河川沙塘鳢胚胎、仔鱼发育过程中孵化腺的超微结构及其孵化酶（HCE）mRNA的定量研究
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摘要
河川沙塘鳢(Odontobutis potamophila),隶属鲈形目(Perciformes),沙塘鳢科(Odontobutidae),是我国重要的淡水经济鱼类之一。在河川沙塘鳢的生物学、胚胎发育、器官发育等相关研究基础上,本文采用透射电镜对河川沙塘鳢胚胎发育过程中孵化腺的超微结构进行了观察,并利用分子生物学的研究方法进一步对河川沙塘鳢孵化酶(HCE)基因的部分序列进行了克隆和分析,测定了河川沙塘鳢胚胎、仔鱼发育过程中孵化酶(HCE)基因mRNA的转录水平,同时探究了其与孵化腺发生之间的时空关系。
河川沙塘鳢的孵化腺为单细胞腺体,最早可在眼晶体形成期观察到孵化腺细胞(Hatching gland cells,HGCs),主要分布于头部腹面、头部与卵黄囊连接处及其卵黄囊前腹面;当胚胎发育至血液循环期时,HGCs不仅在形态上达到最大,数量上急剧增多,而且分布更广泛;胚胎发育进入眼黑色素出现期时,可以观察到处于不同合成时期的酶原颗粒,每个酶原颗粒外面均有膜包围,颗粒之间有明显的界限;当胚胎发育至孵化前期时,HGCs在数量上达到最多,并且可以观察到呈圆球形、直径约为0.25～1.00μm的孵化酶颗粒自HGCs顶部的开口处向外分泌,这些颗粒有些以单体形式存在,有些则粘结成团;初孵仔鱼期,已完成分泌作用的HGCs离开胚胎表面,转移至表皮细胞下方并逐渐衰退。以上结果表明,河川沙塘鳢孵化酶颗粒有一个成熟过程,成熟的孵化酶颗粒主要通过外倾方式向卵周隙中分泌以帮助胚胎的孵化破膜。
用Trizol提取到河川沙塘鳢的总RNA,测得A280 / A260均在1.8～2.0之间,通过AMV逆转录体系获得其cDNA。以RNA为模板,根据相关文献设计的引物进行PCR得到了长度为84bp的基因特异性cDNA扩增片段。所获得的扩增片段经过序列比对、同源性分析,表明所获扩增片段的确为孵化酶(HCE)基因的cDNA片段。
随后,采用实时荧光定量PCR(Real-time fluorescence quantitative polymerase chain reaction, RFQ-PCR)的方法研究了河川沙塘鳢胚胎、仔鱼发育过程孵化酶(HCE)基因mRNA表达量的动态变化。研究结果显示,从受精卵期到眼晶体形成期的早期发育阶段,孵化酶(HCE)基因mRNA的表达量较低(以孵化前期表达量为标准1);在眼黑色素形成期时,其表达量达到最大值;随着胚胎的进一步发育,仔鱼的出膜,孵化酶(HCE)基因mRNA的表达量逐渐降低;但当仔鱼发育到一日龄时,孵化酶(HCE)基因mRNA的表达量又有所增长,随后孵化酶(HCE)基因mRNA的表达量又一次降低。孵化酶(HCE)基因mRNA表达量的变化趋势与孵化腺细胞在超微结构上的形态变化结果大体一致。这表明在河川沙塘鳢的胚胎、仔鱼发育过程中,孵化酶(HCE)基因mRNA的表达存在着动态的变化,且与孵化腺的发生以及孵化酶的分泌过程存在着时空相关性。
The Odontobutis potamophila belongs to Perciformes, Odontobutidae. It is an important freshwater commercial fish in China. As far as we known, the biology, embryonic development and organ development of the Odontobutis potamophila had been well-studied. In this study, the hatching gland (HG) of the Odontobutis potamophila were examined with transmission electron microscopy (TEM) during its embryonic development; Subsequently, in order to investigation the space-time connection between hatching gland (HG) and hatching enzyme (HE) of the Odontobutis potamophila, the part sequence of hatching enzyme (HCE) were isolated and the quantities of mRNA expression of HE gene during the embryonic and larval development were analysed with the method of the Real time PCR.
