CN112592925B - Stable genetic transformation system for lycium ruthenicum and application thereof - Google Patents

Abstract

The invention discloses a stable genetic transformation system of lycium ruthenicum and application thereof, and a thornless lycium ruthenicum sucrose synthaseSUSThe sequence of the gene is shown in a sequence table SEQ ID NO. 1; a transgenic plant expression carrier plasmid is prepared through inserting the plant expression carrier in itSUS+XFusion genes; a stable genetic transformation system of lycium ruthenicum: pre-culturing the lycium ruthenicum apex explant, and adding recombinant agrobacterium for infection; inoculating and dark culturing; after co-cultivation, washing, inoculating the explant to a selection medium, and culturing until buds start to grow to form complete resistant plantlets; cutting off the stems with terminal buds, inoculating the stems into a resistant rooting culture medium, and culturing until the buds can root and grow normally; the beneficial effects are that: hormone-free and low-concentration Kana screening resulted in good and robust growth of resistant plants; the fusion gene reduces the toxicity of green fluorescent protein to lycium ruthenicum, and the plant can grow normally and stably carry out genetic transformation with high transformation efficiency; the callus stage is not needed, the transformation period is shortened, and the agrobacterium pollution is reduced.

Description

A stable genetic transformation system of Lycium barbarum and its application

technical field

The invention belongs to the fields of forestry biotechnology and molecular biology, and specifically relates to a stable genetic transformation system of Lycium barbarum and its application.

Background technique

Black fruit wolfberry (Lycium ruthenicum Murr.) is a perennial shrub belonging to the Solanaceae Lycium genus, with a plant height of about 20-50 cm. It is mainly distributed in northwest desert areas such as Ningxia, Qinghai, Gansu, and Xinjiang. It is the dominant and constructive species of desert communities in the Ejina area of the Heihe River Basin, and plays an important role in soil and water conservation, windbreak and sand fixation. Lycium barbarum fruit is rich in active ingredients such as amino acids, anthocyanins, polysaccharides and polyphenols, among which the content of proanthocyanidins is the highest in the fruit, which has high medicinal value and is commonly used in Uyghur and Tibetan medicine. As the ecological value and economic value of Lycium barbarum are gradually paid attention to, the research on the gene function of Lycium barbarum is getting more and more in-depth. At present, the research on Lycium barbarum black fruit mainly focuses on fruit nutritional components, medicinal components, pigment and polysaccharide extraction technology and pharmacological analysis. Studies have found that its fruit components have the functions of anti-radiation, regulating intestinal flora, anti-oxidation, anti-cancer, anti-fatigue, enhancing immunity, and anti-aging. In addition, recent studies have found that Lycium barbarum polyphenols can delay the occurrence and progression of oxidative stress-related neurodegenerative diseases; Lycium barbarum polysaccharides have neuroprotective effects on primary cortical neuron damage induced by oxygen glucose deprivation/reoxygenation in rats . Black fruit wolfberry is also known as "the king of natural proanthocyanidins". Lycium barbarum pigment is a non-toxic grade substance with good food safety. The pigment has the characteristics of strong tinting strength, good stability, and simple processing, and can be widely used in medicine, food, textile and other industries. In conclusion, Lycium barbarum is an important ecological and economical shrub with broad prospects for development and application.

Despite the above-mentioned many advantages, black fruit wolfberry also has production disadvantages such as many thorns, thin peel and easy root rot. The fast and effective stable genetic transformation system of Lycium barbarum can not only be used to reveal the functions of genes related to its key good traits, but also can achieve the purpose of rapid molecular breeding to improve unfavorable traits.

The currently reported stable genetic transformation systems of Lycium barbarum black fruit are all callus-based regeneration systems, and all use Agrobacterium strain GV3101; there are no reports of successful transformation of Lycium barbarum with Agrobacterium strains EHA105 and LBA4404, let alone rapid and direct organogenesis. The successful report of stable genetic transformation of Lycium barbarum black fruit. The screening methods for Agrobacterium-mediated plant stable genetic transformation include positive selection and negative selection. Negative selection based on antibiotics and herbicides is commonly used. However, due to the principle of negative selection, untransformed cells/tissues die on the resistant medium, and part of the cell death usually causes damage to the surrounding successfully transformed cells, resulting in the inability to obtain resistant plants even if there are successfully transformed cells .

Contents of the invention

The purpose of the present invention is to provide a stable genetic transformation system of Lycium barbarum and its application.

The base sequence of the sucrose synthase SUS gene of Lycium barbarum Lycium barbarum is shown in SEQ ID NO.1 of the sequence table.

The utility model relates to a transgenic plant expression vector plasmid, in which SUS is inserted into the plant expression vector.

A transgenic plant expression vector plasmid, in which a SUS + X fusion gene is inserted into the plant expression vector, X represents the transgenic target gene, and the SUS gene is the gene shown in the sequence table SEQ ID NO.1;

The X is the GFP gene;

The plant expression vector is pRI101AN.

A recombinant Agrobacterium, which has transformed the expression vector plasmid of a transgenic plant;

The Agrobacterium is EHA105 or LBA4404.

A stable genetic transformation system of Lycium barbarum, comprising:

1) Pre-cultivate the explants of Lycium barbarum leaf tips for 1-2 days, and then add the recombinant Agrobacterium to infect. The pre-culture medium consists of 4.74 g/l MS dry powder, 40 g/l sucrose and 4.8 g/l of agar composition;

2) After infection, inoculate into the co-cultivation medium and culture in dark until white Agrobacterium grows around the leaf explants; the co-cultivation medium consists of 4.74 g/l MS dry powder, 40 g/l sucrose , 4.8g/l agar and 100 μM AS;

3) After co-cultivation, the explants of Lycium barbarum leaves were washed with sterile water containing 500 mg/l Cef; the explants were inoculated into the selection medium, and cultured until budding began to form complete resistant plantlets; The selection medium of 4.74g/l MS dry powder, the sucrose of 40 g/l, the agar of 4.8g/l, the Kana of 3 mg/l and the Cef of 300mg/l;

4) The buds of the resistant plantlets described in step 3) grow to a height of 4.5~5.5cm, cut off 1.5~2.5cm stems with terminal buds, inoculate them into the resistant rooting medium, and cultivate until the buds can root and can be normal Growth; the resistant rooting medium is composed of MS dry powder of 2.37g/l, sucrose of 20g/l, agar of 4.8 g/l, Kana of 5 mg/l, and Cef of 300 mg/l.

The invention provides the SUS gene of sucrose synthase from Lycium barbarum thorn, its base sequence is shown in the sequence table SEQ ID NO.1; a transgenic plant expression vector plasmid, which is inserted into the plant expression vector with SUS + X A fusion gene, X represents the transgenic target gene, and the SUS gene is the gene shown in the sequence table SEQ ID NO.1; a stable genetic transformation system of Lycium barbarum black fruit, which includes: taking the leaf tip explants of Lycium barbarum black fruit and pre-cultivating them for 1d , adding the recombinant Agrobacterium to infect; after inoculation, inoculate into the co-cultivation medium, and cultivate in dark until white Agrobacterium grows around the leaf explants; after co-cultivation, wash the black wolfberry with sterile water Leaf explants; inoculate the explants into the selection medium, and cultivate until buds start to form complete resistant plantlets; when the buds grow to a height of 4.5-5.5 cm, cut 1.5-2.5 cm stems with terminal buds and inoculate them into In the resistant rooting medium, cultivate until the buds can take root and grow normally;

Beneficial effects of the present invention: Agrobacterium strains EHA105 and LBA4404 are used to achieve stable genetic transformation of Lycium barbarum black fruit, and the transformation efficiency is as high as 8.33-16.65%; thornlessSUS comes from Lycium barbarum thornless; no exogenous hormone is needed in the whole process; no callus is required stage, shorten the transformation cycle, and reduce the possibility of Agrobacterium contamination; low-concentration Kana can achieve the screening effect and reduce the damage to plant materials; hormone-free and low-concentration Kana screening results in good and robust growth of resistant plants; high expression of green fluorescence was found The protein is toxic to Lycium barbarum, and the plants have unfavorable traits such as slow growth , but the fusion gene SUSGFP that highly expresses GFP and thornlessSUS can reduce this toxicity, and the plants can grow normally. It has the characteristics of thick stem, strong ability to divide roots, and a large increase in the number of fibrous roots.

