根瘤菌87-1-1诱导刺槐根表皮传递细胞特异性表达的cDNA文库构建及序列分析  

cDNA Library Construction and Analysis of Differentially Expressed in Roots of Robinia pseudoacacia Induced by Rhizobium 87-1-1

作　　者：赵银娟[1] 孙敦平 韩素芬[1] 吴小芹[1] 

机构地区：[1]南方现代林业协同创新中心,南京林业大学林学院,江苏省有害生物入侵与控制重点实验室,江苏南京210037

出　　处：《南京师大学报（自然科学版）》2014年第4期76-82,共7页Journal of Nanjing Normal University(Natural Science Edition)

基　　金：国家自然科学青年基金(31100448);高校博士点基金(2113204120004);江苏省博士后基金

摘　　要：探寻根瘤菌诱导刺槐后根表皮细胞分化成传递细胞的特异性表达基因及其分子机制.采用抑制差减杂交(suppression subtraction hybridization,SSH)技术构建刺槐根系被接种根瘤菌和未被接种根瘤菌二者间的正反两个c DNA文库.各挑选正反文库中500个克隆进行测序,在Blastn、Blastp、Swiss Prot、KEGG、COG、Interpro以及Gene ontology(GO)数据库中进行比对注释.结合生理过程对ESTs进行分析,共获得725条非冗余序列(uni EST),正反向文库分别为385条和340条,其中包括674个单一序列(singlets),51个拼接序列(contigs).在Nt库比对中有674条uni ESTs与已知基因匹配,占比93%;在Nr库比对中有648条序列有匹配蛋白,占比89%.有正向文库213个uni ESTs和反向文库156个uni ESTs能进行Geney ontology(GO)功能注释,在细胞组分被注释了270次,分子功能被注释了448次,生物过程被注释了484次.uni ESTs的功能分析显示,正向文库中多与细胞翻译后修饰、转录因子、细胞信号通路、细胞壁/细胞膜/内膜系统以及细胞骨架相关蛋白有关,部分是结瘤相关基因.而反向文库中与细胞生长、代谢物形成的基因相关较多,这与根瘤菌处理刺槐后的生理过程一致.在根瘤菌诱导的刺槐根组织文库中发现特异性基因如MYB类转录因子、囊泡相关膜蛋白等与传递细胞相关的基因,结果有助于进一步研究此类传递细胞的形成分化机制.To find special genes in transfer cells of Locust which was differentiated from root epidemic cells induced by rhizobium, a forward and reverse suppression subtraction hybridization （ SSH ） cDNA library were constructed successfully. Using cDNA from the roots of Robinia pseudoacacia induced by rhizobium as the tester and cDNA from roots of Locust untreated as driver seperately,suppression subtraction hybridization libraries was constructed. 500 colonies in both libraries were isolated and sequenced. Functions of ESTs were analyzed by blast in NCBI including database Nt,Nr, Swissprot,COG,Interpro and GO after removing repeat and redundancy sequences. 725 uniESTs including 385 uniESTs in forward library and 340 uniESTs in reverse library were obtained. The assembling provided a total of 51 contigs and 674 singletons by cluster analyses of the uniESTs. Nucleotide homology searched with Blastn in NCBI non-redundant nucleotide database and 674 uniESTs（93% of total uniESTs） were homologous with known genes. Protein homology searched with Blastx in NCBI non-redundant protein database and 648 uinESTs（89% of total uniESTs）were homologous with known proteins. The results of gene ontology（ GO） annotation showed that 369 uinESTs were involved in biological process with 270 times,molecular function with 448 times and cell component with 484 times respectively. Among these ESTs, putative proteins were related to posttranslational modification, transcription factors, cell signals, cell wall/cell＆amp;nbsp;membrane/endomembrane system, cytoskeleton and nodulin genes in positive library, and related to growth and metabolites biosynthesis in reverse library. These results were generally consistent with physiological processes when the Robinia pseudoacacia were induced by rhizobium. Such as MYB transcription factor,Vesicle-associated membrane protein could be found in positive library which might be special expression in the transfer cells to aid the understanding of de-velopment of transfer cells.

关 键 词：根瘤菌 刺槐 传递细胞 SSH 功能分析 

分 类 号：S718[农业科学—林学]