Lycium barbarum efficient genetic transformation system and application

Abstract

The invention relates to the field of plant genetic transformation, in particular to a wolfberry efficient genetic transformation system and application. The method comprises the following steps: by taking root tissues of sterile Chinese wolfberry seedlings as explants, carrying out mediation infection on a visual plant gene expression vector through an agrobacterium GV3101 strain, and adding melatonin into culture media for co-culture, callus induction, adventitious bud and adventitious root differentiation and selection and the like, so as to differentiate and regenerate plant cells, balance ion penetration and promote the growth of the plant cells. The method plays a key role in plant hormone regulation and the like, and the genetic transformation efficiency of the lycium barbarum is remarkably improved. The visual plant gene expression vector used in the invention comprises three genes CYP76AD1, DODA and GT optimized by solanaceae plant codons and a connecting sequence capable of carrying out self-cleavage among the genes, has the advantages of naked eye visualization, no damage and the like, can be used as a marker gene to track gene expression or visualize a gene transformation process, optimizes a gene transformation system, and improves the expression efficiency of solanaceae plants. The genetic transformation efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the field of plant tissue culture and genetic (gene) transformation, and in particular to a wolfberry efficient genetic transformation system and application. This application claims the priority of CN2024108389191. Background Art

[0002] Ningxia wolfberry (Lycium barbarum L.) is a plant of the genus Lycium in the Solanaceae family. It has become a traditional precious Chinese medicinal material in my country. It has the effects of nourishing the kidney and liver, moistening the lungs and improving eyesight, and is highly valued by Chinese and foreign medical scientists and diet therapy experts. Using genetic transformation, gene editing and other technologies to cultivate special high-quality new varieties for fresh consumption, medicinal use, leaf use, and processing, and accelerating the high output value of Ningxia wolfberry, is the key to achieving high-quality development of Ningxia's wolfberry industry. To this end, a stable and efficient wolfberry tissue culture and genetic (gene) transformation system was established.

[0003] At present, the efficiency of the reported wolfberry genetic transformation system is generally very low, and the marker genes used in plant genetic transformation at this stage are mostly limited to β-glucuronidase (GUS) and green fluorescent protein (GFP) genes. GUS chemical tissue staining requires the addition of expensive substrate X-Gluc, and is invasive, and the observed materials cannot be further cultured; GFP requires a specific light source to visualize the fluorescent signal, which is not practical under natural conditions. Therefore, selecting appropriate marker genes and improving the efficiency of the genetic transformation system are major problems that need to be solved in the genetic transformation of wolfberries. Summary of the invention

[0004] In order to achieve efficient and stable genetic transformation of wolfberry, the present invention has established a technical system that uses the root tissue of wolfberry as the genetic transformation material (explant), uses Agrobacterium GV3101 containing the visualized plant gene expression vector pNULPGE706 as the infection strain, and obtains wolfberry genetic transformation regeneration positive plants through co-cultivation and selection cultivation.

[0005] The present invention also constructs a visualized plant gene expression vector pNULPGE706 ( Figure 1), namely CYP76AD1 (P450oxygenase, cytochrome P450 monooxygenase), DODA (L-3,4-dihydroxyphenylalanine 4,5-dioxygenase, L-3,4-dihydroxyphenylalanine 4,5-dioxygenase) and GT (glucosyltransferase, glucosyltransferase) and the connection sequence between genes that can perform self-cleavage. The expression efficiency and stability of the genetic transformation system provided by the present invention in wolfberry are effectively improved, and the three gene sequences are also codon-optimized for Solanaceae plants. After the three genes are introduced into plant cells, they can catalyze tyrosine to produce betaine, presenting a very bright red color, and have the advantages of being visible to the naked eye and damage-free without the need for chemical staining or special light sources. They can be used as marker genes to track gene expression or visualize the gene transformation process, and optimize the Ningxia wolfberry gene transformation system to improve the genetic transformation efficiency (such as Figure 2-Figure 5 shown).