The invention establishes a screening transformation system with very low concentration of kanamycin (Kana). Under this concentration, the non-transformed explants will not form plants, but they will not die quickly; the transformed explants can quickly form healthy resistant plants; a fast, efficient, low-cost, and easy-to-operate black fruit Lycium barbarum stable genetic transformation system, including screening and establishing target gene groups, successfully cloning the full-length CDS of the thornless black fruit sucrose synthase gene ( thornlessSUS ), constructing an overexpression vector containing a single gene GFP and thornlessSUS , and constructing a fusion gene thornlessSUS- GFP overexpression vector, screening and establishment of recipient plant populations, transformation of Lycium barbarum to quickly obtain plants with resistance and expression of target genes, and identification of traits of successfully transgenic plants, etc.

Description of drawings

Figure 1 Structural diagram of the expression unit of the expression vector; (A) pRI-GFP; (B) pRI-SUS; (C) pRI-SUSGFP;

Figure 2 Screening of Kana concentration in leaf explants of Lycium barbarum; A. Rooted leaf explants 14 days after inoculation (control); B. Rooted explants began to sprout 28 days after inoculation (control); C. Kana 30 days after inoculation Concentration is the leaf explant of 3mg/l; D. the leaf explant of Kana concentration higher than 3mg/l after inoculation 30d;

Figure 3 Transgenic plants of Lycium barbarum black fruit transformed with Agrobacterium; A. Untransformed Lycium barbarum leaf tip explants (control) at 30 days of screening culture; B. Rooting leaf tip explants of transformed Lycium barbarum fruit at 17 days of screening culture C-E. Transformed sprouting leaf explants at 30 days of selection; F, G. Transgenic plants inoculated on the rooting medium containing Kana and Cef for 20 days; H. Inoculated on the rooting medium containing Kana and Cef Untransformed plants after 20 days; I. Rooting situation of transgenic plants (left) and untransformed plants (right);

Figure 4 PCR verification of pRI-SUS vector transgenic plants; M: DL2000 DNA Plus Marker; 1-4: Transgenic resistant plants; 5, 6: Negative control, untransformed plants; 7: Positive control, pRI-SUS vector; 9: Blank control, water;

Figure 5 PCR verification of pRI-SUSGFP vector transgenic plants. M: DL2000 DNA Marker; 1-4: Transgenic resistant plants; 5, 6: Negative control, untransformed plants; 7: Positive control, pRI-SUSGFP vector plasmid; 8: Blank control, water;

Figure 6 Semi-quantitative PCR verification of pRI-SUS vector transgenic plants. M: DL2000 DNA Marker; 1: water (control); 2-3: roots of untransformed plants; 4-5: stems of untransformed plants; 6-7: leaves of untransformed plants; 8-9: roots of transformed resistant plants; 10-11: Stems of transformed resistant plants; 12-13: Leaves of transformed resistant plants

Fig. 7 Laser confocal microscope observation of the leaves of Lycium barbarum with fusion gene SS-GFP .

Detailed ways

Example 1 Cloning of thornlessSUS gene

Raw experimental materials: Mature seeds were collected from several thorny adult Lycium barbarum berries, germinated at 37 ℃ for 24 hours under humid conditions, and then sterilized on the surface in an ultra-clean workbench (75% alcohol treatment for 30-60 s, 0.1% mercury liter treatment for 1- 2 min, rinse with sterile water 3-5 times), blot the surface moisture and inoculate horizontally into 1/2MS medium (1/2MS macroelements + 1/2MS trace elements + 1/2MS iron salt + 1/2MS organic components +2% sucrose +0.45-0.5% agar powder, pH 5.8-6.0). After germination, the seeds were cultured under a photoperiod of 12 h light/12 h dark at 24-26 °C in complete darkness. Light is provided by LED tubes with an intensity of 48 μmol/m ² /s. When the aseptic seedlings develop 10-20 leaves, take young stem segments (leaves removed) containing 1-2 leaf axils as explants, and insert 2-3 mm into the stem medium with the vertical lower end facing down. The above-mentioned stem medium is MS medium containing 4% (w/v) sucrose, 0.50% (w/v) agar powder and 0.1-0.2 mg/L 6-BA, pH value 5.8. The photoperiod and temperature conditions are the same as the above-mentioned species Seedling cultivation. Under this culture condition, some of the explants obtained from seedlings inserted into the stem incision of the medium first produced nodular callus, and then the callus formed a large number of adventitious bud clusters on the same medium; at the same time, the axillary buds also rapidly germinated clustered buds . When the height of the adventitious bud cluster produced by the callus exceeds 1 cm, cut/cut the bud body and inoculate it into 1/2MS medium containing 0.2 mg/L IBA to induce rooting. When the buds take root and the stem develops to contain 10-20 leaves, take the stem segment as an explant again according to the above method, induce adventitious buds, and cut the buds to induce rooting. Carrying out 2-6 generations of regenerative culture in this way can obtain a group of tissue-cultured clones from a mature seed of black barbarum. Each rooting clone group in the bottle was marked separately, placed on the window sill under natural light for domestication, the temperature was 23-28 °C, and when the stems were strong and the leaves were dark green, they were transplanted into flowerpots with a diameter of 15 cm. The transplanting substrate is 1 peat: 1 humus: 2 vegetable garden soil. Before transplanting the substrate, put it into a polyethylene edible fungus bag, seal it, and sterilize it at 121 ℃ for 30-60 minutes under humid and hot conditions. Clean the root medium of the tissue culture seedlings and soak them in the broad-spectrum antibacterial working solution for 5-10 minutes, then transplant them into the substrate in the pot, and cover the substrate with perforated plastic film with water. Note that a bottle is moved into a basin. The water content of the pot soil was kept at 100% of the field capacity within 10 days, and the covered plastic film could be gradually uncovered after 10 days, and the water content of the pot soil was kept at 90-100% of the field capacity. Transplanting was carried out in a greenhouse, the ambient temperature was 25±2°C, and the light was scattered natural light. When the potted seedlings start to take out new stems, they are gradually transferred to direct natural light, and the field water holding capacity of the potted soil remains above 90%. A potted water control test (under natural light in the greenhouse) was carried out on each tissue culture clone that had been transplanted and had just resumed growth. The transplanted seedlings of each clone were divided into two groups, and their field water holding capacity was controlled at more than 90% and Below 60%. Select a typical clone that remains thornless at >90% field capacity. Take the young, elongated and completely thornless stems that have not been lignified, remove the leaves, and cut the top three stem nodes as the original experimental materials for high-throughput transcriptome sequencing (RNA-Seq) and gene cloning.

