Research On The Molecular Mechanisms Of Summer Diapause In The Galeruca Daurica Adults By RNA-seq

Galeruca daurica?Joannis?is a new pest which has had a serious impact on the Inner Mongolian grasslands in recent years.It enters summer diapause as adults,and overwinters as eggs.Diapause plays an important role in enhancing the survival of insects,but little is known about the regulation mechanisms of summer diapause in G.daurica.In this study,the transcriptomes in pre-diapause,diapause,and post-diapause of G.daurica were assembled by RNA-Seq,the diapause-associated genes were screened,and the characterization and expression analysis of three genes were conducted,meanwhile.The main physiological and biochemical indexes were determined in the G.daurica adults at different diapause stages,hoping to clarify the molecular mechanisms of summer diapause in the G.daleruca adults,and to provide a necessary foundation for further investigation on the disaster mechanism and comprehensive management of G.daleruca.The main results are as follows:1.The characteristics of the major physiological and biochemical indexes were investigated in the G.daurica adults at different diapause stages.The contents of water and total proteins were significantly higher in pre-diapause and post-diapause stage than those in diapause,indicating that metabolism in diapause was low.The change trends of the lipid and glycogen contents was the opposite,indicating that lipid and glycogen are main energy materials in diapause of G.daurica adults.The trehalose content fluctuated greatly,which was closely related to the energy demand and transformation in the G.daurica adults at different diapause stages.2.A total of 82,292 unigenes were assembled using the Illumina HiSeq2500platform.Through pairwise comparisons,3,099 differentially expressed genes?DEGs?were found between pre-diapause and diapause.Further analysis revealed that the enrichment of GO was related to metabolic process,carbohydrate metabolic process and lipid metabolic process and so on.By comparing diapause with post-diapause,81 DEGs were found,enriched in lipid biosynthetic,regulation of lipid metabolic and cellular lipid metabolic and so on.Eleven DEGs were selected by qRT-PCR in order to validate the RNA-Seq data,.The results indicated that the change trends of the 11 genes were consistent with the qRT-PCR and RNA-Seq data.3.Trehalase?Tre?,a key enzyme in trehalose metabolism of insects,plays an important role in the energy regulation and development of insects.A soluble trehalase gene,named GdTre1,was cloned from G.daleruca by using the rapid amplification of cDNA ends?RACE?-PCR.The full-length of c DNA is 1,933 bp containing a 1,704 bp open reading frame?ORF?that encodes 567 amino acids with the predicted molecular weight of 66.56 kD and pI of 6.62.The coded protein contains typical functional domains in trehalase superfamily with a signal peptide without transmembrane domain.Homology and phylogenetic analysis showed that GdTre1 has the highest amino acid sequence identity?70.25%?with Leptinotarsa decemlineata Tre1b.qRT-PCR results revealed that GdTre1 was expressed throughout various developmental stages of G.daleruca with higher expression levels in the egg and diapause adult,and lower expression levels in the larva,pre-pupa,pupa and pre-diapause adult.Tissue expression profiles revealed that the expression level of GdTre1 was the highest in the abdomen,and then in the thorax and head of adult.The expression level of GdTre1 increased as the stress temperature increased,with the highest level at 30?,and then decreased a little as the temperature increased.The expression levels of Gd Tre1 and tre activity were significantly different among different day-old adults and showed the same change trends.These results suggested that trehalase has a close relationship with the development and summer diapause in G.daleruca.The results provide a necessary foundation for further investigation on the molecular mechanisms of summer diapause in insects.4.Heat shock proteins?Hsps?are a kind of protein induced by the adverse environmental conditions.Hsps are considered to be most closely related genes in diapause.A heat shock protein Hsp10,named GdHsp10a,was cloned from G.daleruca by using RACE-PCR.The full-length of cDNA is 526 bp containing a 333 bp ORF that encodes 110amino acids with the predicted molecular weight of 11.97 kDa and pI of 9.74.GdHsp10a was found without signal peptide and transmembrane domain.Homology and phylogenetic analysis showed that the amino acid sequence of GdHsp10a has the highest identity?53.15%?with that of Anoplophora glabripennis Hsp10.qRT-PCR revealed that GdHsp10a was expressed at all developmental stages of G.daleruca,and the expression levels in the egg and adult were significantly higher than those in the larva,pre-pupa and pupa.The expression levels reached the highest on the 25^th day,followed by the 100^th,10^thh and 7^th day.Temperature had a significant effect on the expression of GdHsp10a,with the maximum at 30?,followed by 35?,whereas the minimum at 15,20,25 and 40?without significant differences.These results suggested that GdHsp10a may play multiple roles in the development of G.daleruca and its adult summer diapause,and this study provides a necessary foundation for further investigation on the functions of GdHsp10a.5.Juvenile hormone?JH?plays an important role in diapause regulation.Juvenile hormone binding protein?JHBP?is beneficial for protection and transportion of JH.A JHBP,named GdJHBP,was cloned from G.daleruca by using RACE-PCR.The full-length of cDNA is 826 bp containing a 714 bp ORF that encodes 237 amino acids with the predicted molecular weight of 26.58 kDa and pI of 4.37.The coded protein contains typical functional domains in JHBP superfamily,and it contains a signal peptide without transmembrane domain.Homology and phylogenetic analysis showed that the amino acid sequence of GdJHBP has the highest identity?30%?with that of Leptinotarsa decemlineata JHBP 3p2.qRT-PCR revealed that GdJHBP has higher expression levels in the larva and adult,and lower expression levels in the egg and pupa.Tissue expression profiles revealed that the expression level of GdJHBP was the highest in the abdomen and thorax and then in the head of adult.The GdJHBP was obviously influenced by temperature,with the highest level at 35?,and then decreased a little as temperature increased.These results suggested that JHBP has a close relationship with the development and summer diapause in G.daleruca,providing a necessary foundation for further investigation on the molecular mechanisms of summer diapause in insects.