Study On The Mechanism Of Silkworm Strain MF Cannot Lay Diapause Eggs With Injecting DH

Sericulture is an important part of agriculture in China.It plays an important role to increase farmers’ income in western China. And the Comprehensive utilization of Sericulture have made a great development in recent years.As the key role of Sericulture- the silkworm, its growth and development process embodied resistance, dormancy and other habits are closely related to the quality cocoon production, manufacturing and quality silkworm silkworm protection.It is important to make reasonable development、utilization and conservation about our germplasm.when Our laboratory explored the winter preservation methods about the multivoltine silkworm,we found most multivoltine strains of non-diapause can lay diapause eggs by injecting diapause hormone and then solved the difficult problem of winter saved,But one silkworm strain MF cannot lay diapause eggs with injecting DH,whicn is a very interesting and unique phenomenon.In this study,this particular phenomenon as a starting point, based on the diapause hormone signaling pathways regulating diapause, the diapause hormone gene and the diapause hormone receptor gene were cloning and then analysising these genes structure, then Study on the Expression Pattern of diapause hormone gene and diapause hormone receptor gene in in different tissues from the 5th instar silkworm larvae of different varieties and different sexes by semi-quantitative RT-PCR and qRT-PCR.in addition,the candidate genes were using RNA interference technology to explore their functions.The research could improve the gene regulation system about diapause hormone gene in silkworm and clarify themechanism about the pest diapausee,.in addition it can provid important reference on pest control in agricultural production.1、 Molcecular cloning and expression pattern of diapause hormone gene(DH) from the multivoltine silkworm MF 、the multivoltine and bivoltine strainsIn order to elucidate the reason for silkworm strain MF cannot lay diapause eggs with injecting DH,The diapause hormone gene(Bm-MF-DH) was cloned from the silkworm strain MF using reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(RACE).Full-length cDNA of the gene was 781 bp long, consisting of 44 bp 5’untranslated region, 173 bp 3’untranslated region and 564 bp open reading frame(ORF) which encodes for 183 amino acid. Structural analysis showed that this gene was not significantly different from the silkworm strains DAZAO.In this study,the expression level of bmDH gene was determined in different tissues(head, fat body, skin, silk gland, midgut and hemolymph)from the 5th instar silkworm larvae of different varieties and different sexes by semi-quantitative RT-PCR and qRT-PCR. The resultes showed that the gene was expressed in three tissues but not expressed in five tissues from the 5th instar silkworm MF. In addition,the expression level of BmDH gene was different both different silkworm strains and two multivoltine non-hibernating strains,the bivoltine silkworm C108 was highest,MF followed and Nistali was lowest.2、Molcecular cloning and expression pattern of diapause hormone gene(DHR) from the multivoltine silkworm MF 、the multivoltine and bivoltine strainsThe diapause hormone receptor gene DHR was cloned from the silkworm strain MF using reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(RACE).Sequence analysis showed that the DHR gene is 2204 bp long and contains a 371 bp 5’-UTR(untranslated region) and a 370 bp 3’-UTR.It’s open reading frame(ORF) is of 1309 bp, encoding 436 amino acids.Structural analysis showed that this gene was significantly different from other silkworm strains.In this study,the expression level of DHR gene was determined in different tissues(blood head, fat body, silk gland, midgut and hemolymph)from the 5th instar silkworm larvae of different varieties by semi-quantitative RT-PCR. The resultes expression level of DHR gene was different both different silkworm strains and two multivoltine non-hibernating strains, showed that the gene was highest expressed in ovarian and blood tissues from the 5th instar silkworm MF. In addition,the there were significantly different from the expression trends of DHR gene and the expression level of that in the two multivoltine non-hibernating strains.3、The research of Silkworm’s resistance by using RNAi technology to interference the expression of DHR geneBased on the middle sequences in the ORF of DHR gene, the 276 bp long dsRNA was designed and synthesized in vitro and the female pupal stages of different silkworm strains were micro injected by using different concentrations of dsRNA,then Observed the results that these female pupas lay eggs after moth mating and the results that these eggs hatched offspring. the relative changes of DHR gene between the injection groups and control groups was determined by semi-quantitative RT-PCR. The resultes showed that the expression trends of DHR gene in the injection groups were lower than the control groups, and the silkworm C108 lay diapause eggs by using RNAi technology to interference the expression of DHR gene,These results suggested that the lower expression of DHR gene is associated with the phenomenon about silkworm strain MF cannot lay diapause eggs with injecting DH.