CN101307318A - Application of GLDH Gene in Non-heading Chinese Cabbage to Increase Vc Content in Wutaicai - Google Patents

Abstract

The invention relates to the application of GLDH gene of non-heading Chinese cabbage to improve the Vc content of Wutaicai, and belongs to the field of molecular biology and biotechnology. The complete coding region sequence of GLDH gene was obtained by RT-PCR technology, and its plant overexpression vector was further constructed and introduced into Agrobacterium LBA4404. Optimize the technical scheme including medium formula and operation process, and establish the transgenic genetic transformation system of Wutaicai. Agrobacterium-mediated method was used to transform the plant overexpression vector into Utah cabbage. The transgenic plants were obtained through kanamycin resistance screening, and PCR technology was used to identify the integration of the target gene into the genome of Utami. The present invention has verified the important role of the gene in the Vc synthesis pathway, and successfully obtained transgenic plants overexpressing GLDH. Compared with non-transgenic plants, the GLDH gene expression of the transgenic plants was 28.5 times that of the control, and the highest Vc content of the plants was 1.8 times that of the control. It provides a theoretical basis and practical experience for improving vegetable quality through genetic engineering in the future.

Description

Application of GLDH Gene in Non-heading Chinese Cabbage to Increase Vc Content in Wutaicai

1. Technical field

The invention relates to the application of the non-heading Chinese cabbage GLDH gene to increase the Vc content of Wutaia plants, belongs to the field of molecular biology and biotechnology, and is specially used for the transgenic molecular breeding of Wutaia.

2. Background technology

Vitamin C (vitamin C, hereinafter referred to as Vc) in plants, also known as L-ascorbic acid (AsA), not only has very important physiological functions for the plant's own antioxidant system, photosynthetic protection, and regulation of growth and development, but also It provides a rich and convenient source of Vc for humans (humans cannot rely on their own synthesis and storage of AsA, they need to ingest it from food). At present, the market of natural AsA is very broad, while the industrial synthesis of AsA has high economic cost and huge energy consumption in terms of environment. Researchers have also hoped to use microorganisms to directly convert cheap feed into AsA, but so far no biosynthesis method with commercial development value has been obtained. The growing market requires new methods for the production of plant-derived AsA that are more efficient, environmentally friendly, and price-competitive. Therefore, increasing plant AsA content through genetic engineering technology, improving plant nutritional quality and resistance have attracted more and more attention.

Now the biosynthetic pathway of plant AsA has been relatively clear, mainly including D-mannose pathway and D-galacturonic acid pathway. L-galactonic acid-1,4-lactone dehydrogenase GLDH is a key enzyme that catalyzes the last step of Vc biosynthesis in plants, and its important role has not been questioned. At present, researchers have cloned the gene encoding the enzyme from various plants such as cauliflower, tobacco, prickly pear, melon, tomato, pepper, strawberry, Arabidopsis, and observed that the activity of GLDH enzyme in Arabidopsis is closely related to that of AsA in plants. levels showed similar diurnal variations. People hope to achieve the purpose of enhancing AsA content by regulating gene expression, but there are not many studies on the effect of enhanced expression of GLDH gene on AsA content and plant phenotypic traits, and the best strategy for genetic modification of this pathway has not been obtained so far. .

Non-heading Chinese cabbage (Brassica campestris.ssp.chinensis.Makino), also known as Chinese cabbage, green vegetables, and rapeseed, is a vegetable cultivated in four seasons in the middle and lower reaches of the Yangtze River in my country and in the south of the Yangtze River. In recent years, it has also grown in a large area in northern my country. The introduction, cultivation and promotion of Chinese vegetables play an important role in the annual supply of vegetables in our country. At present, genetic engineering is used to introduce the target gene into non-heading cabbage to improve the genetic characteristics and improve the quality of its products, which provides a new technology for the genetic breeding of non-heading cabbage. Wutaicai is a kind of non-heading cabbage, and there is no report about the successful transgenic of Wutaicai.

3. Contents of the invention

technical problem

The purpose of the present invention is to achieve the purpose of regulating the Vc content in plants by overexpressing the key enzyme GLDH gene in the Vc synthesis pathway, and to establish an Agrobacterium-mediated transgenic genetic transformation system for non-heading Chinese cabbage Utami to obtain transgenic plants. Use genetic engineering technology to improve plant quality and stress resistance of plants.

