 mL of YEB liquid medium containing 50 mg / mL Rif to the bacterial solution b obtained in S202, and culture until OD 600 When the pH reaches 0.8, centrifuge at 2250 g for 5 min at 4 °C, remove the supernatant, and add 50 mL of sterile water to resuspend the precipitate to obtain a bacterial suspension;

[0015] S204, adding AS (acetosyringone) with a final concentration of 100 μmol / L, MgCl2 with a final concentration of 10 mmol / L, and MES (2-morpholineethanesulfonic acid) with a final concentration of 10 mmol / L to the suspended bacterial solution obtained in S203 to obtain an Agrobacterium rhizogenes infection solution;

[0016] S3. Infection with Agrobacterium rhizogenes:

[0017] S301, cutting off the root system at the bottom of the hypocotyl of the Xanthoceras sorbifolia seedling obtained in S103, and soaking the remaining part in the infection solution of Agrobacterium rhizogenes obtained in S204 for 10 minutes to obtain the infected Xanthoceras sorbifolia seedling;

[0018] S302, inserting the infected Xanthoceras sorbifolia seedlings obtained in S301 into a culture pot filled with nutrient soil, maintaining a humidity of more than 70%, and cultivating for 15 days to obtain infected plants;

[0019] S4. Induction and cultivation of transgenic hairy roots by Agrobacterium rhizogenes:

[0020] S401, transferring the infected plants obtained in S302 to a plant light culture room at a temperature of 23° C. and culturing for 1 week to obtain infected plants after light culture;

[0021] S402, the infected plants obtained after light culture in S401 were measured by OD 600 Add calcium chloride with a final concentration of 20 mg / L and 6-BA with a final concentration of 10 mg / L to the Agrobacterium rhizogenes infection solution with a concentration of 0.7, and water along the base. The watering time is 3, 6, and 9 days after the light-cultured infected plants obtained in S401. The watering amount for each plant is 2-4 mL, and light is also applied during watering. After the third watering, continue to culture under light for 21 days. After culture, observe the red root system or use handheld fluorescence to observe the expression of GFP to screen transgenic hairy roots. The Agrobacterium rhizogenes infection solution in S402 is the same as the Agrobacterium rhizogenes infection solution in S204. If the OD of the bacterial solution in S204 is 600 If the OD value is less than 0.7, the strain is cultured on a temperature-controlled shaker at 300 rpm and 28°C until it reaches OD 600 If the value exceeds 0.7, dilution culture should be performed.

[0022] Preferably, the preparation method of the TY solid culture medium containing kanamycin described in S201 is: 10g / L peptone, 4g / L yeast extract, 10g / L glucose, 6g / L KH2PO4, 2g / L NaCl, 12.5g / L agar, the pH is adjusted to 7.0, after sterilization, a final concentration of 10mmol / L calcium chloride solution and 50mg / L kanamycin are added and evenly coated.

[0023] Preferably, the preparation method of the kanamycin-resistant liquid TY culture medium described in S201 is: 10 g / L peptone, 4 g / L yeast extract, 10 g / L glucose, 6 g / L KH2PO4, 2 g / L NaCl, the pH is adjusted to 7.0, and after sterilization, a final concentration of 10 mmol / L calcium chloride solution and 50 mg / L kanamycin are added.

[0024] Preferably, the TY solid culture medium plate containing Rif antibiotic in S202 is composed of raw materials of the following concentrations, and the pH is adjusted to 7.0: 10 g / L peptone, 4 g / L yeast extract, 10 g / L glucose, 6 g / L KH2PO4, 2 g / L NaCl, 15 g / L agar powder, and 25 mg / mL Rif antibiotic.

[0025] Preferably, the YEB liquid culture medium containing 25 mg / mL Rif in S202 is composed of the following raw materials at the following concentrations, and the pH is adjusted to 7.0: 10 g / L peptone, 4 g / L yeast extract, 10 g / L glucose, 6 g / L KH2PO4, 2 g / L NaCl, and 25 mg / mL Rif.

