菲律宾蛤仔附着变态过程中的差异基因表达与基因克隆
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摘要
菲律宾蛤仔(Ruditapes philippinarumAdamsetReeve)又称蛤仔,广泛分布在我国黄、渤海海区,其肉味鲜美,营养丰富,为我国四大养殖贝类之一,是捕捞和人工养殖的对象。随着人们生活水平的日益提高,市场对该贝苗种的需求亦日益增加。附着变态过程是贝类生活史中的关健时期,是幼虫向成体转变的重要环节。蛤仔苗种的产量和质量在很大程度上取决于幼苗的附着变态过程,幼苗附着变态的成功与否往往决定着出苗量和育苗的成败。因此研究其受精生物学、胚胎发生、附着、变态等基础生物学的工作已引起科研工作者的重视,然而从分子水平方面阐明附着变态机理的研究还不太深入。本论文以菲律宾蛤仔附着变态时期的幼苗为实验材料,采用分子生物学技术,试图从分子水平上找出与菲律宾蛤仔附着变态及发育相关的基因,为贝类幼苗发育生物学,生理学及分子生物学的发展提供参考,同时为苗种业的开发提供一定的基础理论依据。主要结果如下:
1.采用DDRT-PCR技术,以受精后壳顶前期、壳顶期、眼点期、稚贝期的菲律宾蛤仔幼虫为实验对象,研究菲律宾蛤仔幼虫不同发育时期的基因表达,进而找出与菲律宾蛤仔附着变态相关的基因。
2.研究了肾上腺素、去甲肾上腺素、多巴胺、γ-氨基丁酸、KCl和CaCl_2,在不同剂量和不同的诱导持续时间下,对菲律宾蛤仔眼点幼虫附着变态的诱导作用。在此基础之上,选取出最佳而又经济的神经递质类物质。肾上腺素(使用浓度为10~(-6)M,诱导时间为4h),同样也采用DDRT-PCR技术,6组引物组合,扩增出343条带,67条具有明显表达差异,其中的18条在对照组中有高表达,而另外的49条差异带在实验组中有高表达,表明这些差异带所代表的基因都和。肾上腺素诱导有关,同时也从分子生物学水平上证明了肾上腺素参与了贝类附着变态机理的调控过程。
3.以菲律宾蛤仔眼点幼虫为实验材料,应用RACE技术,克隆了菲律宾蛤仔的replacement histone H3.3的全长cDNA序列(AY916800):菲律宾蛤仔H3.3的基因组序列扩增结果表明:序列L[AY916802]大小为1214bp,含有一个长803bp的内含子;另一序列S[AY916803]长约411bp,不具有内含子。序列L和S编码和H3.3完全相同蛋白质序列,这表明菲律宾蛤仔的H3.3 cDNA
Ruditapes philippinarum is one of four most important bivalve species cultured in China. With the expansion of cultural scale, the need of clam seeds is continuously increasing. The quality and quantity of clam seeds rely greatly on the settlement metamorphosis precession. Settlement and Metamorphosis is an important stage from larva to juvenile in bivalvia. Therefore, more and more researching interests have been put emphases on its fertilization, embryogenesis, settlement and metamorphosis biology. However, molecular regulation and control theory of settlement and metamorphosis biology is not clear up to date.In this study, pediveliger larvae, veliger larvae (pro-settlement and metamorphosis), eye spot larvae and juvenile (post-settlement metamorphosis) and DDRT-PCR and other molecular biology technique are used to find out the genes related with larvae settlement metamorphosis development process, including providing some fundamental understandings helpful for the improvement of scallop seed raising industry. The main results are as follows:1. The mRNA differential display (DDRT-PCR) technique is adopted to find out the genes related with settlement metamorphosis development process of R. philippinarum larvae. Three novel genes are found after analyzed these differential display bands. Their accession numbers are AY916799, AY916798, and AY916797 separately. We thought the novel genes are possibly relative to the early embryo development of R. philippinarum larvae and can provide some fundamental
understandings helpful for the improvement of scallop seed raising industry.2. The effects of different chemical cues on the settlement and metamorphosis of the larva of R.philippinarum are studied. The chemical cues chosen are EPI, NE, L-DOPA, GABA, Kcl and Cacl2, respectively. And according to the experiment results mentioned above, Epinephrine (EPI) is used to induce the metamorphosis of R. philippinarum larvae. We consider that the differential expression gene (49) is relative to the EPI inducement. So it is proved from molecular biology that EPI may be a member during the regulation and control procession of R. philippinarum settlement and metamorphosis.3. A full-length cDNA (accession number: AY916800) encoding a new replacement histone H3 variant, PR-H3.3, has been identified in R. philippinarum embryo by 5' RACE. By polymerase chain reaction (PCR) to amplify the genomic DNA of R. philippinarum, two different histone H3. 3 encoding sequences are obtained: one with intron (AY916802) , 1214bp in length; the other without intron (AY916803), 411bp in length, which may be the H3L-like gene of R. philippinarum. We also cloned the histone H3 gene of R. philippinarum in this study, named RP-H3. RP-H3. 3 and RP-h3 are very similar at protein level, only four amino acids difference. Therefore, we discuss the possible evolution relation between H3. 3 and H3. We predict that H3. 3 is the possible ancestor gene from which the major histone H3 gene is derived and H3L-like is the transit gene in the evolution process from H3. 3 to H3. H3.3 gene has a higher expression in gill and mantle of R. philippinarum by in situ. The prokaryotic expression vector pGEX-4T-H3.3 is constructed and expressed in DH5a . In the preparing experiments, we try to probe the embryos of different developent period and more tissue of R. phiJippinarum by H3. 3 cDNA probe and anti-H3.3 antibody in order to
prove the biology function of H3. 3 and its relationship with settlement and metamorphosis process of scallop, including to provide some fundamental understandings helpful for the improvement of molecular biology, development biology and scallop seed raising industry.
