Rana chensinensis functional gene Rd-RNase3 sequence, construction method and amino acid sequence and application thereof
A technology of functional genes and construction methods, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc.
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Embodiment 1
[0104] Embodiment 1: The specific construction method of the Chinese Rana functional gene Rd-RNase3 sequence of the present invention:
[0105] 1. Primer design
[0106] Gene-specific primers:
[0107] Forward primer N275: TAT TTG CAG TTG TTT TGA GTC TCA C
[0108] Reverse primer C715: CTG CTT ATC ACA TCC CTG TTG TC
[0109] 2. PCR amplification of the target gene
[0110] (1) Genomic DNA was extracted from the fresh blood of Rana using a blood genome extraction kit, and the molecular weight and concentration were detected by 1% agarose electrophoresis.
[0111] (2) Add 100ng of genomic template, 1μL of dNTPs (10mM), 5μl of 10×Taq Buffer, 3U of Taq DNA Polymerase and 30pmol of primers (N275, C715) into a sterile PCR tube, and add sterile ultrapure water to a total volume of 50μl ,well mixed;
[0112] (3) Put the PCR tube containing the sample into the preheated PCR instrument for PCR reaction. PCR amplification conditions: 94°C for 30s, 48°C for 30s, 72°C for 50s, 35 cyc...
Embodiment 2
[0162] Embodiment 2: Biological experiment of the Chinese forest functional gene Rd-RNase3 sequence and amino acid sequence constructed by the present invention
[0163] 1. Materials and methods
[0164] 1. Materials
[0165] (1) Strain and carrier
[0166] Escherichia coli BL21 (DE3) competent cells were purchased from TIANGEN Biotechnology Co., Ltd., and pFLAG-CTS vector was purchased from sigma-aldrich.
[0167] (2) Tool enzymes and biochemical reagents:
[0168] Tryptone, Yeast Extract, Agarose, ampicillin, X-Gal, IPTG, etc. (all purchased from TIANGEN Biotechnology Co., Ltd.), and other reagents were commercially available analytical grade.
[0169] (3) Equipment and equipment
[0170] equipment
[0171] THZ-D desktop constant temperature oscillator, Taicang Experimental Equipment Factory;
[0172] SPN202F electronic balance, Mettler-Toledo Weighing Equipment Systems Co., Ltd.;
[0173] LRH-250 biochemical incubator, Shanghai Yiheng Technology Co., Ltd.;
[0174] ...
Embodiment 3
[0185] Embodiment 3: Preparation of the recombinant fusion protein of the Chinese Rana functional gene Rd-RNase3 sequence of the present invention
[0186] 1. Materials and equipment
[0187] 1. Strain and carrier
[0188] Escherichia coli BL21 (DE3) competent cells were purchased from TIANGEN Biotechnology Co., Ltd., and pFLAG-CTS vector was purchased from sigma-aldrich.
[0189] 2. Tool enzymes and biochemical reagents
[0190] Tryptone, Yeast Extract, Agarose, ampicillin, X-Gal, IPTG, etc. (all purchased from TIANGEN Biotechnology Co., Ltd.), mouse anti-FLAG monoclonal antibody (purchased from Sigma Company), goat antibody linked to alkaline phosphatase Mouse secondary antibody, BCIP / NBT chromogenic kit (purchased from Beijing Zhongshan Jinqiao Biological Co., Ltd.), and other reagents were commercially available analytical grade.
[0191] 3. Equipment
[0192] THZ-D desktop constant temperature oscillator, Taicang Experimental Equipment Factory;
[0193] LRH-250 bioch...
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