[0026] Preferably, the YEB liquid culture medium containing 50 mg / mL Rif in S203 is composed of the following raw materials at the following concentrations, and the pH is adjusted to 7.0: 10 g / L peptone, 4 g / L yeast extract, 10 g / L glucose, 6 g / L KH2PO4, 2 g / L NaCl, and 50 mg / mL Rif.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] The genetic transformation method of Xanthoceras sorbifolia hairy roots of the present invention uses a Xanthoceras sorbifolia seedling receptor material suitable for infection with Agrobacterium rhizogenes to rapidly culture, a red label gene RUBY and a green fluorescent gene GFP as reporter genes, and a non-tissue culture infection and induction culture method to induce and transform hairy roots mediated by Agrobacterium rhizogenes, thereby reducing the cultivation period and cost of traditional plant hairy root induction, and establishing a genetic transformation method of hairy roots suitable for Xanthoceras sorbifolia.

[0029] 2. The present invention successfully achieves the induction of Xanthoceras sorbifolia hairy roots and the establishment of a genetic transformation system for the first time; the artificial germination and cultivation of Xanthoceras sorbifolia seedlings of the present invention have extremely high adaptability to infection by Agrobacterium rhizogenes, thereby ensuring the survival rate of the subsequent hairy root genetic transformation steps; the Xanthoceras sorbifolia hairy root induction and genetic transformation step of the present invention does not require a tissue culture system and a sterile environment, and the operation is simple and fast, thereby ensuring the success rate of infection while reducing material waste and improving efficiency.

[0030] 3. The present invention fills the gap in the genetic transformation system of hairy roots of Xanthoceras sorbifolia. Compared with other existing genetic transformation technologies for woody plants, the artificially grown seedlings of Xanthoceras sorbifolia are used as the recipients, and the transformation is mediated by Agrobacterium rhizogenes. During the transformation, the transformed Agrobacterium rhizogenes is only subjected to two resistance screenings, and there is no need to use tissue culture systems and sterile links, which greatly reduces the genetic transformation cycle and operation steps of hairy roots of woody plants, and has a high positive transformation rate. The technology can be used to quickly and effectively verify the function of Xanthoceras sorbifolia genes, laying a research method foundation for breakthroughs in the research of Xanthoceras sorbifolia gene functions and molecular breeding technology.

[0031] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a picture of the hairy roots of Xanthoceras sorbifolia after transformation with RUBY transgene in Example 1 of the present invention.

[0033] Figure 2 This is a diagram of screening transgenic hairy roots using handheld fluorescence to observe GFP expression in Example 1 of the present invention. DETAILED DESCRIPTION

[0034] The standard formula of 1 / 4MS salt solution (Murashige and Skoog) used in the following examples is: KNO3475mg / L, KH2PO442.5mg / L, CaCl2110mg / L, H3BO31.2mg / L, ZnSO42.6mg / L, CuSO40.025mg / L, Na2-EDTA 9.3mg / L, inositol 88mg / L, VB60.5mg / L, VB10.1mg / L, NH4NO3410 mg / L, MgSO491mg / L, KI 0.23mg / L, MnSO4 5.3mg / L, Na2MoO40.05mg / L, CoCl20.025mg / L, FeSO47.8mg / L, glycine 0.5mg / L, C6H5NO20.1mg / L, D(+)-sucrose 8g / L.

[0035] 1 / 4MS salt solution was purchased from Beijing Solebow Company.

[0036] The preparation method of TY solid culture medium containing kanamycin is as follows: 10g / L peptone, 4g / L yeast extract, 10g / L glucose, 6g / L KH2PO4, 2g / L NaCl, 12.5g / L agar, pH adjusted to 7.0, after sterilization, add a final concentration of 10mmol / L calcium chloride solution and 50mg / L kanamycin and spread evenly.

[0037] The preparation method of kanamycin-resistant liquid TY medium is as follows: 10 g / L peptone, 4 g / L yeast extract, 10 g / L glucose, 6 g / L KH2PO4, 2 g / L NaCl, pH is adjusted to 7.0, after sterilization, a final concentration of 10 mmol / L calcium chloride solution and 50 mg / L kanamycin are added.