Technical solutions

Increase the Vc content of Wutaicai by overexpressing the GLDH gene of non-heading Chinese cabbage.

1) Construction of plant overexpression vector

According to the cDNA sequence of non-heading cabbage L-galactonic acid-1,4-lactone dehydrogenase gene GLDH, gene accession number: AY899298, design primers to amplify its complete coding reading frame, and on the upstream and downstream primers The restriction endonuclease sites XbaI and SmaI were introduced respectively, and the designed primers were:

S1: 5′-GCTCTAGACGCCTGAACTAAAACAAAAATGC-3′ XbaI

A1: 5′-TCCCCCGGGCGGCTTCACTCTTCTTACAAACACT-3′SmaI

Using the total RNA of leaves of non-heading Chinese cabbage 'Suzhouqing' as a template, the first strand of cDNA was synthesized by reverse transcription, and PCR was amplified with ExTaq enzyme. The PCR program was as follows: 94°C for 3 min; 94°C for 1 min, 45°C for 1.5 min , 72°C for 3min, 5 cycles; 94°C for 45s, 64.4°C for 1min, 72°C for 2.5min, 30 cycles; 72°C for 10min; stored at 4°C, the amplified 1938bp GLDH gene cDNA was recovered and purified and cloned into the cloning vector PMD18 In -T, use the XbaI and SmaI restriction sites introduced by primers S1 and A1 to further clone into the plant expression vector pCAMBIA2301, sequence and identify to ensure that the reading frame of the coding region in the expression vector is correct, and obtain the plant overexpression vector plasmid pCAMBIA2301-GLDH;

2) Plant expression vector plasmid pCAMBIA2301-GLDH transformed into Agrobacterium

Add the plant overexpression vector pCAMBIA2301-GLDH to the competent Agrobacterium, mix well, and ice-bath for 30 minutes, then quickly freeze in liquid nitrogen for 1.5 minutes, quickly move to a 37°C water bath for 5-6 minutes, add 1mL of fresh YEB liquid medium, and store at 28°C , 250rpm shaking culture for 4h, then centrifuge at 12000rpm for 6min at 4°C, discard most of the supernatant, then add 200μL YEB liquid medium to suspend the bacterial solution, and spread it on the culture medium containing 50mg/L kanamycin and 50mg/L lizard. Fuping’s YEB solid medium, cultivated in the dark at 28°C for 2-3 days until a single colony grows on the plate, and obtained Agrobacterium LBA4404 carrying the vector pCAMBIA2301-GLDH after PCR identification;

3) Agrobacterium-mediated transformation of Wutaia

① Preparation of Agrobacterium infection solution

Pick a single colony of Agrobacterium LBA4404 carrying the plant overexpression vector pCAMBIA2301-GLDH, inoculate in 5mL YEB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin, shake at 28°C and 200rpm Overnight, the next day, take 500 μL of the bacterial liquid and transform it into 50 mL of fresh YEB liquid medium containing 50 mg/L kanamycin and 50 mg/L rifampicin, shake overnight at 28 °C and 200 rpm, until the OD ₆₀₀ of the bacterial liquid is 0.5 -0.6, centrifuge the bacterial solution at 4000rpm for 10min, discard the supernatant, resuspend with pH 5.2 MS liquid medium and dilute to 50mL, add acetosyringone to a final concentration of 100μmol/L, shake at 28°C for 4h at 200rpm for use Infection and transformation of explants;

② Pre-cultivation, infection and co-cultivation of explants

Sterilize the seeds of Utah with 70% alcohol for 1 min, then treat them with mass volume ratio 0.1% HgCl ₂ for 18 min, wash them with sterile water 4 times, dry the seeds on sterile filter paper and inoculate them in 1/2MS medium. Illumination for 12 hours, light intensity 2000-3000lx, temperature 25°C, after 4-5 days, when the cotyledon is fully expanded, cut the cotyledon with stalk of Utah as an explant and insert it into the pre-culture medium. The cultured petioled cotyledons were taken out from the culture medium and placed in the prepared Agrobacterium-infected bacteria solution for 5 minutes of shaking infection, and the excess Agrobacterium solution was absorbed with sterile filter paper, and another piece of sterilized filter paper was spread on the co-culture On the substrate, soak it with MS liquid medium, place the infected cotyledon with petiole on it for co-cultivation, and culture in dark at 25°C for 24-36h;

