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以葡萄抗性品种‘左优红’组培苗为材料,利用同源克隆法克隆VvWRKY13、VvWRKY45和VvWRKY71,测序结果显示,VvWRKY13扩增片段大小为681bp,编码226个氨基酸;VvWRKY45扩增片段大小为549bp,编码182个氨基酸;VvWRKY71扩增片段大小为936bp,编码311个氨基酸序列。利用生物信息学分析VvWRKY13、VvWRKY45和VvWRKY71序列显示,这3个蛋白均含有保守的WRKY结构域;分子量分别为25.7、20.8和34.8kDa,pI分别为9.08、9.41和6.94。实时定量PCR结果表明,3个WRKYs均在叶片中表达量最高。逆境相关的信号物质,如水杨酸、脱落酸、茉莉酸和一氧化氮以及高盐、低温和渗透等逆境胁迫均可诱导VvWRKY13、VvWRKY45和VvWRKY71的表达。推测3个基因均参与了葡萄抵御逆境胁迫的过程。
VvWRKY13, VvWRKY45 and VvWRKY71 were cloned by homologous cloning using the tissue culture plantlet Zuoliuhong as the material. The sequence of VvWRKY13 amplified fragment was 681bp and encoded 226 amino acids. VvWRKY45 amplified fragment size Was 549bp, encoding 182 amino acids; VvWRKY71 amplified fragment size of 936bp, encoding a 311 amino acid sequence. Bioinformatics analysis of the sequences of VvWRKY13, VvWRKY45 and VvWRKY71 revealed that all three proteins contained a conserved WRKY domain; the molecular weights were 25.7, 20.8 and 34.8 kDa, respectively, and pI was 9.08, 9.41 and 6.94, respectively. Real-time PCR results showed that all three WRKYs expressed the highest level in leaves. Adversity-related signal substances, such as salicylic acid, abscisic acid, jasmonic acid and nitric oxide, as well as high salt, low temperature and osmotic stress can induce the expression of VvWRKY13, VvWRKY45 and VvWRKY71. It is speculated that all three genes are involved in the process of grapes against stress.