[0006] To achieve the technical purpose of the present invention, the present invention provides an efficient genetic transformation system for wolfberry, using root tissue as the material for gene transformation, using Agrobacterium GV3101 containing a visualized plant gene expression vector pNULPGE706 as the infection strain, and sequentially using a co-culture medium and a selection culture medium added with melatonin to induce callus tissue and differentiate adventitious buds and adventitious roots, thereby obtaining wolfberry genetically transformed regenerated positive plants.

[0007] In particular, the tissue culture regeneration system using root tissue as explant has the advantages of high callus induction, adventitious bud differentiation efficiency, short cycle, and large reproduction coefficient.

[0008] In particular, melatonin was added to the culture medium for Agrobacterium infection and co-cultivation, callus induction, adventitious bud and adventitious root differentiation and selection. Melatonin plays a key role in regulating plant cell differentiation and regeneration, ion permeation balance, and plant hormone regulation, thus improving the efficiency of wolfberry genetic transformation.

[0009] To achieve the technical purpose of the present invention, the second aspect of the present invention provides a method for constructing a wolfberry genetic transformation system, comprising: taking the root tissue of wolfberry and placing it into an infection solution prepared by GV3101 Agrobacterium with a pNULPGE706 visualized plant gene expression vector for infection to obtain infected root tissue; after co-culturing the infected root tissue, transferring it to a screening culture medium for selection, and finally subculturing the selected positive adventitious buds to obtain wolfberry gene transformation positive plants.

[0010] To achieve the technical purpose of the present invention, the present invention provides a visualization plant gene expression vector pNULPGE706 ( Figure 1 ), including three gene fragments that have been codon-optimized in Solanaceae plants, namely CYP76AD1 (P450oxygenase, cytochrome P450 monooxygenase), DODA (L-3,4-dihydroxyphenylalanine 4,5-dioxygenase, L-3,4-dihydroxyphenylalanine 4,5-dioxygenase) and GT (glucosyltransferase, glucosyltransferase) and a connection sequence between genes that can self-cleave. The expression efficiency and stability of the genetic transformation system provided by the present invention in wolfberry are effectively improved. After the three genes are introduced into plant cells, they can catalyze tyrosine to produce betaine, showing a very bright red color. It has the advantages of being visible to the naked eye and non-damaged under conditions without chemical staining or special light sources. It can be used as a marker gene to track gene expression or visualize the gene transformation process, optimize the wolfberry gene transformation system, and improve the genetic transformation efficiency (such as Figure 2-Figure 5 shown).

[0011] In particular, the GC percentage of the Solanaceae codon-optimized CYP76AD1 sequence was 36.35%, and the codon adaptation index (CAI) was 0.9.

[0012] Specifically, the CYP76AD1 sequence optimized by Solanaceae codons is shown in SEQ ID NO.1 (eg Figure 6 shown).

[0013] In particular, the GC percentage of the Solanaceae codon-optimized DODA sequence was 39.86%, and the codon adaptation index (CAI) was 0.91.

[0014] Specifically, the DODA sequence optimized by the Solanaceae codon is shown in SEQ ID NO.2 (eg Figure 7 shown).

[0015] In particular, the GT sequence after Solanaceae codon optimization had a GC ratio of 38.06% and a codon adaptation index (CAI) of 0.91.

[0016] Specifically, the GT sequence optimized by Solanaceae codon is shown in SEQ ID NO.3 (eg Figure 8 shown).

[0017] It should be noted that the codon optimization referred to in the present invention can be achieved by conventional bioinformatics software, and the present invention does not impose any particular limitation.

[0018] In particular, after CYP76AD1, DODA and GT of the visualized plant gene expression vector are introduced into plant cells, tyrosine in the plant can be converted into bright red betalain.

[0019] To achieve the technical purpose of the present invention, the fourth aspect of the present invention provides a PCR identification primer for screening the above-mentioned visualized plant gene expression vector and genetically transformed regenerated plants, and the primer sequences are shown in SEQ ID NO.4 and SEQ ID NO.5:

[0020] SEQ ID NO.4 (Sense): 5'-CCCAAGAGGAAATCAAGCAGGTC-3';

[0021] SEQ ID NO. 5 (Anti-sense): 5'-GTGTAGTTGCGGAGGATGGAGAAG-3'.

