Silencing The HaHR3Gene Of Helicoverpa Armigera By RNAi And Research On The Insect-resistant Mechanism Of Transgenic Plants

RNA interference (RNAi) caused by exogenous double-stranded RNA(dsRNA) has developed into a powerful technique in functional genomics, and to date it is widely used to down-regulate crucial physiology-related genes to control pest insects.A molt-regulating transcription factor gene, HaHR3, of cotton bollworm (Helicoverpa armigerd) was selected as the target gene in our research. Four different fragments covering the coding sequence (CDS) of HaHR3were cloned into vector L4440to express dsRNAs in Escherichia coli. The most effective silencing fragment was then cloned into a plant over-expression vector to express a hairpin RNA (hpRNA) in transgenic tobacco (Nicotiana tabacum). When H. armigera larvae were fed the E. coli or transgenic plants, the HaHR3mRNA and protein levels dramatically decreased, resulting developmental deformity and larval lethality. To investigate the existence of systemic RNAi mechanism in H. armigera, degenerate primers were designed to amplify the SID gene, and two sid-1-like genes (Ha-sil-1; Ha-sil-2) were identified in H. armigera for the first time, suggesting the existence of systemic RNAi in H. armigera. The results demonstrate that both recombinant bacteria and transgenic plants could induce HaHR3silence to disrupt H, armigera development, transgenic plant-mediated RNAi is emerging as a powerful approach for controlling insect pests.The main research results are as summarized as follows:1. dsRNAs expression in E. coli(HT115)The HaHR3mRNA secondary structure was predicted by the Sfold web server to design interference fragments. Four different interference fragments were cloned into the L4440vector. After we transformed the constructed vector L4440-HaHR3into HT115competent cells, gel separation showed that dsRNAs were extracted effectively by both methods and that every dsRNA fragment, including dsRNA-EGEP, was expressed successfully in HT115cells. After feeding H. armigera the artificial diet containing dsRNAs for7days, the bioassay results showed that the four different dsRNA-HaHR3fragments increased H, armigera mortality. The mortality plateaued on day6-7. dsFrag.l, which caused the highest mortality, showed a significant difference (P<0.01) with dsEGEP starting on day5. qRT-PCR result indicated that HaHR3was silenced by dsRNAs ingestion48h later, the relative expression level were10%control group.2. Generation of transgenic plants expressing hpRNA-HaHR3Because the dsRNA-HaHR3-fragment1expressed by bacteria caused the highest mortality, only fragment1was selected for expression in transgenic plants. After transforming pCAMBIA-RNAi-HaHR3to tobacco calli and one month of tissue culture,106regenerated plants were obtained. The cotton hypocotyls (Coker201) were transformed by Agrobacterium tumefaciens, and21regenerated cotton seedlings were obtained successfully. Cotton cultivar F20, F25and F33were transformed with pollen-tube pathway method at the same time, several transformed cotton boll and～80,000seeds were obtained successfully.3. Molecular analysis of transgenic tobacco plantsPCR amplification analysis of the sense fragment and antisense fragment showed that58of those regenerated plants were positive. The PCR results in Ti-generation plants (No.10) showed that the ratio of positive plants was in accordance with Mendelian law. Southern blots indicated that exogenous gene fragments were integrated into the genomes of the tobacco plants at different locations. Several single-copy lines were identified at the same time.4. HaHR3gene silencing effect of H. armigeraafter feeding with transgenic tobaccoThe average net weight of H, armigera was suppressed after feeding with hpRNA-HaHR3-expressing tobacco leaves. After5days of feeding, the three treatment groups were similar to each other in mortality (25.00%-30.00%) but significantly different (P<0.05) from the two control groups (5.00%). In addition to higher mortality, developmental deformity at metamorphosis was observed in the hpRNA-HaHR3treatment groups, the pupation and emergence deformity rate were significantly different (P<0.05) from control groups.After feeding H, armigera with transgenic tobacco leaf plates for several days, quantitative RT-PCR showed HaHR3mRNA the HaHR3gene was silenced successfully, especially after48h. After feeding with hp-HaHR3for60h, the relative expression level of HaHR3under both treatments decreased by～85%. When the recombinant vector pET-28a-HaHR3, pET-30a-HaHR3, pET-32a-HaHR3or pGEX-6p-1-HaHR3was transformed into Rosetta(DE3) competent cells, SDS-PAGE analysis showed that the HaHR3protein was expressed successfully in the first three groups. Recombinant protein was purified for antibody preparation. Western blot analysis revealed that expression of HaHR3protein was suppressed in the high-expression period of this protein after feeding with transgenic tobacco plants expressing hpRNA-HaHR3. Not only in the total protein from the whole larval body of H. armigera, but also in the protein of the female adult ovary, HaHR3protein expression was repressed at the same time.5. systemic RNAi in H. armigeraThe partial sequenceof Ha-sil-1and Ha-sil-2genes were identified with degenerated primers, This suggests that systemic RNAi might exist in this insect; The expression patterns of sil-l and sil-2showed that these two genes were present in all life stages of H. armigera, both genes were consistently expressed during all developmental stages.