[0038] TY solid medium plates containing Rif antibiotics were composed of the following raw materials at the following concentrations and the pH was adjusted to 7.0: 10 g / L peptone, 4 g / L yeast extract, 10 g / L glucose, 6 g / L KH2PO4, 2 g / L NaCl, 15 g / L agar powder, and 25 mg / mL Rif antibiotics.

[0039] YEB liquid medium containing 25 mg / mL Rif was composed of the following raw materials at the following concentrations and the pH was adjusted to 7.0: 10 g / L peptone, 4 g / L yeast extract, 10 g / L glucose, 6 g / L KH2PO4, 2 g / L NaCl, 25 mg / mL Rif.

[0040] YEB liquid medium containing 50 mg / mL Rif was composed of the following raw materials at the following concentrations and the pH was adjusted to 7.0: 10 g / L peptone, 4 g / L yeast extract, 10 g / L glucose, 6 g / L KH2PO4, 2 g / L NaCl, 50 mg / mL Rif.

[0041] Agrobacterium rhizogenes R1601 was purchased from the British company BioVector as a commercial competent Agrobacterium rhizogenes, and the plasmid vector transformation was performed according to the competent Agrobacterium rhizogenes transformation instructions.

[0042] Example 1

[0043] RUBY gene expression system for infection and transformation of hypocotyls of Xanthoceras sorbifolia seedlings

[0044] (1) Cultivation of Xanthoceras sorbifolia seedlings

[0045] Fresh, full-grained seeds of the current year were selected to set the concentrated sulfuric acid soaking concentration to 65, 70, and 75% and the soaking time to 1, 2, and 3 minutes; the sodium bicarbonate soaking concentration to 40, 50, and 60 g / L and the soaking time to 5, 10, and 15 minutes, as shown in Table 1. The thick shell of the seeds was soaked in concentrated sulfuric acid to increase the permeability, and the germination rate of the seeds was promoted by neutralization of concentrated sulfuric acid with sodium bicarbonate. Finally, it was determined that the germination rate was the highest when the seeds were soaked in 75% concentrated sulfuric acid for 1 minute, and then the seeds were taken out and soaked in concentrated sulfuric acid with 50 g / L sodium bicarbonate solution for 10 minutes (Table 1), and the germination rate was relatively high. After that, wash with clean water, put the seeds in water-soaked defatted cotton overnight to fully absorb water, then spread the seeds on a seedling tray padded with gauze, and culture them under light conditions with a light intensity of 1000-5000 lux and a temperature of 24°C for 12 hours, and then culture them under dark conditions at a temperature of 18°C ​​for 12 hours. In an artificial incubator with alternating light and dark conditions, maintain a day and night temperature difference of 6°C for artificial cultivation to promote germination and harden the seedlings for 15 days. After the artificial cultivation of germination and hardening the seedlings is completed, the addition concentration and culture cycle of calcium chloride and 6-BA added for seedling growth and development are investigated at the same time. The calcium chloride addition concentration is set to 10, 15, and 20 mg / L, and the 6-BA is 20, 30, and 40 mg / L, respectively. The continuous culture time after germination is 10, 15, and 20 days. Finally, the best method to promote germination and seedling growth is obtained (Table 2): spray 20 mg / L calcium chloride and 20 mg / L The moisturizing germination culture was carried out in 1 / 4MS salt solution of 6-BA for 15 days, and the Xanthoceras sorbifolia materials with good growth status suitable for later root-generating Agrobacterium infection were obtained.

[0046] Table 1 Effects of germination-promoting factor variables on germination rate

[0047]

[0048] Table 2 Effect of nutrient solution on plant survival rate after Agrobacterium infection

[0049]

