栉孔扇贝（Chlamys farreri）性别分化相关基因的筛选以及两个相关基因的表达分析
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摘要
性别分化是发育生物学研究的重要内容之一,性别分化相关基因的研究在脊椎动物特别是哺乳动物中较为深入,目前已经发现了Sry、Sox9、Wnt4等诸多与性别决定和分化有关的基因;但在无脊椎动物特别是海洋贝类中相关的研究尚处于起步阶段。本研究选取我国重要的水产经济贝类——栉孔扇贝(Chlamys farreri)为研究对象,采用Solexa测序技术对其增殖期精卵巢的差异表达谱进行了分析,获得了295262个在精卵巢中差异表达的Tag,其中1354个Tag可以在GenBank数据库中得以注释。选择其中的5条核酸序列进行了半定量RT-PCR验证,发现solexa测序结果与半定量结果的一致率为40%。
采用RACE技术对其中的CL3256序列进行全长cDNA扩增,序列比对初步判定其为核糖体蛋白24基因,定名为Cf-rlp24。该基因全长cDNA为965 bp,开放阅读框为525 bp,编码175个氨基酸,该推测蛋白在不同物种之间同源性很高。Real-time PCR检测Cf-rlp24基因在精、卵巢之间以及不同发育时期的精巢中均存在表达差异,其中生长期和成熟期精巢Cf-rlp24基因的表达量显著高于相应时期的卵巢,分别高出2.200±0.446倍和6.036±1.283倍;在精巢的发育过程中,该基因的表达呈逐渐升高的态势,至成熟期时达最高值,而后迅速回落;但在卵巢的发育过程中,始终处于较低的表达水平,且未见该基因的显著性表达差异。我们得出,Cf-rlp24基因参与栉孔扇贝性腺的发育,且在精巢发育中的作用大于卵巢。
本研究我们还采用Real-time PCR技术,分析了栉孔扇贝卵黄蛋白原基因(Cf-vtg)在两性性腺发育周期以及肝胰腺中的表达,得出该基因主要在卵巢中表达,且其表达量随着卵巢的发育成熟而逐渐升高,生长期和成熟期Cf-vtg基因的表达量分别为增殖期的1.917±0.035和2.570±0.252倍。休止期卵巢该基因几乎没有表达。进一步我们发现Cf-vtg基因在肝胰腺中也有少量的表达,其表达量为增殖期卵巢中的0.045±0.009。该基因在精巢中几乎没有表达。进一步我们采用注射的方法,检测了17β-E2对扇贝Cf-vtg基因的诱导表达,real-time PCR结果显示,17β-E2可明显诱导Cf-vtg基因的表达。因此推测17β-E2可以促进栉孔扇贝扇贝Cf-VTG的合成。
Sex differentiation is one of the most important research fields in development biology. The study about sex differentiation genes in vertebrates, especially in mammal, is deeper than in other species. Up to now, many genes related to sex determination and differentiation, such as Sry, Sox9, Wnt4, have been identified. But the research in invertebrates especially in marine bivalves is limited. In the present study, we selected the important commercial species-Chlamys farreri as the research object. Based on solexa sequencing, we analyzed the differentially expressed transcripts between femal and male gonads at proliferative stage. We totally obtained 295262 tags which are differentially expressed between sexes, and 1354 of which could be annotated with informations in GenBank. 5 of theses genes were selected and analyzed with RT-PCR. We find the concordance rate of Solexa sequencing results and RT-PCR results is about 40%.
We amplified the full-length cDNA sequence of CL3256 gene using RACE technology. Sequence aligiment indicated it as the ribosomal protein L24 gene (Cf-rlp24). The full-length cDNA of Cf-rlp24 gene is 965 nucleotides which could encodes 175 amino acid residues. Alignment analysis indicated that the RLP24 protein shares a high homology among different species. Real-time PCR analysis showed that the expression of Cf-rlp24 gene is different between testis and ovary, and between different stages of testis. The expressions of Cf-rlp24 gene in the testis of growing and mature stage are significant higher than that of ovary, 2.200±0.446 fold and 6.036±1.283 fold, respectively. In the development of testis, the expression of Cf-rlp24 increased with the maturation of testis and reached the peak value at mature stage, then declined rapidly. However, the expression level maintained a low and steady state in ovary. We conclude that Cf-rlp24 gene may play a positive role in gonad development and is more effective in testis.
