CN118421695A - Establishment method and application of Ningxia wolfberry stable genetic transformation system - Google Patents

Abstract

The invention provides a method for establishing a stable genetic transformation system of Ningxia wolfberry and an application thereof, belonging to the technical field of plant genetic engineering. The method successfully clones the full-length CDS sequence of the Ningxia wolfberry sugar metabolism gene LbGALA3 , constructs an overexpression vector CAM-FLAG-GFP containing a single gene fused with GFP, then introduces a recombinant plasmid containing a target gene into an explant of the Ningxia wolfberry through an Agrobacterium-mediated method, and finally obtains a transgenic plant through subculture. A fluorescent protein excitation light source is used to quickly screen positive plants and verify them through PCR. The successfully transformed Ningxia wolfberry is propagated and transplanted, and it is found that the plants can grow normally and are stably genetically transformed, indicating that the method can quickly obtain plants with resistance and expressing the target gene, and provides an important basis for the research on functional genes of active substances related to Ningxia wolfberry.

Description

Establishment and application of a stable genetic transformation system for Ningxia wolfberry

Technical Field

The invention belongs to the technical field of plant gene engineering, and specifically relates to a method for establishing a stable genetic transformation system of Ningxia wolfberry and an application thereof.

Background technique

Ningxia wolfberry (Lycium barbarum L.) is a perennial deciduous shrub of the Lycium genus of the Solanaceae family. It has strong adaptability and its fruit contains a variety of biologically active substances, including sugars, multiple amino acids, flavonoids, carotenoids, etc., and has significant health and medicinal value. As a characteristic economic forest tree species in my country, Ningxia wolfberry has strong salt-alkali resistance and drought resistance. It is most widely cultivated, and the production areas are mainly concentrated in Ningxia, Gansu, Qinghai, Xinjiang, Hebei and other places. It is reported that the planting area of Ningxia wolfberry in the country currently exceeds 3 million mu, of which the planting area in Ningxia accounts for 45%, the fresh fruit output is nearly 300,000 tons, there are more than 100 kinds of deep-processing products, and the comprehensive output value exceeds 27 billion yuan. After decades of large-scale development, Ningxia wolfberry has formed a unique market economy model, combining ecological, economic, cultural and medical health functions, and has become a pillar industry in promoting regional economic development, revitalizing rural economy, and medical research and development applications.

At present, the research on Ningxia wolfberry mainly focuses on the nutritional components of the fruit, medicinal components, polysaccharide extraction technology and pharmacological analysis and application. With the research and development and application of the medicinal value of Ningxia wolfberry, the demand for high-quality fruits rich in main active substances has become a problem faced by industrial development. Therefore, exploring the biosynthetic metabolic mechanism of active substances in Ningxia wolfberry fruit, discovering its key regulatory genes and conducting molecular breeding have become key means of cultivating excellent quality. Establishing a stable genetic transformation system for Ningxia wolfberry is an important means to explore the regulatory mechanism of key functional genes and molecular breeding. It can not only reveal the functional mechanism of genes related to key excellent traits, but also achieve the purpose of rapid molecular breeding to improve unfavorable traits. However, there is currently no method for establishing a rapid, efficient and stable genetic transformation system for Ningxia wolfberry.

Summary of the invention

In view of this, the present invention provides a method for establishing a stable genetic transformation system of Ningxia wolfberry and its application.

A method for establishing a stable genetic transformation system of Ningxia wolfberry comprises the following steps:

(1) Obtaining Ningxia wolfberry explants: cutting the cotyledons of cultured Ningxia wolfberry sterile seedlings that have just unfolded from the middle to obtain Ningxia wolfberry explants;

(2) Construction of overexpression vector: The full-length sequence of Ningxia wolfberry sugar metabolism gene LbGALA3 was amplified by PCR, and the amplified target gene fragment was connected to the vector CAM-FLAG-GFP and transformed into Escherichia coli to obtain a recombinant plasmid. A single clone was selected for colony PCR amplification test, and the plasmid was extracted and sequenced. If the sequencing was correct, the overexpression vector was successfully constructed;

(3) Preparing Agrobacterium resuspension: The recombinant plasmid verified by sequencing was transformed into Agrobacterium GV3101 competent cells, streaked and cultured on LB solid medium containing antibiotics, and inverted cultured at 27°C for 2-3 days. Then, a colony PCR amplification test was performed to screen positive colonies, and a single clone was picked and inoculated into LB liquid medium to prepare an Agrobacterium resuspension with an _OD600 of 0.4-1.2;

(4) Infection: Add the pre-cultured Ningxia wolfberry explants to a culture dish containing Agrobacterium resuspension and infect for 8 to 10 minutes. During the infection process, gently shake the culture dish at regular intervals to promote Agrobacterium infection of the explants.