③ Adventitious bud regeneration, selection and plant formation

Rinse cotyledons with stipe after co-cultivation with sterilized distilled water for 4 times, blot dry with sterilized filter paper, and transfer to differentiation medium. After 25 days, take differentiated adventitious buds from explants and transfer them to screening medium for 2 days. -Three times of kanamycin screening culture, transfer the surviving resistant seedlings to the subculture medium to expand the plants and restore growth, and finally undergo rooting culture until complete regenerated plants are produced, and transplant them as seedlings Normal management to obtain T ₀ generation plants;

Invention medium formula used is as follows:

Seed germination medium: 1/2MS+0.8% agar pH 5.8;

Pretreatment medium: MS+0.1mg/L 2,4-D+1mg/LNAA+0.8% agar pH 5.8;

Co-culture medium: MS+0.1mg/L 2,4-D+2mg/L 6BA+100μmol/L acetosyringone+0.8% agar pH 5.2;

Differentiation medium: MN+4mg/L 6-BA+0.5mg/LNAA+5mg/LAgNO ₃ +500mg/L carbenicillin+0.9% agar pH 5.8;

Screening medium: MS+2mg/L 6-BA+0.3mg/LNAA+500mg/L carbenicillin+50mg/L kanamycin+0.8% agar pH 5.8;

Subculture medium: MS+0.5mg/L 6-BA+0.05mg/L NAA+0.5mg/L KT+0.8% agar pH5.8;

Rooting medium: B5+0.2mg/L NAA+0.7% agar pH5.8,

MN medium is half of NH ₄ ⁺ in MS medium;

④ Acquisition of transgenic plants and PCR identification

Genomic DNA was extracted from the plants of the T ₀ generation, and the plants with positive PCR detection results were the transgenic plants of Wutaicai.

Beneficial effect:

Vc is a very important compound both to the human body and to the plant itself. It is one of the new development directions of molecular breeding to increase the content of Vc or AsA in plants through genetic engineering technology, and to improve the nutritional quality and resistance of plants.

The invention improves the Vc content in the plant through the transgenic method, which has the advantages of small workload, strong operability, short experiment period, single target trait and the like compared with conventional cross breeding. Compared with the non-transgenic control, the expression of GLDH gene in the obtained transgenic plants has a significant difference, the maximum difference can be 28.5 times, and the highest Vc content of the plants is 1.8 times that of the control. important role in the synthetic pathway.

The successful acquisition of the transgenic plants of Wutaia provides new ideas and technical support for us to improve the quality and resistance of cabbage vegetables through genetic engineering technology in the future, which has important practical significance.

This study will not only provide a technical basis for improving the nutritional quality of vegetables, but also provide a scientific basis for the comprehensive and systematic study of the formation mechanism of the nutritional quality of non-heading Chinese cabbage, and further research on the regulation mechanism of Vc in plant growth and development.

4. Description of drawings

Figure 1 Plant overexpression vector pCAMBIA2301-GLDH

Figure 2 PCR detection of transgenic plants 1: DL 2000bp Marker; 2: Vector pCAMBIA2301-GLDH plasmid positive control; 3: Non-transgenic negative control; 4-12: GLDH overexpression transgenic plants

5. Specific implementation

(1) Construction of GLDH gene overexpression vector

1. Cloning of the coding region sequence of the GLDH gene

A pair of specific primers S1/A1 were designed according to the full-length cDNA sequence of non-heading Chinese cabbage GLDH gene (Genbank Accession No AY899298):

S1: 5′-GC TCTAGA CGCCTGAACTAAAACAAAAATGC-3′ XbaI

A1: 5′-TCC CCCGGG CGGCTTCACTCTTTCTTACAAACACT-3′SmaI

Total RNA was extracted from the leaves of non-heading Chinese cabbage 'Suzhouqing' (a local variety in Jiangsu, purchased in the market) as a template, and the first strand of cDNA was synthesized by reverse transcription, and PCR amplification was performed with ExTaq enzyme to amplify the complete GLDH Coding sequence. The reaction conditions are: 94°C for 3 min; 94°C for 1 min, 45°C for 1.5 min, 72°C for 3 min, 5 cycles; 94°C for 45 s, 64.4°C for 1 min, 72°C for 2.5 min, 30 cycles; 72°C for 10 min; 4°C for storage.

