苋菜凝集素基因的克隆及在转基因烟草中抗蚜性研究

通过ＰＣＲ从苋属植物千穗谷（Ａmaranthus hypochondriacus)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２４５３bp，含有－１５３８bp的内含子和两个分别为２１２bp和７０３bp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了化烟草，转化再生植株的ＰＣＲ和Southern blot分析表明，ＡＨＡ基因已整合到烟草的染色体中，有单贝和多拷贝的整合。用与ＡＨＡ蛋白高度同源的ＡＣＡ蛋白的抗血清进行了免疫斑点（Immunodot blot)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５７．２％和４８．８％，有的高达９０％以上，含内含子和不含内含子的ＡＨＡ基因在转基因植株中的抗蚜性不同。

Cloning of AHA Gene from Amaranthus hypochondriacus and it

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.