| The Chinese mitten crab(Eriocheir sinensis)is an important freshwater cultured species in China,and the gonadal development has always been a difficult problem in crab breeding,which seriously affects the improvement of river crab breeding benefits and the healthy development of the river crab industry.Therefore,how to use endocrine control methods to fundamentally control the abnormal development of gonads of river crabs has become one of the major problems that need to be solved urgently in the process of breeding river crabs.The methyl farnesoate MF(Methyl farnesoate)is an important sesquiterpenoid hormones,which can stimulate crustacean ovaries maturation and gonadal development.Met(Methoprene-tolerant)and Kr-h1(Krüppel-homolog 1)as important transcription factors participate in the MF-mediated signal transduction pathway to regulate crustacean ovarian development.There,our study aims to explore the role of Es-Met and Es-Kr-h1 genes in MF-mediated ovarian development in Eriocheir sinensis.In this study,the full-length c DNA of Es-Met and Es-Kr-h1 was cloned using RACE technology from E.sinensis.GC/MS method was used to hemolymph MF titer.The technology of tissue culture in vitro,MF injection in vivo,and ESA surgery were comprehensively utilized to explore the effect of MF on Met(Methoprene-tolerant),Kr-h1(Krüppel-homolog 1),and Vg(Vitellogenin)gene expression in the female E.sinensis.The main results are as follows:1.The deduced amino acid sequence of Met in E.sinensis contains three domains,b HLH,PAS-A and PAS-B,which indicates that Met belongs to b HLH-PAS transcription factor family.Kr-h1 contains 7 conserved zinc finger motifs(Zn2-Zn8),which indicate that it belong to the Krüppel-like factors(KLFs)transcription factor family.In addition,q PCR results showed that Met and Kr-h1 was highest expressed in the hepatopancreas compared with other tissues.2.Tissues expression profile showed that the Es-Met and Es-Kr-h1 genes are both highest expressed in hepatopancreas.QPCR and GC/MS results showed that the expression pattern of Es-Met,Es-Kr-h1 and Es-Vg in the hepatopancreas was strongly positively correlated with the hemolymph MF titer during ovarian development.Further more,the trends of Es-Met and Es-Kr-h1 expression are completely consistent.However,compared with the hepatopancreas,Es-Met,Es-Kr-h1 and Es-Vg were low expressed in ovary,and there is no significant change at each period,in addition,there is no obvious correlation with hemolymph MF titer.3.QPCR results shows that the expression of Es-Met,Es-Kr-h1 and Es-Vg in hepatopancreas can be simultaneously and significantly increased with the addition of higher concentrations of MF in vitro,MF injection in vivo,but there was no obvious change in ovarian tissue.After ablation of the eyestalk,the expression of Es-Met,Es-Kr-h1,and Es-Vg in hepatopancreas were also significantly up-regulated after ESA on Days of 3 d,5 d,7 d,and no significantly changes was found in Es-Met and Es-Kr-h1 in the ovary.In summary,our results indicate that Es-Met and the downstream transcription factor Es-Kr-h1 may be involved in the MF-mediated process of exogenous vitellogenesis and ovarian development in E.sinensis.Our study proposed that Met and Kr-h1 in crustaceans may pass JH/MF-Met-Kr-h1 signaling pathway realizes the regulation of yolk generation and ovarian development in E.sinensis,and provides a theoretical basis for solving the problem of precocious puberty in mitten crab breeding through endocrine control measures. |
