葡萄中RPW8.2同源基因克隆与表达分析

Molecular Cloning and Expression Analysis of RPW8.2 Homologous Genes in Grapevine

利用同源克隆技术分别从白河35-2、广西-1、黑比诺、五月紫4种葡萄中克隆得到了拟南芥广谱抗病基因RPW8.2的同源基因,分别命名为VpR8 H-BH2、VpR8 H-GX1、VvR8 H-PN和VvR8 H-MP,氨基酸多序列比对和系统发育分析显示,该4条序列均属于RPW8.2同源基因。4种葡萄R82 H基因cDNA开放阅读框分别为2 457bp、2 445bp、2 445bp和2 448bp,分别编码819、815、815和816个氨基酸。采用半定量RT-PCR技术分析了R82 H基因在4种葡萄根、茎、成熟叶、幼嫩叶、卷须不同组织中的表达模式,R82 H基因在不同葡萄组织中均有表达,但是表达丰度不尽相同,其中,在白河35-2和五月紫葡萄成熟叶中表达量最高,而在广西-1和黑比诺中幼嫩叶片中表达量高于其他组织。

Four genes designated VpR8H-BH2, VpR8H-GX1, VvR8H-PN and VvR8H-MP were cloned and sequenced by using homology-based cloning method from Chinese wild grapevine Vitis pseudoreticulata accession Baihe-35-2, accession Guangxi-1, and Vitis vinifera cv. Pinot Noir, and Vitis vinifera cv. May Purple, respectively. Amino acid sequences alignment and phylogenesis results showed that four genes were Arabidopsis broad-spectrum disease resistance gene RPW8.2 homologous genes. The full-length ORF of VpR8H-BH2, VpR8H-GX1, VvR8H-PN and VvR8H-MP were 2 457 bp, 2 445 bp, 2 445 bp and 2 448 bp, respectively, and encoded polypeptides of 819, 815, 815 and 816 amino acids, respectively. Semi-quantitative RT-PCR results indicated that four genes were expressed in all of the grapevine tissue types, including root, stem, mature leaves, young leaves and tendrils, but the expressions were different. The VpR8H-BH2 expression level of accession Baihe35-2 and the VvR8H-MP expression level of Vitis vinifera cv. May Purple were the most in mature leaves, but the expression level of young leaves of accession Guangxi-1 and Vitis vinifera cv. Pinot Noir were higher than other tissues.