栉孔扇贝Cf-foxl2基因的克隆、表达模式及功能初探
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摘要
FOXL2是叉头框转录因子家族成员之一，迄今已在多个物种中获得了该基因的序列，然而较深入和系统的研究仅在高等哺乳动物中进行，并确定其参与哺乳动物的卵巢早期发育、分化以及成体卵巢的功能维持，属于雌性相关基因。目前，无脊椎动物中该基因的系统研究还很有限，且尚未见该基因作为无脊椎动物雌性特异基因的相关报道。
本研究以重要的经济养殖物种——栉孔扇贝（Chlamys farreri）为研究对象，克隆获得了foxl2的cDNA全长序列，分析了它的序列特征；半定量RT-PCR揭示了增殖期栉孔扇贝各组织中的空间表达；定量RT-PCR和原位杂交技术分析了该基因在个体发育过程及成体性腺周期中的表达特征；定量RT-PCR技术分析了外源17β-雌二醇对该基因表达的影响；初步探讨了该基因在贝类中可能的功能。
本研究获得的栉孔扇贝foxl2（Cf-foxl2） cDNA序列全长1824bp,编码369个氨基酸，具有保守的叉头框DNA结合区。保守区C端含NTL（细胞核定位信号）序列（RRRRRMKR），蛋白三维结构预测显示，Cf-FOXL2叉头框含3个α螺旋及2个翼状结构。
RT-PCR结果显示其主要在卵巢中表达；其次在肝脏中有很微弱地表达；在精巢及其他组织中无可见表达；初步显示其在精卵巢中的二态性表达特征。
定量RT-PCR研究发现，Cf-foxl2呈母源性表达；在幼虫结构迅速建立的D形期幼虫表达量较高，之后随着幼虫的发育，表达量显著下降；至个体性别分化后表达量明显上升。原位杂交结果显示阳性信号在担轮幼虫之前皆均匀分布；担轮幼虫出现信号集中，主要集中在腹面的口凹附近，对称存在于虫体两侧；D形幼虫时信号强度明显增强，除集中于内脏团外，在外套膜边缘也有强信号出现，之后信号渐弱直至消失；当个体发育到9mm幼贝时，此时从性腺组织学上刚刚可以分辨雌雄，Cf-foxl2阳性信号仅在雌性生殖细胞及周围的体细胞中出现，在同时期的精巢中未见表达；暗示该基因可能是栉孔扇贝雌性性别表型形成的早期标记基因，可能参与扇贝卵巢的分化。
比较Cf-foxl2mRNA在栉孔扇贝成体性腺发育周期中精卵巢的定量RT-PCR结果发现，Cf-foxl2在各时期卵巢中的表达均显著高于同时期精巢中的表达，呈明显的性别二态性表达模式，其中增殖期卵巢是精巢表达量的63倍。在卵巢中，增殖期卵巢的表达量明显高于生长期和成熟期，前者是后两者的3倍，该表达规律与小鼠及青鳉等物种中foxl2的表达规律类似，推测与其在卵巢发育过程中的作用相关相关；在精巢中，Cf-foxl2mRNA仅在成熟期有非常微弱的表达，增殖期与生长期的精巢中无明显表达。成体性腺的原位杂交结果显示，Cf-foxl2的阳性信号在各时期卵巢中的分布位点与其在幼贝雌性性腺中相同，但在成熟个体的精巢中除精子外的其它细胞（生殖细胞和体细胞）中也有微弱信号出现；有趣的是，与胚胎、幼虫和幼体不同，阴性探针在成体两性性腺中均有表达信号，推测成体性腺中可能存在反义RNA，以调控foxl2基因的表达。
采用闭壳肌注射外源17β-雌二醇的方法，检测其对Cf-foxl2表达的诱导发现，成体卵巢与精巢中的Cf-foxl2表达量较对照组都有明显提高（p＜0.05），推测17β-雌二醇对该基因有反馈作用。
综上，在结构上，Cf-foxl2基因的叉头框与已报道其它物种的foxl2基因叉头框部位高度保守；其在个体发育中的表达图式显示该基因呈母源性表达，是卵巢表型形成的早期标记基因；其在成体中的表达模式与已报道的脊椎动物一样，呈现明显的性别二态性表达；进一步发现，外源17β-雌二醇可诱导Cf-foxl2mRNA的体内合成。该基因在栉孔扇贝卵巢中的功能以及与卵子发育的相关性尚需进一步研究。
FOXL2is one of the forkhead box transcription factor family members, whichhas been detected in many species. However, studies in depth and systematicness onfoxl2only have been carried out in higher mammals, and revealed that FOXL2isinvolved in ovarian determination and/or differentiation as well as functionalmaintenance. Until now, limited data were reported on foxl2genes in invertebrates,and no data indicated the gene is female-related gene in invertebrate.
