Yangbi walnut germin-like protein gene JsGLP1 and application thereof
A technology of Yangbi big bubble walnut and germin, which is applied in application, genetic engineering, plant genetic improvement and other directions to achieve the effect of saving cost, reducing environmental pollution and simple operation.
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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, heated and denatured at 70°C for 5 min, then rapidly cooled on ice for 5 min, then added 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 te...
Embodiment 2
[0026] Embodiment 2: plant overexpression vector construction
[0027] The Escherichia coli plasmid pMD18-T-JsGLP1 inserted into JsGLP1 and the plant expression vector pCAMBIA2300S plasmid were extracted using the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong), and 1 μL was used for agarose gel electrophoresis to detect the extracted The integrity and concentration of the plasmid. with restriction enzymes EcoRI (TaKaRa) and Pst (TaKaRa) carried out double digestion of plasmids pMD18-T-JsGLP1 and pCAMBIA2300S respectively (100 μL system). The reaction system and operation process were as follows: take 20 μL of pMD18-T-JsGLP1 and pCAMBIA2300S plasmids respectively, and add 10 μL of 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 the JsGLP1 fragment and the large fragment of the pCAMBIA2300s ...
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 preserved Agrobacterium LBA4404 strain containing the pCAMBIA2300S-JsGLP1 plasmid from the -80°C refrigerator, take 20 μL and inoculate it into 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampicin, 28 Cultivate until the medium becomes 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 Ag...
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