DNA repair protein hmdrp, its coding gene and its use
A technology for repairing proteins and coding, applied in DNA/RNA fragments, recombinant DNA technology, genetic engineering, etc., can solve problems such as cloning DNA repair protein genes
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Embodiment 1
[0125] Example 1 Cloning of Partial Fragment of cDNA Sequence of DNA Repair Protein HmDRP Gene
[0126] method
[0127] Step 1. Extraction, detection and quantification of total RNA
[0128] 1) Take 0.1g of mycelium, and follow the instructions of the FungalRNAKit kit to extract RNA from jiji mushroom.
[0129] 2) Determine the quality of RNA: add 1 μL RNA to 99 μL DEPC-treated water, dilute 100 times, measure the OD values at 260nm and 280nm with a UV photometer, and calculate their ratio to analyze the concentration and purity of RNA. Total RNA (μg) = OD 260 ×40μg / mL×100 (dilution factor)×volume of total RNA solution (μL) / 1000; OD 260 / 280 In the range of 1.8 to 2.0, the purity of the extracted RNA is considered to be very high.
[0130] 3) Determine the integrity and contamination of the RNA. Take 3 μL of sample solution for agarose gel electrophoresis, and observe the brightness of 28SrRNA and 18SrRNA bands. For example, the brightness of 28SrRNA is about twice th...
Embodiment 2
[0185] Example 2 Cloning of Complete ORF of DNA Repair Protein HmDRP Gene
[0186] method
[0187] Step 1. HmDRP gene cDNA terminal sequence amplification
[0188] According to the partial fragment sequence of the cDNA of the target gene, the Primer Premier 5.0 software was used to design specific primers UP1 (SEQ ID NO.10) and UP2 (SEQ ID NO.11) and the adapter primer provided by the kit for 3' RACE amplification, and the specific primer DP1 ( SEQIDNO.12), DP2 (SEQIDNO.13) and DP3 (SEQIDNO.14) were subjected to 5' RACE amplification with the adapter primers provided in the kit.
[0189] 3', 5' RACE Ready cDNA Synthesis
[0190] 1) Add the following reagents to a 0.2mL PCR thin-walled tube treated with DEPC-treated water:
[0191] For 3'RACEReady cDNA: 1.5 μL total RNA, 1 μL 3'RACECDSPrimer, with RNase-FreeddH 2 O to make up to 4.75 μL.
[0192] For 5'RACEReady cDNA: 1.5 μL total RNA, 1 μL 5'RACECDSPrimer, with RNase-FreeddH 2 O to make up to 3.75 μL.
[0193] 2) Mi...
Embodiment 3
[0258] Example 3 DNA repair protein HmDRP gene DNA sequence amplification
[0259] method
[0260] Step 1. Genomic DNA Extraction from Shijiji mushroom
[0261] Scrape 0.1 g of mycelia, and use the CTAB method to extract DNA. For specific steps, refer to the third edition of "Molecular Cloning Experiment Guide". The DNA purity was checked by 1% agarose gel electrophoresis.
[0262] Step 2. HmDRP gene DNA sequence amplification
[0263] Using Genomic DNA of Mycelium jijiji as a template, ORF1 / ORF2 (sequence shown in SEQ ID NO.15-16) as primers, PCR amplification was carried out using the KOD-Plus high-fidelity enzyme of TOYOBO Company to amplify the DNA of the HmDRP gene sequence. Add the following components to the PCR thin-walled tube respectively:
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[0265] PCR amplification program:
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[0268] Set the gradient of the extension temperature and explore the reaction system to obtain the optimal reaction system. After the amplification,...
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