The partial expression sequence of the sucrose synthase gene ( thornlessSUS ) of Lycium barbarum thornless was obtained by the RNA-Seq of Biomec. According to this sequence, use the software Primer Premier5 to design primers for thornless SUS cloning; the primers are designed as follows:

SUS-F: 5'-GGAAAGAATGGCAGCCAGTAG-3'

SUS-R: 5'-CCAGTGTCGGGATAACCAAG-3';

Using the above-mentioned thorn-free stem nodes as materials, the Ultrapure RNA Kit was used to extract total RNA and synthesize the first-strand cDNA. The specific method is as follows:

1) Take 100-200mg stem node material and grind it fully in liquid nitrogen, add 1ml TRIzon Reagent, and mix well;

2) After adding TRIzon Reagent, pipette repeatedly several times to fully lyse the sample; place it at room temperature for 5 minutes to completely separate the protein-nucleic acid complex;

3) Add 200 μl chloroform, cover the tube cap, shake vigorously for 15 seconds, and place at room temperature for 2 minutes;

4) Centrifuge at 12,000 rpm for 10 min at 4°C, and transfer the RNA in the upper aqueous phase to a new RNaseFree centrifuge tube;

5) Add 200 μl of 70% ethanol (prepared with RNase-free water), invert and mix;

6) Add all the solution obtained in the previous step into the adsorption column that has been loaded into the collection tube. Centrifuge at 12000 rpm for 20 s, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

7) Add 700 μl Buffer RW1 to the adsorption column, centrifuge at 12000 rpm for 20 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

8) Add 500 μl Buffer RW2 (add absolute ethanol before use) to the adsorption column, centrifuge at 12000 rpm for 20s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

9) Repeat step 8);

10) Centrifuge at 12000 rpm for 2 min, and discard the waste liquid in the collection tube. Place the adsorption column at room temperature for 5-10 minutes and dry it thoroughly;

11) Put the adsorption column in a new RNase-free centrifuge tube, add 20-50 μl RNase-Free Water to the middle part of the adsorption column, place at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, collect the RNA solution, electrophoresis and use micropipette Nucleic acid protein analyzer to determine the concentration, integrity and purity meet the experimental requirements. Store RNA at -70°C—-80°C to prevent degradation.

12) Use M-mlV Reverse Transcriptase (M1701) to synthesize cDNA: The entire reaction system is carried out in a 0.2ml PCR tube, and the reaction system is as follows:

RNA 5μl

Oligo d(T) ₁₅ 1μl

DEPC water 9μl

Total volume 15μl

The reaction process is as follows: 70°C for 5 minutes, quickly put it on ice, and ice bath for 2 minutes; add 1 μl M-mlV, 5 μl M-mlV 5×Reaction Buffer, 5 μl dNTP, 1 μl RNase, 3 μl sterile water (RNase free ); 60min at 42°C, and store at 4°C.

Use the cDNA synthesized above as a template, and use primers to perform PCR on SUS-F and SUS-R. The system is as follows:

cDNA 1μl

SUS-F 0.5μl

SUS-R 0.5μl

Taq 0.25μl

Taq Buffer 2.5μl

dNTP 0.5μl

Ultrapure water 19.75 μl

Total volume 25μl

The PCR reaction program was: 95°C for 5 min; 95°C for 30 s; annealing temperature 56-60°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 5 min.

The PCR product was electrophoresed on a 1% agarose gel, observed by a gel imaging system, photographed, the ideal fragment was recovered from the gel, transformed with a T vector (pUCm-T Vector kit), and sent to Beijing BGI for sequencing. Sequencing results proved that the obtained thornlessSUS sequence has no stop codon. Therefore, 3'RACE was used to obtain the complete CDS of thornlessSUS gene. According to the partial sequence of the thornlessSUS gene obtained above, the upstream primer C751: 5'-GGGAAACACTGCTCAACG-3' was designed; the downstream primer was the universal primer B26 of 3'RACE: 5'- GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3'; the 3' of the thornlessSUS gene was amplified by PCR Area; PCR reaction system is as follows:

cDNA 1μl

C751 0.5μl

B26 0.5 μl

Taq 0.25μl

Taq Buffer 2.5 μl

dNTP 0.5 μl

Ultrapure water 19.75 μl

Total volume 25 μl

The PCR reaction program was: 95 °C for 5 min; 95 °C for 30 s; 46 °C for 30 s, annealing time of 30 s, 72 °C for 2 min, 35 cycles; 72 °C for 5 min;

The PCR product was electrophoresed on a 1% agarose gel to observe the gene amplification, and the gel imaging system was used to observe and take pictures. Select the PCR product with a single and clear band, recover from the gel, connect to the T vector for transformation, and send it to Beijing Huada Gene for sequencing. According to the sequencing results, the full-length CDS sequence of the gene was spliced. The sequence was compared and analyzed on NCBI, and the full-length CDS of the thornlessSUS gene was expected to be obtained. According to the full-length sequence, the upstream primer F: 5'-ATGGCAGCCAGTAGTCTTAGCA-3' and the downstream primer R-2: 5'-TAAATCCCAGATAATGTCATCACTT-3' were designed using the software Primer Premier5, and the full-length CDS of the thornlessSUS gene was amplified by PCR. The PCR reaction system is as follows:

cDNA 1μl

F 0.5μl

R-2 0.5μl

Taq 0.25μl

Taq Buffer 2.5μl

dNTP 0.5μl

Ultrapure water 19.75μl

Total volume 25μl

The PCR reaction program was: 95 °C for 5 min; 95 °C for 30 s; set 5 annealing gradients, 61.4-55.6 °C for 30 s, 72 °C for 5 min, 35 cycles; 72 °C for 10 min; 4 °C storage;

The PCR products were electrophoresed on a 1% agarose gel to observe the gene amplification, and the gel imaging system was used to observe and take pictures. The target PCR product with a single and clear band was selected, recovered by cutting the gel, connected to the T vector for transformation, and then sent to Beijing Huada Gene for sequencing. The obtained full-length CDS sequence of thornlessSUS is shown in the sequence table SEQ ID NO.1.

The gel used in the above experiments was recovered using the DNA gel recovery kit of Kangwei Century Biotechnology Co., Ltd. The specific method is as follows:

1) Cut out the single target DNA band, put it into two 2 ml centrifuge tubes, and weigh the gel;

2) Add one volume of Buffer PG to the centrifuge tube containing the gel block;

3) In a water bath at 50°C, gently invert the centrifuge tube up and down every 2-3 minutes until the glue is fully dissolved and the solution is yellow;

4) Add 200 μl Buffer PS to the adsorption column loaded into the collection tube, centrifuge at 13000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube;

5) Add the solution obtained in step 3) into the adsorption column, place at room temperature for 2 minutes, centrifuge at 13,000 rpm for 1 minute, discard the waste liquid, and put the adsorption column back into the collection tube;

6) Add 450 μl Buffer PW (add absolute ethanol in advance) to the adsorption column, centrifuge at 13000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube;

7) Repeat step 6);

8) Centrifuge at 13000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube;

9) Put the adsorption column into a new 1.5 ml centrifuge tube, add 50 μl Buffer EB dropwise in the air, place at room temperature for 2 minutes, centrifuge at 13,000 rpm for 1 minute, and store at -20°C.

10) Take 1 μl of the purified DNA fragments and electrophoresis on 1% agarose gel, observe and take pictures with the gel imaging system.

The transformation of the ligated T vector is all connected with the pUCm-T vector, and the specific method is as follows: take 0.2 pmol of the purified and diluted DNA fragment and connect it with 50 ng of the pUCm-T vector, and the reaction system is as follows:

Purify 5 μl of diluted DNA fragment (adjust volume according to length and concentration)

pUCm-T Vector 1 μl

10 x Ligation Buffer 1 μl

50% PEG 4000 1 μl

T4 DNA Ligase 1 μl

ddH2O 1 μl

10 μl total volume

Ligate overnight at 16°C.

The ligated products in the above experiments were all transferred into Escherichia coli DH5α competent for sequencing. The specific operation steps are as follows:

1) Thaw the competent cells in an ice-water bath, add 5 μl of the ligation product to 50 μl of competent cells, and mix gently;

2) Ice-water bath for 30 minutes, do not shake;

3) Heat shock at 42°C for 60 s without shaking;

4) Ice-water bath for 2 minutes, do not shake;

5) Add 500 μl sterile LB liquid medium;

6) 37 ℃, 180 rpm shaking recovery for 60 minutes;

7) Centrifuge at 4000 rpm for 1 min, take 100 μl of the bacterial liquid and spread it on the LB solid medium with Amp resistance (100 mg/l), and culture it upside down at 37 °C.

