Construction Of Plant Expression Vector Of HaCDA HpRNA And Genetic Transformation Analysis Of Tobacco

RNAi is a gene silencing technique,which can cause the degradation of homologous mRNA in cells,and exists in eukaryotes.The construction of dsRNA fragments of important genes related to insect physiological and biochemical processes by genetic engineering can significantly improve plant resistance to insects.Helicoverpa armigera is a worldwide agricultural pest.In this paper,four CDA genes of H.armigera were constructed into hpRNA plant expression vectors,and transformed into tobacco by Agrobacterium-mediated method,and kanamycin resistant plants were obtained.Then the resistant plants were identified by PCR and RT-PCR,and the germination rate of T1 transgenic tobacco plants was tested by chi-square test.Finally,single copy of T0 plant leaves were selected to study insecticidal resistance.The specific contents of the study are as follows:1.Construction of hpRNA plant expression vector containing the target gene CDAFour fragments of CDA gene of H.armigera were obtained by PCR amplification using the pUCm-HaCDA plasmids as templates in our laboratory.They were cloned into pUCBXK plasmids modified in our laboratory by means of TA cloning.After golden-gate clone the target gene fragments were cloned into hpRNAi plant expression vector,after PCR and sequencing identification we obtained four binary expression vector.The plasmid was electroporated into Agrobacterium tumefaciens competent cell LBA4404,and successful Agrobacterium strains containing four cotton bollworm CDA gene were obtained.2.Acquisition and identification of transgenic tobacco plantsFour CDA genes of H.armigera were introduced into tobacco plants by Agrobacterium mediated transformation.The number of kanamycin resistant positive plants obtained by tissue culture was HaCDA5 a 11,HaCDA5 b 11,HaCDA1 10 and HaCDA2 16.The positive transgenic tobacco plants identified by PCR and RT-PCR were HaCDA5 a 9,HaCDA5 b 8,transgenic HaCDA1 8 and HaCDA2 11.The T0 seeds of 5 of the positive plants with every CDA gene were randomly planted in MS solid medium containing kanamycin,and the rate of germination of the T1 generation plants was tested by chi-square test.The number of single copy plants in line with Mendelian isolation ratio was HaCDA5 a 5,HaCDA5 a 5,HaCDA5 a 5 and HaCDA5 a 4 respectively.3.Analysis of insecticidal resistance of transgenic tobacco plants with HaCDASingle copy HaCDA tobacco leaves and wild-type tobacco leaves(control group)were fed with H.armigera larvae.The results showed that the larva bite degree of transgenic HaCDA5 b tobacco leaves was less than that of the other three transgenic cotton bollworm genes after 24 hours of feeding.And they were lighter than the control group.The larval mortality of tobacco leaves fed with HaCDA5 b for five days was the highest,while that of control group was the lowest,and there was significant difference between the two groups.The silencing efficiency of the target gene was detected by qRT-PCR.The results showed that the relative expression of the target gene in the larvae fed on the leaves of 4 H.armigera CDA tobacco plants showed a decrease to a certain extent.The specific silencing efficiency of HaCDA5 b leaves of H.armigera was the highest.The silencing efficiency of transgenic tobacco leaves to larval target gene was significantly different from that of control group.This study provides some experimental basis for screening new insecticide target genes,and provides some new suggestions for using RNAi technology to control cotton bollworm and other agricultural pests.