[0050] (2) Preparation of Agrobacterium rhizogenes infection solution

[0051] The expression vector was selected to contain kanamycin and rifampicin (Rif) resistance sites. The expression system vector containing the RUBY reporter gene (NM_001288889.1) was transformed into Agrobacterium rhizogenes R1601 competent cells (purchased from BioVector, UK). The transformation steps were carried out according to the instructions for plasmid transformation of R1601 competent cells. The cells were cultured in a solid TY medium containing kanamycin at 26°C for 3 days, and a single clone was cultured in a kanamycin-resistant liquid TY until the Agrobacterium OD 600 The value is 0.5-0.8, and the OD is optimized by orthogonal experiment in Table 3600 The value is 0.8. Take about 200 μl of bacterial solution and apply it to TY solid medium plate containing Rif antibiotics and continue to culture for 18 hours until Agrobacterium rhizogenes forms large-scale single colonies. Pick up single colonies of Agrobacterium rhizogenes again and place them in 30 mL (containing Rif concentration 25 mg / mL) YEB liquid medium for 10 hours. When OD 600 When it reaches about 0.5-0.8, the orthogonal test results in Table 3 are preferably OD 600 0.7. Add 20 mL of YEB liquid medium (containing 50 mg / mL Rif) again and culture for several more hours until the OD 600 Reach 0.7-1, according to the orthogonal test results in Table 3, OD 600 The mixture was then poured into a 50 ml centrifuge tube and centrifuged at 4°C and 2250 g for 5 minutes. After slowly discarding the supernatant, 50 ml of sterile water was added to resuspend the precipitate, and then AS (acetosyringone) with a final concentration of 100 μmol / L, MgCl2 with a final concentration of 10 mmol / L, and MES (2-morpholineethanesulfonic acid) with a final concentration of 10 mmol / L were added to prepare the Agrobacterium rhizogenes infection solution.

[0052] Table 3 Effect of Agrobacterium concentration on plant growth status and positive transformation rate

[0053]

[0054]

[0055] (3) Agrobacterium rhizogenes infects hypocotyls of Xanthoceras sorbifolia seedlings

[0056] Directly cut off the root system at the bottom of the hypocotyl of the artificially germinated Xanthoceras sorbifolia seedlings, and soak the remaining part in the infection solution of Agrobacterium rhizogenes for 10 minutes. Insert the infected Xanthoceras sorbifolia seedlings into a culture pot filled with nutrient soil, maintain the humidity above 70%, and cultivate for 15 days. The infected plants;

[0057] (4) Induction and cultivation of hairy roots by Agrobacterium rhizogenes

[0058] The infected plants were transferred to a plant light culture room at a temperature of 23-26°C, cultured for 12 hours under light conditions with a light intensity of 1000-5000 Lux and a temperature of 24°C, and then cultured for 12 hours under dark conditions at a temperature of 18°C. The light and dark conditions were alternated for 1-2 weeks. The hairy root induction rate was the highest after culturing at 23°C for 1 week (Table 4), and the infected plants after light culture were obtained. After the infected plants were cultured under periodic light, the OD 600Add 10-20 mg / L calcium chloride and 10-20 mg / L 6-BA to the 0.5-0.7 agrobacterium rhizogenes infection solution and irrigate along the base. The irrigation cycle is 3, 6, and 9 days after light culture. The irrigation amount per plant is about 2-4 mL. Light is also applied during irrigation. Through experiments, it is preferred to use OD 600 Add 20mg / L calcium chloride and 10mg / L 6-BA to the 0.7 Agrobacterium rhizogenes infection solution and water 4mL along the base. The hairy root regeneration efficiency is relatively high and the root system grows well. After the third watering, continue to culture under light for 21 days. After cultivation, observe the red root system or use handheld fluorescence to observe the expression of GFP to screen transgenic hairy roots. ( Figure 1-2 ).

[0059] Figure 1 In the middle, the red ones are the successfully transformed RUBY transgenic hairy roots. Figure 2 The middle is a picture of ultraviolet irradiation of the GFP fluorescent marker gene. The red arrow points to the green transgenic hairy roots produced by irradiating with a handheld fluorescent flashlight.

[0060] Table 4 Effects of temperature and photoperiod on the induction rate of hairy roots

[0061]

[0062] Table 5 Investigation of hairy root regeneration factors

[0063]

[0064] Example 2

[0065] Transformation of GFP reporter gene into hypocotyls of Xanthoceras sorbifolia seedlings

[0066] (1) Cultivation of Xanthoceras sorbifolia seedlings

[0067] Select fresh, full-grained seeds grown in the current year and soak them in 75% concentrated sulfuric acid for 1 min. Take out the seeds, neutralize them with 50g / L sodium bicarbonate solution and soak them in concentrated sulfuric acid for 10 min, wash them with clean water, put the seeds in water-soaked defatted cotton overnight to facilitate full absorption of water, then spread the seeds flat on a seedling tray padded with gauze, put them in an artificial incubator at 24℃ during the day and 18℃ at night to maintain a day and night temperature difference of 6℃, spray 1 / 4MS salt solution containing 20mg / L calcium chloride and 20mg / L 6-BA every day for moisturizing and germination culture for 15 days.

