CN105695506A - Method for improving cottonseed nutritional quality and application thereof - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for improving the nutritional quality of cottonseed and its application. The technical problem to be solved by the invention is to provide a new option for increasing the carotenoid content of cottonseed and improving the nutritional quality of cottonseed. The technical scheme of the invention is to construct a plant expression vector for driving a phytoene synthase gene PSY by a seed-specific promoter, and transform cotton to increase the carotenoid content in cotton seeds, and the PSY gene is the cotton GhPSY2 gene. After the target gene is regulated by the invention, the carotenoid content in the mature cottonseed is significantly increased. The method of the invention is simple and easy to implement, has remarkable effects and has an application prospect of generating huge economic benefits.

Description

A method for improving the nutritional quality of cottonseed and its use

technical field

The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for improving the nutritional quality of cottonseed and its application.

Background technique

Carotenoids belong to isoprenoid compounds and are an important class of plant natural pigments. Carotenoids are a class of fat-soluble pigments with bright colors, mainly yellow, orange and red. Carotenoids are important health-protecting substances for animals and humans. As the only source of vitamin A in humans and animals, vitamin A formed by the transformation of carotenoids is an indispensable nutrient for humans and animals, and can effectively play a role in the health care of the vision and skin systems. In addition, carotenoids have antioxidant effects, and their presence can delay aging and reduce the incidence of cardiovascular diseases and cancer. However, the animal body itself cannot directly synthesize carotenoids, and the required carotenoids must be ingested from plants or microorganisms through the food chain. Only a few carotenoids such as β-carotene and astaxanthin can be obtained through microbial fermentation, which is far from meeting the demand. The carotenoids directly extracted from plants are safe and reliable, and have obvious physiological and pharmacological activities. However, the content of carotenoids in plants is limited, and it is of great application value to cultivate crops rich in carotenoids.

Cotton is one of the most important agricultural crops in the world and is the main source of natural fibers for human beings. As the largest by-product in the cotton production process, cottonseed has huge utilizable value. Cottonseed is rich in nutrients such as protein and oil, and is an important raw material for oil extraction and feed production. The content of carotenoids in cottonseed is about 15 μg/g. Cultivating cotton varieties with high carotenoids in cottonseed and increasing the comprehensive utilization of cottonseed will be beneficial to improve the economic benefits of cotton planting.

In recent years, the main goal of carotenoid metabolic engineering in plants is to regulate the expression of structural genes in the synthetic pathway. At present, there have been many successful reports on the regulation of carotenoid synthesis by the expression of carotenoid synthase gene in many plants. For example, in "Golden Rice", Lcyb, Psy, and CrtI (Pds) genes were co-transformed into a japonica rice variety to obtain golden endosperm. The analysis showed that the endosperm contained a variety of carotenoids and was rich in content. The Chinese patent application with the patent publication number CN101979601A discloses a method for increasing the carotenoid content of tomato, which uses genetic engineering to construct cryptochrome gene 1, the carboxyl end of cryptochrome gene 1 and the carboxyl end of cryptochrome gene 2. The overexpression vector is used to transform the tomato to obtain the transgenic tomato, and the content of lycopene and total carotenoids in the transgenic tomato fruit is greatly increased.

Phytoene synthase (PSY) is an important rate-limiting enzyme involved in the carotenoid biosynthetic pathway, which catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to form the first carotenoid - Phytoene. Overexpression of phytoene synthase in tomato, rapeseed, Arabidopsis and potato significantly increased the level of accumulated carotenoids in the corresponding tissues. It can be seen that changing the expression level of phytoene synthase will profoundly affect the carotenoid content in plants. But so far, there is no report about the improvement of cottonseed carotenoid content.

Contents of the invention

The technical problem to be solved by the invention is to provide a new option for cottonseed nutrition improvement.

The technical solution of the present invention is a method for improving the nutritional quality of cottonseed, constructing a plant expression vector for driving the phytoene synthase gene PSY by a seed-specific promoter, and transforming cotton to increase the carotenoid content in cottonseed, said PSY The gene is cotton GhPSY2 gene.

Specifically, the GhPSY2 gene has the nucleotide sequence shown in SEQ ID No.1.

Specifically, the promoter regulating the expression of the PSY gene is a kidney bean seed-specific promoter.

Preferably, the seed-specific promoter is the promoter pV of the bean storage protein β-phaseolin gene, which has the nucleotide sequence shown in SEQ ID No.2.

Specifically, the plant expression vector has the nucleotide sequence shown in SEQ ID No.3.

Specifically, the transformation is genetic transformation mediated by Agrobacterium tumefaciens.