[0022] In order to achieve the technical purpose of the present invention, the fifth aspect of the present invention provides an application of the above-mentioned expression vector, and the application is the establishment and optimization of a plant gene transformation system.

[0023] To achieve the technical purpose of the present invention, the sixth aspect of the present invention provides an application of a plant gene transformation system, comprising the steps of using the above-mentioned vector, the above-mentioned application, and the above-mentioned primers.

[0024] In particular, the plant in the method is wolfberry.

[0025] Preferably, the plant in the method is Ningxia wolfberry.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention establishes a complete and efficient tissue culture regeneration system using the root tissue of the aseptic wolfberry seedlings as explants. Compared with the current tissue culture that mostly uses cotyledons, hypocotyls, leaves, petioles, and stem segments as explants, the present invention has the advantages of high callus induction, adventitious bud differentiation efficiency, short cycle, and large reproduction coefficient. It can be applied to large-scale factory seedling cultivation and solve the urgent problem of asexual reproduction of wolfberry high-quality germplasm resources.

[0028] (2) The present invention uses a multi-gene combination method to construct a visualized plant gene expression vector pNULPGE706 containing three gene fragments of CYP76AD1, DODA and GT. The three gene sequences of the vector are codon-optimized according to the Solanaceae plants to further improve the expression efficiency and stability. After being introduced into plant cells, it can catalyze tyrosine to produce betaine, which presents a very bright red color. It has the advantages of being visible to the naked eye and damage-free without the need for chemical staining or special light sources. It can be used as a marker gene to track gene expression or visualize the gene transformation process, optimize the genetic transformation system, and significantly improve the transformation efficiency. The marker genes commonly used in current plant genetic transformation mainly include GUS (β-glucuronidase) and GFP (green-fluorescent protein) genes. The former can be stained with histochemical methods and monitored during the transient expression stage or the entire differentiation process after the explant is infected with Agrobacterium. Its disadvantage is that the material used can no longer be cultured. Although the latter can use a fluorescence microscope to observe the living cells or tissues of transgenic plants without damaging the materials used, it requires a special visual fluorescent light source and is often affected by false fluorescence signals from the plant cells or tissues themselves.

[0029] (3) The present invention establishes a complete and efficient genetic transformation system of wolfberry mediated by Agrobacterium, and adds melatonin to the culture medium for Agrobacterium infection and co-cultivation, callus induction, adventitious bud and adventitious root differentiation and selection. Melatonin plays a key role in regulating plant cell differentiation and regeneration, ion permeation balance, and plant hormone regulation, improves the genetic transformation efficiency of wolfberry, ensures the uniformity and robustness of the proliferated seedlings, effectively promotes the rooting of tissue culture seedlings, and the callus rate reaches more than 93.33%, and the budding rate reaches 87.50%. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the T-DNA part of the visualized plant gene expression vector pNULPGE706 constructed in the embodiment of the present invention;

[0031] Figure 2 In the embodiment of the present invention, the root tissue of the aseptic wolfberry seedlings is used as the explant, and the Agrobacterium GV3101 is used as the infection strain to induce the callus tissue;

[0032] Figure 3 The differentiation effect of adventitious buds in the embodiment of the present invention;

[0033] Figure 4 The results are as follows: the subculture of adventitious buds and the differentiation of adventitious roots in the embodiments of the present invention;

[0034] Figure 5The positive plants of Lycium barbarum genetic transformation obtained through selection and identification in the embodiment of the present invention;

[0035] Figure 6 The CYP76AD1 sequence SEQ ID NO.1 of the present invention is optimized by codons of Solanaceae plants;

[0036] Figure 7 SEQ ID NO. 2 is the DODA sequence of the present invention that is codon-optimized for Solanaceae plants;

[0037] Figure 8 The GT sequence SEQ ID NO. 3 of the present invention is optimized by Solanaceae plant codons. DETAILED DESCRIPTION

