CAJ | 학술논문

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.
면모우약려시아국근년성공인충적고항역성사용급방풍고사식물.이상온하적면모우약려협편화눈지위대조조,랭협박유도처리후적협편화눈지위시험조,획득량자고질량mRNA후반전록성cDNA,RsaI소화사기평말단화,시험조cDNA적5'말단련접상2개특이접두,연후분별여과량대조조cDNA진행잡교,파량빈잡교양품혼합후,여과량적신선변성적대조조cDNA과야잡교,잡교후적혼합액희석후진행제일륜억제성PCR이선택확증특이표체적cDNA,연후이제일륜PCR산물위모판용소식인물진행이차억제성PCR,획득적부집차이표체cDNA즉위면모우약려랭협박SSH문고.장SSH문고적cDNA삽입도재체중,공획득료362개극륭,위후속면모우약려적항동기인극륭여전기인응용전정료기출.
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.