The invention also provides the application of the method in increasing the carotenoid content of cottonseed.

Beneficial effects of the present invention: Through research and analysis, the present invention uses a specific promoter to specifically express the cotton phytoene synthase gene GhPSY2 during seed development, and obtains transgenic cotton with significantly increased carotenoid content in cotton seeds. The test result proves that after the target gene is regulated by the invention, the content of carotenoid in the improved cottonseed is significantly increased, and the nutrition of the cottonseed is strengthened. The method of the invention is simple and easy to implement, has remarkable effect and has the potential of generating huge economic benefits.

Description of drawings

Figure 1pV: Gene structure diagram of the T-DNA region of the expression vector of GhPSY2

CaMV35S-P, cauliflower mosaic virus 35S promoter; 2×CaMV35S-P, two cauliflower mosaic virus 35S promoters in series; CaMV35S-T, cauliflower mosaic virus 35S terminator; GhPSY2, cotton phytoene synthesis Enzyme gene; GUS: NPTII, fusion gene of β-glucosidase and neomycin phosphotransferase; LB, left border of T-DNA; Nos-T, terminator of Agrobacterium opine synthase gene; pV, kidney bean seed Specific promoter; RB, T-DNA right border.

Figure 2 pLGN-pV: GhPSY2 expression vector construction process

See embodiment 2 for the specific experimental method. CaMV35S-P, cauliflower mosaic virus 35S promoter; 2×CaMV35S-P, two cauliflower mosaic virus 35S promoters in series; CaMV35S-T, cauliflower mosaic virus 35S terminator; GhPSY2, cotton phytoene synthesis Enzyme gene; GUS: NPTII, fusion gene of β-glucosidase and neomycin phosphotransferase; LB, left border of T-DNA; Nos-T, terminator of Agrobacterium opine synthase gene; pV, kidney bean seed Specific promoter; RB, T-DNA right border. Relevant enzyme cutting sites and positions are marked on each vector.

Fig.3 Wild type (WT) cotton and pV:GhPSY2 transgenic cotton

A: GhPSY2 expression level (Relative expression) detection; B: wild type or transgenic cotton mature seeds; C: wild type or transgenic cotton mature seeds after pulverization; D: cottonseed oil diluted 20 times with ether; E: cottonseed oil Medium carotenoid content. WT: wild-type sample; #1, #2: two lines of pV: GhPSY2 transgenic cotton; * and ** show significant fronto-significant difference from wild-type control (t test, n=3).

detailed description

Routine experimental operations used in the following examples:

1. Extraction of DNA

Genomic DNA was extracted using Plant Genomic DNA Rapid Extraction Kit (Aidlab). See the instructions for detailed steps.

2. Extraction of RNA

RNA was extracted using the EASYspin Plant RNA Rapid Extraction Kit (Aidlab). See the instructions for detailed steps.

3. PCR amplification of DNA fragments

The amplification system is as follows: 10×ExPCRbuffer (Mg ²⁺ free) 2.5 μL, 2.5 mmol/LdNTPs 2 μL, 25 mmol/LMgCl _{2 2} μL, primer 1 (5 μmol/L) 1 μL, primer 2 (5 μmol/L) 1 μL, ExTaq DNA polymerase 1U , about 60ng of genomic DNA, add ddH ₂ O to 25μL.

The amplification program was: 94°C, 5min; 94°C, 30sec, 56°C, 30sec, 72°C, 1.5min, 35 cycles; 72°C extension, 10min.

4. Recovery, ligation and cloning of DNA fragments

DNA fragments were recovered using the BioFlux Gel Recovery Kit. DNA fragment ligation was performed using T4DNAligase. Establish the following ligation system between the recovered fragment and the pUCm-T (Shanghai Sangong) vector: 1 μL of 10×T4 DNA ligation buffer, 1 μL of vector DNA fragment, 1 μL of exogenous ligation product DNA fragment, 1 μL of T4 DNA ligase, and make up the volume with ddH ₂ O to 10 μL. The molar ratio of vector DNA fragments to exogenous ligation product DNA fragments was 1:3, and ligated at 16°C for 12 hours. The ligation product was then transformed into Escherichia coli DH5α. The obtained resistant clones were cultured overnight in liquid, and the plasmids were extracted with BioFlux plasmid extraction kit, and sequenced in Invitrogen after verification by enzyme digestion.

