| 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 fundamentalunderstandings 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 toprove 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. |
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