半夏凝集素基因的克隆及转基因烟草对蚜虫的抑制作用

Cloning and transformation of pta gene and inhibitory effects of the transgenic tobacco plants on development of aphid populations

采用同源序列克隆方法，通过设计特异性引物从甘肃西和半夏叶片基因组DNA中克隆到1条1069bp的半夏凝集素基因pta，与以同科内植物的mRNA为模板扩增得到的凝集素基因序列的同源性很高，达到98％以上，功能区完整，具有1条信号肽和3个甘露糖结合区，GenBank登录号AY725425。用该基因替换pBI121载体的GUS基因，正向插入35S启动子之下，构建了植物表达载体pBI—pta。通过农杆菌介导法转化烟草，从20株Kan抗性植株中得到PCR检测阳性植株14株，证明pta已整合到烟草基因组中。对随机挑取的7株PCR阳性植株进行了抗蚜虫（Myzus persicae）筛选试验，结果表明：不同株系蚜口密度抑制率在22．5％～89．4％，平均蚜口密度抑制率56．2％。

The full-length DNA （1069 bp, AY725425 in GenBank） of P. ternata agglutinin （PTA） was cloned by using DNA extracted from Pinellia ternata leaves as the template and the primers designed according to the con-servative sequences of lectins. Through comparative analysis of PTA gene （pta） and its deduced amino acid sequences with those of the same species, it was found that pta encoded a precursor lectin with signal peptide andthree mannose-binding boxes like lectins from other species. The sequence identity between them is 98%. A plant expression vector pBI-pta was constructed by replacing GUS gene in pBI121 with pta. pta was then transferredinto tobacco mediated by Agrobacterium tumefaciens and 20 kanamycin-resistance transformants were obtained. PCR analysis indicated that pta had been integrated into the tobacco genome in 14 transformants. The resultsfrom resistance tests to the green peach aphid Myzus persicae with randomly selected 7 transformants showed that the transgenic plants were aphid-resistant, evidenced by 22.5%-89.4% reduction in insect population density,and the average inhibition rate was 56.2%.