The HG of the Odontobutis potamophila is unicellular gland, and was detected first at the stage of lens formation. The hatching gland cells (HGCs) were located in the ventral of the head, the joint region between the head and yolk sac, and the ventral of the yolk sac. At the blood circulation stage , the number and the volume of HGCs increased significantly, and HGCs spread more widely with the development of the embryo; When the embryo reached the eye pigmentation stage,zymogen granules were detected, and every one of the zymogen granule was wrapped by membrane, so that the limit between others can be distinguished significantly; When the embryo reached the pre-hatching stage, the hatching enzyme granules were secreted into the perivitelline space from the top of HGCs. The secreted granules were spherical and 0.25– 1.00μm in diameter. Some of the secreted granules were monomer and the others got together. The hatching enzyme was dissolved in the perivitelline fluid ,where it gained access to the egg envelope and digested the inner layer of egg envelope;At the larval fish just after hatching stage, the secretion of HGCs have finished and HGCs gradually disappeared from epidermis. The results showed that the hatching enzyme granules of the Odontobutis potamophila gradually maturated during its embryonic and larval development, and the mature hatching enzyme granules were excreted from the open of the HGCs to the perivitelline space for the embryo hatching.
The total RNA of the Odontobutis potamophila was extracted with TRIZOL reagent, and the ratio of A280 / A260 was detected. The results showed that all of the rations were between 1.8 and 2.0. The cDNA was obtained by AMV reverse transcription systems. The cDNA sequence of the HE of the Odontobutis potamophila were amplified with primers designed based on the homologous sequence of relative fish. And the cDNA fragment of 84bp base pairs was obtained.Then the homologies of the cDNA have been compared with other fishes. The results showed that the cDNAs which we obtained were the HE expressing genes.
For further study the quantities of mRNA expression of HE gene during the embryonic and larval development of the Odontobutis potamophila, the Real-time PCR method was used with SYBR Green as reporter dye and the product of PCR as standard curve template. The result showed that the expression of HE mRNA was low from fertilized egg stage to lens formation stage; Subsequent the expression of HE mRNA increased rapidly, and the expression of HE mRNA riched the highest level at the eye pigmentation stage. After the eye pigmentation stage, the expression of HE mRNA decreased. However, the expression of HE mRNA increased a few at one day after hatching. After that, the expression of HE mRNA also decreased. The results suggested that the expression of HE mRNA was a dynamic course during its embryonic and larval development, and there was close relationship of the HE gene expression and the development of HG.
河川沙塘鳢的孵化腺为单细胞腺体,最早可在眼晶体形成期观察到孵化腺细胞(Hatching gland cells,HGCs),主要分布于头部腹面、头部与卵黄囊连接处及其卵黄囊前腹面;当胚胎发育至血液循环期时,HGCs不仅在形态上达到最大,数量上急剧增多,而且分布更广泛;胚胎发育进入眼黑色素出现期时,可以观察到处于不同合成时期的酶原颗粒,每个酶原颗粒外面均有膜包围,颗粒之间有明显的界限;当胚胎发育至孵化前期时,HGCs在数量上达到最多,并且可以观察到呈圆球形、直径约为0.25～1.00μm的孵化酶颗粒自HGCs顶部的开口处向外分泌,这些颗粒有些以单体形式存在,有些则粘结成团;初孵仔鱼期,已完成分泌作用的HGCs离开胚胎表面,转移至表皮细胞下方并逐渐衰退。以上结果表明,河川沙塘鳢孵化酶颗粒有一个成熟过程,成熟的孵化酶颗粒主要通过外倾方式向卵周隙中分泌以帮助胚胎的孵化破膜。
用Trizol提取到河川沙塘鳢的总RNA,测得A280 / A260均在1.8～2.0之间,通过AMV逆转录体系获得其cDNA。以RNA为模板,根据相关文献设计的引物进行PCR得到了长度为84bp的基因特异性cDNA扩增片段。所获得的扩增片段经过序列比对、同源性分析,表明所获扩增片段的确为孵化酶(HCE)基因的cDNA片段。
随后,采用实时荧光定量PCR(Real-time fluorescence quantitative polymerase chain reaction, RFQ-PCR)的方法研究了河川沙塘鳢胚胎、仔鱼发育过程孵化酶(HCE)基因mRNA表达量的动态变化。研究结果显示,从受精卵期到眼晶体形成期的早期发育阶段,孵化酶(HCE)基因mRNA的表达量较低(以孵化前期表达量为标准1);在眼黑色素形成期时,其表达量达到最大值;随着胚胎的进一步发育,仔鱼的出膜,孵化酶(HCE)基因mRNA的表达量逐渐降低;但当仔鱼发育到一日龄时,孵化酶(HCE)基因mRNA的表达量又有所增长,随后孵化酶(HCE)基因mRNA的表达量又一次降低。孵化酶(HCE)基因mRNA表达量的变化趋势与孵化腺细胞在超微结构上的形态变化结果大体一致。这表明在河川沙塘鳢的胚胎、仔鱼发育过程中,孵化酶(HCE)基因mRNA的表达存在着动态的变化,且与孵化腺的发生以及孵化酶的分泌过程存在着时空相关性。
