Transgenic Overexpression Of The DH And Its Receptor DHR1 In The Silkworm,Bombyx Mori

Diapause is considered to be a physiological state of arrest of development at stage of the egg,larva,pupa or adult,which is the result of insect adaptation to adverse environment,and has great significance to the survival and reproduction of insects.The model of lepioloptera insect,the silkworm Bombyx mori,is a typical representative of egg diapause,but its molecular mechanism of diapause still remains largely unknown.Previous studies showed that the diapause hormone(DH)secreted by the suboesophageal ganglion of the B.mori is the key inducer of egg diapause.DH transmits signals via diapause hormone receptor(DHR)and determines the diapause of eggs.Strengthening the function and mechanism of DH and DHR will not only help us to better understand the mechanism of egg diapause in B.mori,but also provides theoretical references for silkworm breeding and control of egg diapause pests.The molecular biology research on the diapause of silkworm is mainly focused on the identification and expression of diapause-related genes and physiological and biochemical analysis.There is few study and report based on transgenic technologies.This study aims to overpress DH and DHR1 gene in multivoltine silkworm strain,Nistari(Nis),using transgenic technology and GAL4/UAS dual expression tool,to study their impacts on egg diapause of multivoltine silkworm.The main results are as follows:1.Identification and cloning of DH and DHR1 genesWe identified and cloned the coding sequences(CDS)of the DH and DHR1 gene from the B.mori.Sequence analysis showed that the CDS of DH is 579 bp in length,encodes a protein of 192 amino acids containing one transmembrane domain.The CDSof DHR1 is 1311 bp in length,encodes a protein of 437 amino acids containing seven transmembrane domains.2.Generation of original transgenic lines harboring DH and DHR1Three piggyBac-based expression vectors,pB[A4-GAL4,3×P3DsRed],pB[UASDH,3×P3EGFP] and pB[UASDHR1,3×P3EGFP],were constructed to overexpress DH and DHR1 gene in B.mori.After microinjection of three vectors into the early embryos of Nis,corresponding G1 positive bloods were obtained by fluorescence screening,designated as A4G4,UASDH,and UASDHR1,respectively.The transgenic lines were further confirmed by inverse PCR and genomic PCR.3.Generation of transgenic lines overexpressing DH and DHR1The A4G4 individuals were crossed with UASDH and UASDHR1,respectively.The positive offsprings were obtained by double fluorescent screening,and designated as A4G4/UASDH and A4G4/UASDHR1,respectively.qRT-PCR analysis showed that expression of DH and DHR1 was significantly increased in individuals of A4G4/UASDH and A4G4/UASDHR1,demonstraing the success of generation of transgenic lines overespressing DH and DHR1.4.Phenotype and expression analysis of diapause-related genes in transgenic silkworms overexpresing DHThe m RNA levels of DH in individuals of A4G4/UASDH was analyzed by qRT-PCR.The results showed that DH exhibited peaks of expression at the day-6 and day-9 pupal stage,and reached the highest level at at the stage of day-6 pupa,suggesting that it is a key developmental stage for the determination of egg diapause by DH gene.Furthermore,expression of eight diapause-related genes downstream of the DH gene was detected by qRT-PCR.The results showed that overexpression of DH gene affected at various degrees on the expression of dipause-related genes.Comparatively,overexpression of DH gene had significant effect on the expression of DHR1,TRE1,TRE2,OXR1,SDH1 and SDH2 ab genes,and has little effect on the expression of EA4 and TRPA1.Further investigation showed that 7 of 55 selfcrossed progenies of A4G4/UASDH were diapaused.These results indicate that overespressing DH gene in multivoltine silkworm strain can influnce the expression of diapause-related genes,but it is not enough to lead all selfcrossed parents of A4G4/UASDH lay diapaused eggs.5.Phenotype and expression analysis of diapause-related genes in transgenic silkworms overexpresing DHR1The mRNA levels of DHR1 in individuals of A4G4/UASDHR1 was analyzed by qRT-PCR.The results showed that DHR1 exhibited peaks of expression at two pupal stages,and reached the highest level at the stage of day-6 pupa,suggesting that DHR1 participates in determining the egg diapause at this key developmental stage.Furthermore,expression of seven diapause-related genes downstream of the DHR1 was detected by qRT-PCR The results showed that overexpressjon of DHR1 had different degrees of effects on the expression of dipause-related genes.Comparatively,overexpression of DHR1 had significant effect on the expression of TRE1 and TRE2 genes,while has little effect on the expression of OXR1,SDH1,SDH2 ab,EA4 and TRPA1.Further investigation showed that there were no diapaused eggs in 57 selfcrossed progenies of A4G4/UASDHR1.These results indicate that overespressing DHR1 in multivoltine silkworm strain can influnce the expression of diapause-related genes,but it can not make the selfcrossed parents of A4G4/UASDHR1 lay diapaused eggs.