Pick multiple single colonies with a sterile white tip, add them to 5 ml LB liquid medium supplemented with 100 mg/l ampicillin (Amp) (Shenggong), and culture at 37 °C, 180 rpm for 12-16 h . Take the expanded culture and use M13 primers (M13-F: 5’-GTTGTAAAACGACGGCCAG-3’; M13-R: 5’-CAGGAAACAGCTATGACCATGATTACG-3’) to verify whether the DNA fragment is successfully connected to the T vector by PCR. The PCR reaction system is as follows:

Single colony liquid 1μl

M13-F 0.5μl

M13-R 0.5μl

Taq 0.25μl

Taq Buffer 2.5μl

dNTP 0.5μl

Ultrapure water 19.75μl

Total volume 25 μl

The PCR reaction program was: 95°C for 15 min; 95°C for 30 s; 55°C for 30 s, 72°C for 5 min, 35 cycles; 72°C for 10 min; 4°C for storage. Take 3 μl of PCR products and electrophoresis on 1% agarose gel to observe the gene amplification, observe with gel imaging system, and take pictures. Select the strain corresponding to the PCR product with a single clear band and send it to Beijing Huada Gene for sequencing.

Example 2 Construction of recombinant vector

The original vector is pRI101AN (preserved by Professor Zhang Zhihong’s research group). 5’ Nde I and 3’ Eco RI restriction sites and protective bases were added to both ends of the CDS of the GFP gene by PCR, and then double-digested and ligated. The GFP gene was connected to the pRI101AN vector, and the constructed vector was named pRI-GFP (Figure 1A);

Add 5' Nde I and 3' Eco RI restriction sites and protective bases at both ends of the CDS of the thornlessSUS gene in Lycium barbarum by PCR, and connect the CDS of thornlessSUS through Nde I and 3' Eco RI double restriction and ligation to the pRI101AN vector, and the resulting recombinant vector was named pRI-SUS (Figure 1B);

Add 5' Eco RI and 3' Eco RI at both ends of the GFP gene, and clone the GFP gene into the recombinant vector pRI-SUS through the restriction site 5' Eco RI and 3' Eco RI, and the resulting recombinant The vector was named pRI-SUSGFP (Figure 1C).

2. Transform the recombinant vector plasmid into competent cells

The above-constructed vectors should be transformed into E. coli and verified and sequenced (BGI) to verify the correctness of the sequence and direction; the recombinant vector plasmids are transformed into E. coli competent DH5α. The specific operation steps are as follows:

1) Thaw the competent cells in an ice-water bath, take 3 μl of each of the three expression vector plasmids and add them to 50 μl of competent cells, and mix gently;

2) Ice-water bath for 30 minutes, do not shake;

3) Heat shock at 42°C for 60 s without shaking;

4) Ice-water bath for 2 minutes, do not shake;

5) Add 500 μl sterile LB liquid medium;

6) 37 ℃, 180 rpm shaking recovery for 60 minutes;

7) Centrifuge at 4000 rpm for 1 min, take 100 μl of the bacterial liquid and spread it on the LB solid medium with Kana resistance (50 mg/l), and culture it upside down at 37 °C.

Pick multiple single colonies with a sterile white tip, add them to 5 ml LB liquid medium with Kana resistance (50 mg/l), and incubate at 37 °C, 180 rpm for 12-16 h. Take the expanded cultured bacterial solution, and check whether the recombinant plasmid is transferred into Escherichia coli competent DH5α by PCR method.

The PCR reaction system for verifying the pRI-GFP vector is as follows:

Single colony liquid 1 μl

GFP-F5'-TGCTTCAGCCGCTACCCC-3' 0.5μl

GFP-R 5'-ATCGCGCTTCTCGTTGGG-3' 0.5μl

Taq 0.25 μl

Taq Buffer 2.5 μl

dNTP 0.5 μl

Ultrapure water 19.75μl

Total volume 25μl

The PCR reaction program was: 95°C for 15 min; 95°C for 30 s; 58°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 10 min. Take 3 μl of PCR products and electrophoresis on 1% agarose gel to observe the gene amplification, observe with gel imaging system, and take pictures. The bacterial solution corresponding to the PCR product with a single clear band and the correct length was selected and sent to Beijing Huada Gene for sequencing; the GFP gene sequence is shown in the sequence table SEQ ID NO.2.

The PCR reaction system to verify the transformation effect of pRI-SUS vector is as follows:

Single colony liquid 1 μl

SUS-F 5'-GGAAAGAATGGCAGCCAGTAG-3' 0.5μl

SUS-R 5'-CCAGTGTCGGGATAACCAAG-3' 0.5μl

Taq 0.25μl

Taq Buffer 2.5μl

dNTP 0.5 μl

Ultrapure water 19.75μl

Total volume 25μl

The PCR reaction program was: 95°C for 15 min; 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min; 72°C for 10 min; and storage at 4°C. Take 3 μl of PCR products and electrophoresis on 1% agarose gel to observe the gene amplification, observe with gel imaging system, and take pictures. Select the bacterial liquid corresponding to the PCR product with a single clear band and the correct length and send it to Beijing Huada Gene for sequencing; the obtained sequence is the same as the full-length CDS sequence of the above-mentioned thornlessSUS .

The PCR reaction system for verifying the transformation effect of pRI-SUSGFP vector is as follows:

Single colony liquid 1 μl

SGFP-F5'- ACCCCTCATGGTTATTTCGCT-3' 0.5 μl

SGFP-R5'-TCACCTTGATGCCGTTTCTTCT-3' 0.5 μl

Taq 0.25μl

Taq Buffer 2.5μl

dNTP 0.5μl

Ultrapure water 19.75μl

Total volume 25μl

The PCR reaction program was: 95 °C for 15 min; 95 °C for 30 s; 58 °C for 30 s, 72 °C for 2.5 min, 35 cycles; 72 °C for 10 min. Take 3 μl of PCR products and electrophoresis on 1% agarose gel to observe the gene amplification, observe with gel imaging system, and take pictures. Select the bacterial solution corresponding to the PCR product with a single clear band and the correct length and position, and send it to Beijing Huada Gene for sequencing; the sequence of the SUSGFP fusion gene in the vector is shown in SEQ ID NO.3 in the sequence table.

Recombinant vector (plasmid) extraction method: Streak the DH5α strain with the correct sequence verified above on Kana-resistant (50 mg/l) LB solid medium, culture it at 37 °C until a single colony grows, and pick a single colony. Bacterial colonies were inoculated in liquid LB medium (50 mg/l Kana), activated at 37 °C, 180 rpm, and plasmids were extracted using the plasmid extraction kit from Zhongke Tairui Biotechnology Co., Ltd. The specific operation steps are as follows:

1) Take 4 ml of overnight cultured bacterial solution in a centrifuge tube, centrifuge at 10,000 rpm for 1 min, suck out the supernatant as much as possible with a pipette gun, and collect the bacteria into a centrifuge tube through two centrifuges;

2) Add 250 μl solution PI (with RNaseA added in advance) and 5 μl Lysis Dye to the cell pellet. At this time, the solution is red. Suspend the cells thoroughly with a vortex shaker;

3) Add 250 μl solution P2 to the centrifuge tube, gently turn it up and down 6-8 times to lyse the bacteria;

4) Add 350 μl solution P3 to the centrifuge tube, gently turn it up and down 6-8 times, and mix well. At this time, a yellow flocculent precipitate appears, and centrifuge at 10,000 rpm for 10 min;

5) Use a pipette gun to transfer the supernatant to the adsorption column (the adsorption column is placed in the collection tube), centrifuge at 10000 rpm for 45s, and discard the waste liquid;

6) Add 700 μl PW (add absolute ethanol in advance) to the adsorption column, centrifuge at 10000 rpm for 45 s, and discard the waste liquid;

7) Add 500 μl PW (add absolute ethanol in advance) to the adsorption column, centrifuge at 10000 rpm for 45 s, and discard the waste liquid;

8) Centrifuge at 10000 rpm for 2 min;

9) Put the adsorption column into a clean 1.5 ml centrifuge tube, add 60 μl solution EB, place at room temperature for 2 minutes, and centrifuge at 10,000 rpm for 2 minutes;

The extracted plasmid was verified by electrophoresis and other methods and then stored at -20 °C in preparation for transformation of Agrobacterium competent.

