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在前期获得葡萄白粉病菌应答基因VpSTART的EST基础上,采用RACE和RT-PCR技术克隆该基因cDNA全长序列。VpSTART全长1 321 bp,3′端非编码区114 bp,包含一个1 206 bp开放阅读框,编码401个氨基酸。VpSTART蛋白分子量为45.3 kD,与欧亚种葡萄、玉米、拟南芥、蒺藜状苜蓿和蓖麻的蛋白同源性分别为99%、64%、58%、46%和25%。实时荧光定量PCR表明,VpSTART在‘商–24’葡萄花序、卷须和茎中表达量较高;感白粉病的‘湖南–1’葡萄叶片接种白粉病菌后VpSTART表达量与对照没有显著差异,而抗白粉病的‘商–24’葡萄接种12 h后VpSTART表达量增加,在24~96 h表现显著性差异;用SA、MeJA和Eth等不同信号分子分别处理‘湖南–1’和‘商–24’葡萄叶片1~48 h后,VpSTART基因的表达受SA负调控,受MeJA和Eth正调控。
Based on the ESTs of the VpSTART gene of Vitis vinifera in the early stage, the full-length cDNA of this gene was cloned by RACE and RT-PCR. VpSTART is 1 321 bp in length and 114 bp in 3 ’end. It contains a 1 206 bp open reading frame encoding 401 amino acids. The VpSTART protein has a molecular mass of 45.3 kD and shows 99%, 64%, 58%, 46% and 25% homology with Eurasian grapevines, maize, Arabidopsis thaliana, alfalfa alfalfa and castor. Real-time PCR showed that the expression level of VpSTART was higher in ’Shang-24’ grapevines, tendrils and stems. The expression level of VpSTART in powdery mildew-resistant ’Hunan-1’ grape leaves was not significantly different from that in the control VpSTART expression increased after 12 h of inoculation with powdery mildew-Shang-24 grapevine, and showed significant difference from 24 h to 96 h. Different signaling molecules such as SA, MeJA and Eth were used to deal with the expression of ’Hunan-1’ After 1 ~ 48 h of 24 ’grape leaves, the expression of VpSTART gene was negatively regulated by SA and positively regulated by MeJA and Eth.