(5) Subculture: After infection, the explants are transferred to a co-culture medium for co-culture, and the explants after co-culture are transferred to a differentiation medium for differentiation culture to induce buds. The differentiated resistant buds are transferred to a 1/2MS resistance medium for resistance culture, and then rooted to form complete plants.

(6) Screening positive plants: Use fluorescent protein excitation light source to quickly detect rooted plants and verify them through PCR to screen out Ningxia wolfberry transgenic plants that overexpress the LbGALA3 gene, and then expand and transplant the successfully transformed Ningxia wolfberry plants.

Preferably, in step (2), the primer sequences used for PCR amplification of the target gene fragment are as shown in SEQ ID NO: 1 and SEQ ID NO: 2;

The reaction system is as follows:

2×P515 enzyme 20μL 10 μM upstream primer 1μL 10 μM downstream primer 1μL template 3μL Sterile _ddH2O 15μL total capacity 40μL

The reaction program was: 95°C for 10 min, 95°C for 30 s, 57°C for 30 s, 72°C for 30 s/kb, 30 cycles; 72°C for 10 min.

Preferably, in the step (1), the culturing of Ningxia wolfberry sterile seedlings is as follows: the mature seeds of Ningxia wolfberry are disinfected with 75% alcohol for 1 min, disinfected with sodium hypochlorite solution for 10-20 min, rinsed with sterile water for 4 times, inoculated in 1/2MS solid culture medium, cultured in the dark at 25°C for 3 days, and then cultured in light for one week to obtain Ningxia wolfberry sterile seedlings; the 1/2MS resistance culture medium comprises 2.22 g/L MS powder, 20 g/L sucrose, 1 L/L _H2O , and 7.5 g/L agar.

Preferably, in step (2), the target gene fragment is connected to the vector CAM-FLAG-GFP by adding 5'NdeI and 3'EcoRI double restriction sites and protective bases at both ends of the CAM-FLAG-GFP gene CDS by PCR, and after restriction digestion, the result is detected by agarose gel electrophoresis, the gel corresponding to the band of the large vector fragment is cut, and the restriction digestion product is recovered by the Biotech gel recovery kit, wherein the double restriction digestion reaction system is as follows:

5xTango buffer 8.0μL XOt 1μL EcoRI 1μL Plasmids 10 μg _ddH2O 20μL total capacity 40μL

The reaction procedure is: enzyme digestion at 37℃ for 40min; inactivation at 65℃ for 15min; when the target fragment is connected to the vector, add reaction buffer to the PCR tube, add the target DNA fragment and the linearized vector in a certain molar ratio, add homologous recombinase, and then mix well and keep warm at 50℃ for 15min.

Preferably, in the step (3), in the LB solid culture medium and the LB liquid culture medium, the antibiotic is a mixed antibiotic of spectinomycin and gentamicin, the content of spectinomycin is 100 mg/L, and the content of gentamicin is 100 mg/L.

Preferably, in the step (3), in the LB solid culture medium and the LB liquid culture medium, the antibiotics are a mixed antibiotic of kanamycin and gentamicin, the content of kanamycin is 100 mg/L, and the content of gentamicin is 100 mg/L.

Preferably, the co-culture and differentiation culture medium components are 4.43g/L MS dry powder, 30g/L sucrose, 7.5g/L agar, 1L/L; the 1/2MS resistance medium components are 2.22g/L MS powder, 20g/L sucrose, 1L _H2O , 7.5g/L agar.

Preferably, the variety of Ningxia wolfberry is 'Ningqi No. 1'.

An application of the Ningxia wolfberry stable genetic transformation system in the identification of Ningxia wolfberry gene functions.