Example 3 Preparation of acceptor material

Mature seeds were collected from several prickly adult Lycium barbarum berries, placed in moist sterile gauze at 37 ℃ for 24 h, and then sterilized on the surface in an ultra-clean workbench (75% alcohol for 30-60 s, 0.1% mercuric chloride for 1 -2 min, rinse with sterile water 3-5 times), blot the surface moisture and inoculate horizontally on 1/2 MS medium (MS dry powder 2.37 g/l+20 g/l sucrose+4.5-5.0 g/l agar powder, pH 5.8-6.0). After germination, the seeds were cultured under a photoperiod of 12 h light/12 h dark in complete darkness at 23-27 °C. Light is provided by LED tubes with an intensity of 48 μmol/m ² /s. When the height of the aseptic seedling exceeds 8 cm and is lower than 11 cm, the plant is cut into two parts: the 3-5 cm high stem with the top and the lower half with the root without the top. Put the two parts into freshly prepared 1/2MS medium that does not contain any exogenous phytohormones, and when the upper part takes root, the lower part of side branches or roots germinates, and the height of the plant is 8-11 cm, cut off another 3 -5 cm high stems with tops, and transfer the upper part of the top and the lower part of the root to the freshly prepared hormone-free 1/2MS medium, and the plant height reaches 8-11 cm again, continue to cut and transfer according to the above method . In this way, through more than 4 generations of transfer, the relatively fast strains of rooting, growth and reproduction are retained and marked respectively. Cut off 3-5 mm from the tip part of the young leaves of the clones screened out above as explants, and inoculate them on MS medium (MS dry powder 4.74 g/l+ sucrose 40 g/l+ agar 4.8- 5.0 g/l, pH=5.8). Under these conditions, the clonal lines capable of forming complete plants through the direct organogenesis pathway were retained, and the 3-5 mm tips of the fully expanded young leaves of the plants obtained by direct organogenesis were cut again as explants, adaxial Inoculate MS medium face down, and after the leaf tip explants form plants through direct organogenesis, cut the leaf tip explants again, inoculate, and propagate... In this way, the leaf tip direct organogenesis of more than 10 generations will pass through Young plants with direct organogenesis serve as recipient donors for genetic transformation. Except that the seeds were placed under dark conditions before germination, the photoperiod and other light conditions for other cultures were the same as those after seed germination, and the temperature was 25±2 °C. The MS dry powder and agar powder used were purchased from Jinan Zhongtian Tissue Culture Gardening Supplies Co., Ltd., and the sucrose was purchased from Shenggong. 1M NaOH (Sanko) was used to adjust the pH value, and the medium was autoclaved at 121°C for 20 minutes and then placed in the culture room for observation for 7 days. It was confirmed that it was sterile and normal before it could be used for inoculation.

The determination of kanamycin screening concentration in the selection medium of embodiment 4

Select the clone seedlings of Lycium barbarum in good condition and basically the same development, and use sterile scissors to cut half of the tip of the young leaves in an ultra-clean bench as explants. The explants were inoculated backside up into MS medium containing different concentrations of kanamycin (Kana, Sanko) (MS dry powder 4.74 g/l+ sucrose 40 g/l+ agar 4.8 g/l, pH=5.6-6.0 ), the Kana concentration of the medium was set to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 100 mg/l , at least 90 explants were inoculated per treatment. The temperature of the tissue culture room was 25±2°C, and the pH value was adjusted using 1M NaOH (Sanko). After the medium was autoclaved at 121°C for 20 minutes, filter-sterilized (0.22 μm) Kana mother solution was added before solidification. The light source in the culture room was an LED tube, and the photoperiod was 12 h light/12 h dark. The results showed that the control group without Kana added to the medium and the concentration of Kanna at 1 mg/l, 2 mg/l of Lycium barbarum leaf tip explants began to take root after 7 days, and the rooting rate reached the maximum after 14 days, and the highest up to 100%. After 10 days, the leaf explants of Lycium barbarum with a Kanna concentration of 3 mg/l began to take root, and the rooting rate was 18.89%, and the rest of the concentration remained unchanged. After 28 days, the control group without Kana and the explants of Lycium barbarum berries with Kanna concentrations of 1 mg/l and 2 mg/l began to sprout, and the germination rates were 52.22 %, 35.56 %, and 32.22 %, respectively. After rooting, the leaf explants of Lycium barbarum with a Kanna concentration of 3 mg/l stopped growing when the root length reached about 1 cm, and no buds were produced subsequently. After inoculation for a period of time, except for the budding control and the leaf explants of Lycium barbarum with Kanna concentration of 1-2 mg/l, the leaf explants of Lycium barbarum of other treatments died gradually. Although the leaf explants of Lycium barbarum with a Kanna concentration of 3 mg/l produced short roots, the elongation and growth of the roots were inhibited, and no buds could be produced to form plants, and finally the leaf explants gradually turned yellow and died (Fig. 2). Therefore, MS medium supplemented with Kanna concentration of 3 mg/l was selected as the initial selection medium in this experiment. This concentration can achieve the purpose of screening, and can reduce the damage to the plant material, so as to achieve the purpose of making the successfully transformed leaf vein incision cells take root and sprout as much as possible.

Embodiment 5 Determination of the use concentration of cefotaxime sodium

Agrobacterium tumefaciens strains EHA105 and LBA4404 preserved by the research group of Professor Zhang Zhihong of Shenyang Agricultural University were used. Put the Agrobacterium stored in the -80°C refrigerator on ice and let it melt, dip a small amount of bacterial liquid with a sterile inoculation wire in the ultra-clean workbench, and culture it in a line containing 50 mg/l rifampicin (Rif) On the YEP solid medium, cultivate it upside down at 27-28 ℃ for 2-3 days; pick a single colony of Agrobacterium with a sterile white tip in the ultra-clean workbench, and add it to the YEP liquid medium containing 50 mg/l Rif , 27-28 ℃, 180-200 rpm shaking culture for 10 h until OD600 is about 0.5, pipette about 100 μl of bacterial liquid and spread evenly on YEP solid medium containing 50 mg/l Rif. Soak the circular small filter paper pieces with 100, 150, 200, 250, 300, 350, 400, 450 and 500 mg/l cefotaxime sodium (Cef) solutions respectively, and place them on the above-mentioned uniformly smeared solid culture cultured overnight at 27-28°C. Overnight observation found that filter paper soaked with ≥300 mg/l Cef solution could completely inhibit the growth of Agrobacterium EHA105 and LBA4404. Therefore, 300 mg/l was selected as the concentration of Cef in the screening medium to inhibit the outbreak of Agrobacterium again. 500 mg/l was selected as the concentration of Cef in the washing solution. In the experiment of using Cef alone to inhibit Agrobacterium, if it is found that Agrobacterium grows, 300 mg/l carbenicillin and 300-700 mg/l Cef can be used in combination to achieve a stronger inhibitory effect.