In order to facilitate the construction of the vector, according to the multiple cloning site of the plant binary vector pCAMBIA2301, restriction sites XbaI and SmaI were introduced at the 5' end and 3' end of the target gene, respectively.

The PCR product was recovered and purified by agarose gel and connected to the cloning vector PMD18-T, and the resulting recombinant plasmid was named pMD-GLDH.

2. Construction of plant overexpression vector

The recombinant plasmid pMD-GLDH was double-digested with XbaI and SmaI to recover a target fragment of about 1930 bp, and the vector pCAMBIA2301 plasmid was double-digested with XbaI and SmaI to recover a large fragment of about 12000 bp. The recovered product was dephosphorylated and ligated with T ₄ DNA ligase, and the ligated product was transformed into Escherichia coli DH5α to construct the plant overexpression vector pCAMBIA2301-GLDH (Fig. 1).

The plant overexpression vector pCAMBIA2301-GLDH constructed in this study contains the NPT-II (kanamycin resistance) fusion gene and the GUS fusion gene that can be expressed in plants and are connected to the CaMV35S promoter and NOS terminator. As a screening marker for the genetic transformation of the target gene into plant cells. Recombinant plasmid clones were identified by colony PCR and restriction endonuclease digestion.

(2) Transformation of plant expression vector plasmid into Agrobacterium

1. Preparation of Competent Agrobacterium

Pick a single colony of Agrobacterium LBA4404 strain and inoculate it in 5mL of YEB liquid medium, and culture it overnight at 28°C with shaking at 250rpm. The next day, take 1mL of the bacterial liquid and transform it into 50mL of fresh YEB liquid medium, and cultivate it at 28°C with shaking at 250rpm until The OD ₆₀₀ of the bacterial solution is about 0.5, transfer the bacterial solution to a 50mL sterile centrifuge tube, place in an ice bath for 45min, centrifuge at 4000rpm at 4°C for 5min, discard the supernatant, and suspend the precipitate with 10mL pre-cooled 0.15mol/L NaCl Afterwards, centrifuge at 4°C, 4000rpm for 5min, discard the supernatant, suspend the pellet with 2mL of pre-cooled 20mmol/L CaCl ₂ , dispense 150μL of each portion into 1.5mL centrifuge tubes (add 100μL of 50% glycerol), and freeze in liquid nitrogen for 2min , and stored in a -70°C refrigerator for later use.

2. Vector transformation of Agrobacterium

Take out a frozen competent Agrobacterium LBA4404 from the -70°C refrigerator, put it on ice to thaw, absorb 2 μg of the plant overexpression vector pCAMBIA2301-GLDH plasmid and add it to the thawed competent Agrobacterium, mix gently, Ice-bathed for 30 minutes, quick-frozen in liquid nitrogen for 1.5 minutes, and then quickly transferred to a 37°C water bath for 5-6 minutes. Add 1mL of fresh YEB liquid medium, shake and culture at 28°C and 250rpm for 4h to recover the strain, then centrifuge at 4°C and 12000rpm for 6min, discard most of the supernatant, leaving about 50μL, then add 200μL of YEB liquid medium Then suspend the bacterial liquid, spread it on the YEB solid medium containing 50mg/L kanamycin and 50mg/L rifampicin, culture in dark at 28°C for 2-3d until plaques grow on the plate, after PCR identification, obtain the Agrobacterium LBA4404 of the vector pCAMBIA2301-GLDH. The above work needs to be performed aseptically on an ultra-clean workbench.

(3) Agrobacterium-mediated transformation of Wutaia

1. Preparation of Agrobacterium Infection Solution

Pick a single colony of Agrobacterium LBA4404 carrying the plant overexpression vector pCAMBIA2301-GLDH, inoculate it in 5mL YEB liquid medium supplemented with 50mg/L kanamycin and 50mg/L rifampicin, shake overnight at 28°C and 200rpm , with the bacterial solution as a template, PCR was used to verify its correctness. The next day, take 500 μL of the bacterial liquid and transform it into 50 mL of fresh YEB liquid medium containing 50 mg/L kanamycin and 50 mg/L rifampicin, shake overnight at 28°C and 200 rpm until the OD ₆₀₀ of the bacterial liquid is 0.5-0.6, Centrifuge the bacterial solution at 4000rpm for 10min, discard the supernatant, resuspend with pH 5.2 MS liquid medium and dilute to 50mL, add acetosyringone to a final concentration of 100μmol/L, place at 28°C, shake at 200rpm for 4h, and then use it for external use. Plant infection transformation.