The Odontobutis potamophila belongs to Perciformes, Odontobutidae. It is an important freshwater commercial fish in China. As far as we known, the biology, embryonic development and organ development of the Odontobutis potamophila had been well-studied. In this study, the hatching gland (HG) of the Odontobutis potamophila were examined with transmission electron microscopy (TEM) during its embryonic development; Subsequently, in order to investigation the space-time connection between hatching gland (HG) and hatching enzyme (HE) of the Odontobutis potamophila, the part sequence of hatching enzyme (HCE) were isolated and the quantities of mRNA expression of HE gene during the embryonic and larval development were analysed with the method of the Real time PCR.
The HG of the Odontobutis potamophila is unicellular gland, and was detected first at the stage of lens formation. The hatching gland cells (HGCs) were located in the ventral of the head, the joint region between the head and yolk sac, and the ventral of the yolk sac. At the blood circulation stage , the number and the volume of HGCs increased significantly, and HGCs spread more widely with the development of the embryo; When the embryo reached the eye pigmentation stage,zymogen granules were detected, and every one of the zymogen granule was wrapped by membrane, so that the limit between others can be distinguished significantly; When the embryo reached the pre-hatching stage, the hatching enzyme granules were secreted into the perivitelline space from the top of HGCs. The secreted granules were spherical and 0.25– 1.00μm in diameter. Some of the secreted granules were monomer and the others got together. The hatching enzyme was dissolved in the perivitelline fluid ,where it gained access to the egg envelope and digested the inner layer of egg envelope;At the larval fish just after hatching stage, the secretion of HGCs have finished and HGCs gradually disappeared from epidermis. The results showed that the hatching enzyme granules of the Odontobutis potamophila gradually maturated during its embryonic and larval development, and the mature hatching enzyme granules were excreted from the open of the HGCs to the perivitelline space for the embryo hatching.
The total RNA of the Odontobutis potamophila was extracted with TRIZOL reagent, and the ratio of A280 / A260 was detected. The results showed that all of the rations were between 1.8 and 2.0. The cDNA was obtained by AMV reverse transcription systems. The cDNA sequence of the HE of the Odontobutis potamophila were amplified with primers designed based on the homologous sequence of relative fish. And the cDNA fragment of 84bp base pairs was obtained.Then the homologies of the cDNA have been compared with other fishes. The results showed that the cDNAs which we obtained were the HE expressing genes.
For further study the quantities of mRNA expression of HE gene during the embryonic and larval development of the Odontobutis potamophila, the Real-time PCR method was used with SYBR Green as reporter dye and the product of PCR as standard curve template. The result showed that the expression of HE mRNA was low from fertilized egg stage to lens formation stage; Subsequent the expression of HE mRNA increased rapidly, and the expression of HE mRNA riched the highest level at the eye pigmentation stage. After the eye pigmentation stage, the expression of HE mRNA decreased. However, the expression of HE mRNA increased a few at one day after hatching. After that, the expression of HE mRNA also decreased. The results suggested that the expression of HE mRNA was a dynamic course during its embryonic and larval development, and there was close relationship of the HE gene expression and the development of HG.
引文
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