Example 6 Preparation of Competent Agrobacterium

Agrobacterium EHA105 and LBA4404 (preserved by the research group of Professor Zhang Zhihong of Shenyang Agricultural University) stored in the refrigerator at -80 ℃ were left to melt on ice, and a small amount of bacterial liquid was dipped in a sterile inoculation wire in the ultra-clean workbench, and cultured by streaking on the On the YEP solid medium containing 50mg/l Rif, cultivate it upside down at 27-28 ℃ for 2-3 days; use a sterile white tip to pick up a single colony of Agrobacterium EHA105 and LBA4404 in the ultra-clean workbench, and add it to the culture medium containing 50 mg /l Rif’s YEP liquid medium, 28°C, 180 rpm shaking culture for 10 h until the OD ₆₀₀ is about 0.5, take 5 ml of the activated bacterial liquid in the ultra-clean workbench and add it to a sterile 50 ml centrifuge tube Place on ice for 10 min; centrifuge at 5,000 rpm for 10 min at 4°C, and remove the supernatant with a pipette; add 5 ml of 25 mM pre-cooled on ice to the centrifuge tube in an ultra-clean workbench. Resuspend the bacteria in CaCl ₂ solution (sterilized by filter), let stand on ice for 20 min, centrifuge at 5000 rpm for 10 min at 4 °C, discard the supernatant; add 1 ml pre-prepared Cold 25 mM CaCl ₂ solution was used to resuspend the cells again, and let it stand on ice for 20 minutes. Agrobacterium competent cells were quick-frozen in liquid nitrogen and stored at -80°C for later use.

Example 7 Transformation of Agrobacterium with recombinant vector

Pipette 5 μl of recombinant vector (pRI-GFP, pRI-SUS or pRI-SUSGFP) into 50 μl of Agrobacterium EHA105, LBA4404 competent in the ultra-clean bench, mix gently; stand on ice for 5 min , put into liquid nitrogen for 1 min; 37 °C water bath for 5 min, ice bath for 2 min; add 800 μl YEP liquid medium to the centrifuge tube in an ultra-clean workbench, 28 °C, 180 rpm shaking culture for 4 h, 5000 rpm Centrifuge at room temperature for 5 minutes, use a pipette gun to absorb excess liquid medium, leave about 100 μl of liquid, resuspend the bacteria, and spread the bacteria solution on a medium containing 50 mg/l Rif and 50 mg/l Kana on the YEP solid medium, cultured upside down at 27-28 ℃ for 2-3 days; pick a single colony of Agrobacterium with a sterile inoculation silk in the ultra-clean workbench, and evenly smear it on the medium containing 50 mg/l Rif and 50 mg/l Kana YEP solid medium, cultured upside down at 28 ℃ for 1-2 days; pick up the second-transformed Agrobacterium lawn with a white tip in the ultra-clean workbench, and check whether the recombinant plasmid has been transferred into the agrobacterium by PCR. Competent bacillus, PCR reaction system (bacteria lawn replaces bacterial liquid, ultrapure water supplements the volume to 25 μl) and the procedure is the same as that in 1.2 to check whether the recombinant plasmid is transferred into E. coli competent DH5α; according to the electrophoresis results, select the target fragment The double-transformed colony with the same band length was shaken and cultured in 5 ml YEP liquid medium containing 50 mg/l Rif and 50 mg/l Kana on a shaker at 28 °C and 180 rpm, and mixed with sterilized glycerol. The solution was stored at a ratio of 1:1 for subsequent experiments such as sequencing and infection. The correct strains verified by sequencing were used for subsequent genetic transformation, and the engineering bacteria containing various vectors were referred to as EHA105 _pRI-GFP , EHA105 _pRI-SUS , EHA105 _pRI-SUSGFP , LBA4404 _pRI-GFP , LBA4404 _pRI-SUS and LBA4404 _pRI-SUSGFP .

Example 8 Agrobacterium-mediated genetic transformation

1. Preparation and infection of bacterial solution

In the ultra-clean workbench, dip a small amount of EHA105 _pRI-GFP , EHA105 _pRI-SUS , EHA105 _pRI-SUSGFP , LBA4404 _pRI-GFP , LBA4404 _pRI-SUS or LBA4404 _pRI-SUSGFP strains with sterile inoculation silk, and culture them in streaks On the YEP solid medium containing 50mg/l Rif and 50mg/l Kana, culture it upside down at 27-28 ℃ for 2-3 days; in the ultra-clean bench, pick a single colony of Agrobacterium with a white tip, and use 50mg/l Rif and 50mg/l Kana in 5ml YEP liquid culture medium with shaking for 18h; l Continue shaking culture in 5 ml YEP liquid medium of Kana until the OD ₆₀₀ value of EHA105 _pRI-GFP , EHA105 _pRI-SUS and EHA105 _pRI-SUSGFP bacteria liquid is 0.39-0.61, while LBA4404 _pRI-GFP , LBA4404 _pRI-SUS or When the OD ₆₀₀ value of the LBA4404 _pRI-SUSGFP bacterial solution is 0.39-0.80, centrifuge at 5000 rpm for 5 min at 4°C, discard the supernatant, and use sterilized MS liquid medium (MS dry powder 4.74 g/l+ Sucrose 40 g/l, pH=5.8) 1:1 resuspend the bacteria, keep on ice for 1h for later use. Add filter-sterilized acetosyringone (AS) to a final concentration of 100 μM before resuspending the cells in MS liquid medium to increase transformation efficiency.

Pour the resuspended bacterial solution into a sterilized Petri dish, place the leaf tip explants of Lycium barbarum berries that have not been pre-cultivated, pre-cultivated for 1 day, and pre-cultured for 2 days, and put them on filter paper to absorb the water, and then add them to containing Infection in the Petri dish of bacterial liquid. The infection time of the above 6 kinds of bacteria was set at 5-15min. During the infection process, the petri dish was gently shaken at regular intervals to promote the Agrobacterium infection of the leaf explants of Lycium barbarum. After the infection, the leaf tip explants of Lycium barbarum black fruit were transferred to sterile filter paper with tweezers, and the excess bacterial solution was blotted dry. All the above operations were carried out in a clean bench. Pre-cultivation was carried out under dark conditions in a tissue culture room at 25±2°C. Leaf tip explants were also used and inoculated with the back facing up. The pre-cultivation medium was MS medium (MS dry powder 4.74 g/l+sucrose 40 g/l+agar 4.8 g/l), the pH value was adjusted to 5.8 with NaOH, and autoclaved at 121°C for 20 minutes.

2. Co-cultivation

Inoculate the leaf tip explants of Lycium barbarum black fruit that had been sucked dry into the co-cultivation medium (MS dry powder 4.74 g/l+ sucrose 40 g/l+ agar 4.8 g/l+100 μM AS, pH=5.8), place 22-23 explants per dish. Continue the dark culture in the above-mentioned tissue culture room for 2-3 days until white Agrobacterium grows around the leaf explants.

