【作者】 林清芳；

【导师】 王茅雁；

【作者基本信息】 内蒙古农业大学 ， 遗传学， 2010， 硕士

【摘要】 沙冬青是我国西北荒漠区特有的唯一常绿旱生阔叶灌木,具有很强的抗寒和抗旱等抗逆特性。目前对于沙冬青抗逆性分子机理和抗逆基因的分离鉴定集中于抗寒性方面,有关其抗旱基因的分离和鉴定尚未见报道。本论文以蒙古沙冬青(Ammopiptanthus mongolicus)为材料,采用SMART(Switching Mechanism At 5’end of RNA Transcript method)技术构建了其在干旱胁迫下的全长cDNA文库,并对文库中的部分克隆进行了5’端测序和序列分析,为进一步开展表达谱分析和重要抗旱基因的克隆鉴定工作奠定了基础。实验首先对经典热酚法加以改进,解决了蒙古沙冬青叶片总RNA提取中多糖和多酚类物质污染严重的问题,获得符合建库要求的高质量总RNA。此外,还发现Promega公司的PolyATtract? mRNA Isolation Systems更适合于沙冬青mRNA的分离纯化。以不同干旱胁迫处理的蒙古沙冬青混合mRNA为模板合成cDNA,构建了SMART全长cDNA文库。原始文库的滴度和重组率分别为1.88×107 pfu/mL和97.87%,扩增文库的滴度为4.32×1010pfu/mL;插入片段大小在500~2500 bp之间,表明所建文库质量较高。将所构建的λTriplEx2文库转化成pTriplEx2质粒文库,经菌液PCR鉴定获得3500个阳性克隆。将其中960个克隆进行5’端测序,共获得875个有效EST序列。将EST序列去载体后进行拼接,获得97个Contig和509个Singlet,总计606个Unigene。这些Unigene通过功能注释可分成19个大类,其中与蛋白质合成、修饰、转运和降解相关的基因所占比例最高,其次是一些具有普通功能的基因和参与小分子及离子运输与代谢的基因,与能量代谢、基因转录、细胞壁和膜的形成、信号转导、细胞周期、胞内运输和RNA加工与修饰等过程相关的基因也占一定比例。有2.65%的Unigene未得到功能注释。更多还原

【Abstract】 As an only evergreen broad-leaf shrub in the northwest desert of China, Ammopiptanthus mongolicus shows very strong resistance to both drought and cold stresses. At present, studies on the sterss-resistant molecular mechanisms and the involved genes in the plant are limited to its cold resistance only, and report about the isolation and characterization of the drought-resistant genes from the plant has not been found as yet. In the present study, a drought-induced full-length cDNA library of A. mongolicus was constructed by using SMART (switching mechanism at 5’-end of RNA transcript) cDNA library construction method. In addition, 5’-sequencing and the seqnence analyses of some clones from the library were carried out. The work laid a foundation for both the expression profile analysis and the cloning and characterization of drought-resistant genes from the plant in the future.Firstly, the classical hot phenol method was improved and the contamination problems of total RNA by polyphenols and polysaccharides during its extraction from A. mongolicus were solved, and as a result, high qulity total RNA meeted the requirements for SMART cDNA library construction were extracted from the plant. What’s more, the PolyATtract? mRNA Isolation Systems produced by Promega company was proved more suitable for the mRNA isolation from total RNA of A. mongolicus.Subsequently, using mixed mRNA isolated from different A. mongolicus samples treated under different drought conditions as template for cDNA synthesis, we constructed a full-length cDNA library with SMART cDNA library construction method. The titer of the unamplified library is 1.88×107; the recombination percentage of the library reaches up to 97.87%; the titer of the amplified library is 4.32×1010 pfu/ml; the sizes of the insert cDNA fragments ligated toλTriplEx2 vector ranges from 0.5 to 2.5kb. All the results suggest that the library has a higher qulity.Furthermore, part of the unamplifiedλTriplEx2 library was converted to pTriplEx2 library, and 3500 positive clones were selected from the plasmid library by PCR. Up to date, 960 positive clones have been sequenced from their 5’-end, and 875 useful EST sequences were obtained. After removing the vetor sequences and clustering them, we finally obtained 97 contig and 509 singlet, summing into 606 Unigene. All these Unigene can be classified into 19 clusters. Of which, the genes related to synthesis, modification, transport and degradation of proteins are the most, and the genes with genaral functions and the genes associated with the transport or metabolism of small molleculars and irons rank second. The genes associated with energy metabolism, transcription, cell wall and membrane formation, signal transduction, cell cycle, intracellular transportation, RNA processing and modification, and so on, respectively are also involved in the Unigene. Finally, there exist 2.65 percent Unigene which have not been functionally annotated as yet.更多还原