We also performed real-time PCR to analyse the expression regulation in gonads cycle and female hepatopancreas. The result showed that Cf-vtg gene mainly expresses in ovary during the gonad cycle and the level increases as the development of ovary. The expression level is 1.917±0.035 (mean±S.E.) and 2.570±0.252 times higher during the growing and mature stage, respectively, than during the proliferative stage. There was negligible expression during the resting stage. Meanwhile, we observe weak expression in the female hepatopancreas. The level was 0.045±0.009 (Mean±S.E.) of that in the ovary during the proliferative stage. We did not detect any expression in the testis or other tissues. Further researches about the inducing effects of estradiol-17βon Cf-vtg expression with injuction method were performed. Real-time PCR showed that the expression level of Cf-vtg gene significantly increased in the ovary. It was concluded that estradiol-17βcould promote the synthesis of Cf-VTG.
采用RACE技术对其中的CL3256序列进行全长cDNA扩增,序列比对初步判定其为核糖体蛋白24基因,定名为Cf-rlp24。该基因全长cDNA为965 bp,开放阅读框为525 bp,编码175个氨基酸,该推测蛋白在不同物种之间同源性很高。Real-time PCR检测Cf-rlp24基因在精、卵巢之间以及不同发育时期的精巢中均存在表达差异,其中生长期和成熟期精巢Cf-rlp24基因的表达量显著高于相应时期的卵巢,分别高出2.200±0.446倍和6.036±1.283倍;在精巢的发育过程中,该基因的表达呈逐渐升高的态势,至成熟期时达最高值,而后迅速回落;但在卵巢的发育过程中,始终处于较低的表达水平,且未见该基因的显著性表达差异。我们得出,Cf-rlp24基因参与栉孔扇贝性腺的发育,且在精巢发育中的作用大于卵巢。
本研究我们还采用Real-time PCR技术,分析了栉孔扇贝卵黄蛋白原基因(Cf-vtg)在两性性腺发育周期以及肝胰腺中的表达,得出该基因主要在卵巢中表达,且其表达量随着卵巢的发育成熟而逐渐升高,生长期和成熟期Cf-vtg基因的表达量分别为增殖期的1.917±0.035和2.570±0.252倍。休止期卵巢该基因几乎没有表达。进一步我们发现Cf-vtg基因在肝胰腺中也有少量的表达,其表达量为增殖期卵巢中的0.045±0.009。该基因在精巢中几乎没有表达。进一步我们采用注射的方法,检测了17β-E2对扇贝Cf-vtg基因的诱导表达,real-time PCR结果显示,17β-E2可明显诱导Cf-vtg基因的表达。因此推测17β-E2可以促进栉孔扇贝扇贝Cf-VTG的合成。
Sex differentiation is one of the most important research fields in development biology. The study about sex differentiation genes in vertebrates, especially in mammal, is deeper than in other species. Up to now, many genes related to sex determination and differentiation, such as Sry, Sox9, Wnt4, have been identified. But the research in invertebrates especially in marine bivalves is limited. In the present study, we selected the important commercial species-Chlamys farreri as the research object. Based on solexa sequencing, we analyzed the differentially expressed transcripts between femal and male gonads at proliferative stage. We totally obtained 295262 tags which are differentially expressed between sexes, and 1354 of which could be annotated with informations in GenBank. 5 of theses genes were selected and analyzed with RT-PCR. We find the concordance rate of Solexa sequencing results and RT-PCR results is about 40%.
We amplified the full-length cDNA sequence of CL3256 gene using RACE technology. Sequence aligiment indicated it as the ribosomal protein L24 gene (Cf-rlp24). The full-length cDNA of Cf-rlp24 gene is 965 nucleotides which could encodes 175 amino acid residues. Alignment analysis indicated that the RLP24 protein shares a high homology among different species. Real-time PCR analysis showed that the expression of Cf-rlp24 gene is different between testis and ovary, and between different stages of testis. The expressions of Cf-rlp24 gene in the testis of growing and mature stage are significant higher than that of ovary, 2.200±0.446 fold and 6.036±1.283 fold, respectively. In the development of testis, the expression of Cf-rlp24 increased with the maturation of testis and reached the peak value at mature stage, then declined rapidly. However, the expression level maintained a low and steady state in ovary. We conclude that Cf-rlp24 gene may play a positive role in gonad development and is more effective in testis.
We also performed real-time PCR to analyse the expression regulation in gonads cycle and female hepatopancreas. The result showed that Cf-vtg gene mainly expresses in ovary during the gonad cycle and the level increases as the development of ovary. The expression level is 1.917±0.035 (mean±S.E.) and 2.570±0.252 times higher during the growing and mature stage, respectively, than during the proliferative stage. There was negligible expression during the resting stage. Meanwhile, we observe weak expression in the female hepatopancreas. The level was 0.045±0.009 (Mean±S.E.) of that in the ovary during the proliferative stage. We did not detect any expression in the testis or other tissues. Further researches about the inducing effects of estradiol-17βon Cf-vtg expression with injuction method were performed. Real-time PCR showed that the expression level of Cf-vtg gene significantly increased in the ovary. It was concluded that estradiol-17βcould promote the synthesis of Cf-VTG.
引文
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