表达苏云金杆菌δ-内毒素基因的转基因烟草的抗虫性

Insect Resistance of Transgenic Tohacco Plants Expressing δ-endotoxin Gene of Bacillus thuringiensis

苏云金杆菌(Bacillus thuringiensis)HD-1的δ-内毒素基因原始克隆TH12和TH48分别含有5.3kb和6.6kb型两类同源异族基因。经定点突变改造其5′端,酶切对3′端进行缺失后,在大肠杆菌中仍能表达出有杀虫活性的被修饰的毒蛋白。将上述加工过的基因插入到双元载体pBin437(pBin19的派生质粒)中,借助辅助Ti质粒pAL4404转入烟草,获得了抗卡那霉素的再生植株。经Southern印迹和狭槽印迹(Slot blot)杂交证明有1—5个拷贝的毒素基因整合至烟草染色体中。Northern印迹法分析结果表明这两类基因都在转基因植株中得到表达。有4株5.3kb类型,3株6.6kb类型的转基因植株对烟青虫有高抗性,与对照相比,这7株转基因烟草植株的杀虫率可达40—50%,并能显著抑制昆虫蜕皮和生长发育,表现明显的抗虫作用。结果表明这两类毒蛋白基因都可在植物中表达并赋于转基因植株以抗虫性。

The original B.thuringiensis δ-endotoxin (Bt toxin)gene clones TH12and TH48 contain two different classes of homologous genes, the 5.3kbclass and 6.6kb class respectively. Bt toxin genes of both classes modj-fied at the 5'-end and truncated at the 3′-end can still be expressed toproduce the entomocidal, truncated toxin proteins in E.coli. The modi-fied Bt toxin genes were inserted into the plant binary expression vec-tor pBin437 (a derivative plasmid of pBin 19) and were transferedinto tobacco genome by disarmed Ti plasmid pAL 4404 gene transfersystem. Southern blot and DNA slot blot analysis indicate that the Bttoxin genes have been integrated into tobacco genome at a copy numberof 1--5. Northern blot analysis of poly A^+ RNAs from progenies ofthe transgenic plants revealed that Bt toxin genes of both 5.3 and 6.6kb classes were expressed in transgenic plants, though the transcriptswere degraded to RNAs of lower molecular weights. In entomocidal test,5 plants from the progeny of 5.3 kb class gene-transformed SR1 tobaccoplants and 3 plants from that of 6.6kb class gene-transformed plantswere found to be toxic to the testing larvea of H. assulta. In compari-sion with the control, mortality of the insects feeded on transgenicplants reached 40--50% and the growth of the survived insects was re-markably inhibited. These results indicated that the modified Bt genes ofthe 5.3kb and 6.6kb classes were expressed in transgenic plants andcould confer the transgenic plants a new character of insect resistance.