【作者】 马丽；

【导师】 沈昕；

【作者基本信息】 北京林业大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 胡杨是我国西北干旱盐碱的荒漠和戈壁地带能形成森林的唯一高大乔木树种。由于其具有极强的耐盐能力,近年来对胡杨的研究已成为树木抗盐机理研究的热点。本文对胡杨14-3-3蛋白基因、质膜H~+-ATPase基因及Na~+/H~+逆向转运蛋白基因(PeNHA)进行了基因克隆及转化研究,并取得下述研究进展:建立群众杨组织培养再生体系,并在此基础上利用农杆菌介导的遗传转化方法将35S-PeNHA基因导入群众杨,PCR及PCR-Southern杂交检测结果表明PeNHA已整合到群众杨基因组,获得的再生植株为转基因植株;进行了胡杨14-3-3蛋白基因克隆的初步研究,获得了长为999bp的DNA片段,经测序和同源性分析证明该DNA为14-3-3蛋白基因片段;运用噬菌斑原位杂交法对胡杨的cDNA文库进行了质膜H~+-ATPase基因的筛选,并进行了重组质粒λExcell的初步鉴定,得到约1000bp的cDNA片段,推测该片段可能是胡杨质膜H~+-ATPase基因;运用DNA重组技术构建了Na~+/H~+逆向转运蛋白基因(PeNHA)小球藻表达载体。上述研究进展的取得为进一步阐明胡杨耐盐机理奠定了基础。更多还原

【Abstract】 Populus euphratica is the only arbor species which can survive in semi-arid areas in Northwest China. Due to its tolerant to high salinity, Populus euphratica becomes one of the main research interests in the field of plant salt tolerance mechanism. In this dissertation, studies was focused mainly on genes coding for 14-3-3 protein and plasma membrane H~+-ATPase as well as Na~+/H~+ antiporter (PeNHA) from Populus euphratica. Research progress were made mainly on following aspects: Firstly tissue culture and regeneration system of Populus popularis , a salt-sensitive transformation acceptor, was obtained and then based on this system the gene coding Na~+ /H~+ antiporter of Populus euphratica was transferred into Populus popularis by Agrobacerium-mediated transformation approach. The transformation was proved by PCR and PCR-Southern Blotting. Secondly by PCR approach, a 900bp DNA fragment representing the gene coding for 14-3-3 protein was cloned from Populus euphratica . It was then sequenced and proved to be the DNA sequence coding for 14-3-3 protein by Blast analysis. Thirdly screening for gene coding for plasma membrane H~+ATPase of Populus euphratica from cDNA library was conducted and a positive clone with a 999bp insert on the recombinant-vector λ ExCell was obtained, and this insert was speculated to be the plasma membrane H~+-ATPase gene of Populus euphratica. At last, an expression vector containing PeNHA was constructed for expressing PeNHA in Chlorella pyrenoidosa. The results summed up above establish a basis for further thorough illumination of the salt tolerance mechanism of Populus euphratica.更多还原