5. GUS histochemical staining

Since the expression vector used in the laboratory has a GUS reporter gene, GUS histochemical staining is generally used to detect and track the transgene. Specific method: Take a small amount of transgenic cotton leaves (with wounds) and place them in a 96-well plate, add GUS staining solution [0.1mol/LK ₃ Fe(CN) ₆ , 0.1mol/LK ₄ Fe(CN) ₆ , 0.01mol/LNa ₂ EDTA, 500mg/LX-Gluc, 1% TritonX-100 (v/v), 0.14mol/L sodium phosphate buffer (pH7.0)], placed at a constant temperature of 37°C for about 2 hours, and then dyed fully Destain with 75% ethanol. The plants whose leaves can be stained with specific blue by GUS staining solution are positive for the transgene.

6. Extraction of cottonseed oil and determination of its carotenoid content

Cottonseed oil is extracted by Soxhlet extraction method:

1) Put the folded filter paper bag into a glass dish and dry it in an oven at 105°C for 2 hours, take it out and put it in a desiccator to cool to room temperature, and weigh it accurately to record W ₁ .

2) Take about 3g of seeds and pulverize them with a pulverizer, pass through a 40-mesh sieve, take about 1g of samples and put them into the above-mentioned weighed filter paper bag, put them in an oven at 105°C for 3 hours, take them out and put them in a desiccator to cool to room temperature. Accurately weigh W ₂ .

3) The above-mentioned filter paper bag containing the sample and weighed is put into a fat extractor (BUCHIB-811 extraction system), and soaked in a sufficient amount of ether overnight.

4) After overnight, extract at 70-80°C for 8 hours. After the extraction is completed, collect the bottom oil (ie cottonseed oil), put the paper bag in an oven at 105°C to dry for 3 hours, take it out and put it in a desiccator to cool to room temperature, and weigh it accurately Remember W ₃ .

5) Crude fat content in seeds (%)=(W ₂ -W ₃ )/(W ₂ -W ₁ )×100%

Further, dilute the cottonseed oil obtained by the above extraction n times with ether to the detectable range of a microplate reader, take 200 μL on a microplate plate, detect the absorbance at 445 nm with a microplate reader, and substitute the absorbance into the standard curve (refer to β- Carotene standard curve) to calculate the carotenoid content.

7. Preparation of β-carotene standard curve

1) Accurately weigh 1mg of β-carotene standard substance, dilute it in a 10ml brown volumetric flask with ether, the concentration of β-carotene is 100μg/ml;

2) Continue to dilute to 7 concentration gradients: 0, 0.1, 1, 3, 6, 9, 12, 15 μg/mL;

3) Take 200 μL on a microplate plate, and detect the absorbance at 445 nm with a microplate reader;

4) Linear regression, making a standard curve.

Table 1 Primer list

Primer Sequence 5'-3' describe GhPSY2-U GGATCCATGGCTGGTGTTCTTCTTTGGGTG Gene Amplification Upstream Primers GhPSY2-D GAATTCACTAGTGATCTTCAGAACAATTTG Gene Amplification Downstream Primers pV-F AAGCTTGAATTCATTGTACTCCCAGTATC Promoter amplification upstream primers pV-R GGATCCCTCGAGTCTAGAAGTAGTATTG Promoter amplification downstream primers GhPSY2-F(RT) ATGAATTTTGATGAGCTCTAT RT-PCR upstream primers GhPSY2-R(RT) ATTAGCAATTCCGAGGGCTA RT-PCR downstream primers GhACT4-F(RT) TTGCAGACCGTATGAGCAAG RT-PCR upstream primers GhACT4-R(RT) ATCCTCCGATCCAGACACTG RT-PCR downstream primers

Example 1 Acquisition of cotton phytoene synthase gene GhPSY2

On the Phytozome public database (https://phytozome.jgi.doe.gov), using the base sequence of Arabidopsis phytoene synthase gene AtPSY (At5g17230) as bait, BlastN search homologous genes. The results showed that the gene numbered Gorai.006G009400 in Raymond cotton was highly homologous to At5g17230. Using the CDS sequence of Gorai.006G009400 as a reference, design primers (Table 1, GhPSY2-U&GhPSY2-D), use upland cotton cDNA as a template, amplify the obtained fragment, and add BamHI and EcoRI sites at both ends of the fragment. The sequencing results are as follows: SEQ ID NO.1. Cluster analysis showed that this sequence had high homology with known phytoene synthase genes from other species (such as Arabidopsis, tomato, soybean, etc.), so this sequence was considered to be a cotton phytoene synthase gene. Gene encoding erythromycin synthase (GhPSY2).