In this study, scallop Chlamys farreri, a commercially important aquatic productwas chosen as the expereimental animal. The full-length cDNA sequences of the C.farreri foxl2(Cf-foxl2) was cloned and its sequence characteristics was analysed.Furthermore, RT-PCR was used to detected the spatial expression in different tissuesof adult at proliferative stage; real-time PCR and in situ hybridization were employedto analyse the expression pattern in C. farreri during ontogenesis and gonadicreproductive cycle. Moreover, effect of exogenous estradiol-17β on the Cf-foxl2expression level was detected. Finally, Cf-folx2function in mollusks was probedprimarily.
The full-length sequence of Cf-foxl2cDNA has1824bp with an open readingframe of1107bp encoding369amino acid residues containing the conserved domainforkhead box, a putative NLS sequences was characterized by RRRRRMRR and thethree-dimensional protein structure was predicted, containing three α-helices and twowing-like structure.
RT–PCR showed that Cf-foxl2is specifically expressed in ovary of theproliferative stage and a weakly expression in hepatopancreas, but is not detected inother organs including the testis. The result showed primarily that the Cf-foxl2expresses in a sexually dimorphic pattern.
Using quantitative real-time PCR, it was found that Cf-foxl2mRNA is amaternal product and the highest expression during the development is detected inD-shaped larvae in which the larval structure is rapidly established, and then itdeclines significantly. Until the sex of the juvenile is differentiated, the expression ofCf-foxl2is increased significantly. In situ hybridization revealed that Cf-foxl2mRNAare evenly distributed at various stages before trochophore, then concentrate in theventral surface near the mouth concave and exists symmetrically in trochophore.Intensity of the signals enhances significantly in D-shaped larvae, focuses on thevisceral mass and the mantle margin, after that the signals are weakened to disappear.When the shell height reaches to9mm, male and female can be distinguished basedon the histological structure, the signals are located in the cytoplasm of follicle cellsand germ cells in the ovary, but no signal is detected in testis. The result waspresumed that Cf-foxl2is the early marker gene of the ovary phenotype formation inscallop, and participates in regulation of ovary differentiation.
Using quantitative real-time PCR, the Cf-foxl2mRNA expression was comparedbetween the ovary and the testis during the reproductive cycle. The result showed thatthe expression level in the ovary is significantly higher than that in the testis at thesame stage, and Cf-foxl2presents a significant sexually dimorphic expression pattern,such as the highest expression is detected in the ovary of proliferative stage, about62-fold higher than that in the testis; in ovary, about2-fold higher in that ofproliferative stage than in that of growing and mature stages. The expression pattern issimilar to that in mouse (Mus musculus) and medaka (Oryzias latipes). In situhybridization of adult scallop revealed that Cf-foxl2mRNA is located in the same locias that of the juvenile, but weak signals are also detected in testis except in thespermatozoa. Interestingly, Signals of sense probes are presented in the same loci asthat of antisense probes in the adult ovary and testis, which is different from the resultin the embryos, the larvae and the juveniles. It was hypothesized that antisense RNAof Cf-foxl2might be present in adult scallop, and might act in vivo as a factor toinvolve in the regulation of Cf-foxl2in the adult scallop.
By injecting estradiol-17β into adductor muscle of the scallop, the effect of the estradiol-17β on Cf-foxl2mRNA expression level is detected. The result showed theCf-foxl2mRNA expression levels in ovary and testis are significantly increased. Theresult implied that estradiol-17β can promote the Cf-folx2expression at mRNA leveland a feedback relationship may exist.
