小菜蛾中肠氨肽酶N3基因片断克隆技巧及其表达量

本研究通过设计简并引物,并且进行温度梯度PCR和二次PCR,最终筛选出适合扩增小菜蛾Plutella xylostella(L.)中肠APN3(氨肽酶N3,Aminopeptidase N3)片段的引物和温度,这为研究APN3的基因功能、APN3同APN其他同工酶之间的亲缘关系奠定了基础。通过比较电泳图谱,发现APN3在小菜蛾中肠中的表达量远远低于APN1、APN2和APN5的表达量。小菜蛾中肠APN同工酶氨基酸序列分析发现,APN3同APN1的亲缘关系最近,同APN2的亲缘关系最远。以上结果对于揭示Bt毒素作用于小菜蛾的机理以及小菜蛾对Bt毒素的抗性分子机制具有重要理论意义。

Clone technique and expression level of partial APN3 cDNA originated from Plutella xylostella midgut

The APN3 gene from the midgut of Plutella xylostella（L.）was amplified using temperature gradient PCR with degenerate primers.Two weak bands were apparent after electrophoresis of the first PCR products.Therefore,a second PCR was performed with the first PCR product as the template and original primer.The optimal primer was finally selected,which can be used to amplify partial APN3 cDNA at an annealing temperature of 48℃.These results provide techniques to study the gene function of APN3 and phylogenesis among APN isoforms.After comparing electrophoresis bands we speculate that the expression of APN3 is far lower than that of APN1,APN2 and APN5.We conclude that APN3 is most closely related to APN1 and most distantly related to APN2.These results are important to understanding how Bt toxins are toxic to P.xylostella and the molecular resistance mechanism of P.xylostella to these toxins.