2. Pre-cultivation, infection and co-cultivation of explants

Select the seeds of Wutaicai (a local variety in Jiangsu, purchased in the market) to be sterilized with 70% alcohol for 1 min, then treated with 0.1% _HgCl for 18 min, rinsed with sterile water for 4 times, and inoculated on 1/2 2MS solid medium, the culture conditions are light for 12h, light intensity 2000-3000lx, temperature 25°C. After 4-5 days, when the cotyledon is fully unfolded, cut the cotyledon with stalk (the complete cotyledon with 1-2mm cotyledon, and avoid the growth point) as an explant and insert it obliquely into the pre-medium, and the culture conditions are the same as above. . Take the petioled cotyledons that have been pre-cultivated for 2-3 days from the culture medium, place them in the prepared Agrobacterium-infected bacteria solution and infect for 5 minutes, absorb the excess Agrobacterium solution with sterile filter paper, and take another sterilized Bacteria filter paper was laid flat on the co-culture medium, soaked with MS liquid medium, and the infected cotyledon with stalk was placed on it, ensuring that the incision of the cotyledon petiole was in contact with the filter paper, and cultured in the dark at 25°C for 24-36h.

3 Adventitious bud regeneration, selection and plant formation

Rinse cotyledons with stipe after co-cultivation with sterilized distilled water for 4 times, blot dry with sterilized filter paper, and transfer to differentiation medium. After 25 days, take differentiated adventitious buds from explants and transfer them to screening medium for 2 days. -Three times of kanamycin screening culture, 20 days each time. Transfer the surviving resistant seedlings to the subculture medium to multiply the plants and restore growth, and finally undergo rooting culture until complete regenerated plants are produced. Select the plants with good growth to open the bottle and harden the seedlings, wash off the root medium, and transplant To the nutrient pot equipped with peat soil: vermiculite = 3: 1, to obtain T ₀ generation plants.

The medium formula used for transgenics is as follows:

Seed germination medium: 1/2MS+0.8% agar pH 5.8

Pretreatment medium: MS+0.1mg/L 2,4-D+1mg/L NAA+0.8% agar pH 5.8

Co-culture medium: MS+0.1mg/L 2,4-D+2mg/L 6BA+100μmol/L acetosyringone+0.8% agar pH 5.2

Differentiation medium: MN+4mg/L 6-BA+0.5mg/LNAA+5mg/LAgNO ₃ +500mg/L carbenicillin+0.9% agar pH 5.8

Screening medium: MS+2mg/L 6-BA+0.3mg/LNAA+500mg/L carbenicillin+50mg/L kanamycin+0.8% agar pH 5.8

Subculture medium: MS+0.5mg/L 6-BA+0.05mg/L NAA+0.5mg/L KT+0.8% agar pH5.8

Rooting medium: B5+0.2mg/L NAA+0.7% agar pH5.8

MN medium is NH ₄ ⁺ halved in MS medium.

4. Acquisition of transgenic plants and PCR identification

Genomic DNA was extracted from new leaves of regenerated plants, and detected by PCR method. The DNA extracted from the leaves of normal regenerated non-transgenic Utami plants was a negative control template, and the plant expression vector plasmid pCAMBIA2301-GLDH was a positive control template. The primers used were:

GUS-F: 5′-ACGTCCTGTAGAAAACCCCAACC-3′

GUS-R: 5′-TCCCGGCAATAACATACGGCGT-3′

The PCR reaction program was: 94°C for 3min; 94°C for 45s, 59°C for 1min, 72°C for 1.5min, 30 cycles; 72°C for 10min. The plants with positive PCR detection results are the transgenic plants of Wutaicai. (figure 2).