It can be seen from the above technical scheme that the present invention provides a method for establishing a stable genetic transformation system of Ningxia wolfberry and its application. Compared with the prior art, the beneficial effects are as follows: by successfully cloning the full-length CDS sequence of the Ningxia wolfberry sugar metabolism gene L bGALA3, an overexpression vector CAM-FLA G-GFP containing a single gene fused with GFP is constructed, and the recombinant plasmid containing the target gene is introduced into the explant of Ningxia wolfberry by Agrobacterium-mediated method, and finally a transgenic plant is obtained by subculture, and a positive plant is quickly screened by a fluorescent protein excitation light source and verified by PCR, and the successfully transformed Ningxia wolfberry is expanded and transplanted, and it is found that the plant can grow normally and stably genetically transformed, indicating that the method can quickly obtain plants with resistance and expression of the target gene. The method has the advantages of rapidity, high efficiency, low cost, and easy operation, and can be prepared on a large scale, providing a model species for the study of functional genes of active substances related to Ningxia wolfberry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the construction principle of the gene LbGALA3 overexpression vector driven by the CaMV 35S promoter fused with GFP.

FIG. 2 is the PCR amplification result of the full-length sequence of the target gene LbGALA3.

FIG3 is the result of PCR amplification test of single clones.

FIG. 4 is a diagram of the stages of a genetic transformation system.

FIG5 is the result of rapid screening of LbGALA3-positive plants using a fluorescent protein excitation light source.

FIG. 6 is the PCR positive clone identification result of LbGALA3 transgenic wolfberry seedlings and wild-type seedlings.

FIG. 7 shows the status of the T0 generation transgenic positive seedlings of LbGALA3 identified after propagation and transplantation.

Detailed ways

The technical solutions and technical effects of the embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings of the present invention.

Example 1

This embodiment provides a method for establishing a stable genetic transformation system of Ningxia wolfberry, comprising the following steps:

(1) Obtaining Ningxia wolfberry explants: Selecting mature seeds of ‘Ningqi No. 1’ with full morphology, disinfecting them with 75% alcohol for 1 min, disinfecting them with sodium hypochlorite solution for 10-20 min, and rinsing them with sterile water for 4 times, inoculating them in 1/2MS solid culture medium (2.22 g/L MS powder, 20 g/L sucrose, 1 L/L H2O, 7.5 g/L agar), culturing them in the dark at 25°C for 3 days, and then culturing them in the light for one week to obtain sterile seedlings, and cutting the cotyledons of the sterile seedlings that have just unfolded from the middle to obtain Ningxia wolfberry explants;

(2) Construction of overexpression vector: PCR amplification of the full-length sequence of Ningxia wolfberry sugar metabolism gene LbGALA3 was performed, and the amplified target gene fragment was connected to the vector CAM-FLAG-GFP, and transformed into Escherichia coli to obtain a recombinant plasmid. Single clones were selected for colony PCR amplification test, and plasmids were extracted and sequenced. If the sequencing was correct, the overexpression vector was successfully constructed;

In this embodiment, the primer sequences used for PCR amplification of the target gene fragment are as follows:

SEQ ID NO: 1 (LbGALA3-CF-GFP-F:TGGAGAGGACACGCTCGAGATGGC AATGTCCAGTGGC);

SEQ ID NO:2(LbGALA3-CF-GFP-R:CATCCTTGTAGTCGAATTCTTCTATTGAAGTGTGAGTCGCTC);

The reaction system is as follows:

2×P515 enzyme 20μL 10 μM upstream primer 1μL 10 μM downstream primer 1μL template 3μL Sterilized _ddH2O 15μL total capacity 40μL

The reaction program was: 95°C for 10 min, 95°C for 30 s, 57°C for 30 s, 72°C for 30 s/kb, 30 cycles; 72°C for 10 min.

The PCR reaction product is used for agarose gel electrophoresis. First, prepare 1% agarose gel, add the PCR product to the gel well, and place it in the electrophoresis tank for electrophoresis. The voltage is 60-100V, and the sample moves from the negative pole (black) to the positive pole (red). When bromophenol blue moves to about 1 cm from the bottom edge of the gel plate, stop electrophoresis. Observe under ultraviolet light, and fluorescent bands will appear if DNA is present. Use a gel imaging system to take pictures and save. Quickly cut the gel containing the target band under ultraviolet light, transfer it to a 2.0mL centrifuge tube, and recover the target fragment according to the instructions of the Magen gel recovery kit (HiPure Gel Pure DNA Mini Kit), and store it at -20℃ for later use.

