转双抗虫基因烟草的研究

Studies on Transgenic Tobacco Plants Expressing Two Kinds of Insect Resistant Genes

用改造的雪花莲凝集素基因GNAmm与合成的苏云金芽孢杆菌 (Bt)毒蛋白cry1Ac基因构建了带有双价基因的植物表达载体 ,在该表达载体中这两个基因的转录分别受笋瓜PP2启动子 (SPP2P)和CaMV 35S启动子的调控。通过根癌土壤杆菌介导转化法 ,获得了一批抗卡那霉素的转化再生烟草植株。PCR检测及基因组DNASouthernblot、Slotblot杂交分析的结果表明Gna基因和Bt基因已整合到烟草总DNA中。用Bt毒蛋白抗血清进行Westernblot分析 ,转基因植株均有Bt杀虫蛋白的不同程度的表达。对转化再生烟草的虫试结果表明 ,在所受试的 19株烟草中 6 0 %的植株上的棉铃虫在 5天内死亡率达到 10 0 % ,而且存活幼虫的生长发育受到明显抑制 ;蚜虫抑制生长试验表明 ,多数转化再生植株具有较强的抗蚜活性 ,平均能够抑制桃蚜 5 0 %～ 6 0 %的蚜口密度 ,有的高达 80 %以上。

A synthetic Bt cry1Ac gene fussed with a secretary signal coding sequences at 5′end and a modified gna gene were used to construct a plant expression vector pBSGS1M + and this vector was transferred into tabacco ( Nicotiana tabacum L. )by Agrobacterium\|mediated transformation method.Results of PCR,Southern blot and Slot blot analysis indicated that both the chimeric Bt cry1Ac and gna genes were integrated into the genomes of transformed plants.Western blot analysis indicated that at least the cry1Ac protein was produced in transgenic plants.Upon insect bioassay using cotton bollworm ( Heliothis armigera Hubner),the mortality of insect larvae on 60% regenerated plants reached 100% in 5 days post infestation and the growth of the survived larvae was seriously inhibited;The results from insect bioassay with peach aphid ( Myzus persicae ) showed that the transgenic plants were aphid\|resistant,evidenced by a 50%～60% reduction in aphid population density,even over 80% for some individual transgenic plants.These results reflect that the modification of the two insect resistant genes and construction of the expression vector are correct and could be valuable for later application in crop breeding for insect resistance.