In summary, the forkhead box of Cf-foxl2is high conservative with that in otherspecies. Its expression pattern during the development indicated that Cf-foxl2ismaternal gene and is the early marker gene in the formation of ovary phenotype. AndCf-foxl2presents a sexually dimorphic expression pattern in adult scallop which isconserved with vertebrate. Exogenous estradiol-17β can promote the Cf-foxl2RNAsynthesis. More extensive studies should be performed to conclude the function ofCf-foxl2in ovary and oogenesis of the scallop.
本研究以重要的经济养殖物种——栉孔扇贝（Chlamys farreri）为研究对象，克隆获得了foxl2的cDNA全长序列，分析了它的序列特征；半定量RT-PCR揭示了增殖期栉孔扇贝各组织中的空间表达；定量RT-PCR和原位杂交技术分析了该基因在个体发育过程及成体性腺周期中的表达特征；定量RT-PCR技术分析了外源17β-雌二醇对该基因表达的影响；初步探讨了该基因在贝类中可能的功能。
本研究获得的栉孔扇贝foxl2（Cf-foxl2） cDNA序列全长1824bp,编码369个氨基酸，具有保守的叉头框DNA结合区。保守区C端含NTL（细胞核定位信号）序列（RRRRRMKR），蛋白三维结构预测显示，Cf-FOXL2叉头框含3个α螺旋及2个翼状结构。
RT-PCR结果显示其主要在卵巢中表达；其次在肝脏中有很微弱地表达；在精巢及其他组织中无可见表达；初步显示其在精卵巢中的二态性表达特征。
定量RT-PCR研究发现，Cf-foxl2呈母源性表达；在幼虫结构迅速建立的D形期幼虫表达量较高，之后随着幼虫的发育，表达量显著下降；至个体性别分化后表达量明显上升。原位杂交结果显示阳性信号在担轮幼虫之前皆均匀分布；担轮幼虫出现信号集中，主要集中在腹面的口凹附近，对称存在于虫体两侧；D形幼虫时信号强度明显增强，除集中于内脏团外，在外套膜边缘也有强信号出现，之后信号渐弱直至消失；当个体发育到9mm幼贝时，此时从性腺组织学上刚刚可以分辨雌雄，Cf-foxl2阳性信号仅在雌性生殖细胞及周围的体细胞中出现，在同时期的精巢中未见表达；暗示该基因可能是栉孔扇贝雌性性别表型形成的早期标记基因，可能参与扇贝卵巢的分化。
比较Cf-foxl2mRNA在栉孔扇贝成体性腺发育周期中精卵巢的定量RT-PCR结果发现，Cf-foxl2在各时期卵巢中的表达均显著高于同时期精巢中的表达，呈明显的性别二态性表达模式，其中增殖期卵巢是精巢表达量的63倍。在卵巢中，增殖期卵巢的表达量明显高于生长期和成熟期，前者是后两者的3倍，该表达规律与小鼠及青鳉等物种中foxl2的表达规律类似，推测与其在卵巢发育过程中的作用相关相关；在精巢中，Cf-foxl2mRNA仅在成熟期有非常微弱的表达，增殖期与生长期的精巢中无明显表达。成体性腺的原位杂交结果显示，Cf-foxl2的阳性信号在各时期卵巢中的分布位点与其在幼贝雌性性腺中相同，但在成熟个体的精巢中除精子外的其它细胞（生殖细胞和体细胞）中也有微弱信号出现；有趣的是，与胚胎、幼虫和幼体不同，阴性探针在成体两性性腺中均有表达信号，推测成体性腺中可能存在反义RNA，以调控foxl2基因的表达。
采用闭壳肌注射外源17β-雌二醇的方法，检测其对Cf-foxl2表达的诱导发现，成体卵巢与精巢中的Cf-foxl2表达量较对照组都有明显提高（p＜0.05），推测17β-雌二醇对该基因有反馈作用。
综上，在结构上，Cf-foxl2基因的叉头框与已报道其它物种的foxl2基因叉头框部位高度保守；其在个体发育中的表达图式显示该基因呈母源性表达，是卵巢表型形成的早期标记基因；其在成体中的表达模式与已报道的脊椎动物一样，呈现明显的性别二态性表达；进一步发现，外源17β-雌二醇可诱导Cf-foxl2mRNA的体内合成。该基因在栉孔扇贝卵巢中的功能以及与卵子发育的相关性尚需进一步研究。
FOXL2is one of the forkhead box transcription factor family members, whichhas been detected in many species. However, studies in depth and systematicness onfoxl2only have been carried out in higher mammals, and revealed that FOXL2isinvolved in ovarian determination and/or differentiation as well as functionalmaintenance. Until now, limited data were reported on foxl2genes in invertebrates,and no data indicated the gene is female-related gene in invertebrate.