[0068] (2) Preparation of Agrobacterium rhizogenes infection solution

[0069] Transform the competent Agrobacterium rhizogenes R1601 (purchased from BioVector, UK) with the GFP reporter gene vector and culture it on the solid TY medium with corresponding resistance at 28°C for about 2 days. Pick a single clone and culture it in the corresponding resistance liquid TY until the OD600 value of Agrobacterium is about 0.8. Take 200ul and apply it on the solid TY medium plate and continue to culture for 18 hours until the entire plate of Agrobacterium rhizogenes is covered. The formula of TY medium is: 5g / L peptone, 3g / L yeast extract, pH adjusted to about 7.0, add 10mmol / L calcium chloride solution and 40mg / L kanamycin after high-pressure sterilization, and add 15g / L agar powder to the solid. Pick a single colony of Agrobacterium rhizogenes again and place it in 30mL (containing Rif concentration of 25mg / mL) YEB liquid medium for culturing for 10 hours. During this period, wait for OD 600 When the OD reaches 0.7, add 20 mL (containing 50 mg / mL Rif concentration) YEB liquid culture medium and culture for another 2 hours. 600 When the pH reaches 0.8, pour it into a 50 ml centrifuge tube and centrifuge it at 2250 g at 4°C for 5 minutes. After slowly discarding the supernatant, pour in 50 ml of sterile water to resuspend the precipitate, and then add 100 μmol / L AS, 10 mmol / L MgCl2, and 10 mmol / L MES to prepare the Agrobacterium rhizogenes infection solution.

[0070] (3) Agrobacterium rhizogenes infects the roots of Xanthoceras sorbifolia seedlings

[0071] Cut off the roots of Xanthoceras sorbifolia seedlings from the bottom of the hypocotyl to form a wound, soak them in Agrobacterium infection solution for 10 minutes, and insert them into a culture pot filled with nutrient soil, maintaining the humidity above 70%.

[0072] (4) Induction and cultivation of Agrobacterium rhizogenes

[0073] The plants were then transferred to a plant light culture room at 23°C and cultured for 1 week. On days 3, 6, and 9 after light culture, the plants were infected with plasmid-containing Agrobacterium tumefaciens solution at OD 1.0. 600 The irrigation rate was 0.7, and 4 ml of Agrobacterium infection solution supplemented with 20 mg / L calcium chloride and 10 mg / L 6-BA was poured from the base of each plant. After continuing the light culture for 21 days, a handheld fluorescent screen was used to screen the hairy roots that clearly produced green fluorescence.

[0074] Example 3

[0075] Root infection and transformation of Xanthoceras sorbifolia seedlings into RUBY gene expression system

[0076] (1) Cultivation of Xanthoceras sorbifolia seedlings

[0077] Select fresh, full-grained seeds grown in the current year and soak them in 75% concentrated sulfuric acid for 1 min. Take out the seeds, neutralize them with 50g / L sodium bicarbonate solution and soak them in concentrated sulfuric acid for 10 min, wash them with clean water, put the seeds in water-soaked defatted cotton overnight to facilitate full absorption of water, then spread the seeds flat on a seedling tray padded with gauze, put them in an artificial incubator at 24℃ during the day and 18℃ at night to maintain a day and night temperature difference of 6℃, spray 1 / 4MS salt solution containing 20mg / L calcium chloride and 20mg / L 6-BA every day for moisturizing and germination culture for 15 days.

