CAJ | 학술논문

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cDNA文库。各挑选正反文库中500个克隆进行测序,在Blastn、Blastp、SwissProt、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( uniEST),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uniESTs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uniESTs和反向文库156个uniESTs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。 uniESTs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。
탐심근류균유도자괴후근표피세포분화성전체세포적특이성표체기인급기분자궤제。채용억제차감잡교( suppression subtraction hybridization,SSH)기술구건자괴근계피접충근류균화미피접충근류균이자간적정반량개cDNA문고。각도선정반문고중500개극륭진행측서,재Blastn、Blastp、SwissProt、KEGG、COG、Interpro 이급Gene ontology( GO)수거고중진행비대주석。결합생리과정대 ESTs 진행분석,공획득725조비용여서렬( uniEST),정반향문고분별위385조화340조,기중포괄674개단일서렬( singlets),51개병접서렬( contigs)。재Nt고비대중유674조uniESTs여이지기인필배,점비93%；재Nr고비대중유648조서렬유필배단백,점비89%。유정향문고213개uniESTs화반향문고156개uniESTs능진행Geney ontology( GO)공능주석,재세포조분피주석료270차,분자공능피주석료448차,생물과정피주석료484차。 uniESTs적공능분석현시,정향문고중다여세포번역후수식、전록인자、세포신호통로、세포벽/세포막/내막계통이급세포골가상관단백유관,부분시결류상관기인。이반향문고중여세포생장、대사물형성적기인상관교다,저여근류균처리자괴후적생리과정일치。재근류균유도적자괴근조직문고중발현특이성기인여MYB류전록인자、낭포상관막단백등여전체세포상관적기인,결과유조우진일보연구차류전체세포적형성분화궤제。
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 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.