(4) Identification of transcript levels in transgenic Utah plants by fluorescent quantitative PCR

Total RNA was extracted from the new leaves of the same leaf position of the non-transgenic negative control and the transgenic Wutaia (operated according to the instructions of the Simply P Total RNA Extraction Kit kit from Bioflux Company), and the expression of GLDH gene was detected by fluorescent quantitative PCR method: GLDH gene The primers are SP2/AP2, the non-heading Chinese cabbage 3-phosphate glyceraldehyde dehydrogenase gene is used as the internal standard gene, and the primers are GAPDHF/GAPDHR:

SP2: 5'-GGCCTTGGTGGACTTGGA-3',

AP2: 5'-CATCACTCCACCCCTACTC-3';

GAPDHF: 5'-ACTGTCTCGCTCCATTCG-3',

GAPDHR: 5′-AGTTTCCCCTTTGAGGTTAG-3′

The method refers to the instructions of the fluorescent quantitative reagent SYBR Premix Ex Taq ^TM from TaKaRa Company. The PCR reaction program is 95°C for 2min; 95°C for 20s, 52°C for 20s, 72°C for 20s, 45 cycles; 75°C for 1s; 78°C for 1s; 72°C for 10min; Melting curves were determined from 72°C to 95°C. The experimental data were analyzed by Rotor-Gene 6 software. It was found that compared with the control of non-transgenic plants, the expression of GLDH gene in the transgenic plants was significantly increased, the highest being 28.5 times that of the control.

(5) Determination of Vc content in transgenic Wutaia plants

The leaves of non-transgenic negative control and transgenic plants at the same period were used as materials, and the content of AsA in plants was determined by an improved spectrophotometer. Specific method: Weigh 0.3 g of plant leaves, add 3 mL of 5% TCA to grind into a homogenate, then centrifuge at 4000×g for 10 min, and the supernatant is used for determination. During the measurement, 1.0mL 5% TCA, 1.0mL EtOH, 0.5mL 0.4% H ₃ PO ₄ -EtOH, 1.0mL 5% BP-EtOH, 0.5mL 0.03% FeCl ₃ -EtOH and 0.5mL were added to the 4.5mL reaction system Sample extract. React in a water bath at 30°C for 90 minutes, and measure the absorbance at a wavelength of 534 nm.

Compared with the non-transgenic control, the Vc content of the obtained transgenic plants was significantly different, the highest being 1.8 times that of the control, and it was generally positively correlated with the expression of the GLDH gene.

The invention verifies the important role of the GLDH gene in the plant Vc synthesis pathway, and obtains the transgenic plants of Wutah with higher Vc content. It provides new ideas and technical support for us to improve the quality and resistance of cabbage vegetables through genetic engineering in the future, and has important practical significance.

SEQUENCE LISTING

<110> Nanjing Agricultural University

<120> Application of non-heading Chinese cabbage GLDH gene to increase Vc content in Wutaicai

<130> instruction manual

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<141>2008-06-12

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Claims (2)