[0078] (2) Preparation of Agrobacterium rhizogenes infection solution

[0079] The RUBY expression system vector was transformed into Agrobacterium rhizogenes R1601 competent cells (purchased from BioVector, UK) and cultured in solid TY medium with corresponding resistance at 28°C for 2 days. A single clone was selected and cultured in liquid TY with corresponding resistance until the OD of Agrobacterium reached 1. 600 The value is 0.8, take 200ul to apply on solid TY medium plate and continue to culture for 18 hours until the whole plate is covered with Agrobacterium rhizogenes. The formula of TY medium is: 5g / L peptone, 3g / L yeast extract, pH adjusted to 7.0, add 10mmol / L calcium chloride solution and 40mg / L kanamycin after high pressure sterilization, and add 15g / L agar powder to the solid. Pick a single colony of Agrobacterium rhizogenes again and put it into 30mL (containing Rif concentration 25mg / mL) YEB liquid medium for 10 hours. During this period, wait until OD 600 When the OD reaches 0.7, add 20 mL (containing 50 mg / mL Rif concentration) YEB liquid culture medium and culture for another 2 hours. 600 When the pH reaches 0.8, pour it into a 50 ml centrifuge tube and centrifuge it at 2250 g at 4°C for 5 minutes. After slowly discarding the supernatant, pour in 50 ml of sterile water to resuspend the precipitate, and then add 100 μmol / L AS, 10 mmol / L MgCl2, and 10 mmol / L MES to prepare the Agrobacterium rhizogenes infection solution.

[0080] (3) Infection with Agrobacterium rhizogenes

[0081] Select relatively sturdy roots of Xanthoceras sorbifolia, make several small horizontal cuts with a surgical blade, soak the roots directly in the infection solution of Agrobacterium rhizogenes for 10 minutes, bury them in a seedling tray filled with nutrient soil, and seal it with a lid to keep the humidity above 70%.

[0082] (4) Induction and cultivation of Agrobacterium rhizogenes

[0083] The plants were then transferred to a plant light culture room at 23°C and cultured for 1 week. They were irrigated with plasmid-containing Agrobacterium infection solution (OD600 = 0.7) on days 3, 6, and 9 after culture. 4 ml of Agrobacterium infection solution supplemented with 20 mg / L calcium chloride and 10 mg / L 6-BA was watered from the base of each plant. After continuing light culture for 21 days, it was observed whether red transgenic hairy roots were produced.

[0084] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for inducing genetic transformation of Xanthoceras sorbifolia hairy roots, characterized in that: The method is: S1. Cultivation of Xanthoceras sorbifolia seedlings suitable for infection with Agrobacterium rhizogenes: S101. Select fresh seeds with full grains grown in the current year, soak them in a sulfuric acid solution with a mass fraction of 75% for 1 minute, then take out the seeds, soak them in a 50g / L sodium bicarbonate solution for 10 minutes, rinse them with clean water, put the seeds in water-soaked absorbent cotton overnight, and then spread the seeds on a seedling tray padded with wet gauze to obtain seeds to be germinated; S102, culturing the seeds to be germinated obtained from S101 under light conditions at a temperature of 24° C. for 12 h, and then culturing them under dark conditions at a temperature of 18° C. for 12 h, and artificially cultivating and accelerating germination and hardening the seedlings by alternating light and dark conditions for 15 days; S103, after the artificial cultivation of germination and seedling hardening in S102 is completed, 1 / 4MS salt solution containing 20 mg / L calcium chloride and 20 mg / L 6-BA is sprayed every day for moisturizing and germination acceleration for 15 days to obtain Xanthoceras sorbifolia seedlings; S2. Preparation of Agrobacterium rhizogenes infection solution: S201, the expression vector containing RUBY gene was transformed into Agrobacterium rhizogenes R1601 competent cells, cultured on TY solid medium containing kanamycin at 26°C for 3 days, and a single clone was cultured in kanamycin-resistant liquid TY until the Agrobacterium OD 600 The value is 0.8, and bacterial solution a is obtained; S202, take 200 μL of the bacterial solution a obtained in S201 and spread it on a TY solid medium plate containing Rif antibiotics and continue to culture for 18 h, pick a single colony of Agrobacterium rhizogenes again and put it into 30 mL of YEB liquid medium containing 25 mg / mL Rif and culture it until OD 600 Reaching 0.7, bacterial solution b was obtained; S203, add 20 mL of YEB liquid medium containing 50 mg / mL Rif to the bacterial solution b obtained in S202, and culture until OD 600 When the pH reaches 0.8, centrifuge at 2250 g for 5 min at 4 °C, remove the supernatant, and add 50 mL of sterile water to resuspend the precipitate to obtain a bacterial suspension; S204, adding acetosyringone with a final concentration of 100 μmol / L, MgCl2 with a final concentration of 10 mmol / L, and 2-morpholineethanesulfonic acid with a final concentration of 10 mmol / L to the suspended bacterial solution obtained in S203 to obtain an Agrobacterium rhizogenes infection solution; S3. Infection with Agrobacterium rhizogenes: S301, cutting off the root system at the bottom of the hypocotyl of the Xanthoceras sorbifolia seedling obtained in S103, and soaking the remaining part in the infection solution of Agrobacterium rhizogenes obtained in S204 for 10 minutes to obtain the infected Xanthoceras sorbifolia seedling; S302, inserting the infected Xanthoceras sorbifolia seedlings obtained in S301 into a culture pot filled with nutrient soil, maintaining a humidity of more than 70%, and cultivating for 15 days to obtain infected plants; S4. Induction and cultivation of transgenic hairy roots by Agrobacterium rhizogenes: S401, transferring the infected plants obtained in S302 to a plant light culture room at a temperature of 23° C. and culturing for 1 week to obtain infected plants after light culture; S402, the infected plants obtained after light culture in S401 were measured by OD 600 Add calcium chloride with a final concentration of 20 mg / L and 6-BA with a final concentration of 10 mg / L to the 0.7 Agrobacterium rhizogenes infection solution, and water along the base. The watering time is 3, 6, and 9 days after the light culture of the infected plants obtained in S401. The watering amount for each plant is 2-4 mL. After the third watering, continue to culture under light for 21 days, observe the red root system or use a handheld fluorescence to observe the expression of GFP to screen the transgenic hairy roots.

