CAJ | 학술논문

植物对病虫害的抗性主要是由抗病基因(resistance gene,R)决定的。目前已经克隆了20多个R基因，大部分都属于NBS-LRR类，这类基因编码NBS(nucleotide binding site, NBS)和LRR（leucine-rich repeat, 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-27中微分离的附加的单条染色体，这条染色体上携带BYDV(barley yellow dwarf virus, BYDV)抗性基因。PCR扩增产物主要有三条带，200bp, 400bp和950bp，将其中的200bp(nbsA)和400bp(nbsB)克隆到pGEM T-vec tor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，为克隆R基因提供了一条捷径。
식물대병충해적항성주요시유항병기인(resistance gene,R)결정적。목 전이경극륭 료20다개R기인，대부분도속우NBS-LRR류，저류기인편마NBS(nucleotide binding site, NBS)화LRR（leucine-rich repeat, LRR）량개비교보수적결구역。식물적NBS유궤개보수 구 ，본문근거기중적P-loop화GLPL량개보수구설계료간병인물，통과PCR확증NBS서렬。재PCR 확증중사용적DNA모판시종소빙맥이부가계TAI-27중미분리적부가적단조염색체，저조 염색체상휴대BYDV(barley yellow dwarf virus, BYDV)항성기인。PCR확증산물주요유삼 조대，200bp, 400bp화950bp，장기중적200bp(nbsA)화400bp(nbsB)극륭도pGEM T-vec tor상병분별측서。3개nbsA극륭적측서결과완전일치；대nbsB적60다개극륭적불동매절분석 ，지발현료일개극륭적매절특성여기타불동。소이설，이단조염색체위모판PCR확증산물조 성비교간단，성거료RFLP작도적허다공작，가이위극륭R기인제공특이성탐침。본문적실험 설계장염색체미절할화미극륭기술여기인극륭련계재일기，위극륭R기인제공료일조첩경。
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. W e designed the degenerate oligonucleotide primers according to the conserved domai ns-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 t hr ee 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 se q uence 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 ba nds with others. These results indicate that the composition of PCR product from one chromosome DNA is very simple, comparing with the PCR product amplifying fr om the genomic DNA.