Preliminary Study On Molecular And Breeding Function Of Key Genes(MSTN) Regulating Muscle Growth And Development In Chinese Mitten Crab

Molting and increment are closely tied with muscle development in Chinese mitten crab?Eriocheir sinensis?.In this study,we conducted histological observation of muscle tissues from the four different molting stages?Pre-molt,D;Ecdysis,E;Post-molt,A and Inter-molt,C?,and comparative transcriptome analysis of muscle tissues from the two molting stages?Pre-molt and Post-molt?in Chinese mitten crab,then we screened some candidate genes which positively and negatively controlled on muscle development and one key regulated gene?MSTN?.Furthermore,we studied preliminary molecular function of MSTN gene using RNA interference method,and evaluated breeding function of this gene by analyzing association of its single nucleotide polymorphisms?SNP?and growth traits in multi-populations.The main results were presented as follows:Part1.The discovery of key genes regulating muscle growth and development in Chinese mitten crabIn order to investigate phenotypic change and gene expression difference in muscle tissues before and after molting in Chinese mitten crab,histological observation and water content of muscle tissues in walking leg at the four stages(D_3-4?E?A_2d and C_10d)were conducted,and transcriptome analysis at the pre-molt(D_3-4)and post-molt(A_2d)stages were also carried out.It was found that the largest diameter and cross-sectional area of the muscle fibers were observed in the pre-molt stage,and the smallest in the post-molt stage,ranging as D>E>C>A,with significant difference?P<0.01?among them.However,the density of muscle fiber was completely opposite ranging as D<E<C<A.The water content was the highest?86.37%?in the post-molt(A_6h)and the lowest?74.49%?in the pre-molt(D_3-4)with also significant difference?P<0.01?.There were 1387 genes up-expression in the pre-molt stage with negative regulating muscle development such as protein hydrolysis and myotube cutting.Similarly,there were 877 genes down-expression in the post-molt stage with positive regulating muscle growth such as the proliferation of muscle cells and the fusion of muscle cells.Furthermore,the 4 genes with negative regulation?POMT,NEB2,ZCI2A and MSTN?and 4 genes with positive regulation?mTOR,TITIN,XRCC and ICCR?in muscle development were also found.The expression characteristics of MSTN gene indicated that the highest expression in the post-molt(A_6h)and the lowest in the pre-molt?D₀?.The results of this study made a foundation for understanding structure changes and molecular mechanism of muscle at different molting stages.Part2.Preliminary study on molecular function of MSTN gene regulating muscle growth and development in Chinese mitten crabIn order to further explore molecular function of MSTN gene in regulating molting and growth in Chinese mitten crab,RNA interference?RNAi?method was continually employed in the three molting cycles.The results showed that the dsRNA interference efficiency was high,especially,its efficiency was the highest?99%?after 48 hours of interference.In the first molting cycle of RNA interference,the interference group showed lightly short molt-interval?P>0.05?and significantly low growth rate in body weight?P<0.05?compared with the control group.In the second,third molting cycles,the interference group displayed significantly shorter molt-interval and lower growth rate in body weight?P<0.05?than the control group.The results indicated that molt-interval could be significantly shorten and molting should be significantly fasten when MSTN gene was disturbed,which give a guideline of regulating molting in Chinese mitten crab aquaculture.Part3.Preliminary study on breeding function of MSTN gene regulating muscle growth and development in Chinese mitten crab.In this part,the feasibility and potentiality of MSTN gene as breeding function were tested by analyzing association of gene diversity and growth traits?body weight and carapace length?in Chinese mitten crab.Three SNPs were found in the first exon of MSTN gene,which were named as S1,S2and S3,respectively?S1:C714T;S2:G729A;S3:G753T?.These three SNPs were middle or high polymorphism loci,and they were all in hardy-Weinberg equilibrium?P>0.05?in population structure.A general linear model used to analyze the correlation between the three SNPs and growth traits?body weight and carapace length?indicated that S1 but not S2 and S3 had a significant effect on the growth traits?P<0.05?.The TT genotype was the most beneficial genotype on growth in Chinese mitten crab,which could be used as candidate for molecule marker-assisted breeding and has the potential of molecular breeding.