Example 2 Construction of the GhPSY2 expression vector specific to the seed development stage

The process of constructing GhPSY2 into the plant expression vector pLGN-Nos is shown in Figure 2. The pLGN-Nos vector is a binary plant expression vector transformed from the traditional plant expression vector pBI121. Its T-DNA segment (the region between RB and LB, Figure 2) was replaced by a constitutive CaMV35S promoter (CaMV35S-P) controlling the fusion gene expression cassette of the reporter gene GUS and the marker gene NPTII, and another cassette composed of CaMV35S- P-controlled expression cassette.

Use primers (Table 1, pV-F and pV-R) to amplify and clone the promoter pV from the bean genome, and add HindⅢ and BamHI sites at both ends of the promoter. GhPSY2 was excised from the cloning vector with BamHI and EcoRI, and the promoter pV was excised from the cloning vector with HindIII and BamHI. The above two fragments were simultaneously added to the ligation reaction system containing the pLGN-Nos vector fragment digested by HindIII and EcoRI, and ligated to obtain the final expression vector pLGN-pV:GhPSY2. All restriction endonucleases were purchased from Roche Company and operated according to the instruction manual. The recovery, ligation and Escherichia coli transformation of DNA fragments were carried out according to the aforementioned conventional operation methods. Referring to the Bio-RADMicroPulser user manual, the above-mentioned vectors were introduced into Agrobacterium LB4404 by electric shock transformation.

The genetic transformation of embodiment 3 cotton

The cotton genetic transformation of the above expression vector was carried out by the method mediated by Agrobacterium tumefaciens, and the medium formula used is shown in Table 2. The specific method is as follows: select the full wild-type Jimian No. 14 cotton seeds (shelled), place in a sterile conical flask, rinse with 75% alcohol for ₁ minute, and sterilize with 0.1% HgCl for about 10 minutes (shaking the conical flask) ), fully rinsed with sterile water for 6-8 times, placed in a shaker at 30°C (100-110rpm), and waited for the radicle to grow about 1cm (about 24-36h, change the water every 8 hours), take it out, and put the embryo The roots are inserted into the seed germination medium. Culture in a dark room at 30°C until the hypocotyls elongate to 2-3cm (about 48h). A single colony of Agrobacterium carrying the expression cassette vector was inoculated in liquid YEB medium containing 50 mg/LKm and 125 mg/LSm, and placed on a shaker (200 rpm) at 28°C. After about 20 hours, measure the OD value (OD ₆₀₀ ) of the bacterial solution every hour, and the OD ₆₀₀ is preferably 0.8-1.0. Collect the Agrobacterium liquid, centrifuge at 8000 rpm for 1 min, and discard the supernatant. After the co-cultivation liquid medium containing AS (acetosyringone, 100μmol/L) was used to resuspend the pellet at a ratio of 1:1 (v/v), the resuspension was collected in a 100mL Erlenmeyer flask and placed on a shaker at 30°C (100- 110rpm) for about 30min. Cut the hypocotyl into small pieces about 0.8-1cm long, place them in the resuspension solution, infect on a shaker at 30°C for 40 minutes, discard the liquid, spread the hypocotyl on the surface of the co-culture medium, culture in the dark room for about 48 hours, and transfer to In the selection medium, culture at 30°C (16h light/8h dark), subculture once every 15 days or so until callus formation. After the number of calluses increases, transfer to the callus medium for culture. Pick the embryogenic callus in good condition and insert it into the suspension medium, place it on a shaker at 30°C (100-110rpm) for suspension culture for about one week, filter it through a 30-mesh sieve, and spread the filtered fine embryogenic granules on the body Embryo elongation medium until green mature somatic embryos grow. Transfer to the elongation medium and continue to cultivate the somatic embryos to about 1 cm, transfer to the rooting medium until the seedlings grow into (about 10 cm), transplant to the nutrient pot, and continue to cultivate. All the above operations must be done under sterile conditions.

The selected GUS ⁺ and vigorously regenerated cotton seedlings were transplanted into planting pots, and were routinely managed in the greenhouse until the cotton fibers and seeds matured. Harvest T1 generation transgenic cotton seeds, continue planting T1 generation and harvest T2 generation seeds, sow T2 generation seeds and carry out GUS tissue staining (see routine operation method) to the germinated T2 generation seedlings, screen out homozygous transgenic T2 generation lines ( All are GUS-positive or GUS-negative plants), transplant T2 homozygous lines, and detect the expression level of the target gene GhPSY2 and investigate the changes in seed traits.

Table 2 Medium for genetic transformation of cotton mediated by Agrobacterium tumefaciens

MS: Murashige & Skoog, 1962; B5: Gamborg, 1986; Gelrite: Sigma, Cat. No.: G1910; SH: Schenk & Hildebrandt, 1972.

