陆地棉抗病基因同源序列的克隆与分析  

Cloning and Analysis of NBS Type Resistance Gene Analogs in Upland Cotton(Gossypium hirsutum)

作　　者：简桂良[1] 赵磊[1] 张文蔚[1] 齐放军[1] 王升正[1] 

机构地区：[1]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100193

出　　处：《生物技术通报》2011年第10期101-108,共8页Biotechnology Bulletin

基　　金：国家转基因生物新品种培育科技重大专项子课题(2008ZX005-004);国家公益性农业科研专项(3-19)

摘　　要：根据已知抗病基因NBS保守区的P-loop和GLPL区设计一对简并引物F1/R1,以7个抗黄萎病陆地棉品种和2个感黄萎病品种的基因组DNA为模板进行PCR扩增。在9个品种中均扩增出500 bp左右的条带。对目的条带进行回收,连接、转化克隆得到350个阳性克隆,进行测序。在8个棉花品种中克隆到74条具有完整开放读码框的棉花RGAs序列。这74条序列共有64种不同的基因型,有10条与其他品种中的RGAs序列相同。用MEGA软件对8个棉花品种的74条RGA序列以及12个已知的抗病基因的NBS区域进行聚类分析,可分为4类;4类RGAs之间的相似性较低,各类之内的RGAs虽然来自不同品种,氨基酸序列的相似度却非常高。推测各大类中相似性较高的序列分别属于同一个基因家族,从位点上说可能处于同一个基因簇。A specific fragment of about 500 bp was cloned from the genomic DNA of the nine identified upland cotton（Gossypium hirsutum） by PCR degenerate primer designed according to the conserved domains P-loop and GLPL in the NBS region of reported R genes.The fragments were recycled and inserted into pGM-T vector,and then transformed into E.coli DH5α.350 recombinations were obtained through colony RPC and restriction digestion identification.The clones are sequenced and 74 RGAs with complete open reading frames（ORF）from eight varieties of cotton were obtained.Comparing with the reported R genes,the 74 RGAs all contain P-loop（Kinase1a）,Kinase2,Kinase3a,GLPL region and motif RNBS-A,B,C defined by Meyers.Cluster analysis of their putative amino acid showed that the RGAs sorted into two distinct types,TIR-NBS type and nonTIR-NBS type.The TIR type RGAs all contain the RNBS-A-TIR motif while the nonTIR type RGAs all contain RNBS-A-nonTIR motif.This result consists with the early report that NBS-LRR gene has two major groups in dicotyledon.The deduced amino acid of the RGAs have high similarity with the reported R genes.NonTIR type RGAs have asimilarity of 32%-50% with L6,M,Gro1-4,N,while TIR type RGAs have a similarity of 23.2%%-56.5% with I2C-1,Mi-1.1,RPM1,RPP8,RPS2,RPS5,XA1,PRf.Analysis of molecular variance（AMOVA）indicated that the genetic variation mainly exist within varieties rather than among varieties.

关 键 词：棉花 抗病基因同源序列 NBS 

分 类 号：S562[农业科学—作物学]