[0038] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0039] Materials: The Ningxia wolfberry 'Zhongxuan No. 1' cultivated in our laboratory for many years was used as the plant material; the heat shock competent Agrobacterium strains were GV3101, LBA4404, Agl1 and EHA105; Kanamycin sulfate (Kan), carbelicilin sodium (Car), Timentin (Tim), cefotaxime (Cef) and other antibiotics were purchased from Beijing Coolaibo Technology Co., Ltd.; MS (Murashige and Skoog) medium, sucrose (Sucrose), agar (Agar), melatonin (MT), 6-benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA), zeatin (ZT), acetosyringone (Ace) and other reagents were purchased from Beijing Solable Technology Co., Ltd.

[0040] Example 1 Construction of Lycium barbarum genetic transformation system

[0041] 1. Explant culture

[0042] Trim the branches of Lycium barbarum 'Zhongxuan No. 1' into 5-7cm (with 3-4 bud nodes), put them into a sterilized triangular bottle, add 70% alcohol, sterilize the surface for 30 seconds, pour out the alcohol; add sterile water to wash once, pour out the sterile water; then add 1% sodium hypochlorite solution (NaClO, the original solution Cl content is 5%), sterilize for 20 minutes, shake in the middle, pour out the disinfectant; wash with sterile water for more than 5 times, absorb excess water with sterilized filter paper, and insert the branches into sterile seedling culture medium. The composition of sterile seedling culture medium is 1 / 2MS culture medium with sucrose and agar added, specifically: 2.21g·L -1 MS+15g·L -1 Sucrose +7g·L -1 Agar. Incubate in a 25°C, 5000 lux, 16-hour lighting incubator. Subculture once every 4 weeks to obtain wolfberry sterile seedlings containing root tissue.

[0043] It should be noted that the sterile wolfberry seedlings can also be obtained by conventional techniques in the art, and the present invention is not limited thereto.

[0044] 2. Construction of visual plant gene expression vector

[0045] 2.1 Gene codon optimization and multi-gene linkage

[0046] The sugar beet CYP76AD1, DODA and Mirabilis jalapa GT genes were codon-optimized for Solanaceae plants. The GC ratio of the CYP76AD1 sequence after codon optimization for Solanaceae plants was 36.35%, and the codon adaptation index (CAI) was 0.9. The optimized sequence is shown in SEQ ID NO.1. For specific sequences, see Figure 6 The GC ratio of the Solanaceae codon-optimized DODA sequence is 39.86%, and the codon adaptation index (CAI) is 0.91. The optimized sequence is shown in SEQ ID NO.2. For the specific sequence, see Figure 7 The GC ratio of the GT sequence optimized by Solanaceae plants is 38.06%, and the codon adaptation index (CAI) is 0.91. The sequence is shown in SEQ ID NO.3. For the specific sequence, see Figure 8 After the termination codons of the first two genes were removed, they were connected in sequence using P2A (GSGATNFSLLKQAGDVEENPGP) and then constructed into the Agrobacterium binuclear plasmid to obtain a visualized plant gene expression vector named pNULPGE706 (e.g. Figure 1 shown).

[0047] It should be pointed out that any order of connection of the optimized CYP76AD1, DODA and GT genes can achieve the technical purpose of the present invention, and the present invention is not limited thereto.

[0048] It should be noted that codon optimization can be achieved using bioinformatics software commonly used in the art, and the present invention is not limited thereto.

[0049] 2.2 Transformation of Agrobacterium competent cells

[0050] About 200 ng of pNULPGE706 was added to heat shock competent cells of Agrobacterium GV3101, LBA4404, Agl1 and EHA105 for heat shock transformation. The transformed Agrobacterium single colony was tested for PCR positive, and the primer sequences were:

[0051] SEQ ID NO.4 (Sense), 5'-CCCAAGAGGAAATCAAGCAGGTC-3';

[0052] SEQ ID NO. 5 (Anti-Sense), 5'-GTGTAGTTGCGGAGGATGGAGAAG-3'.

