BACKGROUND: Diapause is the arrest of the development of insects and can be used for the development of effective agricultural pest management strategies. Heat shock protein 70 (Hsp70) is reported to be up-regulated during diapause to maintain survival in some insect species. However, its regulatory mechanism is unknown. RESULTS: Expression of hsp70in Helicoverpa armigera was found to be up-regulated in diapause pupal brains. To elucidate the molecular regulatory mechanisms of hsp70, we focused our attention on its transcription factor, heat shock factor 1 (HSF1). Four alternative splicing variants of HSF1 from pupal brains of H. armigera were identified, and subcellular localization analysis indicated that these variants were exclusively expressed in the nucleus. Real-time PCR analysis showed that all of these variants were up-regulated in diapause pupal brains, and their expression patterns were consistent with that of hsp70. Finally, promoter activity assay and Western blotting detection demonstrated that hsp70 was activated and up-regulated by these variants. CONCLUSION: Expression of hsp70 in H. armigera during diapause is regulated by multiple alternatively spliced isoforms of HSF1.The results of this study may provide important information for understanding the regulatory mechanisms of hsps during insect diapause.
Despite the great potential of Serratia marcescens in industrial applications, lack of powerful genetic modification tools limits understanding of the regulatory networks of the useful metabolites and therefore restricts their mass production. To meet the urgent demand, we established a genome-editing strategy for S. marcescens based on Red recombineering in this study. Without host modification in advance, nucA and pigA were substituted by PCRamplified resistance genes. No long homologous arms were required at the two sides of resistance genes. Using this procedure, the fragment at the S. marcescens as large as 20 kb was easily deleted. Then we constructed a counter-selection gene kil constructed under the control of inducible P BAD operon, which demonstrates obvious lethality to S. marcescens. Subsequently, Gm R -kil double selection cassette was inserted into the CDS of pigA gene. Using single-stranded DNA-mediated recombination, this insertion mutation was efficiently repaired through kil counter-selection. A powerful genetic modification platform based on Red recombineering system was successfully established for S. marcescens. Multiple types of modification and multiple recombination strategies can all be performed easily in this species. We hope this study will be useful for the theoretical research and the research of metabolic engineering in S. marcescens.
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