Genetic Transformation And Analysis Of Tobacco With HaRLI Gene

The RNase L pathway is an antiviral pathway induced by interferon,and the RLI gene,as a negative regulator of the RNase L pathway,has been studied more and more.In this study,the HaRLI gene was used as the research object to explore the function of the gene in virus treatment and plant mediated insect RNAi.The main contents are as follows:1.Cloning and sequence analysis of HaRLI geneThe BmRLI homology gene sequence was obtained from the transcriptome of Helicoverpa armigera by homology search.RT-PCR method was used to clone the full-length gene sequence in cotton bollworm,named HaRLI.The gene contain a 1824 bp ORF encoding 608 amino acids.NCBI Blast showed that the gene has 3 conserved domains,namely,one iron sulfur binding domain and two ATP binding cassette.2.Expression of HaRLI gene after treatment with HaNPV virusThe expression of HaRLI gene in normal and virus treated insects was analyzed by RT-qPCR method.The results showed that the HaRLI gene expression after the treatment with HaNPV virus for 24 h was significantly decreased compared with that of the normal feeding insects,reaching about 60%.3.Transgenic plants and genetic analysis of progeniesThere is a BamH I restriction site on the HaRLI gene,therefore,we designed Bsa I site nested BamH I or Sal I cleavage site at the 5 'end of the two primer to construct plant expression vector pBin438-HaRLI,introducing HaRLI gene into tobacco genome through Agrobacterium mediated method.The kanamycin resistant genetic analysis of the transgenic T0 and T1 generation plant seeds showed that the kanamycin resistance of the T0-4 plant seeds was in accordance with the Mendel 3: 1 segregation ratio,which was speculated that the foreign gene is a single copy insertion.Subsequently,the seeds of 17 strains of T1 plants were also screened by Kan.The results showed that 5 strains were all resistant(pure),and the other 10 strains showed a separation ratio of about 3: 1(heterozygous).Among them,2 strains were not in accordance with Mendel's separation ratio,and the resistant seedlings were about 4 times as sensitive as that of the susceptible ones.4.Effects of leaf feeding on RNAi efficiencyThe RNAi vector of chitin synthase 1(Hachs1),chitin synthase(Hachs2)and green fluorescent protein(GFP)gene was constructed and transformed into HT115 competent cells.Wild type and T1-8 plant leaves were coated with the bacteria of expressing dsRNA to feed the larvae for 24 h and then the target genes were detected by RT-qPCR.Hachs2 gene expression was down regulated after feeding wild type leaves,while Hachs1 gene has not been silent,but increased.After feeding transgenic leaves,Hachs1 and Hachs2 gene were reduced,and compared with the control group,the down-regulation of Hachs2 gene has extremely significant difference.It was found that compared with feeding wild type tobacco leaves,the silencing efficiency of Hachs1 and Hachs2 gene was higher in Helicoverpa armigera with feeding transgenic leaves containing bacterial dsRNA.5.Analysis of insect resistance of transgenic HaRLI gene leavesThe leaves of WT,T1-8 and T1-20 were selected for feeding,and no obvious difference was observed among them after 12 h.The biting of WT leaves is relatively serious,but there is no obvious lethal phenomenon after 36 h,.It was that transgenic tobacco had a certain resistance to cotton bollworm.The above studies provide some reference for the study of HaRLI gene function.