Peanut mutant genes, protein coded by peanut mutant genes, and preparation method of peanut mutant
A technique for mutating genes and mutants, applied in biochemical equipment and methods, botany equipment and methods, horticultural methods, etc., can solve problems such as heavy workload, and achieve the effect of solving peanut allergy
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[0054] Accordingly, embodiments of the present invention further provides a method for preparing mutants of peanut, comprising the steps of:
[0055] Sl, genes that provide support and editing embryogenic callus Peanut Peanut allergen gene Arah1, amplification primers;
[0056] S2, peanut allergen Arah1 gene as a template for PCR amplification by the amplification primer to obtain peanut allergen gene coding sequence Arah1a Arah1 peanut allergen gene coding sequence Arah1 Arah1b;
[0057] S3, peanut allergen gene coding sequence Arah1a Arah1 peanut allergen Arah1 gene coding sequence as homologous sequence regions Arah1b CRISPR targets, and to give sgRNA T1 sgRNA T2;
[0058] S4, and sgRNA T1 were synthesized sense strand and an antisense strand sgRNA T2 to obtain a double-stranded and double-stranded DNA T1 DNAT2;
[0059] S5, the double-stranded DNA T1 sgRNA T1 expression is inserted into the box, and then cloned into a gene vector editing, editing a gene vector to obtain Tl;
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Embodiment
[0109] 1. Cloning and sequencing of peanut allergens gene ARAH1
[0110] According to the peanut allergen gene Arah1 (Genbank: AB440237.1), PCR primers Arah1-F and ARAH1-R were designed, and the nucleotide sequences were respectively SEQ ID NO: 7 and SEQ ID NO: 8 Show.
[0111] The peanut vanes, the kit extracts genomic DNA, and the ARAH1 gene fragment was amplified by PCR. The PCR reaction system is as follows:
[0112] 50 μl of reaction systems include: DDH 2 O: 13μL, primers: 5 μL, DNA: 5 μL, 2 × Taq Mix: 25 μL, Mg 2+ : 1 μL, glycerin: 1 μL.
[0113] The reaction conditions were as follows: Pre-95 ° C for 5 min, then at 95 ° C degeneration 30s, 56 ° C for 1 min for 1min for 1min as a cycle, and after 40 ° C, 72 ° C completely extends 10 min.
[0114] The PCR product was sequenced for DNA, and two ARAH1 gene coding regions were obtained, respectively from the two sub-genomic genes Arah1a and ARAH1b of peanut A and B, and the nucleotide sequences were respectively SEQ ID NO: 11 a...
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