The detection of embodiment 4GhPSY2 gene transcription level

The RNA of control and transgenic cotton embryos 36 days after flowering was extracted, and a strand of cDNA was synthesized by reverse transcription, which was used as a template for quantitative PCR detection. The specific operation steps are as follows: use a cDNA one-strand synthesis kit (TaKaRa Company) to synthesize one-strand cDNA of various RNAs, and all operations are carried out according to the instructions of the kit. Quantitative PCR was carried out on the CFX96 quantitative PCR detection system (Bio-Rad). The 25 μL reaction system included 12.5 μL 2×SuperMix (Bio-Rad), 0.2 μmol/L each of the upstream and downstream primers and 1 μL one-strand cDNA. The temperature cycle parameters were pre-denaturation at 95°C for 2 min; 95°C for 10 sec, 57°C for 20 sec, and 40 cycles of amplification. Cotton Actin4 gene was used as internal standard. The relative expression of each gene was calculated with the analysis software Bio-RadCFXManager2.0 that comes with the quantitative PCR instrument. The sequences of the quantitative PCR primers used are listed in Table 1.

Gene expression analysis found that the transcription level of GhPSY2 in transgenic samples was significantly higher than that in wild-type samples, indicating that the pV promoter can promote the expression of GhPSY2 in embryos (Fig. 3A).

The investigation of embodiment 5 transgenic cotton seed character

Carotenoids are a class of important terpenoid natural pigments that appear yellow, orange or red. Observe the mature cotton seeds: wild-type mature seeds are white, while pV:GhPSY2 transgenic cotton seeds are orange (Fig. 3B). The mature seeds were further pulverized, the wild-type samples were light yellow, and the pV:GhPSY2 transgenic samples were orange (Fig. 3C). The cottonseed oil in the seeds was extracted with ether as a solvent, and the extracted cottonseed oil was diluted 20 times in ether, and the wild-type sample was nearly colorless, while the transgenic sample remained obviously yellow (Fig. 3D). The detection of carotenoid content showed (Figure 3E): the carotenoid content in cottonseed oil of the wild-type sample was 38.63±4.02 μg/mL, and the carotenoid content in cottonseed oil of the two transgenic samples was 269.05±4.47 μg/mL and 245.22±6.36 μg/mL, significantly higher than that of the wild-type sample. In summary, promoter pV can promote the expression of phytoene synthase gene GhPSY2 to promote the synthesis and accumulation of carotenoids in cotton seeds.

The above examples show that the method for improving cottonseed nutrition of the present invention can up-regulate the expression of GhPSY2 in the embryo, promote the synthesis of carotenoids, and achieve the purpose of strengthening cottonseed nutrition. In the present invention, a cotton phytoene synthetase gene (GhPSY2) is discovered and cloned through gene sequence homologous comparison. Using the bean seed-specific promoter pV to guide the specific expression of GhPSY2 in cottonseed, increase the level of phytoene synthase in cottonseed, so as to achieve the purpose of increasing the carotenoid content in cottonseed, is a simple and feasible way to strengthen the nutrition of cottonseed method.

references:

Doyle, J.J., Schuler, M.A., Godette, W.D., Zenger, V., Beachy, R.N., & Slightom, J.L. (1986).

Claims (7)

1. the method improveing Semen Gossypii nutritional quality, it is characterized in that: build seed-specific expression promoter and drive the plant expression vector of phytoene synthase gene PSY, and converting cotton, improving carotenoid content in Semen Gossypii, described PSY gene is Cotton Gossypii GhPSY2 gene。

2. the method for claim 1, it is characterised in that: described GhPSY2 gene has the nucleotide sequence as shown in SEQIDNo.1。

3. method as claimed in claim 1 or 2, it is characterised in that: the promoter of regulation and control PSY gene expression is phaseolus vulgaris seeds specific promoter。

4. method as claimed in claim 3, it is characterised in that: described seed-specific expression promoter is the promoter pV of Kidney bean storage protein β-Phaseolin β-phaseolin gene, has the nucleotide sequence as shown in SEQIDNo.2。

5. the method as described in any one of Claims 1 to 4, it is characterised in that: described plant expression vector is the nucleotide sequence shown in SEQIDNo.3 such as。

6. the method as described in any one of Claims 1 to 5, it is characterised in that: described is converted into Agrobacterium tumefaciens mediated genetic transformation。

7. the purposes in improving Semen Gossypii carotenoid content of the method described in any one of claim 1～6。