[0053] Positive monoclonal strains GV3101, LBA4404, Agl1 and EHA105 containing pNULPGE706 vector were screened and stored at -80°C for future use.

[0054] 3. Genetic transformation

[0055] 3.1 Preparation of Agrobacterium infection solution

[0056] Take the positive Agrobacterium GV3101 containing the vector pNULPGE706 selected in step 2.2 and place it on ice to melt, streak it on the solid LB medium with a disposable inoculation loop, and place it in a 37°C incubator for activation for 48 hours to obtain activated bacteria; scrape a small amount of activated bacteria with an inoculation loop and add it to the infection solution (as shown in Table 1), culture at 28°C, 180-200 times / min shaking until the logarithmic growth stage, and collect the bacteria by centrifugation at 4°C, 5500rpm for 10 minutes. Adjust the concentration OD with the infection solution 550 =0.25, and obtain Agrobacterium infection solution.

[0057] Table 1 Agrobacterium infection solution

[0058]

[0059] Melatonin (MT) plays an important role in regulating plant growth and development and biotic and abiotic stresses. For example, melatonin is involved in biological processes such as leaf senescence, flowering, fruit ripening and root development. The present invention uses it as one of the components of the infection solution for genetic transformation of wolfberry for the first time, especially using the root system as an explant to infect in the infection solution with melatonin added, which promotes the transformation of the target gene and its integration into the genome in the root tissue of wolfberry. Adding melatonin can not only reduce the browning, necrosis and vitrification of the explant during the Agrobacterium-mediated transformation, but also enhance the vitality of Agrobacterium, promote the growth of the explant, and realize and improve the efficiency of Agrobacterium-mediated genetic transformation of wolfberry.

[0060] It should be noted that the concentration of Agrobacterium is an important factor affecting the efficiency of genetic transformation. Too high a concentration will cause toxicity and necrosis of the explants, while too low a concentration will affect its transformation efficiency. The present invention determines that an Agrobacterium concentration OD550 of 0.25 can achieve efficient transformation with roots as explants, with significant effects.

[0061] 3.2 Explant infection

[0062] Select the young root tissue of sterile seedlings subcultured for about 45 days as explants, with a length of 0.5-1 cm. Put it into the Agrobacterium infection solution (as shown in Table 1) for 3 minutes, collect the explants and place them on sterile filter paper, and after absorbing the liquid, move them to the co-culture medium (as shown in Table 2), place 15-20 explants in each culture dish, and culture them in the dark at 22°C for 3 days.

[0063] Table 2 Co-culture medium

[0064]

[0065] 3.3 Callus induction, adventitious bud differentiation and selection

[0066] After the co-cultivation, the explants were transferred to the callus induction, adventitious bud differentiation and selection medium (as shown in Table 3) and cultured at 25°C for 16 hours per day. Generally, the formation of red callus can be observed in 7-10 days (as shown in Table 3). Figure 2 After about 20 days, red adventitious buds can be observed to differentiate (as shown in Figure 3 The same culture medium was used for subculture every 4 weeks, during which the adventitious buds with good growth could be transferred to the test tube of adventitious root differentiation medium (as shown in Table 3 and Table 4) for further culture (as shown in Table 3 and Table 4). Figure 4 ) and adventitious root differentiation (as shown Figure 5As shown). From the above genetic transformation process, it can be seen that the tissue culture regeneration system established by the present invention with the root tissue of the aseptic wolfberry seedling as the explant, and the Agrobacterium-mediated wolfberry genetic transformation system are highly efficient; the visualization gene expression vector expresses stably, and the gene expression or visualization gene transformation process can be tracked without the aid of special light source conditions or chemical staining. The regenerated red plants are identified by PCR and are determined to be positive plants with successful gene transformation. The identification primer sequences are:

[0067] SEQ ID NO.4 (Sense): 5'-CCCAAGAGGAAATCAAGCAGGTC-3';

[0068] SEQ ID NO. 5 (Anti-Sense): 5'-GTGTAGTTGCGGAGGATGGAGAAG-3'.

