罗氏沼虾雄性生殖系统特异性分子机制
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摘要
生物的性别、生殖现象是生物学研究的主要内容之一,它涉及到许多重大的生物学问题,比如性别决定、遗传、发育和进化等,因此历来倍受关注。
目前对于性别、生殖的分子机制研究主要集中在人类和模式生物中,而对于一般水生生物这方面的研究很少,尤其在甲壳类中,已知的相关分子信息更加缺乏。虾类作为具有重要经济价值的水生生物,在分子水平的研究开展得相对较少,因此,大量获取水生动物性别和生殖相关基因资源来研究它们的生殖生物学、遗传育种等显得尤为必要。
本论文以罗氏沼虾为研究对象,开展关于性别与生殖相关分子机制的研究。在甲壳动物中首次采用大规模筛选差异基因的方法,即抑制性消减杂交,利用该方法建立了富含雄性特异性基因的消减文库。通过筛选差异性文库并测序,在Genbank通过BLAST比对,发现一个克隆包含两个保守的Kazal型domain,命名为MRPINK,另外三个序列是新的表达序列,其中之一包含有开放阅读框(ORF),命名为Mar-Mrr。
在此基础上应用5'-RACE和3'-RACE的方法,直接体外扩增,从而获得基因全长cDNA。Mar-Mrr cDNA全长为683bp,编码区333bp,编码110个氨基酸,分子量约11.7kDa。Mar-Mrr基因5'和3'非翻译区长度分别是82bp和268bp,其中3'非翻译区具有两个多聚腺苷酸信号,分别位于poly(A)上游64bp和14bp;推演的多肽含有24个氨基酸的信号肽和86个氨基酸的成熟肽,其中Gly、Ser和Ala含量较高,分别为15.4%、10.9%和9.1%。MRPINK cDNA全长为736bp,编码区405bp,编码135个氨基酸,分子量约14.7kDa的多肽分子,MRPINK基因5'和3'非翻译区长度分别是72bp和247bp,其中3'非翻译区具有一个多聚腺苷酸信号;推演的氨基酸序列中含有两个Kazal型domain,且包含21个氨基酸的信号肽。
Mar-Mrr与Genbank已知序列无同源性、相似性,在罗氏沼虾中属于首次克隆到的雄性生殖相关功能基因。MRPINK和已知的Kazal型蛋白酶抑制剂在氨基酸序列上的同源性为16-32%,其Kazal型domain序列上有19-52%的同源性,表明这个蛋白属于Kazal家族中的成员,在无脊椎动物中,这是首次获得与性别生殖相关的Kazal型蛋白酶抑制剂的基因信息。此外含有两个Kazal型结构域并
Sex and reproduction have received much attention in view of their importance in genetics, development, and evolution. Up to now, many studies are mainly focus on dealing with the human and biomodel organism, and a number of critical genes involved in sex differentiation and reproduction have been identified. But a little available and valuable gene information is shown in crustacean sex and reproduction, even the molecular cascade of sex and reproduction remain poorly understood. So it is of the great significance that researches about the molecular mechanism of sex and reproduction should be carried out in crustacean.In our present study, we selected the prawn, Macrobrachium rosenbergii, which is the commercially important organism, as the experimental materials. In crustacean, we firstly used suppression subtraction hybridization (SSH) to generate a subtracted cDNA library enriched for the male-specific transcripts. The subtracted and unsubtracted cDNA mixtures were used as probes in hybridization to screen the library. Thereby, some of male reproductive tract specific genes were obtained. After sequencing and by blast searches, one of them included an obvious open reading frame (ORF) and named as Mar-Mrr. Another one showed significant similarity to Kazal-type proteinase inhibitor and named as MRPINK.Then, full-length cDNA sequences for Mar-Mrr and MRPINK were determined by 3' and 5' RACE. The Mar-Mrr cDNA was found to contain 683 nucleotides including an ORF of 333 bp that encoded 110 amino acids peptide, with a molecular weight of 11.7 kDa, pI 5.574. The cDNA included a 82 bp 5' untranslated region (UTR) and a 268 bp 3' UTR that contained two potyadenylation signals (AATAAA) at 64 bp and 14 bp upstream from poly (A) tail in the mRNA, respectively. The deduced translation products contained a putative signal peptide of 24 amino acids, and a mature peptide of 86 amino acids. The amino acids composition of Gly, Ser and Ala was found to be relatively higher (15.4%, 10.9% and 9.1%). The full-length cDNA of MRPINK was found to contain 736 bp. The cDNA were found to include a 405 bp ORF, a 72 bp 5' UTR and a 259 bp 3' UTR that contained a polyadenylation signal (AATAAA). The ORF of the MRPINK cDNA was conceptually translated into a 134 amino acid residues precursor containing a 21 amino acid signal peptide, a 113 amino acid mature peptide with two Kazal-type domains, and a translation stop signal.
