红耳龟性别分化相关基因的筛选与分析

Screen and analysis of genes related to sexual differentiation in the red-eared slider turtle, Trachemys scripta

温度依赖型性别决定(temperature-dependent sex determination, TSD)是一种奇特的表型可塑性现象,但其背后的分子机制尚不清楚。红耳龟(Trachemysscripta)是一种典型TSD动物,个体性别完全由胚胎发育时所处的环境温度决定。为提供更多能够解释TSD机制的基础数据,选取红耳龟第16期产雄温度(male-producing temperature, MPT)和产雌温度(female-producing temperature, FPT)性腺,采用IlluminaHiseq 2500高通量平台进行转录组测序,筛选出雌雄差异表达基因474个,其中,MPT高表达基因283个,FPT高表达基因191个。通过与NR、Swiss-Prot、GO、COG、KOG、KEGG数据库进行比对,获得60个GO功能注释分类和139条KEGG代谢通路。根据差异表达基因的功能注释分析发现:参与红耳龟性别决定和分化的候选基因,包括Dmrt1、Sox9、Foxl2等高度保守的性别分化基因,Cyp11b1、Hsd11b2等类固醇激素代谢相关基因,以及Hspb6、Dnaja4、Serpinh1等热敏性蛋白编码基因。最后,通过荧光定量PCR和免疫荧光染色对部分候选基因进行表达验证,与转录组结果相一致。该研究挖掘了一些新的温度响应因子和激素代谢相关因子,为破译红耳龟TSD分子机制提供了基础。

Temperature-dependent sex determination is a notable model of phenotypic plasticity;however, its underlying molecular mechanism remains unclear.As a typical TSD species, the red-eared slider turtle(Trachemys scripta) has its offspring gender completely determined by incubation temperature. In order to provide more basic data for illustrating the TSD mechanism, transcriptome sequencing was performed on T. scritpa early embryonic gonads of stage 16 at different temperature(male-producing temperature(MPT) and female-producing temperature(FPT)) in this study. IlluminaHiseq 2500 high-throughput platform was used for transcriptome sequencing. A total of 474 differentially expressed genes were screened out, among which 283 genes showed MPT higher expression and 191 genes showed FPT higher expression. By comparing with NR, Swiss-Prot, GO, COG, KOG and KEGG databases, 60 GO functional annotation classifications and 139 KEGG metabolic pathways were obtained. According to functional annotation of differentially expressed genes, these candidates included: sex determination and differentiation related conserved genes, such as Dmrt1, Sox9, Foxl2, etc;steroid hormone metabolism related genes, such as Cyp11 b1, Hsd11 b2, etc;heat-sensitive protein coding genes, such as Hspb6, Dnaja4, Serpinh1, etc. Several selected candidates were verified by quantitative PCR and immunofluorescence. In conclusion, some new temperature sensitive and hormone metabolism related factors were found out from RNA-seq data of turtle embryonic gonads, providing basic support for illustration of the molecular mechanism of TSD.