3. Selective culture and generation of resistant plants

After co-cultivation, the explants of Lycium barbarum leaves were washed 5-6 times with sterile water containing 500 mg/l Cef (0.22 μm sterile filter membrane filtration), until there was no Agrobacterium around the explants. Transfer the cleaned leaf tip explants to sterile filter paper with tweezers, blot the excess liquid and inoculate the selective medium (MS dry powder 4.74 g/l+ sucrose 40 g/l+ agar 4.8 g/l +3 mg/l Kana+300 mg/l Cef, pH=5.8). Conditions such as light in the culture room are the same as those in the "Determination of Kanamycin Screening Concentration". The selection medium will be replaced every 7 days. During the selective cultivation process, Kana was added to the culture medium for screening to avoid escape plants as much as possible. A strict control group was set up in the experiment. The transformed leaf tip explants of Lycium barbarum black fruit began to root after 10 days of selection and culture, and the rooting rate reached the highest after 17 days, while the leaf tip explants of untransformed Lycium barbarum berries were resistant to Kana (3 mg/l). Gradually yellowed and died on the selective medium; after 28 days of selective culture, the roots of the rooted leaf tip explants began to sprout and form complete resistant plantlets (Fig. 3). Statistics show: (1) EHA105 _pRI-GFP genetic transformation rate is as high as 8.33%; (2) LBA4404 _pRI-GFP genetic transformation rate is as high as 8.33%; (3) LBA4404 _pRI-SUS genetic transformation rate is as high as 16.65%; (4) EHA105 The genetic transformation rate _{of pRI-SUSGFP} was as high as 8.33%; (5) the genetic transformation rate of LBA4404 _pRI-SUSGFP was as high as 8.33%; (6) the genetic transformation rate of EHA105 _pRI-SUS was as high as 8.33%. The above transformation rate=germination rate of leaf tip explants on screening medium/average germination rate of leaf tip explants under non-transgenic conditions (control)*100%. The method obtains resistant plants without a callus stage, reduces the risk of outbreak of Agrobacterium infection again, has a short period, and the resistant plants grow robustly.

4. Secondary screening of resistant plants

In order to avoid the emergence of escaped plants more effectively, we conducted a secondary screening experiment; the specific method is, when the above-mentioned resistant buds grow to a height of about 5 cm, cut about 2 cm of terminal bud stems in an ultra-clean bench, and inoculate into the resistant rooting medium (MS dry powder 2.37 g/l+sucrose 20 g/l+agar 4.8 g/l+5 mg/l Kana+300 mg/l Cef, pH=5.8); after 30 days of culture, the resistant Sexual buds take root and can grow normally (Figure 3), and those that cannot take root are regarded as escaped buds and are eliminated. A strict control group was set in the experiment; the results showed that almost all the resistant materials screened in the previous step (selection culture) could produce roots and form complete resistant plants.

Example 9 Verification of transgenic plants

The resistant plants screened above were identified from the levels of DNA (PCR), RNA (RT-PCR) and protein (observation of GFP by laser confocal microscopy).

1. DNA level detection

The DNA of resistant plants and untransformed control materials was extracted by CTAB method. The specific operation steps are as follows:

1) Grind 100 mg leaves of resistant and control plants in 500 μl lysate, and collect them in 2 ml centrifuge tubes;

2) Water bath at 65 ℃ for 30 minutes, invert and mix several times in the middle;

3) Add an equal volume of 500 μl DNA Extraction Reagent to the centrifuge tube and mix by inverting;

4) Centrifuge at 12000 rpm for 10 min;

5) Use a pipette gun to draw the supernatant into another clean 2ml centrifuge tube, add 0.7 times the volume of 350 μl isopropanol, invert to mix, and ice-bath for 5 minutes;

6) Centrifuge at 12000 rpm for 2 min, discard the supernatant, and invert the tube for 1 min to remove isopropanol;

7) Add 500 μl of 70% absolute ethanol to rinse the pellet, centrifuge at 12000 rpm for 1 min, and discard the supernatant;

8) Repeat step 7);

9) Use a pipette gun to suck out the residual absolute ethanol as much as possible, and leave it at room temperature for several minutes to remove the residual absolute ethanol;

10) Add 50 μl of TE to dissolve the DNA pellet and store at -20°C.

Take 3 μl of DNA and electrophoresis on 1% agarose gel, observe the DNA extraction, observe with a gel imaging system, and take pictures.

Using the extracted DNA as a template, verify whether the transformed plants are positive plants by PCR amplification. The primers, PCR reaction system and PCR reaction procedure used for the PCR verification of plants transformed with the pRI-SUSGFP recombinant vector are the same as those for verifying whether the recombinant vector is transferred into competent cells. The primers used for the PCR verification of the plants transformed with the pRI-SUS recombinant vector are primers designed according to the partial sequence of the Npt II gene. The PCR reaction system is as follows:

DNA 1 μl

Primer-F (Npt II-F) 0.5μl

Primer-R (Npt II-R) 0.5 μl

Taq 0.25 μl

Taq Buffer 2.5 μl

dNTP 0.5 μl

Ultrapure water 19.75 μl

Total volume 25 μl

The PCR reaction program was: 95°C for 5 min; 95°C for 30 s; 58°C for 30 s, 72°C for 2 min, 35 cycles; 72°C for 10 min; 4°C for storage. Take 3 μl of PCR products and electrophoresis on 1% agarose gel to observe the gene amplification, observe with gel imaging system, and take pictures.

The results showed that the pRI-SUS transformed resistant plants amplified a band at 4500 bp, and the length of the band was consistent with that of the positive control (pRI-SUS vector plasmid), while the untransformed control plants had no band at the same position ( Fig. 4), it is preliminarily proved that the transgenic plants of Lycium barbarum overexpressing the SUS gene were obtained; the length of the amplified band of the resistant plants transformed with pRI-SUSGFP was consistent with that of the positive control (pRI-SUSGFP vector plasmid), which was preliminarily proved The transfused gene plants were obtained (lanes 3 and 4 in Figure 5).

2. RNA level detection

The RNA extraction kit Ultrapure RNA Kit was used to extract the total RNA of roots, stems and leaves of resistant and control plants. cDNA was synthesized using M-mlV Reverse Transcriptase (M1701). The specific operation steps are the same as above.

Use a micronucleic acid protein analyzer to detect the cDNA concentration, use the cDNA diluted to the same concentration as a template, and use the internal reference gene to detect whether the cDNA is available. The PCR reaction system is as follows:

cDNA 1 μl

Primer-F (Actin-F) 0.5 μl

Primer-R (Actin-R) 0.5 μl

Taq 0.25 μl

Taq Buffer 2.5 μl

dNTP 0.5 μl

Ultrapure water 19.75 μl

Total volume 25 μl

The PCR reaction program was: 95°C for 5 min; 95°C for 30 s; 30 cycles of 58°C for 30 s and 72°C for 30 s; 72°C for 10 min; and storage at 4°C. Take 3 μl of PCR products and electrophoresis on 1% agarose gel to observe the gene amplification, observe with gel imaging system, and take pictures.

The verified and available cDNA diluted to the same concentration was selected as a template, and the expression of the target gene in the transformed plant was verified by semi-quantitative PCR. The PCR reaction system is as follows:

The PCR reaction program was: 95°C for 5 min; 95°C for 30 s; 58°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 10 min; 4°C storage; 3 μl of PCR products were electrophoresed on 1% agarose gel, Compare and observe the brightness of the target band, observe with the gel imaging system, and take pictures; the results show that the expression level of the thornlessSUS gene in the roots, stems, and leaves of the transgenic resistant plants is significantly higher than that of the untransformed control (Figure 6); , the target gene thornlessSUS was overexpressed (transcribed) in the roots, stems and leaves of Lycium barbarum.

3. GFP fluorescence detection

In the ultra-clean bench, use sterile scissors to cut the leaves of the resistant plants transformed with the vectors pRI-GFP and pRI-SUSGFP and the leaves of the control plants that have not been transformed, and cut the leaves horizontally with a scalpel. GFP expression was observed at a wavelength of 550 nm. Note that the observation conditions for the control control and the resistant material are exactly the same. The results showed that GFP protein was expressed in the resistant plants transformed by GFP and fusion gene SUSGFP (Fig. 7).

