摘要
为了研究蜕皮激素受体（EcR）在中华绒螯蟹蜕皮和性腺发育过程中的作用,本实验采用逆转录聚合酶链反应（RT-PCR）以及cDNA末端快速扩增（RACE）技术,克隆了中华绒螯蟹EcR基因全长cDNA序列。运用实时荧光定量PCR（qRT-PCR）方法,分析该基因在不同组织、蜕皮过程及卵巢发育阶段的表达特征,并研究了甲基法尼酯（MF）对卵巢中EcR的调控作用。结果显示,中华绒螯蟹EcR cDNA全长2 156 bp,编码422个氨基酸,其序列与招潮蟹相似性最高,为89%。荧光定量PCR结果显示,EcR在Y器官中表达量最高;在卵巢和肌肉中少量表达;在肝胰腺、鳃、心脏、胃、肠、胸神经节和脑神经节中微量表达。在中华绒螯蟹不同蜕皮时期中,Y器官中EcR表达量在D期和E期的表达量最高;肝胰腺中EcR表达量从AB期到D期有上升的趋势,到E期有所降低;肌肉中EcR表达量在AB期最高;鳃中EcR表达量在D期最高。在中华绒螯蟹卵巢发育过程中,EcR在卵巢发育Ⅰ-Ⅳ期的表达量有上升趋势,到Ⅴ期表达量降低;体外注射实验中,MF1（注射1μg）组的EcR表达量与对照组和生理盐水组无显著差异,而MF2（注射2μg）组的EcR表达量显著上升;离体培养实验中,MF1组（10^-8mol/L）和MF2组（10^-7mol/L）显著高于对照组和生理盐水组。研究表明,EcR基因在中华绒螯蟹蜕皮和卵巢发育过程有重要作用,MF可以调控EcR基因在卵巢中的表达。
To study the regulatory role of ecdysteroid receptor（ EcR） in molting and ovarian development of Chinese mitten crab,the full-length EcR gene of Eriocheir sinensis was cloned by using reverse transcriptase polymerase chain reaction（ RT-PCR） and rapid-amplification of cDNA ends（ RACE）. The full-length cDNA sequence of EcR was 2 156 bp,and included a 1269bp ORF which encoded 422 amino acid residues. The alignment of EcR amino acid sequence of Eriocheir sinensis shared 89% identity with Uca pugilator. Quantitative real-time PCR（ qRT-PCR） was used to quantify the relative expression level of EcR in different tissues,molting process,ovarian process and regulation of MF on ovarian EcR in E. sinensis The result showed the EcR mRNA were expressed in all tissues examined and highly in Y-organ,with small amount in ovary and muscle,and trace in hepatopancreas,gill,heart,stomach,intestine,thoracic ganglion and cerebral ganglion. During the molting process,the expression levels of EcR mRNA of Y-organ remained lowfrom AB period to C period,then significantly increased in D period and E period.（ P〈0. 05）; the amount of EcR expression in hepatopancreas was rising from AB period to D period,and decreased in E period; EcR expression in muscle was highest in AB period（ P〈0. 05）; EcR expression in gill was highest in D period（ P〈0. 05）. In the process of Eriocheir sinensis ovarian development,the levels of EcR expression in ovary gradually increased to the maximum from stage Ⅰ to Ⅳ,lowin stage Ⅴ. In injection experiment,EcR expression of the MF1（ 1 μg / crab） group had no significant difference with the control group and physiological saline group,MF2（ 2 μg / crab） group（ P〈0. 05） had a marked increase; In vitro experiment, EcR expression was significantly increased in MF1（ 10-8mol / L） group and MF2（ 10-7mol / L） group（ P〈0. 05）. The results indicated that EcR might play an important role during molting process and ovarian development in Eriocheir sinensis,and MF could regulate EcR gene expression in ovarian. However,further studies are required to explore the underlying molecular mechanisms and clarify the function of EcR mRNA.
出处
《水产学报》
CAS
CSCD
北大核心
2014年第5期651-661,共11页
Journal of Fisheries of China
基金
国家"八六三"高技术研究发展计划(2012AA10A409-5)
国家农业科技成果转化项目(2012GB2C000147)
国家自然科学基金(40806068
41276158)
上海教委知识服务平台项目(ZF1206)
上海市科委非政府合作项目(11320706400
13340721500)
