核盘菌诱导下甘蓝型油菜宁RS-1的SSH文库构建

Construction of SSH cDNA library from Ning RS-1 after inoculation of Sclerotinia sclerotiorum

以甘蓝型油菜宁RS-1为材料,用核盘菌(Sclerotinia sclerotiorum(Lib.)de Bary)对幼苗进行接种,提取经核盘菌诱导前和诱导后的宁RS-1叶片mRNA,经反转录成cDNA,以及经过酶解后加接头和两次杂交,构建差异表达的抑制消减(SSH)cDNA文库。正向消减文库是以诱导后的宁RS-1 cDNA为tester,以正常的宁RS-1 cD-NA为driver。所构建的正向文库重组率达到96.2%,反向文库重组率达到94.5%,文库容量均达到了2 190和2 780,表明所构建的文库质量良好。对从文库中随机挑取的克隆进行测序分析,获得了与抗病和代谢有关的基因,如γ-谷氨酰半胱氨酸酶基因、抗真菌蛋白基因、铁蛋白基因、ACC氧化酶基因等,为开展油菜抗菌核病重要功能基因的克隆和鉴定奠定了基础。

Total RNA was extracted from the leaves of Ning RS-1 before and after inoculation by Sclerotinia sclerotiorum（Lib.） de Bary.Leaves＇ mRNA were purified and transcribed into cDNA.An adaptor was added on to the cDNA by enzymatic hydrolysis,followed by two rounds of hybridization.Finally differential expression of suppression subtracted cDNA library of the induced Ning RS-1 was built.In subtractive hybrid library,the induced Ning RS-1 cDNA was used as a tester and the normal Ning RS-1 cDNA was used as a driver.In reverse library,they were used in opposite way.The subtractive library had a recombination rate of 96.2% with a base capacity of 2 190.And the reversed one had a recombination rate of 94.5% with capacity of 2 780.It indicated that the library quality could meet the requirements of screening resistance genes to Sclerotiorum.Genes related to metabolism of resistance to the disease were identified by sequencing of the clones picked randomly from the cDNA library.They included γ-glutamylcysteine synthetase gene,anti-fungal protein,iron protein and ACC oxidase,etc.