绵毛优若藜冷诱导SSH文库构建研究

Construction of cold-stressed SSH library of Ceratoides lanata and sequence analysis of its differentially expressed cDNA

绵毛优若藜是我国近年成功引种的高抗逆性饲用及防风固沙植物。以常温下的绵毛优若藜叶片和嫩枝为对照组,冷胁迫诱导处理后的叶片和嫩枝为试验组,获得两者高质量mRNA后反转录成cDNA,RsaI消化使其平末端化,试验组cDNA的5’末端连接上2个特异接头,然后分别与过量对照组cDNA进行杂交,把两份杂交样品混合后,与过量的新鲜变性的对照组cDNA过夜杂交,杂交后的混合液稀释后进行第一轮抑制性PCR以选择扩增特异表达的cDNA,然后以第一轮PCR产物为模板用巢式引物进行二次抑制性PCR,获得的富集差异表达cDNA即为绵毛优若藜冷胁迫SSH文库。将SSH文库的cDNA插入到载体中,共获得了362个克隆,为后续绵毛优若藜的抗冻基因克隆与转基因应用奠定了基础。

As a high stress resistance psammophyte with the characteristic of windbreak and sand fixation, Ceratoides lanata had been successfully introduced into China in recent years. The leaves and twigs of Ceratoides lanata in room temperature were used as the driver and those treated by cold stress were used as tester. The tester and driver ds cDNA were prepared from their high quality mRNA which were purified from their total RNA. The tester and driver cDNA were separately digested to obtain shorter, blunt-ended molecules by RasI. Then two tester populations were created with different adaptors, while the driver cDNA has no adaptors. Differentially expressed genes were equalized and enriched by two round subtractive hybridizations using excess driver population as compared with tester population. The differentially expressed cDNAs were exponentially amplified by first round suppression PCR using the diluted hybridization product as template, and then the secondary PCR was performed using the first PCR product as template by nested primers to finally enrich the differentially expressed cDNAs, which consists of the SSH library of Ceratoides lanata. These cDNAs were inserted into vectors and 362 cDNA clones were obtained. The result lays a foundation for the cDNA cloning of antifreeze gene from Ceratoides lanata, and their transgenic application.