By blast searches, the sequences of Mar-Mrr and the deduced protein had no significant homology with any known gene or protien in DDBJ/EMBL/GenBank database. This is the first time that a novel functional gene was found to relate with male reproduction in the prawn. As for MRPINK, comparison with several Kazal-type double-headed proteinase inhibitors revealed 15% - 32% identities. The two domains of MRPINK showed 19% - 52% identities with Kazal-type domains from other species. This is the first occasion in invertebrate in which male reproductive tract specific Kazal-type proteinase inhibitor was identified.By Northern blotting analysis, significant accumulation of Mar-Mrr transcript was observed in male reproductive tracts. The transcription of the gene was observed in all three parts of the male reproductive tract and the highest expression of the gene was found to be in the vas deferens. The expression level of Mar-Mrr was examined throughout the developmental process from the post-larva to the adult every two weeks. Results revealed the Mar-Mrr mRNA transcript appeared from the fourth week after post-larva development and the expression level evidently increased following 6, 8,10 and 12 weeks after the post-larva stage. Based on these results, we examined the expression of Mar-Mrr during the androgenic gland maturation. The result indicated the Mar-Mrr mRNA expression was significantly increased during the androgenic gland maturation. Thus, transcription of Mar-Mrr was shown to be a development-dependent regulated expression pattern. MRPINK mRNA expression was not detected in any other tissues, only in male reproductive tracts. Expression of MRPINK was detected in all the tested stages once the male reproductive tract formed. The semi-quantitative RT-PCR indicated that the expression of the gene was to be continuous in the shaped male reproductive tract. The results of Northern blotting showed that the MRPINK gene expression was dominant in vas deferens and quite low in the testis.In situ hybridization (ISH) was used to determine which cells within the male reproductive tract express the gene mRNA. We demonstrated Mar-Mrr and MRPINK mRNAs were localized only in the secretory epithelial cells of vas deferens. Using PCR with genomic DNA of M. rosenbergii as template, the results suggested that the two genes contained no introns. Southern blotting analysis indicated the existence of multiple gene copies of Mar-Mrr and MRPINK.In general, this is the first time to clone the two male specific genes from prawns, and to analyze their expression and localization. Moreover, it was proposed
that Mar-Mrr would play key roles in the male reproductive tract and MRPINK would play roles in maintaining internal surroundings stability rather than in directly regulating the onset of a specific stage in the male reproductive tract. These results will provide theoretical basis to have a better understanding of the molecular mechanism in male reproductive tract of the prawn.
目前对于性别、生殖的分子机制研究主要集中在人类和模式生物中,而对于一般水生生物这方面的研究很少,尤其在甲壳类中,已知的相关分子信息更加缺乏。虾类作为具有重要经济价值的水生生物,在分子水平的研究开展得相对较少,因此,大量获取水生动物性别和生殖相关基因资源来研究它们的生殖生物学、遗传育种等显得尤为必要。
本论文以罗氏沼虾为研究对象,开展关于性别与生殖相关分子机制的研究。在甲壳动物中首次采用大规模筛选差异基因的方法,即抑制性消减杂交,利用该方法建立了富含雄性特异性基因的消减文库。通过筛选差异性文库并测序,在Genbank通过BLAST比对,发现一个克隆包含两个保守的Kazal型domain,命名为MRPINK,另外三个序列是新的表达序列,其中之一包含有开放阅读框(ORF),命名为Mar-Mrr。
在此基础上应用5'-RACE和3'-RACE的方法,直接体外扩增,从而获得基因全长cDNA。Mar-Mrr cDNA全长为683bp,编码区333bp,编码110个氨基酸,分子量约11.7kDa。Mar-Mrr基因5'和3'非翻译区长度分别是82bp和268bp,其中3'非翻译区具有两个多聚腺苷酸信号,分别位于poly(A)上游64bp和14bp;推演的多肽含有24个氨基酸的信号肽和86个氨基酸的成熟肽,其中Gly、Ser和Ala含量较高,分别为15.4%、10.9%和9.1%。MRPINK cDNA全长为736bp,编码区405bp,编码135个氨基酸,分子量约14.7kDa的多肽分子,MRPINK基因5'和3'非翻译区长度分别是72bp和247bp,其中3'非翻译区具有一个多聚腺苷酸信号;推演的氨基酸序列中含有两个Kazal型domain,且包含21个氨基酸的信号肽。
Mar-Mrr与Genbank已知序列无同源性、相似性,在罗氏沼虾中属于首次克隆到的雄性生殖相关功能基因。MRPINK和已知的Kazal型蛋白酶抑制剂在氨基酸序列上的同源性为16-32%,其Kazal型domain序列上有19-52%的同源性,表明这个蛋白属于Kazal家族中的成员,在无脊椎动物中,这是首次获得与性别生殖相关的Kazal型蛋白酶抑制剂的基因信息。此外含有两个Kazal型结构域并
Sex and reproduction have received much attention in view of their importance in genetics, development, and evolution. Up to now, many studies are mainly focus on dealing with the human and biomodel organism, and a number of critical genes involved in sex differentiation and reproduction have been identified. But a little available and valuable gene information is shown in crustacean sex and reproduction, even the molecular cascade of sex and reproduction remain poorly understood. So it is of the great significance that researches about the molecular mechanism of sex and reproduction should be carried out in crustacean.In our present study, we selected the prawn, Macrobrachium rosenbergii, which is the commercially important organism, as the experimental materials. In