中国野生刺葡萄抗白腐病NBS-LRR类抗病基因 同源序列的分离与鉴定

【目的】通过同源克隆法从刺葡萄‘高山2号’叶片中得到抗白腐病基因的同源片段，为筛选葡萄抗白腐病基因奠定基础。【方法】根据已知植物抗病基因NBS-LRR保守区设计简并引物，从高抗葡萄白腐病刺葡萄‘高山2号’的基因组DNA与cDNA上得到抗病基因同源片段(RGAs)，并对其表达分析进行检测。【结果】从刺葡萄‘高山2号’上获得了10个NBS-LRR类抗病基因同源片段，10条葡萄RGAs间在氨基酸水平上的同源性表现出丰富的多态性，进一步分析发现，在10条葡萄RGAs中，4个推测所属基因为non-TIR-NBS-LRR类抗病基因，6个所属基因为TIR-NBS-LRR类抗病基因。定量PCR分析表明，NB7基因受到白腐菌的诱导，而且NB7基因在刺葡萄叶片中为低丰度表达，NBS6基因受到白腐菌的诱导后为下调表达。【结论】在刺葡萄上成功获得了抗病基因同源序列，通过荧光定量PCR分析发现，NB7基因与NBS6基因都受到白腐菌的诱导表达，为最终克隆得到葡萄抗白腐病基因奠定基础。

Isolation and Identification of NBS-LRR Resistance Gene Analogs Sequences from Vitis davidii

【Objective】 The purpose of this study was to get white rot disease resistant homologous clips from Vitis davidii ‘Vitis davidii cv. Gaoshan 2’, which will provide an effective means for screening white rot disease resistant gene. 【Method】 According to the known plant resistance genes NBS-LRR conservative area, degenerate primers were designed and used to clone targeted resistance gene analogs using templates of genomic DNA and cDNA from white rot disease resistant variety of Vitis davidii ‘Vitis davidii cv. Gaoshan 2’. Then the gene expression analysis was performed. 【Result】 Ten NBS-LRR kinds of resistance gene analogs from Vitis davidii ‘Vitis davidii cv. Gaoshan 2’ were obtained, and their amino acid sequences showed rich polymorphism. Further analysis found that among the ten NBS sequences, four genes belong to non-TIR-NBS-LRR kind of resistance genes, six belong to TIR-NBS-LRR kind of resistance genes. Quantitative PCR analysis showed that NB7 gene was induced by white rot fungus, and NB7 gene was less abundantly expressed in the leaves of Vitis davidii, NBS6 gene is down-regulated expression after induced by white rot disease. 【Conclusion】 Ten RGAs were successfully obtained from Vitis davidii, and fluorescence quantitative PCR analysis showed that, both NB7 gene and NBS6 gene expression are induced by white rot fungus. The results of this study have layed a foundation for final cloning white rot disease resistant gene from Vitis.