The germination protein gene jsglp1 of walnut-like walnut in Yangbi big bubble and its application
A technology of Yangbi big bubble walnut and germin, which is applied in the direction of application, genetic engineering, plant genetic improvement, etc., to achieve the effect of shortening the breeding cycle, reducing the use, and broad market application prospects.
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Embodiment 1
[0022] Example 1: JsGLP1 Full-length cDNA cloning and sequence analysis
[0023] The walnut was inoculated with G. anthracnose, and the total RNA was extracted from the leaves 4 hours after inoculation. The treated leaves of walnut were ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and treated with isothiocyanate. Total RNA was extracted by acid guanidine method. The reverse transcriptase M-MLV (promega) was used to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process were as follows: take 5 μg total RNA, add 50 ng oligo (dT), 2 μL dNTP Mix (2.5 mM each), the reaction volume was made up to 14.5 μL with DEPC water; after mixing, heat denaturation at 70°C for 5 min, then rapidly cool on ice for 5 min, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin ( 200U), 1 μL M-MLV (200U), mix well and centrifuge briefly, incubate at 42°C for 1.5 h, take it out and heat at 70°C for 10 min to terminat...
Embodiment 2
[0026] Embodiment 2: plant overexpression vector construction
[0027] Use the SanPrep column type plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert JsGLP1 coli plasmid pMD18-T- JsGLP1 As well as the plant expression vector pCAMBIA2300S plasmid, 1 μL was used for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. restriction endonuclease Eco RI (TaKaRa) and Pst (TaKaRa) for plasmid pMD18-T- JsGLP1 and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD18-T- JsGLP1 and pCAMBIA2300S plasmid, add 10 μL 10×K buffer, 5 μL EcoRI , 5 μL Pst , 60 μL ddH 2 O, after mixing, centrifuge for a short time, and place at 37°C for overnight reaction. All digested products were subjected to agarose gel electrophoresis, and then SanPrep Column DNA Gel Extraction Kit (Shanghai Shenggong) was used for JsGLP1 The fragments and the large ...
Embodiment 3
[0030] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants
[0031] The transgenic recipient in this experiment was tobacco ( Nicotiana tabacum L.). Tobacco seeds were soaked in 75% alcohol for 30 s, washed with sterile water and washed with 0.1% HgCl 2 Soak for 8 minutes, then wash several times with sterile water, sow on 1 / 2 MS medium, culture in dark at 28°C for 5-8 days, transfer to light incubator after germination (25°C, 16 h / d light) , and then subculture once a month with MS medium.
[0032] Take out the stored pCAMBIA2300S-containing pCAMBIA2300S- JsGLP1 Agrobacterium LBA4404 strain of the plasmid was inoculated in 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampin in 20 μL, and cultured at 28°C until the medium was turbid. Pipette 1 mL of turbid bacterial solution onto LB solid medium containing 50 mg / L Km, and incubate at 28°C for 48 h. Then scrape off an appropriate amount of Agrobacterium on ...
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