The process of connecting the target gene fragment with the vector CAM-FLAG-GFP is as follows: 5'NdeI and 3'EcoRI double restriction sites and protective bases are added to both ends of the CDS of the CAM-FLAG-GFP gene by PCR. After restriction digestion, the results are detected by agarose gel electrophoresis, and the gel corresponding to the band of the large vector fragment is cut out. The restriction digestion product is recovered by the Biotech gel recovery kit. The double restriction digestion reaction system is as follows:

5×Tango buffer 8.0μL XOt 1μL EcoRI 1μL Plasmids 10 μg _ddH2O 20μL total capacity 40μL

The reaction procedure is: enzyme digestion at 37℃ for 40min; inactivation at 65℃ for 15min; when the target fragment is connected to the vector, add reaction buffer to the PCR tube, add the target DNA fragment and the linearized vector in a certain molar ratio, add homologous recombinase, and then mix well and keep warm at 50℃ for 15min.

The transformation process of E. coli is as follows: (1) Thaw a tube of 50 μL of E. coli competent cells on ice, the competent cell efficiency is >5×10 ⁶ cfu/μg, and gently tap the tube wall to resuspend the cells; add 10 μL of recombinant plasmid to the competent cells, tap a few times, and incubate on ice for 30 min; (2) Heat shock in a 42°C water bath for 45 s and quickly put on ice for 2 min; (3) Add 70 μL LB liquid culture medium and incubate on a shaker at 37°C for 60 min; (4) Take 400 μL of bacterial solution in a clean bench and evenly spread it on a plate (str resistance, final concentration is 50 mg/L). After the bacterial solution is absorbed by the solid culture medium, invert it at 37°C overnight, and use colony PCR to identify positive clones.

(3) Preparing Agrobacterium resuspension: The recombinant plasmid verified by sequencing is transformed into Agrobacterium GV3101 competent cells, and streaked on LB solid medium containing 100 mg/L spectinomycin or kanamycin and 100 mg/L gentamicin. After inverted culture at 27°C for 2-3 days, a colony PCR amplification test is performed to screen positive colonies, and a single clone is picked and inoculated into LB liquid medium to prepare an Agrobacterium resuspension with an _OD600 of 0.4-1.2, which is stored at -80°C for later use;

(4) Infection: Add the Ningxia wolfberry explants to a culture dish containing Agrobacterium resuspension and infect for 8 to 10 minutes. During the infection process, gently shake the culture dish at intervals to promote Agrobacterium infection of the explants.

(5) Subculture: After infection, the explants were transferred to sterile filter paper, and the excess bacterial liquid was sucked off and inoculated into co-culture medium (4.43 g/L MS dry powder, 30 g/L sucrose, 7.5 g/L agar, 1 L H ₂ O) for co-culture until callus tissue was formed. The explants after co-culture were washed with sterile water for 5 to 6 times, transferred to sterile filter paper, and the excess liquid was sucked off and inoculated with the back side facing up into differentiation medium (4.43 g/L MS dry powder, 30 g/L sucrose, 7.5 g/L agar, 1 L H ₂ O) for differentiation culture to induce buds. The differentiated resistant buds were transferred to 1/2MS resistance medium (2.22 g/L MS powder, 20 g/L sucrose, 1 L/L H ₂ O, 7.5 g/L agar) for resistance culture, and roots were formed to form complete plants;

(6) Screening positive plants: Use fluorescent protein excitation light source to quickly detect rooted plants and verify them through PCR to screen out Ningxia wolfberry transgenic plants that overexpress the LbGALA3 gene, and then expand and transplant the successfully transformed Ningxia wolfberry.

FIG. 1 is a schematic diagram of the construction principle of the gene LbGALA3 overexpression vector driven by the CaMV 35S promoter fused with GFP.

Figure 2 is the result of PCR amplification of the full-length sequence of the target gene LbGALA3. The results show that the present invention successfully cloned the full-length CDS of the Ningxia wolfberry sugar metabolism gene LbGALA3. The LbGALA3 gene sequence is shown in (SEQ ID NO: 3).

FIG3 is the result of PCR amplification test of a single E. coli colony. Lane M in the figure is DL2000, and lanes 1-8 are transformed E. coli colonies. The result shows that the LbGALA3 gene has been successfully transferred into E. coli.

Figure 4 is a diagram of the stages of the genetic transformation system, in which A is an explant prepared from sterile seedling leaves obtained from the seeds of Ningxia wolfberry 'Ningqi No. 1', B is the induction of callus formation in the explant culture medium, C is the induction of bud differentiation, and D is the transfer of the differentiated resistant buds to the 1/2MS resistance culture medium to form a complete plantlet.

