黄萎病菌诱导的海岛棉抗病反应的SSH文库构建及分析

Construction and Analysis of SSH Library of Gossypium barbadense upon Infection with Verticillium dahliae

在Pima90幼苗接种黄萎病病原菌后2、4、8、12、24、48、72和96h分批取幼根以抽提棉花总RNA。以未接种病原的棉花cDNA为驱动方,利用SSH法对接种病原后的棉花cDNA进行差减杂交。对杂交后的cDNA进行T/ A克隆并转化到大肠杆菌中完成文库构建,共获得了534个克隆。以M13引物对扩增插入片段进行PCR扩增,电泳结果显示插入片段大小从0.2～1.2kb不等,平均大小为0.5kb。将克隆中的插入片段点种于尼龙膜上,分别以病原诱导前后的总cDNA为探针进行反向Northern杂交。对78个在海岛棉的抗病反应中呈上升表达的克隆进行了测序并对测序结果去载体后在NCBI上利用Blastn和Blastx进行序列相似性分析。结果表明,大部分阳性克隆与拟南芥等多个物种不同逆境条件下诱导的相关基因或表达序列相同或同源,如棉花病程相关蛋白家族10,拟南芥抗病反应家族蛋白等。SSH文库的构建和分析将有助于了解棉花抗黄萎病反应的分子机制。

Roots were collected from the seedlings inoculated with pathogen Verticillium dahliae after 2,4,8,12,24,48,72 and 96 hours for total RNA extraction.The cDNAs from the inoculated seedlings were used as the tester and those from the control seedlings as the driver.SSH method was employed to find the differently expressed cDNAs responding to the pathogen.T/A clone library was constructed containing 534 clones.The cDNA inserts were amplified from the bacterial clones directly with M13 primers by PCR.The size of the products ranged 0.2~1.2 kb with an average size of 0.5 kb.The SSH products were dotted on nylon filters,and the positive clones were screened by virtual Northern blotting with probes of the two kinds of initiative cDNAs.Totally 78 clones which were up-regulated and putatively involved in the defense response of G.barbadense were identified and sequenced.Sequence similarity searches were performed with the Blastn and Blastx.Most of them showed high or partial homology to genes or ESTs induced by different stresses in Arabidopsis thaliana and other species,such as the pathogenesis-related 10 family of G.hirsumtum and disease resistance-responsive family protein in Arabidopsis thaliana.The results would be helpful to understand the molecular mechanisms of disease response in cotton.