| Aspongopus chinensis Dallas,1851 is an important resource insect that is not only a traditional Chinese medicine,but also a delicious food,with many function of strong anti-cancer,antibacterial,anti-clotting,health care et al.In recent years,it was paid more and more attention,and its social demand is increasing day by day.Because of its destructive collection and environmental pollution,the quantity of A.chinensis in the wild decreased year by year.Artificial breeding of A.chinensis is an important way to solve the problem of short supply.However,there is a severe problem about overwintering diapause in A.chinensis,which causes it happening only one generation per year.In order to realize its large-scale artificial propagation,it is first necessary to solve the key scientific problem of A.chinensis diapause.Therefore,this study was performed to make clear the changes of the contents of fat,carbohydrate,protein,Juvenile hormone ?(JH?)and 20-hydroxyecdysone(20E)during the diapause and its termination of A.chinensis.At the same time to clarify the effects of exogenous JH? and 20 E on its diapaus,they were injected into A.chinensis of diapause.In addition,in order to reveal the physiological mechanism of A.chinensis diapause,transcriptome sequencing was used to analyze the difference in gene expression during overwintering diapause of A.chinensis.The specific research results are as follows:1.The content changes of fat,carbohydrate and protein in A.chinensis during diapauseTo explore the relationship between the change of nutrient and diapause in A.chinensis,the soxhlet extraction method,anthrone colorimetric method and BCA protein assay kit were used to determine the fat,carbohydrate and protein of A.chinensis in the diapause period(October-April)and diapause termination(May).The results showed that the fat gradually decreased with time,and the lowest content happened in the late diapause(April)(19.51%±0.47%)compared with 40.55%±1.48% in the early diapause(October),with a significant difference(P<0.05).The carbohydrate accumulated from the early stage(October)to November,then decreased till April,and increased again significantly in the diapause termination(May)(P<0.05).The protein was slowly increasing from the early stage of diapause(October),while began to decline in March.And the content of protein was significantly lower at diapause termination than that during diapause(P<0.05).2.Determination of JH? and 20 E during diapause and diapause termination of A.chinensis.To study the contents of JH? and 20 E in diapause and its termination of A.chinensis,high performance liquid chromatography was used to determine their contents.The results showed that JH? in diapause(4.3016±0.0449 ng/?L)was lower significantly than that in diapause termination(9.6873±1.6157 ng/?L).There was no 20 E detected in both diapause and its termination.3.Effects of exogenous JH? and 20 E on diapause A.chinensis.In order to verify the effects of JH? and 20 E on diapause A.chinensis,different concentrations of exogenous JH? and 20 E were injected under the wings respectively.30 days later of exogenous JH? treatment,A.chinensis appeared mating behavior,and the highest mating rate was happened in the 50 ng/?L experimental group(91.60%±0.038%),which was significantly higher(P<0.05)than other groups,whose mating rates were 39.33%±0.78%(100 ng/?L),21.88%±0.09%(200 ng/?L)and 21.70%±0.10%(10 ng/?L)respectively.For exogenous 20 E,only 150 and 200 ng/?L experimental groups appeared mating phenomenon after 37 days of treatment,and the mating rates were 14.44%±0.10% and 16.88%±0.08% respectively,without significant difference(P?0.05).4.Transcriptomics analysis of A.chinensis during diapause and diapause terminationIn order to explore the physiological mechanism,transcriptome sequencing analysis of A.chinensis during diapause and its termination were performed to screen differentially expressed genes,and explore diapause-related pathways.Illumina HiSeq X Ten sequencing platform was used to deal with high-throughput sequencing,and a total of 336,230,260 clean reads were assembled into 80769 unigenes.Futher analysis of these unigenes using the six major database(NR,Swiss-Prot,Pfam,COG,GO and KEGG databases)caculated that a total of 44.78%(36169)of the genes were annotated in these databases.Analysis of the differentially expressed genes of diapause and its termination among the transcriptome data showed that there were 3524 differentially expressed genes,of which 1350 were up-regulated and 2174 were down-regulated.Using KEGG functional annotation,these unigenes annotaed in physiological processes,such as carbohydrate metabolism,amino acid metabolism,fat metabolism,signal transduction and translation.According to KEGG enrichment analysis,the differentially expressed genes were enriched in insect hormone biosynthesis,carbohydrate metabolism,lipid metabolism,insulin signaling pathway and other physiological processes that may be related to diapause.This study provides a basis and reference for further research on the function of diapause-related genes and solving the diapause problem of A.chinensis. |
PDF Full Text Request