The rooted bottle seedlings were quickly tested using a fluorescent protein excitation light source and positive plants were screened by PCR verification. Figure 5 shows the results of rapid screening of LbGALA3 positive plants using a fluorescent protein excitation light source. In the figure, A is a fluorescent light source for photographing LbGALA3 transgenic callus, B is a fluorescent light source for photographing LbGALA3 transgenic induced buds, and C is a fluorescent light source for photographing LbGALA3 transgenic plants. The results show that positive plants have obvious green fluorescence.

The genomic DNA of the resistant plants and the untransformed control materials was extracted by SDS method, and then agarose gel electrophoresis was performed, and then EB staining was used for observation and photography in a gel imaging system. The specific operation method is as follows: 1) 100 mg of resistant and control plant leaves were respectively ground in 500 μL lysis buffer and collected in a 2 mL centrifuge tube; 2) 65°C water bath for 30 minutes, inverting several times in the middle to mix; 3) Add an equal volume of 500 μL of DNA extractant, invert to mix; 4) centrifuge at 12000rpm for 10min; 5) pipette the supernatant into another clean 2mL centrifuge tube, add 0.7 times the volume of 350μL isopropanol, invert to mix, and place on ice for 5min; 6) centrifuge at 12000rpm for 2min, discard the supernatant, invert the tube with the mouth downward for 1min to remove the isopropanol; 7) add 500μL of 70% anhydrous ethanol to rinse the precipitate, centrifuge at 12000rpm for 1min, and discard the supernatant; 8) repeat step 7); 9) use a pipette to suck out as much residual anhydrous ethanol as possible, leave at room temperature for several minutes, and remove the residual anhydrous ethanol; 10) add 50μl TE to dissolve the DNA precipitate, and store at 20℃. Take 3μL DNA for electrophoresis on 1% agarose gel, observe the DNA extraction, observe with gel imaging system, and take pictures.

The results are shown in Figure 6, where lane M is DL2000, lanes 1 to 15 are LbGALA3-T0 generation strains, lane WT is a negative control (wild type), lane CK is a blank control (ddH ₂ O), and lane P is a positive control (Agrobacterium liquid). It can be seen that the resistant plants transformed with the LbGALA3 gene amplified a band at 480bp, and the band length was consistent with the positive control. Nine positive seedlings were obtained in the LbGALA3-T0 generation strains, namely 2, 3, 4, 7, 8, 10, 11, 11, 12, and 13, respectively, and the transformation rate reached 60%. The untransformed control plants had no bands at the same position, which preliminarily proved that the Ningxia wolfberry transgenic plants with overexpression of the LbGALA3 gene were obtained.

FIG. 7 shows the status of the identified T0 generation transgenic positive seedlings of LbGALA3 after propagation and transplantation, indicating that the plants can grow normally.

The above disclosure is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. A person skilled in the art can understand that all or part of the processes of the above embodiments and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims (9)

1. A method for establishing a stable genetic transformation system of Ningxia wolfberry is characterized by comprising the following steps: the method comprises the following steps:

(1) Acquiring an Ningxia wolfberry explant: cutting the cotyledon of the aseptic seedling of the cultivated Ningxia wolfberry from the middle to obtain an explant of the Ningxia wolfberry;

(2) Constructing an over-expression vector: carrying out PCR amplification on the full-length sequence of the Lycium barbarum sugar metabolism gene LbGALA, connecting the amplified target gene fragment with a vector CAM-FLAG-GFP, and then converting the target gene fragment into escherichia coli to obtain a recombinant plasmid, selecting a monoclonal to carry out colony PCR amplification test, extracting plasmid sequencing, and successfully constructing an over-expression vector if sequencing is correct;

(3) Preparing agrobacterium resuspension: transforming recombinant plasmid with correct sequencing verification into agrobacterium GV3101 competent cells, streaking and culturing on LB solid medium containing antibiotics, performing inversion culture at 27 ℃ for 2-3 days, performing colony PCR amplification test, screening out positive colonies, picking up monoclonal and inoculating to LB liquid medium, and preparing agrobacterium heavy suspension with OD ₆₀₀ = 0.4-1.2;

(4) Dip dyeing: adding the precultured Ningxia wolfberry explant into a culture dish containing agrobacterium tumefaciens heavy suspension to infect for 8-10 min, and slightly shaking the culture dish at intervals in the infection process to promote the agrobacterium tumefaciens to infect the explant;

(5) Subculture: transferring the explant after infection to a co-culture medium for co-culture, transferring the co-cultured explant to a differentiation medium for differentiation culture, inducing bud growth, transferring the differentiated resistant bud to a 1/2MS resistant medium for resistance culture, and forming a complete plant after rooting;

(6) Screening positive plants: and (3) rapidly detecting the rooted plants by using a fluorescent protein excitation light source, verifying by PCR, screening LbGALA gene over-expressed Ningxia wolfberry transgenic plants, and propagating and transplanting the successfully transformed Ningxia wolfberry plants.

