小麦抗赤霉病品种NBS同源序列克隆与分析

根据二穗短柄草NBS-LRR类基因的保守序列设计同源引物,以小麦抗赤霉病品种苏麦3号、宁7840和望水白基因组DNA为模板,通过PCR扩增,得到43条序列,其中4条为非编码序列或结构域不完整;39条与植物抗病基因同源,其中的7条内部存在终止密码子,可能是假基因,经过比对分析,其余32条具有连续的开放阅读框和保守结构域,推导的氨基酸序列均具有Kinase-1a、Kinase-2和Kinase-3a及GLPL区等几个保守区,在GenBank中均能找到与之高度同源的其他物种的核酸序列,并且Kinase-2的最后一个氨基酸均为色氨酸(W),属于non-TIR类NBS基因。32条序列可分为4大类,它们之间核苷酸同源性为64%-98%,编码氨基酸同源性为22%-98%。根据序列分析随机设计5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,7-1、s-3、s-4和w-2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;7-13不表达,再次证明属于假基因。32条序列在之前未被报道过,这些RGA可以作为筛选赤霉病功能性抗病基因的候选序列。

Isolation and Analysis of NBS Analogs from Wheat Fusarium Head Blight Resistant Cultivater

The homologous primers were designed for Brachypodium distachyon NBS-LRR genes.Resistance gene analogs(RGA) sequences were cloned from wheat genomic DNA of FHB resistant lines Sumai 3,Ning 7840 and Wangshuibai.Out of 43 RGA,4 were non-coding sequences or incomplete domain while 39 sequences had homology with plant resistance genes.Seven of them were interrupted by stop codons,which may be pseudogenes.The blast analysis of remaining 32 sequences showed continuous open reading frame and conserved domains.The deduced amino acids(aa) have Kinase-1a,Kinase-2,Kinase-3a and GLPL domains,which could find high homology in GenBank with other plant nucleic acid sequences.Moreover,the last amino acid of Kinase-2 domain of these sequences is Trp(W),so all belong to the non-TIR-NBS.The 32 sequences could be diverse 4 classes and showed 64%-98% homology in nucleotides and 22%-98% in amino acid sequences.To further analyze the expression o f these RGAs,RT-PCR was preformed.The results showed that 7-1,s-3,s-4 and w-2 could express,which indicated that these fragments might be partial sequence of functional R genes;whereas 7-13 didn’t express,which indicated it might be pseudogene again.These 32 RGAs are new and can be used as candidate sequences to confer functional resistance to the FHB.