Gene and method for regulating gossypol traits of cotton
A technology of cotton and gossypol, applied in the field of botany
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Embodiment 1
[0060] Embodiment 1, tissue expression characteristic
[0061] After extensive and in-depth screening, the inventors cloned and obtained multiple EST sequences that may be related to gossypol synthesis according to the data of differential expression of glandular cotton and non-glandular cotton, and obtained the full-length sequence by using the method of RACE. The expression characteristics of CYP82D113, DH1 and gossypol synthesis pathway-related enzyme genes in different tissues are as follows: figure 1 A-D and F.
[0062] After further research, the inventors found that the expression of one cytochrome P450 monooxygenase (CYP82D113) and one alcohol dehydrogenase (DH1) in the cotton leaves was suppressed, and then the leaves of gossypol (Gossypol) and semi-gossypol The accumulation of ketones (Hemigossypol) decreased significantly. Determination of gossypol content in cottonseed as figure 1 e.
Embodiment 2
[0063] Example 2, Cotton Total RNA Extraction, PCR Amplification of Target Gene DH1 and CYP82D113
[0064] A. Extraction of cotton total RNA and preparation of cDNA reverse transcription
[0065] Cotton material (upland cotton variety "Jinmian R15", purchased from the Cotton Research Institute of Shanxi Academy of Agricultural Sciences) was ground with liquid nitrogen, and 0.5ml plant total RNA extraction reagent (RNAplant plus Reagent, Tiangen) was added to each 100mg material, and shaken until thoroughly Mix well and place at room temperature for 5min. Centrifuge at 12,000 rpm at 4°C for 1 min, transfer the supernatant to a new RNase-free centrifuge tube, add 0.1ml 5M NaCl, and mix gently. Add 0.3ml of chloroform and mix by inverting up and down. Centrifuge at 12000 rpm for 10 min at 4°C. LiCl was added to the supernatant to a final concentration of 2M. Stand at -20°C for 3 hours, then centrifuge at 13000g for 10min. The pellet was washed once with 70% ethanol, dried in...
Embodiment 3
[0074] Embodiment 3, vector construction, Escherichia coli transformation and yeast transformation
[0075] A. Vector construction
[0076] high fidelity enzyme The full-length cDNA fragment of DH1 amplified by HS DNA polymerase was digested with BamHI and HindIII and ligated into pET-32a vector; the full-length cDNA fragment of CYP82D113 was digested with SmaI and KpnI and ligated into pYeDP60 yeast expression vector.
[0077] B. Preparation of Escherichia coli Competent Cells
[0078] Escherichia coli BL-21 stored at -70°C was streaked on a solid LB plate and cultured overnight at 37°C; a single colony was picked and placed in 5 mL of liquid LB medium and cultured overnight at 250 rpm. The next day, inoculate into 500mL liquid LB medium at a ratio of 1 / 50, culture at 18-22°C until OD600≈0.5 (about 5-6h), and cool on ice for 10min. Centrifuge at 2,500 g for 10 min at 4°C, resuspend the cells with 160 mL of transformation buffer, discard the supernatant, and finally resusp...
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