crustacean, we firstly used suppression subtraction hybridization (SSH) to generate a subtracted cDNA library enriched for the male-specific transcripts. The subtracted and unsubtracted cDNA mixtures were used as probes in hybridization to screen the library. Thereby, some of male reproductive tract specific genes were obtained. After sequencing and by blast searches, one of them included an obvious open reading frame (ORF) and named as Mar-Mrr. Another one showed significant similarity to Kazal-type proteinase inhibitor and named as MRPINK.Then, full-length cDNA sequences for Mar-Mrr and MRPINK were determined by 3' and 5' RACE. The Mar-Mrr cDNA was found to contain 683 nucleotides including an ORF of 333 bp that encoded 110 amino acids peptide, with a molecular weight of 11.7 kDa, pI 5.574. The cDNA included a 82 bp 5' untranslated region (UTR) and a 268 bp 3' UTR that contained two potyadenylation signals (AATAAA) at 64 bp and 14 bp upstream from poly (A) tail in the mRNA, respectively. The deduced translation products contained a putative signal peptide of 24 amino acids, and a mature peptide of 86 amino acids. The amino acids composition of Gly, Ser and Ala was found to be relatively higher (15.4%, 10.9% and 9.1%). The full-length cDNA of MRPINK was found to contain 736 bp. The cDNA were found to include a 405 bp ORF, a 72 bp 5' UTR and a 259 bp 3' UTR that contained a polyadenylation signal (AATAAA). The ORF of the MRPINK cDNA was conceptually translated into a 134 amino acid residues precursor containing a 21 amino acid signal peptide, a 113 amino acid mature peptide with two Kazal-type domains, and a translation stop signal.
By blast searches, the sequences of Mar-Mrr and the deduced protein had no significant homology with any known gene or protien in DDBJ/EMBL/GenBank database. This is the first time that a novel functional gene was found to relate with male reproduction in the prawn. As for MRPINK, comparison with several Kazal-type double-headed proteinase inhibitors revealed 15% - 32% identities. The two domains of MRPINK showed 19% - 52% identities with Kazal-type domains from other species. This is the first occasion in invertebrate in which male reproductive tract specific Kazal-type proteinase inhibitor was identified.By Northern blotting analysis, significant accumulation of Mar-Mrr transcript was observed in male reproductive tracts. The transcription of the gene was observed in all three parts of the male reproductive tract and the highest expression of the gene was found to be in the vas deferens. The expression level of Mar-Mrr was examined throughout the developmental process from the post-larva to the adult every two weeks. Results revealed the Mar-Mrr mRNA transcript appeared from the fourth week after post-larva development and the expression level evidently increased following 6, 8,10 and 12 weeks after the post-larva stage. Based on these results, we examined the expression of Mar-Mrr during the androgenic gland maturation. The result indicated the Mar-Mrr mRNA expression was significantly increased during the androgenic gland maturation. Thus, transcription of Mar-Mrr was shown to be a development-dependent regulated expression pattern. MRPINK mRNA expression was not detected in any other tissues, only in male reproductive tracts. Expression of MRPINK was detected in all the tested stages once the male reproductive tract formed. The semi-quantitative RT-PCR indicated that the expression of the gene was to be continuous in the shaped male reproductive tract. The results of Northern blotting showed that the MRPINK gene expression was dominant in vas deferens and quite low in the testis.In situ hybridization (ISH) was used to determine which cells within the male reproductive tract express the gene mRNA. We demonstrated Mar-Mrr and MRPINK mRNAs were localized only in the secretory epithelial cells of vas deferens. Using PCR with genomic DNA of M. rosenbergii as template, the results suggested that the two genes contained no introns. Southern blotting analysis indicated the existence of multiple gene copies of Mar-Mrr and MRPINK.In general, this is the first time to clone the two male specific genes from prawns, and to analyze their expression and localization. Moreover, it was proposed
that Mar-Mrr would play key roles in the male reproductive tract and MRPINK would play roles in maintaining internal surroundings stability rather than in directly regulating the onset of a specific stage in the male reproductive tract. These results will provide theoretical basis to have a better understanding of the molecular mechanism in male reproductive tract of the prawn.
引文
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