4. Changes in traits of transgenic plants

The present invention finds that under the control of the CaMV 35S promoter, the GFP gene is highly constitutively expressed heterologously in Lycium barbarum, which can cause toxic and side effects on the plants, and the plants in the bottle have unfavorable traits such as slow growth. However, when GFP is fused with thornlessSUS to form SUS -GFP, and stably transformed Lycium barbarum, the successful transgenic plants can grow normally, which proves that GFP fused with thornlessSUS, even if it is still constitutively high expression, has significantly reduced the toxic and side effects of Lycium barbarum. Lycium barbarum plants with high constitutive expression of thornlessSUS showed the characteristics of thick stems, strong ability to divide roots, and a large increase in the number of fibrous roots (Fig. 3D).

sequence listing

<110> Shenyang Agricultural University

<120> A Stable Genetic Transformation System of Lycium barbarum Black Fruit and Its Application

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<213> Lycium ruthenicum Murr.

<400> 3

atggcagcca gtagtcttag cattaaggac cgtttggagg aatccatttt ggctcgtcca 60

gatgaaattt cggcactcaa gtcaagggtt gaaactgaag ggaaaggggt catgaaacca 120

cttgatctct tgaaccattt gatttctgtg aatactaaga agaatggagt aaatgttggt 180

gccagtgcac tcgtggaagt tctcagttgc agccaagaag ctgtgattgt accaccacaa 240

cttgcactag ctgtacgtcc aaggcccggt gtgtgggagt atgtgtcact gaatcttaag 300

acaaagaaag tggctgaatt gagcatcccc aaataccttc aattgaaaga gaacgctgtc 360

gatgaaagtg gaaacgtctt ggaaatggat tttgagccat ttactactgt aactcctcca 420

aagacacttt ctgactccat tggtaatggt ttggagtttc ttaatcgcca tattgcttca 480

acaatgttcc atgacaagga gattgccaag tgcctccttg actttctcag acagcataac 540

tacaaaggaa agtcattgat ggtgaaagaa agcatccaaa gcctggaaag tttccaatat 600

gtcctgaaaa aagcagagga atatctgcac tctctgaatc cagaaactcc atactctaac 660

tttgaatcga aatttgaaga gattggcttg gaaagagggt ggggaaacac tgctcaacgc 720

gtgcaagaaa caatctgtca tttgttgcac ctccttgagg ctcctaatgc atcttccttg 780

gaaaaattcc ttggaagaat cccattggtt ttcaatgttg tcattctcac ccctcatggt 840

tatttcgctc aagaaaacgt ccttggttat cccgacactg gtggccaggt tgtgtacatt 900

cttgatcaag ttccagccat ggagcgtgag atgcttctcc gtttgaagct tcaaggactt 960

gatgatatcc tcccccggat ccttgttgta acaaggctgc tgcctgatgc agttggaacc 1020

acttgtggtg agcgcatgga gaaagtatat ggggcagagc attctcatat tattcgtgtt 1080

ccatttagaa ctgagaaagg aatgttgcgc aaatggatct caagattcga agtctggcca 1140

tacatggaaa ctttcactga ggatgtcgcg gaagaacttg tcaaagagct gcaagctaaa 1200

ccagacttga tcattggaaa ctacagtgag ggaaaccttg ctgcctcctt gttggctaag 1260

aaatttgggg ctactcaatg cacaatagct catgccttgg agaaaaccaa gtatccaaac 1320

tctgaccttt actggaagaa atttgatgac aagtatcatt tctcaagtca gttcagtgct 1380

gatctttttg cgatgaatca cactgatttc atcatcacca gcactttcca agaaatcgct 1440

ggaagcaaga atactgtagg gcagtatgag agccaacactg cttttaccat gcctggattg 1500

taccgagtag tccatggaat tgattcattt gatccaaaat tcaacattgt ctcccctggg 1560

gctgatatgt cgatctactt cccttacaca gaaaaggaaa aaaggctaaa caatttccac 1620

ccggaaattg aagaactact ctacagtcct gttgagaaca aagagcacct atgtgtgttg 1680

aaggaccgga acaagccaat tctcttcacc atggcaaggc tagatcgcgt gaagaatcta 1740

acagggctcg ttgaatggta tgccaagaat gcaaggctga gagagcttgt taaccttgtg 1800

gttgttggtg gagacagaag gaaagaatcc aaggatttgg aagagcaagc agagatgaaa 1860

aaaatgtatg accttattga aacctacaac ctgaatggac aattcaggtg gatttcttct 1920

cagatgaatc gtataaggaa cggagagctt taccgatata ttgcagacac gaggggtgct 1980

ttcgttcagc cagctttcta cgaggctttc ggtttgacag ttgttgaatc catgacttgt 2040

ggtttgccaa cttttgctac ttgtaatggt ggaccatttg agattatagt gcatggaaaa 2100

tctggattcc acattgaccc taatcagggt gacaggaatg ctgatctttt ggtcaatttc 2160

tttgagaaat ccaaagaaga tccaagttat tgggataaca tttccaaggg aggcctacaa 2220

cgcatcattg agaagtatac atggcaaatt tattcacaga aagtgatgac attatctggg 2280

atttatggat tctggaagta tgcaaccaca aatgacaaag ttgctagtgc aaagaagcgc 2340

tatctcgaaa tgttttatga acttatgttt aagaaagctg ctgagaaagt tccactggct 2400

attgatgaaa tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 2460

gagctggacg gcgacgtgaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 2520

gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 2580

tggcccaccc tcgtgaccac cttcacctac ggcgtgcagt gcttcagccg ctaccccgac 2640

cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 2700

accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 2760

gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 2820

ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 2880

cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 2940

cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 3000

gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 3060

cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctcacggcat ggacgagctg 3120

tacagatctt aa 3132

Claims (8)

1. The SUS gene of the sucrose synthase of the lycium ruthenicum has a base sequence shown in a sequence table SEQ ID NO. 1.

2. A transgenic plant expression vector plasmid, which is obtained by inserting the SUS gene of the sucrose synthase of lycium ruthenicum in a plant expression vector according to claim 1.

3. A transgenic plant expression vector plasmid is characterized in that SUS+X fusion genes are inserted into a plant expression vector, X represents a transgenic target gene, and the SUS genes are genes shown in a sequence table SEQ ID NO. 1.

4. A transgenic plant expression vector plasmid according to claim 3, characterized in that: x is GFP gene.

5. A transgenic plant expression vector plasmid according to claim 2, 3 or 4, characterized in that: the plant expression vector is pRI101AN.

6. A recombinant agrobacterium transformed with a transgenic plant expression vector plasmid according to claim 2 or 3.

7. A recombinant agrobacterium according to claim 6 wherein: the agrobacterium is EHA105 or LBA4404.

8. A stable genetic transformation system for lycium ruthenicum, comprising:

1) Taking an explant of lycium ruthenicum apex, pre-culturing for 1-2 d, adding the recombinant agrobacterium infection described in claim 5, wherein the pre-culturing culture medium consists of 4.74 g/l MS dry powder, 40g/l sucrose and 4.8 g/l agar;

2) After infection, inoculating the plant to a co-culture medium, and culturing in a dark way until white agrobacterium grows around the leaf explants; the co-culture medium consists of 4.74 g/l MS dry powder, 40g/l sucrose, 4.8 g/l agar and 100 mu M AS;

3) After co-cultivation, the lycium ruthenicum leaf explants were washed with sterile water containing 500 mg/l Cef; inoculating the explant to a selection medium, and culturing until buds start to form complete resistant plantlets; the selection medium consists of 4.74 g/l of MS dry powder, 40. 40g/l of sucrose, 4.8 g/l of agar, 3 mg/l of Kana and 300mg/l of Cef;

4) Step 3), the buds of the resistant plantlets grow to 4.5-5.5 cm in height, 1.5-2.5 cm of stems with terminal buds are cut, inoculated into a resistant rooting culture medium, and cultured until the buds can root and grow normally; the resistant rooting medium consists of 2.37 g/l of MS dry powder, 20g/l of sucrose, 4.8 g/l of agar, 5 mg/l of Kana and 300mg/l of Cef.

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