2. The method for establishing a stable genetic transformation system of Ningxia wolfberry according to claim 1, wherein the method comprises the following steps: in the step (2), the primer sequence adopted by the target gene fragment amplified by PCR is shown as SEQ ID NO. 1 and SEQ ID NO. 2;

the reaction system is as follows:

2 XP 515 enzyme 20μL 10 Mu M of upstream primer 1μL 10 Mu M downstream primer 1μL Template 3μL Sterilizing ddH ₂ O 15μL Total volume of 40μL

；

The reaction procedure is: 95℃for 10min,95℃for 30s,57℃for 30s,72℃for 30s/kb,30 cycles; and at 72℃for 10min.

3. The method for establishing a stable genetic transformation system of Ningxia wolfberry according to claim 1, wherein the method comprises the following steps: in the step (1), the cultivation of the aseptic seedlings of the Ningxia wolfberry is as follows: sterilizing mature seeds of Ningxia wolfberry with 75% alcohol for 1min, sterilizing with sodium hypochlorite solution for 10-20 min, washing with sterile water for 4 times, inoculating into 1/2MS solid culture medium, dark culturing at 25deg.C for 3 days, and light culturing for one week to obtain aseptic seedlings of Ningxia wolfberry; the 1/2MS resistant culture medium comprises 2.22g/L MS powder, 20g/L sucrose, 1L/L H ₂ O and 7.5g/L agar.

4. The method for establishing a stable genetic transformation system of Ningxia wolfberry according to claim 1, wherein the method comprises the following steps: in the step (2), the connection process of the target gene fragment and the carrier CAM-FLAG-GFP is as follows: and adding a 5'NdeI and 3' EcoRI double cleavage site and a protective base at two ends of the CDS of the CAM-FLAG-GFP gene by PCR, performing enzyme digestion, performing result detection by agarose gel electrophoresis, cutting gel of a corresponding strip of a large carrier fragment, and recovering enzyme digestion products by using a Baitaike gel recovery kit, wherein a double enzyme digestion reaction system is as follows:

5xTango buffer 8.0μL XhoI 1μL EcoRI 1μL Plasmid(s) 10μg ddH₂O 20μL Total volume of 40μL

；

The reaction procedure is: enzyme cutting at 37 ℃ for 40min;65 ℃ C: inactivating for 15 min; when the target fragment is connected with the carrier, a reaction buffer solution is added into a PCR tube, the target DNA fragment and the linearization carrier are added according to a certain molar ratio, homologous recombinase is added, and then the mixture is uniformly mixed and then is kept at 50 ℃ for 15min.

5. The method for establishing a stable genetic transformation system of Ningxia wolfberry according to claim 1, wherein the method comprises the following steps: in the step (3), in the LB solid culture medium and the LB liquid culture medium, the antibiotics are mixed antibiotics of spectinomycin and gentamicin, the content of the spectinomycin is 100mg/L, and the content of the gentamicin is 100mg/L.

6. The method for establishing a stable genetic transformation system of Ningxia wolfberry according to claim 1, wherein the method comprises the following steps: in the step (3), in the LB solid medium and the LB liquid medium, the antibiotics are mixed antibiotics of kanamycin and gentamicin, the content of kanamycin is 100mg/L, and the content of gentamicin is 100mg/L.

7. The method for establishing a stable genetic transformation system of Ningxia wolfberry according to claim 1, wherein the method comprises the following steps: the components of the co-culture and differentiation culture medium are 4.43g/L MS dry powder, 30g/L sucrose, 7.5g/L agar and 1L/L; the 1/2MS resistant culture medium comprises 2.22g/L MS powder, 20g/L sucrose, 1LH ₂ O and 7.5g/L agar.

8. The method for establishing a stable genetic transformation system of Ningxia wolfberry according to claim 1, wherein the method comprises the following steps: the variety of Ningxia wolfberry is Ningqi No. 1.

9. Use of a stable genetic transformation system of lycium barbarum obtained by the method for establishing the stable genetic transformation system of lycium barbarum according to any one of claims 1-8 in the identification of the functions of the genes of the lycium barbarum.