小冰麦异附加系TAI-27中附加染色体上NBS同源序列的扩增

Cloning of the NBS Sequences from the Addition Chromosome in Wheat wheatgrass Alien Addition Line TAI-27

植物对病虫害的抗性主要是由抗病基因 (resistancegene ,R)决定的。目前已经克隆了 2 0多个R基因 ,大部分都属于NBS LRR类 ,这类基因编码NBS(nucleotidebind ingsite ,NBS)和LRR(leucine richrepeat ,LRR)两个比较保守的结构域。植物的NBS有几个保守区 ,本文根据其中的P loop和GLPL两个保守区设计了简并引物 ,通过PCR扩增NBS序列。在PCR扩增中使用的DNA模板是从小冰麦异附加系TAI 2 7中微分离的附加的单条染色体 ,这条染色体上携带BYDV (barleyyellowdwarfvirus ,BYDV)抗性基因。PCR扩增产物主要有三条带 ,2 0 0bp ,4 0 0bp和 950bp ,将其中的 2 0 0bp(nbsA )和4 0 0bp(nbsB)克隆到 pGEMT vector上并分别测序。 3个nbsA克隆的测序结果完全一致 ;对nbsB的 60多个克隆的不同酶切分析 ,只发现了一个克隆的酶切特性与其他不同。所以说 ,以单条染色体为模板PCR扩增产物组成比较简单 ,省去了RFLP作图的许多工作 ,可以为克隆R基因提供特异性探针。本文的实验设计将染色体微切割和微克隆技术与基因克隆联系在一起 。

Most of the cloned R genes encode proteins containing a nucleotide binding site and leucine rich repeats (NBS LRR). There are some conserved domains in NBS. We designed the degenerate oligonucleotide primers according to the conserved domains P loop and GLPL. The template of PCR is the addition chromosome DNA that we microdissect from the wheat wheatgrass alien addition line TAI 27. There are three main bands from the PCR amplification: 200bp (nbsA), 400bp (nbsB) and 950bp. The band of 200bp and 400bp are cloned into pGEM T vector and sequenced. The sequence of three nbsA clones is the same. 60 nbsB clones are digested with Eco R V and Nco I, Cla I and Sac I. And we just find only one clone showing different bands with others. These results indicate that the composition of PCR product from one chromosome DNA is very simple, comparing with the PCR product amplifying from the genomic DNA.