掌叶半夏凝集素基因PPA2抗蚜功能分析  

Insect Resistance Analysis of an Agglutinin Gene PPA2 Cloned from Pinellia pedatisecta

作　　者：刘玎[1,2] 陈劲[1,2] 刘志[1] 朱生伟[2] 

机构地区：[1]湖南农业大学生物科学技术学院,长沙410128 [2]中国科学院植物研究所植物分子与生理学重点实验室,北京100093

出　　处：《生物技术通报》2016年第10期180-187,共8页Biotechnology Bulletin

基　　金：国家自然科学基金-新疆联合基金项目(U1403284)

摘　　要：掌叶半夏凝集素(Pinellia pedatisecta Agglutinin,PPA)基因属于单子叶甘露糖结合凝集素家族,具有抗虫活性。采用反转录聚合酶链式反应(RT-PCR)技术从掌叶半夏幼叶中克隆到1个新的凝集素基因,命名为PPA2,其开放阅读框长408 bp,编码135个氨基酸,有一个甘露糖保守结合域,包含3个甘露糖专一结合位点。分别构建了由35S启动子和韧皮部特异性启动子At PP2驱动的植物表达载体,农杆菌介导的叶盘法转化烟草后,经鉴定获得了单拷贝插入高表达量的纯合转基因烟草株系。通过Western-blot分析,将获得的转基因纯合株系按蛋白表达水平分成高、中、低3类并进行抗蚜试验,结果表明这些转基因烟草均具有明显抗蚜效果,35S-PPA2转基因植株平均抑蚜率极高,而3种蛋白表达类型的At PP2-PPA2转基因植株普遍低于35S启动子驱动下的转基因植株。PPA2可以作为高效抗蚜候选基因,具有重要的应用价值。Pinellia pedatisecta Agglutinin（PPA）belonging to mannose-binding lectin muti-family has insect resistant activity. A new gene,named PPA2,encoding a PPA protein was cloned by RT-PCR from young leaves of pinellia pedatisecta. The PPA2 consists of an ORF of 408 bp coding 135 amino acids（14.7 kD）,and contains a mannose conservative domain with three mannose-binding sites. The transgenic tobacco plants mediated by Agrobacterium with leaf disc method were obtained under the control of Ca MV 35 S promoter and Arabidopsis thaliana phloem promoter At PP2. A few of homozygotic transgenic plants with single copy insertion and high expression level were indentified in T3 generation,and then these plants were divided into high,middle,and low classes according to the level of expression protein detected by western-blot analysis. The results of aphid bioassay showed that all transgenic plants had obviously insecticidal activity against the tobacco aphids（Myzus nicotianae）,and the Ca MV 35S-PPA2 transgenic plants had the highest insecticidal activity,while the aphid inhibition was much lower in At PP2-PPA2 transgenic plants than in Ca MV 35S-PPA2 ones. These findings suggest that PPA2 is a suitable candidate gene for high efficient aphid resistance and has important application prospect.

关 键 词：掌叶半夏凝集素 基因克隆 转基因烟草 抗蚜基因 

分 类 号：Q943.2[生物学—植物学] S432.23[农业科学—植物病理学]