Activity Analysis Of Enzymes Related To Carbohydrate Metabolism In The Early Embryonic Stage Of Differently Treated Eggs From The Bivoltine Bombyx Mori Strain

The diapause of Bombyx mori has been paid attention by people for a long time,and a lot of research has been done,but the molecular mechanism of diapause is not fully understood.The initiation and release of diapause in Bombyx mori is accompanied with a large number of metabolic activities,including carbohydrate metabolism.Studies have shown that there are significant differences in activities related to carbohydrate metabolism between diapause-destined eggs and non-diapause eggs.The diapause of silkworm eggs laid by the bivoltine Bombyx mori strain is regulated by environmental conditions in the embryonic period of the parent eggs.The progeny eggs are diapause-destined?DD?when the parent eggs incubating under an over 20? temperature with a long period of lighting in the embryonic stage.And the diapause of DD eggs can be broken by hydrochloric acid immersion.When the parent eggs are incubated under a temperature below 15? with a short period of lighting,their offspring are non-diapause eggs?ND?.Then,what is the relationship between enzymes related to carbohydrate metabolism in the embryonic stage of silkworm eggs and diapause? To answer this question,the bioltine Bombyx mori Strain Qiufeng was used as material to explore the relationship between expression level of carbohydrate metabolism related enzymes and diapause in the early embryonic development stage both in the RNA and protein levels.The main results are as follows.1.Bioinformatics analysis of enzymes related to carbohydrate metabolism in silkworm.The protein structure prediction was performed for three enzymes related to the sugar metabolism,hexokinase?HK?,pyruvate kinase?PK?and phosphofructokinase?PFK?by using the Swiss Model software.And their phylogenetic developments were analyzed in B.mori and other more than 10 species by using MEGA6.0 and other bioinformatics software.The results showed that a copy of Bm HK and Bombyx mandarina HK were clustered together,indicating that the system classification is close to homology;Bm PK,Papilio Machaon PK,Papilio Xuthus PK were clustered on a branch,indicating a close relationship;The copy of Bm PFK?XP₀12549268.1?and the Bombyx mandarina PFK are gathered in the same branch,indicating a close relationship.2.Analysis of RNA expression level of enzymes related to carbohydrate metabolism in Bombyx mori.The activated hibernating eggs of the the bivoltine B.mori strain Qiufeng were used as materials,and incubated under 25? in natural brightness or under 17? in darkness to prepare the diapause-destined eggs?DD?and non-diapause-destined eggs?ND?,respectively.Then a part of DD eggs were treated with hydrochloric acid at 20 h after oviposition?set as the starting point?to obtain the immediately acid treated eggs?IA?.Samples were obtained at 0,2,6,12,24,48,72 and 96 h separately and stored at-80? for use.The q RT-PCR technology was used to detect the expression level of 8 enzyme genes related to carbohydrate metabolism including hexokinase?HK?,pyruvate kinase?PK?,phosphofructokinase?PFK?,triose phosphate isomerase?TPI?,trehalase?TRE?,sorbitol dehydrogenase?SDH?,glucose dehydrogenase?GCDH?and phosphoenolpyruvate carboxykinase?PEPCK?in DD,ND and IA three kinds of silkworm eggs.It is shown that within 96 h of the measurement,the expression levels were generally higher in the ND and IA group than that in the DD group,including glycolysis-related enzyme genes Bm HK,Bm PK,Bm PFK,Bm TPI,pentose phosphate pathway-related enzyme gene Bm GCDH and other metabolic pathway-related enzyme genes Bm SDH,Bm TRE.While the expression level of gluconeogenesis-related enzyme gene Bm PEPCK in DD group was generally higher than that in ND and IA group.3.Activity analysis of enzymes related to carbohydrate metabolism in B.moriThe enzymatic activity of the eight enzymes,HK,PK,PFK,TPI,TRE,SDH,GCDH,and PEPCK related to carbohydrate metabolism in three kinds of B.mori eggs were detected by ultraviolet-visible spectrophotometry.The results show that within 96 h of the measurement,the enzyme activities are higher in the ND and IA groups compared to the DD group including glycolysis-related enzymes Bm HK,Bm PK,Bm PFK,Bm TPI,pentose phosphate pathway-related enzyme Bm GCDH,and other metabolic pathway-related enzymes Bm SDH,Bm TRE.The enzymatic activity of the gluconeogenesis-related enzyme Bm PEPCK in the DD group is generally higher than that in the ND group and the IA group,which was in consensus with the results of RNA expression.4.Analysis of Bm HK gene structure and expression profile in the early embryonic stageThe 4 transcripts of Bm HK gene were retrieved from NCBI website and called Bm HK-a,Bm HK-b,Bm HK-c,Bm HK-d.The specific sequences of four transcripts were used to design primers,and their expression levels in three kinds of silkworm eggs were determined by q RT-PCR technology.The results showed that the expression levels of these 4 transcripts presented the characteristic of periodic change;And in the same kind of eggs,the expression level of Bm HK-a was higher than that of the other three transcripts at the same time point within 96 h,indicating that Bm HK-a played an important role in the function of the Bm HK gene.In summary,in early embryonic development stage of the DD group eggs,carbohydrate metabolism is mainly towards energy and material storage for diapause,while in eggs of ND and IA groups carbohydrate metabolism is mainly towards material catabolism due to fast embryonic development.The results preliminary uncover the relationship between carbohydrate metabolism and diapause of B.mori,which is beneficial to better understand the molecular mechanism of diapause of B.mori.