[0069] Table 3 Callus induction, adventitious bud differentiation and selection medium

[0070]

[0071] Figure 4 This is the seedling stage of the genetically transformed wolfberry plant. Figure 5 It is the seedling stage of the genetically transformed wolfberry plants. Figure 4 and Figure 5 It can be observed that at different growth stages of transgenic positive plants, tissues such as leaves, stem segments and roots all show a deep red color visible to the naked eye, indicating that a large amount of betalain is accumulated in all tissues of the entire plant. The three gene fragments CYP76AD1, DODA and GT in the pNULPGE706 visualized plant gene expression vector constructed by the present invention can be used as marker genes to track gene expression or visualize the gene conversion process, which is convenient and fast, reduces costs, improves the accuracy of screening and cultivating positive plants, and shortens the screening cycle.

[0072] Table 4 Adventitious root differentiation medium

[0073]

[0074] The present application adds melatonin (as shown in Tables 1-3) to culture media for Agrobacterium infection and co-cultivation, callus induction, adventitious bud differentiation and selection, which plays a key role in regulating plant cell differentiation and regeneration, ion permeation balance, and plant hormone regulation, thereby improving the genetic transformation efficiency of wolfberry.

[0075] In order to screen and obtain a system capable of constructing a genetic transformation of wolfberry and achieving efficient expression, the inventors conducted a large amount of experimental processing work. The following are some comparative experiments.

[0076] Comparative Test Example 1 Test of Different Explants

[0077] Theoretically, plant cells are omnipotent. At present, cotyledons, hypocotyls, embryos, stem segments and leaves are commonly used as explants for tissue culture and genetic transformation. However, due to different plant species, the degree of difficulty and transformation efficiency vary. In a comparative test of the present invention, leaves, petioles, stem segments and roots are used as explants, Agrobacterium GV3101 is used as the infection strain, and the others are compared according to the method in the embodiment. Specifically, sterile seedlings that have been subcultured for about 45 days are also selected, and the leaves are cut into 5×5mm, and the petioles and stem segments are cut into a length of about 0.5-1cm.

[0078] The culture status and genetic transformation status of each explant were observed, and the callus induction rate and adventitious bud differentiation rate of various explants were statistically analyzed (as shown in Table 5).

[0079] Table 5 Callus induction rate and adventitious bud differentiation rate of various explants

[0080]

[0081] The experimental observation found that although the callus induction rate of root and leaf explants was above 90%, the adventitious bud differentiation rate of leaves was only 35%, far lower than the 87.50% of the root. In addition, the callus obtained by root differentiation was denser and in good condition, making it easier to differentiate into a large number of adventitious buds, which grew vigorously and easily obtained regenerated seedlings.

[0082] The present invention uses root tissue as tissue culture and gene transformation material (explant) of wolfberry for the first time, and its induction rate and adventitious bud differentiation rate are much higher than other explants, achieving unexpected results.

[0083] Comparative Test Example 2 Tests with Different Infecting Strains

[0084] In a comparative test of the present invention, roots were used as explants, and Agrobacterium GV3101, Agl1, LBA4404, and EHA105 were used as infection strains. The rest was carried out according to the method in the embodiment, and the callus induction rate and adventitious bud differentiation rate after infection with various strains were statistically analyzed (as shown in Table 6).

[0085] Table 6 Callus induction rate and adventitious bud differentiation rate after infection with various Agrobacterium strains

[0086]

[0087] The experimental observation found that when Agrobacterium EHA105 was used as the infection strain, the explants grew bacteria immediately after infection, and no callus tissue or adventitious buds appeared. Other strains could produce callus tissue and adventitious buds; the Agrobacterium GV3101 strain had a higher callus induction rate and adventitious bud differentiation rate, reaching 95% and 87.5% respectively.

[0088] Comparative Test Example 3 Tests with Different Culture Media

[0089] The present invention conducted a comparative test on the concentrations of hormones ZT, BAP and NAA in the culture medium (as shown in Table 7). The root system was used as the explant, Agrobacterium GV3101 was used as the infection strain, and the rest was carried out according to the method in the embodiment.

