细极链格孢菌蛋白激发子诱导棉苗基因表达差减文库的构建及EST分析  

Construction of Suppression Subtractive Hybridization Library with cDNA from Seedling Cotton Induced by Protein Elicitor from Alternaria tenuissima and Analysis of EST

作　　者：张志刚[1] 邱德文[2] 杨秀芬[2] 曾洪梅[2] 袁京京[2] 刘峥[2] 杨晓萍[3] 官春云[1] 

机构地区：[1]湖南农业大学,长沙410128 [2]中国农业科学院植物保护研究所,北京100081 [3]湖南省棉花科学研究所,常德415000

出　　处：《棉花学报》2007年第4期248-254,共7页Cotton Science

基　　金：国家973重点基础研究发展计划(2003CB114204);国家863重点基础研究发展计划(2003AA241130)

摘　　要：利用细极链格孢菌蛋白激发子粗提蛋白液诱导棉花苗期相关基因表达，以清水喷施为对照样本，进行抑制差减杂交，构建棉花苗期基因表达文库。结果显示，插入片段的大小主要集中在200～1000bp。随机选取150个克隆进行菌液PCR检测与测序，并将所测序列与Gen-Bank dbEST库进行比对，共得到104个单一序列。其中，78个EST与其它物种已知基因部分区域的同源性为48．54％～100％，占总EST的75％；6条EST序列能在数据库中检索到同源性序列，但其功能尚不清楚，占5．77％；10个EST能在数据库中发现为推测蛋白，占9．6％；10个EST在GenBank中没有查到对应的同源序列，可能是新基因，占9．6％。同时，将所得到的104条EST序列在WEGO网站上进行功能分类，通过Gene ontology确定具体EST的分子功能。其中，在生物途径分类中，与生理途径和细胞过程相关的EST最多，分别为48个和45个；在细胞组份功能分类中，与细胞相关的EST最多，达到44个；在分子功能分类中，催化功能与分子绑定相关的EST数量最多，分别达到了30个和28个。由此可见，细极链格孢菌蛋白激发子诱导棉苗时涉及到植物的生理生化的多条途径，受到多个基因的调控，可能包括抗病与防御蛋白、信号传导蛋白、转录因子、能量代谢相关蛋白、抗逆相关蛋白、细胞保护机制蛋白、功能未知及其它蛋白等相关蛋白。The gene expression of seedling cotton induced by protein elicitor from Alternaria tenuissima, the construction of suppression subtractive hybridization （SSH） library with cDNA from seedling cotton, and analysis of EST were described. The results demonstrated that the insertion fragments of SSH library were ranging from 200 bp to 1 kb. 150 clones were randomly picked for PCR and sequencing. After matching the result in GenBank dbEST, 104 sequences were obtained. Of them, 78 had 48.54%～100% similarity with known genes, which is 75% of the total EST. Six sequences have their homology with the sequences in GenBank with unknown function. 10 have no similarity with the sequences in GenBank, which might be new genes. The function of all the 104 EST was determined by classified being in WEGO and gene ontology. Most of the EST were related to physiological process and cell process, with the number of 48 and 45, respectively. 44 of them related to cell component, 30 related to catalytic activity, 28 related to binding. It is suggested that several pathways were induced when the seedling cotton was treated with protein elicitor, including resistance, cell signaling, transcription, energy metabolism, et al.

关 键 词：细极链格孢菌蛋白激发子 棉花 抑制性差减杂交(SSH) EST 

分 类 号：S562[农业科学—作物学]