野生稻NBS类抗病基因同源序列的分离与序列分析  

Isolation and Sequence Analysis of NBS Resistance Gene Analogues in Wild Rice

作　　者：张向明[1] 韦靖鸾[1] 张丹[1] 王春台[1] 刘学群[1] 刘新琼[1] 

机构地区：[1]中南民族大学生命科学学院/生物技术国家民委重点实验室,湖北武汉430074

出　　处：《植物遗传资源学报》2010年第6期741-748,共8页Journal of Plant Genetic Resources

基　　金：国家自然科学青年基金项目(30600400);湖北省自然科学杰出青年基金项目(2009CDA125);中南民族大学校级教学改革项目(JYX09031)

摘　　要：为了挖掘野生稻中的抗病资源,根据已克隆的植物抗病基因核苷酸结合位点序列中的保守结构域设计3对简并引物,从疣粒、药用、高秆、宽叶和斑点野生稻基因组DNA中分离出13条NBS类抗病基因类似物,其中11条具有连续的ORF,具有NBS类R基因的保守基元P-loop、kinas-2、kinas-3a和GLPL。在NCBI上进行同源性搜索发现,其中12条RGAs的核苷酸序列与水稻已知的NBS类R基因具有66%～94%的同源性,与其他植物已知R基因具有67%～84%的同源性;其对应的氨基酸序列与水稻已知的NBS类R基因具有43%～93%的同源性,与其他植物已知R基因具有37%～79%的同源性。另外1条的核苷酸序列与水稻假定的NBS类R基因具有76%的同源性,其氨基酸序列与水稻假定的NBS类R基因具有74%的同源性。根据序列分析结果设计6对不同基因特异性引物,并利用RT-PCR技术进行表达分析,结果表明,RN1BD5、RN1BD10、RN1GG2和RN1YY6均能表达,说明这些片段可能是功能性抗病基因的部分序列;而RN1KY9和RN1GG5没有表达,可能是假基因。To explore resistance genes from wild rice,three sets of degenerate primers were designed based on the conservative domain of the nucleotide binding site（NBS） region from the cloned plant disease resistance genes（R） to isolate resistance gene analogues（RGAs） from genomic DNA of Oryza granulata,O.officinalis,O.alta,O.latifolia and O.punctata.After sequencing and analyzing by alignment,13 NBS RGAs were detected,11 of which had uninterrupted open reading frames（ORF） and contained the conserved motifs of NBS R genes such as P-loop,kinas-2,kinas-3a and GLPL,and the other two sequences were with immature stop codon.At the nucleotide level,the sequence identity of 12 RGAs ranged from 66% to 94% with the cloned NBS R genes from O.sativa,and between 67% and 84% with the cloned R genes from other plants.While the 12 RGAs deduced amino acid sequences showed identity with the cloned NBS type R gene from O.sativa ranging from 43% to 93%,and with the cloned R gene from other plants ranging from 37% to 79%.As for the other RGA,its nucleotide sequence identity with O.sativa putative NBS class R genes was 76%,and the deduced amino acid sequence identity was 74%.To further analyze the expression of these RGAs,RT-PCR was preformed.The results showed that RN1BD5,RN1BD10,RN1GG2 and RN1YY6 could express,which indicated that these fragments might be partial sequence of functional R genes;whereas RN1KY9 and RN1GG5 didn′t express,which indicated they might be residual arti

关 键 词：野生稻 抗病基因类似物 基因分离 序列分析 

分 类 号：S511.9[农业科学—作物学]