CAJ | 학술논문

基于接种华癸中慢生根瘤菌(Mesorhizobium huakuii) 7653R 的紫云英(Astragalus sinicus L.) 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 cDNA ends)方法获得了其基因全长序列,利用 BLAST 在线软件和 GenBank,GenBank 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-adenosglmethiomine,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.
기우접충화계중만생근류균(Mesorhizobium huakuii) 7653R 적자운영(Astragalus sinicus L.) 감염근여미접충대조근재전록수평상적차이,이용억제차감잡교기술(suppression subtractive hybridization,SSH),건립료자운영결류고담과정중적차이표체 cDNA 문고,공획득527 개유효극륭,기중증강혹특이성표체적상조문고중포함 341 개극륭,억제표체적하조문고포함 186 개극륭.대기중적1개상조표체 cDNA 극륭 AsB6 이용 RACE(rapid amplification of cDNA ends)방법획득료기기인전장서렬,이용 BLAST 재선연건화 GenBank,GenBank EST화Tigr Porcine EST등수거고진행동원서렬비교,표명 AsB6 여목숙적β-동지선합성매급의뢰우SAM (S-adenosglmethiomine,S-선감단안산) 적최갑기전이매구유 66% 적동원성,동시이용실시형광정량 PCR 연구료해기인적시공표체특정.결과현시,접충근류균7653R후,재자운영감염근화근류중해기인적표체량현저증강,차재접충후근류형성조기화고담중만기표체량상대교고.추측기가능여근류균전기감염화근류형성、근류대사공능유관.
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.