胡杨PeXTH基因的克隆及其转基因烟草的抗镉性分析  

Cloning of Populus euphratica PeXTHgene and its effect on Cd^(2+) tolerance of transgenic tobacco

作　　者：韩彦莎[1] 鲁彦君[1] 于瑞[1] 赵瑞[1] 沈昕[1] 陈少良[1] 

机构地区：[1]北京林业大学生物科学与技术学院,北京100083

出　　处：《西北农林科技大学学报（自然科学版）》2014年第1期37-45,50,共10页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31270654;31170570;31200470);北京市自然科学基金项目(6112017);北京市优秀博士学位论文指导教师专项基金项目(YB20081002201);高等学校学科创新引智计划项目(B13007);中央高校基本科研业务费专项(JC2011-2;TD-2012-04)

摘　　要：【目的】克隆胡杨木葡聚糖内转糖苷酶/水解酶基因PeXTH,并研究该基因和植物抗Cd2+性之间的关系。【方法】利用RT-PCR方法从胡杨中克隆一个木葡聚糖内转糖苷酶/水解酶基因PeXTH,将该基因的全长cDNA克隆到植物表达载体pGreen0029上,然后通过农杆菌介导法,将重组表达载体转入烟草,并对转基因烟草的抗Cd2+能力进行初步分析。【结果】PeXTH基因的开放阅读框(ORF)长867bp,含ATG起始密码子和TAG终止密码子,编码由288个氨基酸组成的蛋白;多重氨基酸序列比对结果显示,PeXTH蛋白含有木葡聚糖内转糖苷酶/水解酶(XTHs)的保守催化活性域DEIDFEFLG和N-糖基化位点NLSG;Real-Time PCR检测结果表明,PeXTH基因已经成功在2个转基因株系(L6和L14)中过表达;Cd2+胁迫后,转基因烟草根组织中积累的Cd2+含量显著高于野生型,而叶组织中积累的Cd2+含量明显低于野生型;Cd2+胁迫下,转基因烟草叶片的相对电导率显著低于野生型,但抗氧化酶活性、营养元素含量、叶片净光合速率和蒸腾速率等生理指标都要明显高于野生型。【结论】PeXTH基因的过表达提高了烟草对Cd2+的抗性,为进一步深入研究PeXTH基因在植物抗Cd2+机制中的作用奠定了良好基础。[Objective] The aim of this study was cloning a putative xyloglucan endotransglucosylase/ hydrolase gene （PeXTH） of Populus euphratica and analyzing its role in Cd2＋ tolerance of transgenic to- bacco plants. [Method] The PeXTH gene was isolated from Populus euphratica leaves by RT-PCR. The full-length cDNA of PeXTH was cloned into the expression vector pGreen0029,and then the recombinant pGreenOO29-PeXTH was transferred into tobacco plants through Agrobacteriurn-mediated method. Anti- Cd2＋ characters of the transgenic lines were analyzed. [Result] The PeXTH gene contained an open read- ing frame of 867 bp encoding a putative protein with 288 amino acids with the start codon of ATG and the termination codon of TAG. The putative amino acids sequence of PeXTH contained the conserved DEI-DFEFLG domain and the N-glycosylation motif NLSG. Real-Time PCR analysis showed PeXTH gene was overexpressed in two transgenic tobacco lines. Compared to wild plants,transgenic tobacco had higher Cd2＋ concentrations in roots and lower Cd2＋ concentrations in leaves. Under Cd2＋ stress,the relative electrolytic leakage in leaves of transgenic plants was significantly lower than that of wild plants,while antioxidant en- zyme activities, nutrient element contents, net photosynthetic rates and transpiration rates in leaves of transgenic tobacco plants were significantly higher. [Conclusion] Overexpression of PeXTH enhanced Cda＋ tolerance of tobacco plants.

关 键 词：胡杨 烟草 PeXTH Cd2+ 抗Cd2+性 

分 类 号：S722.36[农业科学—林木遗传育种] Q785[农业科学—林学]