参与紫云英共生固氮的1个上调表达结瘤素基因AsB6的分离与鉴定  

Isolation and Identification of an Up-Regulated Nodulin Gene AsB6 Involved in Symbiotic Nitrogen Fixation of Astragalus sinicus L

作　　者：吴梅[1] 丑敏霞[1] 李一星[1] 陈大松[1] 李友国[1] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070

出　　处：《华中农业大学学报》2010年第2期169-174,共6页Journal of Huazhong Agricultural University

基　　金：国家"973"计划课题(2010CB126502);华中农业大学人才启动基金(2005XRC026)资助

摘　　要：基于接种华癸中慢生根瘤菌(Mesorhizobiumhuakuii)7653R的紫云英(Astragalus sinicusL.)感染根与未接种对照根在转录水平上的差异,利用抑制差减杂交技术(suppression subtractive hybridization,SSH),建立了紫云英结瘤固氮过程中的差异表达cDNA文库,共获得527个有效克隆,其中增强或特异性表达的上调文库中包含341个克隆,抑制表达的下调文库包含186个克隆。对其中的1个上调表达cDNA克隆AsB6利用RACE(rapid amplification of cDNAends)方法获得了其基因全长序列,利用BLAST在线软件和GenBank,Gen-Bank EST和Tigr Porcine EST等数据库进行同源序列比较,表明AsB6与苜蓿的β-酮酯酰合成酶及依赖于SAM(S-adenosgl methiomine,S-腺苷蛋氨酸)的羧甲基转移酶具有66%的同源性,同时利用实时荧光定量PCR研究了该基因的时空表达特征。结果显示,接种根瘤菌7653R后,在紫云英感染根和根瘤中该基因的表达量显著增强,且在接种后根瘤形成早期和固氮中晚期表达量相对较高。推测其可能与根瘤菌前期感染和根瘤形成、根瘤代谢功能有关。A cDNA library of Astragalus sinicus L.genes specifically expressed in infected roots by Mesorhizobium huakuii 7653R was generated by using a PCR-based suppressive subtractive hybridization （SSH） technique with two mRNA populations of infected and uninfected roots.A total number of approximately 527 SSH cDNA clones were obtained,including 341 up-regulated gene clones and 186 down-regulated gene clones.The full-length cDNA sequence of up-regulated AsB6 gene was obtained by RACE and analyzed through Blasting,GenBank,GenBank EST and Tiger Porcine EST.AsB6 gene showed 66% similarity with a-etoacyl-ketoacyl synthase and SAM dependent carboxyl methyltransferase in Medicago truncatula.The temporal and spatial expression pattern of AsB6 was estimated using quantitative fluorescence real-time RT-PCR analysis.It was found that its expression level,induced by M.huakuii 7653R,in infected roots and nodule was significantly enhanced,and it reached the peak level during the early period of nodulation.The results suggested the symbiotic function of AsB6 gene might involved in the rhizobium infection,nodulation and maintenance of nodule metabolism.

关 键 词：抑制差减杂交 紫云英 上调基因 β-酮酯酰合成酶 共生固氮 

分 类 号：S154.381[农业科学—土壤学]