克氏原螯虾sex-lethal基因的克隆与表达分析

Cloning and expression analyses of sex-lethal gene in Procambarus clarkii

为探究克氏原螯虾sex-lethal基因(Sxl)的功能与分子机制,采用RACE(rapid amplification of cDNA ends)技术克隆获得4个克氏原螯虾Sxl cDNA序列,利用荧光定量PCR技术检测其在不同组织以及早期发育时期的表达情况。生物信息学分析发现,PcSxlβ和PcSxlδ比PcSxlγ多了一段77 bp的碱基序列,而PcSxlβ和PcSxlδ预测所得的氨基酸序列较PcSxlγ短。克氏原螯虾的Sxl氨基酸序列与红螯螯虾的序列相似性较高(75.76%)。保守结构域分析显示,这4种转录异构体序列预测的氨基酸序列都具有2个相同的高度保守的RRM结构域。表达分析结果显示,PcSxl在成年雄性克氏原螯虾的触角腺和鳃中表达量较高,在成年雌性中则是中肠和前肠的表达量较高,雌性卵巢中的表达量要显著高于雄性精巢。早期发育时期中,无节幼体时期的表达量最高,在蚤状幼体期表达量出现下降,但在出膜后第1天表达量又有所升高,随后整体则呈现出逐渐下降的趋势。以上结果表明克氏原螯虾PcSxl与其性别分化有关。

In order to explore the function and molecular mechanism of sex-lethal gene in Procambarus clarkii,four Sxl cDNA sequences from P.clarkii were obtained using rapid amplification of cDNA ends,and their expression in different tissues and at early developmental stages were detected by quantitative fluorescence PCR.Bioinformatics analyses revealed that PcSxlβand PcSxlδwere 77 bp longer than PcSxlγ,and the predicted amino acid sequence of PcSxlβand PcSxlδwere shorter than that of PcSxlγ.The PcSxl sequences was highly similar to that of Cherax quadricarinatus(75.76%).The analysis of conservative domain showed that all the predicted amino acid sequences of the four transcriptional isomers had two highly conservative RRM domains.The expression analyses showed that PcSxl was highly expressed in the antennal glands and gills of adult males,and in the midgut and foregut of adult females,and was significantly higher in the female ovaries than the male testis.During the early developmental period of the crayfish,the expression of the genes was the highest at the nauplius stage,but decreased at the zoea stage;however,the expression level increased at the first day after hatching,and then showed a gradually decreasing trend.The results of this study suggested that PcSxl was involved in sexual differentiation in P.clarkii.