In this study, scallop Chlamys farreri, a commercially important aquatic productwas chosen as the expereimental animal. The full-length cDNA sequences of the C.farreri foxl2(Cf-foxl2) was cloned and its sequence characteristics was analysed.Furthermore, RT-PCR was used to detected the spatial expression in different tissuesof adult at proliferative stage; real-time PCR and in situ hybridization were employedto analyse the expression pattern in C. farreri during ontogenesis and gonadicreproductive cycle. Moreover, effect of exogenous estradiol-17β on the Cf-foxl2expression level was detected. Finally, Cf-folx2function in mollusks was probedprimarily.
The full-length sequence of Cf-foxl2cDNA has1824bp with an open readingframe of1107bp encoding369amino acid residues containing the conserved domainforkhead box, a putative NLS sequences was characterized by RRRRRMRR and thethree-dimensional protein structure was predicted, containing three α-helices and twowing-like structure.
RT–PCR showed that Cf-foxl2is specifically expressed in ovary of theproliferative stage and a weakly expression in hepatopancreas, but is not detected inother organs including the testis. The result showed primarily that the Cf-foxl2expresses in a sexually dimorphic pattern.
Using quantitative real-time PCR, it was found that Cf-foxl2mRNA is amaternal product and the highest expression during the development is detected inD-shaped larvae in which the larval structure is rapidly established, and then itdeclines significantly. Until the sex of the juvenile is differentiated, the expression ofCf-foxl2is increased significantly. In situ hybridization revealed that Cf-foxl2mRNAare evenly distributed at various stages before trochophore, then concentrate in theventral surface near the mouth concave and exists symmetrically in trochophore.Intensity of the signals enhances significantly in D-shaped larvae, focuses on thevisceral mass and the mantle margin, after that the signals are weakened to disappear.When the shell height reaches to9mm, male and female can be distinguished basedon the histological structure, the signals are located in the cytoplasm of follicle cellsand germ cells in the ovary, but no signal is detected in testis. The result waspresumed that Cf-foxl2is the early marker gene of the ovary phenotype formation inscallop, and participates in regulation of ovary differentiation.
Using quantitative real-time PCR, the Cf-foxl2mRNA expression was comparedbetween the ovary and the testis during the reproductive cycle. The result showed thatthe expression level in the ovary is significantly higher than that in the testis at thesame stage, and Cf-foxl2presents a significant sexually dimorphic expression pattern,such as the highest expression is detected in the ovary of proliferative stage, about62-fold higher than that in the testis; in ovary, about2-fold higher in that ofproliferative stage than in that of growing and mature stages. The expression pattern issimilar to that in mouse (Mus musculus) and medaka (Oryzias latipes). In situhybridization of adult scallop revealed that Cf-foxl2mRNA is located in the same locias that of the juvenile, but weak signals are also detected in testis except in thespermatozoa. Interestingly, Signals of sense probes are presented in the same loci asthat of antisense probes in the adult ovary and testis, which is different from the resultin the embryos, the larvae and the juveniles. It was hypothesized that antisense RNAof Cf-foxl2might be present in adult scallop, and might act in vivo as a factor toinvolve in the regulation of Cf-foxl2in the adult scallop.
By injecting estradiol-17β into adductor muscle of the scallop, the effect of the estradiol-17β on Cf-foxl2mRNA expression level is detected. The result showed theCf-foxl2mRNA expression levels in ovary and testis are significantly increased. Theresult implied that estradiol-17β can promote the Cf-folx2expression at mRNA leveland a feedback relationship may exist.
In summary, the forkhead box of Cf-foxl2is high conservative with that in otherspecies. Its expression pattern during the development indicated that Cf-foxl2ismaternal gene and is the early marker gene in the formation of ovary phenotype. AndCf-foxl2presents a sexually dimorphic expression pattern in adult scallop which isconserved with vertebrate. Exogenous estradiol-17β can promote the Cf-foxl2RNAsynthesis. More extensive studies should be performed to conclude the function ofCf-foxl2in ovary and oogenesis of the scallop.
引文
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