2. A method for inducing genetic transformation of Xanthoceras sorbifolia hairy roots according to claim 1, characterized in that: The preparation method of the TY solid culture medium containing kanamycin described in S201 is: 10g / L peptone, 4g / L yeast extract, 10g / L glucose, 6g / L KH2PO4, 2g / L NaCl, 12.5g / L agar, the pH is adjusted to 7.0, after sterilization, a final concentration of 10mmol / L calcium chloride solution and 50mg / L kanamycin are added and evenly coated.

3. A method for inducing genetic transformation of Xanthoceras sorbifolia hairy roots according to claim 1, characterized in that: The preparation method of the kanamycin-resistant liquid TY culture medium described in S201 is: 10 g / L peptone, 4 g / L yeast extract, 10 g / L glucose, 6 g / L KH2PO4, 2 g / L NaCl, the pH is adjusted to 7.0, and after sterilization, a final concentration of 10 mmol / L calcium chloride solution and 50 mg / L kanamycin are added.

4. A method for inducing genetic transformation of Xanthoceras sorbifolia hairy roots according to claim 1, characterized in that: The TY solid culture medium plate containing Rif antibiotic described in S202 is composed of raw materials with the following concentrations, and the pH is adjusted to 7.0: 10g / L peptone, 4g / L yeast extract, 10g / L glucose, 6g / L KH2PO4, 2g / L NaCl, 15g / L agar powder, and 25mg / mL Rif antibiotic.

5. A method for inducing genetic transformation of Xanthoceras sorbifolia hairy roots according to claim 1, characterized in that: The YEB liquid culture medium containing 25 mg / mL Rif described in S202 is composed of the following raw materials at the following concentrations, and the pH is adjusted to 7.0: 10 g / L peptone, 4 g / L yeast extract, 10 g / L glucose, 6 g / L KH2PO4, 2 g / L NaCl, and 25 mg / mL Rif.

6. A method for inducing genetic transformation of Xanthoceras sorbifolia hairy roots according to claim 1, characterized in that: The YEB liquid culture medium containing 50 mg / mL Rif described in S203 is composed of the following raw materials at the following concentrations, and the pH is adjusted to 7.0: 10 g / L peptone, 4 g / L yeast extract, 10 g / L glucose, 6 g / L KH2PO4, 2 g / L NaCl, and 50 mg / mL Rif.

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