Identification Of Membrane-Bound Trehalase In Diapause Eggs Of Silkworm And Expression Pattern In Early Embryonic Development

Diapause is a survival strategy for insect,which can enable them to survive in unfavorable environments,such as cold winters,hot summers,and dry seasons.Insect diapause is a physiological process,and its main feature is the temporary stagnation of development in the physiology.In recent years,insect diapause has been a hot topic for researchers,mainly from the perspectives of metabolic physiology,molecular mechanisms,and genetic factors.Trehalase is an important enzyme in the diapause process of insects.It mainly catalyzes the decomposition of trehalose.As the blood sugar of insects,trehalose plays an important role in the diapause process.Silkworm is an important economic insect.The previous researcher performed transcriptome sequencing on the diapause and non-diapause eggs of the silkworm at 24 h and 36 h after oviposition.The analysis found that the membrane-bound trehalase（BmTreh-2）gene has a greater effect on the diapause.Therefore,in this study,the bivoltine silkworm Dazao was used as the experimental material,and we carried out on the 0-9days postpartum diapause silkworm eggs and non-diapause silkworm eggs by measuring the physiological index determination,q PCR detection,western blot,immunofluorescence localization,silkworm egg interference,etc.In this way,the expression pattern of BmTreh-2 gene in the early embryonic development of silkworm diapause eggs was explored.The main research results obtained are as follows:（1）BmTreh-2 gene bioinformatics analysis and T-cloning of partial gene sequenceAccording to the existing transcriptome sequencing data and NCBI database,the CDS sequence of the BmTreh-2 gene was obtained.The main bioinformatics prediction result conclude that the ORF of BmTreh-2 gene is 1929 bp,which encodes 642 amino acids,and the protein molecular weight is 73 k Da,the isoelectric point p I is 5.65,and the instability index is 41.18.It is speculated that it may be an unstable protein.It is predicted that the N-terminal of the protein has a signal peptide of 18 amino acids,containing a transmembrane domain,a hydrophilic protein,and the 45^th-555^thamino acid fragment is the functional domain of trehalase,so the protein belongs to the trehalose family.Using the c DNA of Dazao diapause eggs at 3th day as a cloning template,a T-clone recombinant vector containing part of the target fragment was successfully obtained.（2）BmTreh-2 gene Prokaryotic expression,protein purification and polyclonal antibody preparation in silkworm,Bombyx moriA non-signal peptide part of the CDS sequence of the silkworm BmTreh-2 gene was selected,and two restriction sites of Bam HI and Xhol were added to the upstream and downstream primers.The T-clone recombinant plasmid was used as a template for PCR amplification.The p ET-28a-BmTreh-2 prokaryotic expression plasmid at the restriction site was transferred to BL21（DE3）E.coli competent cells for expansion,and IPTG was used to induce protein expression,then successfully expressing a protein with a molecular weight of 35 k Da.After the protein was purified and concentrated by Ni-column affinity chromatography,polyclonal antibodies were prepared and was detected by Elisa that the titers of immune antibodies were all over1:50K.After the result of western blot with recombinant antigens showed it’s qualified.（3）Analysis of the expression pattern of silkworm BmTreh-2 gene in the early stage of egg diapause embryo developmentBy measuring the trehalase activity,it was found that the trehalase activity was higher within 3 days after oviposition of diapause eggs,and then the enzyme activity dropped sharply to a lower level.The non-diapause eggs had a vigorous trehalase activity after 6 days of greening.The content of trehalose and glycogen gradually decreased after the laying of diapause and non-diapause eggs.RT-q PCR and q PCR detected the expression level of the trehalase gene at 0-9 d post-laying silkworm eggs.The results showed that the overall expression trend of the trehalase gene in the diapause group was high in the early stage,and entered the trehalase gene with the diapause.The expression is down-regulated,and the expression level is very low.With the deepening of diapause,its expression level tends to be stable;the expression level of trehalase gene is lower after non-diapause eggs are laid.As the embryo develops,after the 7th day of greening,The expression of the trehalase gene is extremely high and continues until the silkworm eggs hatch.Western Blot results showed that there was a peak expression of BmTreh-2 at the 3rd day of diapause eggs,and then decreased,and almost no expression at 9 d.The non-diapause eggs showed a gradual upward-regulation trend after spawning and continued until the ants emerged.Paraffin sections and HE staining showed that the embryos of diapause eggs basically formed around 2 days,and there was basically no change in their morphology,and there were always abundant yolk particles in the diapause eggs;the embryos of non-diapause eggs continued to develop after laying,and silkworms could be silkworms in 5 days.The body segmentation is clearly visible,and the head-thoracic differentiation is obvious.With the continuous growth of the embryo,a large amount of nutrients and metabolites are needed,so the yolk particles are gradually absorbed,so the yolk particles basically disappear after 7 days of greening.The results of immunofluorescence localization showed that BmTreh-2 protein was widely distributed in embryos and yolks of diapause eggs,and the fluorescence signal was the strongest 3 d postpartum;BmTreh-2protein of non-diapause eggs was mainly concentrated in embryos.（4）Study on the expression of genes in the main pathways of sugar metabolism in the early embryonic development of silkworm diapause eggsThe content of trehalose,glucose,glycogen,and sorbitol in the early embryonic development of diapause eggs was measured,and it was found that the contents of trehalose and glycogen in diapause eggs were gradually decreased,and the content of sorbitol in diapause eggs and non-diapause eggs The eggs gradually increased after laying,and the glucose content reached the highest value 2 days after the diapause eggs were laid,then dropped sharply afterwards,and increased sharply on the 7 days after the non-diapause eggs were laid.Using q PCR technology to detect the m RNA levels of genes in the main pathways of sugar metabolism,it was found that the enzyme genes involved in respiratory metabolism in non-diapause eggs were significantly up-regulated within 2 days after birth,indicating that violent physiological metabolism occurred in the eggs during this period;The expression levels of enzyme genes related to glycogenolysis in eggs were higher within 2 days after delivery,and the expression of aldose reductase,which catalyzes the conversion of trehalose to sorbitol,gradually increased after 2 days.The RNAi technology was used to inject ds BmTreh-2 into the newly laid diapause eggs.After 48 h,the expression of BmTreh-2 was found to be significantly down-regulated.After 72 h,it was found that BmTreh-2 returned to normal levels and related genes on the BmTreh-2 pathway.Significant changes have also taken place.In summary,this project mainly analyzes the expression pattern of the silkworm BmTreh-2 gene in the early postpartum embryonic development of the silkworm diapause eggs（non-diapause eggs as a control group）from various aspects such as physiological indicators,transcription levels,and protein levels.At the same time,RNAi technology was used to intervene the newly laid diapause eggs.The changes of BmTreh-2 gene were detected at two important time nodes of 2 d and 3 d,and some of the sugar metabolism pathways where BmTreh-2 was located were analyzed.The expression changes of main genes have deeply explored the function of silkworm BmTreh-2 gene in the early stage of embryonic development of diapause eggs and the expression pattern of related carbohydrate metabolism pathways.