1.采用DDRT-PCR技术,以受精后壳顶前期、壳顶期、眼点期、稚贝期的菲律宾蛤仔幼虫为实验对象,研究菲律宾蛤仔幼虫不同发育时期的基因表达,进而找出与菲律宾蛤仔附着变态相关的基因。
2.研究了肾上腺素、去甲肾上腺素、多巴胺、γ-氨基丁酸、KCl和CaCl_2,在不同剂量和不同的诱导持续时间下,对菲律宾蛤仔眼点幼虫附着变态的诱导作用。在此基础之上,选取出最佳而又经济的神经递质类物质。肾上腺素(使用浓度为10~(-6)M,诱导时间为4h),同样也采用DDRT-PCR技术,6组引物组合,扩增出343条带,67条具有明显表达差异,其中的18条在对照组中有高表达,而另外的49条差异带在实验组中有高表达,表明这些差异带所代表的基因都和。肾上腺素诱导有关,同时也从分子生物学水平上证明了肾上腺素参与了贝类附着变态机理的调控过程。
3.以菲律宾蛤仔眼点幼虫为实验材料,应用RACE技术,克隆了菲律宾蛤仔的replacement histone H3.3的全长cDNA序列(AY916800):菲律宾蛤仔H3.3的基因组序列扩增结果表明:序列L[AY916802]大小为1214bp,含有一个长803bp的内含子;另一序列S[AY916803]长约411bp,不具有内含子。序列L和S编码和H3.3完全相同蛋白质序列,这表明菲律宾蛤仔的H3.3 cDNA
Ruditapes philippinarum is one of four most important bivalve species cultured in China. With the expansion of cultural scale, the need of clam seeds is continuously increasing. The quality and quantity of clam seeds rely greatly on the settlement metamorphosis precession. Settlement and Metamorphosis is an important stage from larva to juvenile in bivalvia. Therefore, more and more researching interests have been put emphases on its fertilization, embryogenesis, settlement and metamorphosis biology. However, molecular regulation and control theory of settlement and metamorphosis biology is not clear up to date.In this study, pediveliger larvae, veliger larvae (pro-settlement and metamorphosis), eye spot larvae and juvenile (post-settlement metamorphosis) and DDRT-PCR and other molecular biology technique are used to find out the genes related with larvae settlement metamorphosis development process, including providing some fundamental understandings helpful for the improvement of scallop seed raising industry. The main results are as follows:1. The mRNA differential display (DDRT-PCR) technique is adopted to find out the genes related with settlement metamorphosis development process of R. philippinarum larvae. Three novel genes are found after analyzed these differential display bands. Their accession numbers are AY916799, AY916798, and AY916797 separately. We thought the novel genes are possibly relative to the early embryo development of R. philippinarum larvae and can provide some fundamental
understandings helpful for the improvement of scallop seed raising industry.2. The effects of different chemical cues on the settlement and metamorphosis of the larva of R.philippinarum are studied. The chemical cues chosen are EPI, NE, L-DOPA, GABA, Kcl and Cacl2, respectively. And according to the experiment results mentioned above, Epinephrine (EPI) is used to induce the metamorphosis of R. philippinarum larvae. We consider that the differential expression gene (49) is relative to the EPI inducement. So it is proved from molecular biology that EPI may be a member during the regulation and control procession of R. philippinarum settlement and metamorphosis.3. A full-length cDNA (accession number: AY916800) encoding a new replacement histone H3 variant, PR-H3.3, has been identified in R. philippinarum embryo by 5' RACE. By polymerase chain reaction (PCR) to amplify the genomic DNA of R. philippinarum, two different histone H3. 3 encoding sequences are obtained: one with intron (AY916802) , 1214bp in length; the other without intron (AY916803), 411bp in length, which may be the H3L-like gene of R. philippinarum. We also cloned the histone H3 gene of R. philippinarum in this study, named RP-H3. RP-H3. 3 and RP-h3 are very similar at protein level, only four amino acids difference. Therefore, we discuss the possible evolution relation between H3. 3 and H3. We predict that H3. 3 is the possible ancestor gene from which the major histone H3 gene is derived and H3L-like is the transit gene in the evolution process from H3. 3 to H3. H3.3 gene has a higher expression in gill and mantle of R. philippinarum by in situ. The prokaryotic expression vector pGEX-4T-H3.3 is constructed and expressed in DH5a . In the preparing experiments, we try to probe the embryos of different developent period and more tissue of R. phiJippinarum by H3. 3 cDNA probe and anti-H3.3 antibody in order to
prove the biology function of H3. 3 and its relationship with settlement and metamorphosis process of scallop, including to provide some fundamental understandings helpful for the improvement of molecular biology, development biology and scallop seed raising industry.
引文
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