1. The application of non-heading Chinese cabbage GLDH gene to increase the Vc content of Wutaicai. 2. the non-heading Chinese cabbage GLDH gene according to claim 1 improves the application of Wutaicai Vc content, it is characterized in that: 1) Construction of plant overexpression vector According to the cDNA sequence of non-heading Chinese cabbage L-galactonic acid-1,4-lactone dehydrogenase gene GLDH, gene accession number: AY899298, design primers to amplify its complete coding reading frame, and on the upstream and downstream primers The restriction endonuclease sites XbaI and SmaI were introduced respectively, and the designed primers were: S1: 5′-GCTCTAGACGCCTGAACTAAAACAAAAATGC-3′ XbaI A1: 5′-TCCCCCGGGCGGCTTCACTCTTCTTACAAACACT-3′SmaI Using the total RNA of non-heading cabbage 'Suzhouqing' leaves as a template, the first strand of cDNA was synthesized by reverse transcription, and PCR amplification was performed with ExTaq enzyme. The PCR program was as follows: 94°C for 3 minutes; 94°C for 1 minute, 45°C for 1.5 minutes , 72°C for 3min, 5 cycles; 94°C for 45s, 64.4°C for 1min, 72°C for 2.5min, 30 cycles; 72°C for 10min; stored at 4°C, the amplified 1938bp GLDH gene cDNA was recovered and purified and cloned into the cloning vector PMD18 In -T, the XbaI and SmaI restriction sites introduced by primers S1 and A1 were used to further clone into the plant expression vector pCAMBIA2301, and the sequence identification ensured that the reading frame of the coding region in the expression vector was correct, and the plant overexpression vector plasmid pCAMBIA2301-GLDH was obtained; 2) Plant expression vector plasmid pCAMBIA2301-GLDH transformed into Agrobacterium The plant overexpression vector pCAMBIA2301-GLDH was added to the competent Agrobacterium, mixed evenly, ice-bathed for 30 minutes, then quick-frozen in liquid nitrogen for 1.5 minutes, quickly moved to a 37°C water bath for 5-6 minutes, added 1 mL of fresh YEB liquid medium, and incubated at 28°C , 250rpm shaking culture for 4h, then 4°C, 12000rpm centrifugation for 6min, discard most of the supernatant, and then add 200μL YEB liquid medium to suspend the bacterial solution, and spread it on the culture medium containing 50mg/L kanamycin and 50mg/L lizard. Fuping’s YEB solid medium, cultivated in the dark at 28°C for 2-3 days until a single colony grows on the plate, and obtained Agrobacterium LBA4404 carrying the vector pCAMBIA2301-GLDH after PCR identification; 3) Agrobacterium-mediated transformation of Wutaia ① Preparation of Agrobacterium infection solution Pick a single colony of Agrobacterium LBA4404 carrying the plant overexpression vector pCAMBIA2301-GLDH, inoculate in 5mL YEB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin, shake at 28°C and 200rpm Overnight, the next day, take 500 μL of the bacterial liquid and transform it into 50 mL of fresh YEB liquid medium containing 50 mg/L kanamycin and 50 mg/L rifampicin, shake overnight at 28 °C and 200 rpm, until the OD ₆₀₀ of the bacterial liquid is 0.5 -0.6, centrifuge the bacterial solution at 4000rpm for 10min, discard the supernatant, resuspend and dilute to 50mL with pH5.2 MS liquid medium, add acetosyringone to a final concentration of 100μmol/L, shake at 28°C for 4h before use Infection and transformation of explants; ② Pre-cultivation, infection and co-cultivation of explants Sterilize the seeds of Utah with 70% alcohol for 1 min, then treat them with mass volume ratio 0.1% HgCl ₂ for 18 min, wash them with sterile water 4 times, dry the seeds on sterile filter paper and inoculate them in 1/2MS medium. Illumination for 12 hours, light intensity 2000-3000lx, temperature 25°C, after 4-5 days, when the cotyledon is fully expanded, cut the cotyledon with stalk of Utah as an explant and insert it into the pre-culture medium. The cultured petioled cotyledons were taken out from the culture medium and placed in the prepared Agrobacterium-infected bacteria solution for 5 minutes of shaking infection, and the excess Agrobacterium solution was absorbed with sterile filter paper, and another piece of sterilized filter paper was spread on the co-culture On the base, soak it with MS liquid medium, place the infected cotyledons with petioles on it for co-cultivation, and culture in dark at 25°C for 24-36h; ③ Adventitious bud regeneration, selection and plant formation Rinse cotyledons with stipe after co-cultivation with sterilized distilled water for 4 times, blot dry with sterilized filter paper, and transfer to differentiation medium. After 25 days, take differentiated adventitious buds from explants and transfer them to screening medium for 2 days. -Three times of kanamycin screening culture, transfer the surviving resistant seedlings to the subculture medium to expand the plants and restore growth, and finally undergo rooting culture until complete regenerated plants are produced, and transplant them as seedlings Normal management to obtain T ₀ generation plants; Invention medium formula used is as follows: Seed germination medium: 1/2MS+0.8% agar pH5.8; Pretreatment medium: MS+0.1mg/L 2,4-D+1mg/L NAA+0.8% agar pH5.8; Co-culture medium: MS+0.1mg/L 2,4-D+2mg/L 6BA+100μmol/L acetosyringone+0.8% agar pH5.2; Differentiation medium: MN+4mg/L 6-BA+0.5mg/LNAA+5mg/LAgNO ₃ +500mg/L carbenicillin+0.9% agar pH5.8; Screening medium: MS+2mg/L 6-BA+0.3mg/LNAA+500mg/L carbenicillin+50mg/L kanamycin+0.8% agar pH5.8; Subculture medium: MS+0.5mg/L6-BA+0.05mg/LNAA+0.5mg/L KT+0.8% agar pH5.8; Rooting medium: B5+0.2mg/L NAA+0.7% agar pH5.8, MN medium is half of NH ₄ ⁺ in MS medium; ④ Acquisition of transgenic plants and PCR identification Genomic DNA was extracted from the plants of the T ₀ generation, and the plants with positive PCR detection results were the transgenic plants of Wutaicai.

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