中国野生葡萄抗霜霉病相关基因VpPR4b及其启动子的克隆和功能分析

Cloning and Functional Analysis of the CDS and Promoter of VpPR4b Gene Response to Downy Mildew in Chinese Wild Grape

以中国野生葡萄(Vitis piasezkii)‘留坝-8’(抗霜霉病)和欧亚种葡萄(V.vinifera)‘黑比诺’(易感霜霉病)为材料,克隆得到抗霜霉病相关基因VpPR4b和VvPR4b。序列分析发现VpPR4b和VvPR4b核苷酸序列相似性为98.61%。VpPR4b编码区为432 bp,编码143个氨基酸,含有BARWIN保守结构域,属于典型的Ⅱ型PR4蛋白。VpPR4b和VvPR4b均可在霜霉菌侵染后诱导表达。亚细胞定位表明VpPR4b定位于细胞膜上。分别从‘留坝-8’和‘黑比诺’中克隆得到了VpPR4b和VvPR4b的启动子,其序列相似性高达95.65%,并且含有相似的顺式作用元件。通过酵母单杂交验证了转录因子WRKY40和WRKY75可以结合VpPR4b启动子。本研究的结果表明PR4b可能在葡萄抵御霜霉病侵染过程中发挥一定作用。

VpPR4b and VvPR4b were cloned form Vitis piasezkii‘Liuba-8’(resistant to downy mildew)and V.vinifera‘Pinot Noir’(susceptible to downy mildew).Sequence analysis showed that the identity between VpPR4b and VvPR4b nucleotide was 98.61%.The coding region of VpPR4b is 432 bp,which encodes 143 amino acids and contains the BARWIN conserved domain,indicating it is a typical typeⅡPR4 protein.Both VpPR4b and VvPR4b are highly expressed after inoculation with Plasmopara viticola.Subcellular localization analysis indicated that VpPR4b was localized in the membrane.The promoters of VpPR4b and VvPR4b were cloned from‘Liuba-8’and‘Pinot Noir’,respectively.It showed that VpPR4b and VvPR4b promoter sequence similarity is as high as 95.65%,and contains similar cis-acting elements.It has been verified that the transcription factors WRKY40 and WRKY75 can bind the VpPR4b promoter in yeast.This study showed that PR4b may play a role in the resistance to downy mildew in grape.