苋菜凝集素基因的克隆及在转基因烟草中抗蚜性研究  

Cloning of AHA Gene from Amaranthus hypochondriacus and it's Aphid Inhibitory Effect in Transgenic Tabacco Plants

作　　者：周永刚[1] 田颖川[1] 莽克强[1] 

机构地区：[1]中国科学院微生物研究所,北京100080

出　　处：《生物工程学报》2001年第1期34-39,共6页Chinese Journal of Biotechnology

基　　金：国家高技术研究与发展计划项目!(Z 17 0 1 0 1);ICSC的世界实验室的部分资助&&

摘　　要：通过PCR从苋属植物千穗谷 (Amaranthushypochondriacus)的总DNA中扩增出苋菜凝集素 (AHA)的核基因片段。序列分析结果表明该基因为 2 45 3bp ,含有一 15 38bp的内含子和两个分别为 2 12bp和 70 3bp的外显子。采取反向PCR的方法获得仅含该基因的编码区克隆。以此为基础与二元表达载体 pBin438构建含内含子与不含内含子AHA基因的植物表达载体 pBAHAg和pBAHAc并通过土壤农杆菌介导转化了烟草。转化再生植株的PCR和Southernblot分析表明 ,AHA基因已整合到烟草的染色体中 ,有单拷贝和多拷贝的整合。用与AHA蛋白高度同源的ACA蛋白的抗血清进行了免疫斑点 (Immunodotblot)检测 ,结果初步表明转基因烟草有AHA蛋白的表达。虫试结果表明转pBAHAg和 pBAHAc烟草对蚜虫的平均抑制率分别达 5 7 2 %和 48 8% ,有的高达 90 %以上。Using total DNA isolated from \%Amaranthus hypochondriacus\% as template,\%Amaranthus hypochondriacus\% agglutinin \%AHA\% gene was amplified by PCR and cloned.Sequence analysis results showed that this gene is consisted of 2453 base pairs including one 1538 bp intron and two exons of 212 bp and 703 bp respectively.After inverse PCR amplification,coding region of \%AHA gene was obtained.\%AHA\% gene with it′s intron ( AHAg ) and withou intron ( AHAc ) were inserted downstream of 35S promotor in the binary vector pBin438 resulting in the construction of two plant expression vectors pBAHAg and pBAHAc repectively.Leave explants of \%Nicotinana tabacum\% var.SR1 were transformed with \%A.tumefaciens\% LBA4404 harbouring the above expression vectors.Results from PCR and Southern blot analysis showed that \%AHA\% genes were inserted into the genome of transformed tobacco plants.Immunodot blot analysis indicated that \%AHA\% was expressed in transgenic plants.The results from insect bioassay with peach aphid (\%Mizus persicae\%) showed that the transgenic plants of pBAHAg and pBAHAc were aphid resistant, evidenced by a 57%～48% reduction in insect population density,some plants were more than 85%.The aphid resistance of transgenic plants transformed with \%AHAg\% gene as judged by aphid inhibition rate was higher than that of plants transormed with \%AHAc\% gene indicating that the intron in \%AHAg\% may be favorable for expression of \%AHA\% in transgenic plants.

关 键 词：千穗谷苋菜AHA基因 转基因烟草 抗蚜性 凝集素基因 

分 类 号：Q789[生物学—分子生物学]