Genetic Transformation Of Tobacco With Haace1 HpRNA Gene And Helicoverpa Armigera RNAi Analysis

The Helicoverpa armigera is an important agricultural pest.In recent years,the control of H.armigera has attracted widely attention.In this study,the acetylcholinesterase gene in H.armigera was used as the target,the plant that expressing hpRNA of Haace1 gene was obtained by transgenic methods in order to explore its insect-resistant value.In addition,the vector that expressing hpRNA in yeast was constructed and been compared with the feeding dsRNA RNAi experiment based on the bacterial expression system?L4440/HT115?.In order to explore whether the yeast expression system is suitable for screening the target gene of plant insect RNAi.The details are as follows:1.Transgenic plants and genetic analysispRNAi-GG-ace1f and pRNAi-GG-ace1r of plant expression vectors were transformed into the LBA4404 competent cells of Agrobacterium,the target fragments were transferred into tobacco genome by Agrobacterium mediated method,and regenerated seedlings were obtained by plant tissue culture technology.The kanamycin resistant genetic analysis and PCR verification showed that 13 strains of T₀ Haace1f transgenic tobacco and 9 strains of T₀ Haace1r transgenic tobacco were obtained.The genetic analysis of the transgenic T₀ generation plant seeds showed that:kanamycin resistance of the 4 strains seeds of Haace1f transgenic was in accordance with the Mendelian 3:1 segregation ratio,which was inferred that the foreign gene is a single copy insertion,but there was a separation ratio of 2.08:1,does not conform to Mendelian separation ratio;Of the 5 strains of Haace1r transgenic tobacco,they all conformed to the separation ratio of Mendelian 3:1,which was presumed to be single copy insertion.2.Effects of leaf feeding on RNAi efficiencyThe transgenic tobacco and the wild type tobacco leaf were fed to the two-age larva of H.armigera,the leaf bites and larva growth status were observed,and the expression of Haace1 gene was detected by RT-qPCR.The results showed that Haace1f transgenic tobacco and Haace1r transgenic tobacco had inhibitory effect on Haace1 gene expression of larva,and the inhibition effect of Haace1r transgenic tobacco was more significant.3.Feeding-based RNAi analysis of dsRNA expression vectors of yeast and bacterialThe construction of the hpRNA expression vector of yeast was referred to pRNAi-GG-ace1f plasmid and PYES2 plasmid by designing suitable enzyme-cutting sites.Then the new vector was transformed into competent cells of INVSc1.Compared with the interference effects of yeast and bacterial dsRNA expression system feeding on H.armigera,we find that the inhibitory effect of the yeast strain INVSc1 expressing dsRNA on the target gene was less than that of Escherichia coli strain HT115 after RT-qPCR result analysis.This study provides a theoretical basis for plant genetic engineering and biological control of H.armigera based on the target genes of Haace1.