白菜类富含亮氨酸重复(LRR)抗病蛋白基因BcLRR cDNA克隆及其介导的植物软腐病抗性分析  

cDNA Cloning of an Leucine-rich Repeats (LRR)-Like Disease Resistance Protein Gene BcLRR from Chinese Cabbage and its Functional Analysis in Mediating Plant Resistance to Soft-rot Disease

作　　者：谢华[1,2] 陈绪清[1] 朱朗[3] 贾云鹤[4] 姚磊[1,2] 崔崇士[4] 马荣才[1,2] 

机构地区：[1]北京市农林科学院北京农业生物技术研究中心,北京100097 [2]首都师范大学生命科学院,北京100037 [3]中国农业大学生物学院,北京100193 [4]东北农业大学园艺学院,哈尔滨150030

出　　处：《农业生物技术学报》2010年第3期416-423,共8页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展规划(863计划)(No.2006AA100108-3-7和No.2006AA10Z123);北京市自然科学基金重点项目(No.5051002)共同资助

摘　　要：利用前期构建的软腐欧文氏菌(Erwinia carotovora subsp.carotovora)菌株BC1侵染诱导表达的大白菜(Brassica campestrissubsp.pekinensis)SSHcDNA文库和cDNA芯片。在芯片分析的基础上,选取一个与拟南芥(Arabidopsis thaliana)类富含亮氨酸重复(LRR)抗病蛋白Cf-5高度同源的EST序列(GenBank登录号:DN962361)进行进一步分析。用5'-和3'-RACE方法克隆了该基因全长1286bp的cDNA序列,包含一个完整ORF和3'UTR,并具有Poly(A)加尾信号,将其命名为BcLRR基因(GenBank登录号:EU424347)。BcLRR推导蛋白序列由327个氨基酸组成,除N端跨膜螺旋结构域外,大部分为LRR结构域,包含8个胞外LRR重复单元,由7个规范的LXXLXLXN结构和1个不规范LXXLXXXN组成。氨基酸序列比对分析发现,BcLRR蛋白与拟南芥类LRR抗病蛋白Cf-5同源性高达96.6%,与棉花(Gossypium hirsutumL.)类LRR抗病蛋白GhLRR同源性为82.3%,它们之间大部分变异限定在N端信号肽区。系统发育关系表明,在三种植物中功能均未知的这一蛋白属于一个新的胞外LRR蛋白类型,在结构上不同于其它抗性已知的胞外型LRR蛋白。将受CaMV35S启动子驱动的BcLRR基因完整ORF序列转化拟南芥Col-0,PCR鉴定、Southern杂交和RT-PCR分析表明,BcLRR基因已整合到拟南芥基因组中,并在拟南芥中获得了转录。软腐病病情指数分析表明,转BcLRR植株提高了对软腐欧文氏菌的抗性。An unique expressed sequence tag （EST） which has the highest similarity to the sequence of Arabidopsis thaliana LRR （leucine-rich repeats） Cf-5 disease resistance protein-like, was found from an SSH cDNA library constructed using mRNA isolated from leaves of Chinese cabbage （Brassica campestris ssp. pekinensis） after Erwinia carotovora subsp. carotovora （Ecc） infection. RACE was used to extend the 5＇ and 3＇ unknown sequence of this EST. The cloned cDNA, named BcLRR, was 1 286 bp including a complete open reading frame （ORF） and a complete 3＇ UTR with a putative poly（A） signal （GenBank accession No. EU424347）. The deduced polypeptide contained 327 amino acids and consisted of transmembrance domain at the N terminus and 8 extra-cytoplasmic LRR motifs, 7 of which had canonical LXXLXLXXN repeat units and one had non-canonical LXXLXXXXN unit. Amino acid sequence alignment analysis revealed that BcLRR displayed high homology of 96.6 % with Arabidopsis Cf-5 and of 82.3% with upland cotton （Gossypium hirsutum L.） LRR disease resistance protein-like GhLRR; The majority of their differences were confined to their N terminal signal peptide regions. Phylogenetic analysis further demonstrated that these three functionally unknown proteins were significantly distinct from other known resistance extra-cytoplasmic LRR proteins （Cf-2, Cf-4, Cf-5, and Cf-9 from tomato （Solanum lycopersicon）, Hs1pro-1 from sugarbeet （Beta vulgaris L.） and Xa21 from rice （Oryza sativa L.）） and belonged to a new type of extra-cytoplasmic LRR protein. The complete ORF sequence of BcLRR was amplified and cloned into modified vector pGreen0029 under CaMV35S promoter, forming plant expressed vector pGreen0029-BcLRR, which was then transformed into Arabidopsis thaliana Col-0 via Agrobacterium GV3101/Soup. The results of PCR, southern blot and RT-PCR showed that BcLRR was integrated into the Arabidopsis genome and expressed in the transgenic plants. BcLRR protein enhanced the resistance to Ecc BC1 in th

关 键 词：大白菜 BcLRR CDNA克隆 遗传转化 软腐欧文氏菌抗性 

分 类 号：S852.65[农业科学—基础兽医学]