豆科凝集素基因Le4的克隆及其表达产物对蚜虫的抗性

Cloning of Lectin Le4 Gene from Leguminosaeand the Aphid Resistance Effect of Its Protein Production

植物凝集素是含有一个或者多个非催化结构,并能可逆结合特异单糖或寡糖的蛋白,它具有凝集细胞、沉淀多糖和糖蛋白的功能。近年来,植物凝集素因其独特的生理功能,在农作物抗虫工程中获得了广泛的关注。本研究采用RT-PCR的方法,从浙江苞萝黄大豆叶片中成功克隆了一个新的豆科凝集素Le4基因,利用p ET-28a质粒构建了Le4基因的原核表达载体,成功表达了Le4蛋白。通过对诱导温度及异丙基-β-D-硫代半乳糖苷(IPTG)终浓度等条件进行优化,利用Western blotting技术,在Le4表达菌株超声破碎后的上清液中检测到了可溶性的Le4蛋白。通过涂抹法将含有可溶性Le4表达蛋白的大肠杆菌菌液涂抹在小麦叶片上,与涂抹空载体菌液相比,小麦叶片麦长管蚜数目减少了约50%。以上结果初步表明来源于大豆的凝集素基因Le4蛋白具有杀虫效应。

Plant lectins are plant proteins possessing one orseveral non-catalytic domain which can reversibly bind mono- or polysaccharides. Plant lectins have the functions of agglutinate cells, precipitate polysaccharides and glycoproteins. In recent years, plant lectins have attracted more attention in insect-resistant crop genetic engin- eering due to its unique physiological functions. In this study, Le4, a new plant lectin gene, was successfully cloned from soybean by means of RT-PCR strategy. Plasmid ofpET-28a was used to constructed prokaryotic expression vector of Le4, and Le4 protein was successfully expressed in E. coli. Le4 protein was detected in the content of soluble supernatant using western blotting strategy under the optimized conditions of induction temperatures and final concentration of Isopropyl-beta-D-thiogalactopyranoside （IPTG）. In order to preliminaryinvestigate the aphid resistance effect of Le4 protein, the transformants producing soluble Le4 after ultrasonic decomposition was spread on wheat leaves, the number of aphids in the wheat leaves was reduced about 50% compared with the number of aphids in wheat leaves spread on empty vector bacteria total protein. These results preliminarily indicated that Le4 protein derived from soybean had insecticidal effect.