[0090] Table 7 Hormone concentration ratio of culture medium

[0091]

[0092] After repeated experiments, it was found that culture medium Lb8 had the best effect, with callus tissue growing larger, firmer and greener, and with the most adventitious buds; followed by culture medium Lb4, Lb5, Lb17, Lb26, and Lb27, with callus tissue smaller in volume, poor in color, and with a small number of adventitious buds; culture medium Lb13, Lb15, and Lb18 had the smallest callus tissue volume, and almost no adventitious buds were generated.

[0093] Comparative Test Example 4 Test of Different Antibiotics

[0094] The present invention conducted a comparative test on the concentration of antibiotic kanamycin in the culture medium (as shown in Table 8). The root system was used as the explant, Agrobacterium GV3101 was used as the infection strain, and the rest was carried out according to the method in the embodiment.

[0095] Table 8 Antibiotic Concentration Ratio

[0096]

[0097] After repeated experiments, it was found that when the kanamycin concentration was 6.25 mg·L -1 When the concentration was 25 mg·L -1 When the concentration was 12.5 mg·L -1 When the genetically transformed wolfberry explants grew moderately, some genetically transformed positive plants were successfully selected, so the final concentration was set at 12.5 mg·L -1 is the optimal screening concentration of the antibiotic kanamycin.

[0098] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0099] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A wolfberry efficient genetic transformation system, characterized in that: Root tissue was used as the material for gene transformation, and Agrobacterium GV3101 containing the visualized plant gene expression vector pNULPGE706 was used as the infection strain. Co-culture medium and selection medium with melatonin added were used in sequence to induce callus tissue and differentiate adventitious buds and adventitious roots, thereby obtaining positive plants for genetic transformation and regeneration of wolfberry.

2. A method for constructing a wolfberry genetic transformation system, characterized in that: include: Taking the root tissue of wolfberry and placing it in the infection solution prepared by GV3101 Agrobacterium containing the visualized plant gene expression vector pNULPGE706 for infection, to obtain the infected root tissue; After co-cultivation of the infected root tissue, the infected root tissue is transferred to a screening medium for selection, and finally the selected positive adventitious buds are subcultured to obtain Lycium barbarum gene-transformed positive plants.

3. A visualized plant gene expression vector, characterized in that: It contains three gene sequences of CYP76AD1, DODA and GT that have been optimized by codons, as well as the connecting sequences between genes that can be self-cleaved; Preferably, the codon optimization is Solanaceae codon optimization.

4. The visualized plant gene expression vector according to claim 3, characterized in that: The CYP76AD1 gene sequence optimized by codons of Solanaceae plants is shown as SEQ ID NO.1; or / and the DODA gene sequence optimized by codons of Solanaceae plants is shown as SEQ ID NO.2; or / and the GT gene sequence optimized by codons of Solanaceae plants is shown as SEQ ID NO.

3.

5. A visualized plant gene expression vector as claimed in any one of claims 3 to 4, characterized in that: After the codon-optimized CYP76AD1, DODA and GT genes are introduced into plant cells, tyrosine in the plant can be converted into bright red betalain.

6. A PCR identification primer for screening genetically transformed regenerated plants, the sequences of which are shown in SEQ ID NO.4 and SEQ ID NO.5; in, The plant is wolfberry, preferably, the plant is Ningxia wolfberry.

7. A PCR identification primer for screening the visualized plant gene expression vector according to any one of claims 3-4, characterized in that: The primer sequences are shown in SEQ ID NO.4 and SEQ ID NO.

5.

8. An application of the visualized plant gene expression vector as claimed in any one of claims 3 to 4, characterized in that: The application is the establishment and optimization of a plant gene transformation system.

9. An application of a plant gene transformation system, characterized in that: The method comprises the steps of using the visualized plant gene expression vector according to any one of claims 3 to 4, the primers according to claims 6 to 7, and the application according to claim 8.

10. The use according to claim 9, characterized in that: The plant is wolfberry; preferably, the plant is Ningxia wolfberry.

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