华东葡萄VpWDR基因的克隆、表达及亚细胞定位分析  

Cloning, expression and subcellular localization analysis of the VpWDR gene from Vitis pseudoreticulata

作　　者：武杰[1] 曾爱玲[1] 张军科[1] 

机构地区：[1]西北农林科技大学园艺学院.农业部西北地区园艺作物生物学与种质创制重点实验室,陕西杨凌712100

出　　处：《果树学报》2013年第3期354-360,I0002,共8页Journal of Fruit Science

基　　金：西北农林科技大学基本科研业务费专项(QN2009014);国家自然科学基金(No30771493)

摘　　要：【目的】探索中国葡萄野生种抗白粉病候选基因VpWDR与白粉病抗性的相关性。【方法】以中国野生华东葡萄(Vitis pseudoreticulata)叶片为植物材料,采用RACE技术对华东葡萄抗白粉病株系‘白河-35-1’叶片cDNA文库测序获得的WDR基因EST序列片段进行全长克隆,利用实时荧光定量PCR技术分析VpWDR在华东葡萄抗白粉病株系‘白河-35-1’叶片中受白粉菌侵染后的表达变化,采用基因枪介导的瞬时转化技术检测VpWDR表达产物在洋葱表皮细胞内的定位。【结果】获得了华东葡萄VpWDR mRNA全长1 577 bp,开放阅读框1 377 bp,3'UTR 138 bp,5'UTR 62 bp,编码458个氨基酸,理论等电点为5.07,预测分子量为51.7 kDa;核酸和编码的氨基酸序列同源性分析结果表明,VpWDR属于WD40蛋白基因家族,并命名为VpWDR;在人工接种葡萄白粉菌后的葡萄叶片进行基因表达分析,表明该基因在葡萄叶片中受白粉菌诱导先上调表达,再回到原始水平,最大值出现在接种后12 h,表达量约为内参基因β-Actin的2.8倍;此外,亚细胞定位结果表明该基因编码的蛋白定位于细胞核、细胞质和细胞膜,无亚细胞特异性分布。【结论】VpWDR蛋白定位于细胞核、细胞质和细胞膜位。并且VpWDR基因对白粉菌产生响应,从而推测该基因可能参与葡萄白粉菌侵染过程中的抗病反应。[Objective ]The objective of the study was to explore the relation between the gene expression of VpWDR and its function during grapevine powdery mildew infection. [Method]Using the leaves of Chi- nese wild grapevine species Vitis pseudoreticulata ＇Baihe-35-1＇ as material, full lengths of the WDR gene was cloned by RACE technique based on the EST sequence and designated as FpWDR. The expres- sion of VpWDR was analyzed by Quantitative RT-PCR after the infection of Erysiphe necator. Sub-cellu- lar localization of VpWDR was carried out by transient expression in onion epidermal cells using particle bombardment. [Result]The full length of VpWDR cDNA was 1 577 bp and contained 3UTR 138 bp, 5UTR 62 bp and an ORF of 1 377 bp which encoded a 458 amino acid polypeptide with a calculated molecular mass of 51.7 kDa and an isoelectric point of 5.07. Phylogenetic analysis showed that VpWDR belonged to WD40 protein family. Quantitative RT-PCR analysis of the transcript levels during the infec- tion process in the leaves showed that VpWDR was induced after inoculation in grapevine leaves. The transcript level of VpWDR reached a peak 12 h after inoculation, which was 2.8 times higher than that of the reference gene, then decreased to original level. In addition, localization results showed that the Vp-WDR protein was localized in cell nucleus, cytoplasm and cell membrane, which had no specific distri- bution. [Conclusion]The activity of VpWDR was induced by E. necator in leaves of Chinese wild V. pseu- doreticulata ＇Baihe-35-1 , suggesting that the gene could participate in plant-pathogen defense response during E. necator infection.

关 键 词：华东葡萄 WD40基因 RACE克隆 抗病基因 亚细胞定位 

分 类 号：S663.1[农业科学—果树学]