文蛤（Meretrix meretrix）幼虫生长发育相关基因的克隆和功能分析

IOCAS-IR > 海洋环流与波动重点实验室

	文蛤（Meretrix meretrix）幼虫生长发育相关基因的克隆和功能分析
其他题名	Molecular Cloning and Functional Analysis of Genes involved in Larval Development in Clam Meretrix meretrix
	王晓梅
学位类型	博士
	2008-06-06
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	文蛤幼虫铁蛋白组织蛋白酶b Caspase Real Time Pcr 整体原位杂交免疫组化特异性抑制剂
摘要	贝类养殖是我国海水养殖业中最重要的组成部分之一。贝类人工大规模养殖中的关键环节是种苗的人工繁育，早期幼虫能否正常生长发育，对幼虫变态和变态后生长率及成活率有很大的影响，直接关系到生产产量和经济效益，所以，了解幼虫发育机制对于生产实践具有重要的指导意义。文蛤（Meretrix meretrix）是一种在东亚各国沿海和滩涂地区广泛分布的双壳贝类，是我国一种重要的经济品种。本论文以文蛤幼虫为研究对象，分别对文蛤幼虫发育过程中贝壳形成相关的铁蛋白（MmeFer）、营养及变态相关的组织蛋白酶B（MmeCB）及变态过程中细胞凋亡相关的caspase三个基因进行了克隆，分析了基因及编码蛋白在担轮幼虫期（L1）、D形幼虫期（L2）、壳顶幼虫期（L3）和稚贝期（L4）的时空表达特征，解析了其可能的功能，并研究了相应酶类的特异性抑制剂作用对幼虫发育过程的影响，进行了目标蛋白的功能验证，详述如下：研究结果显示，在文蛤胚胎发育到原肠胚时放入不含铁离子的人工海水中培养，发育成无壳的畸形，随着人工海水中铁离子添加浓度的升高，幼虫长出壳状组织接近正常状态；而发育到L1期幼虫放入不含铁离子的人工海水中培养却可以发育出正常的壳，推测铁和铁代谢相关蛋白在幼虫贝壳初始形成有重要的作用。根据构建的文蛤幼虫cDNA文库中提供的序列信息，从文蛤中克隆了与铁离子代谢密切相关的铁蛋白（MmeFer）的全长cDNA 序列；通过Real time PCR发现，MmeFer mRNA的表达量在贝壳形成前后有明显改变；整体原位杂交结果显示MmeFer mRNA在L1期的表达部位刚好是贝壳生成的起始部位，推断文蛤铁蛋白与文蛤幼虫贝壳初始形成密切相关。利用文蛤幼虫cDNA文库中的EST信息在幼虫中克隆到文蛤组织蛋白酶B（MmeCB）全长cDNA序列；通过整体原位杂交分析发现MmeCB mRNA在L2至L4期幼虫的消化腺部位表达，而且在L2期的幼虫表皮也有表达，说明MmeCB可能和幼虫消化相关，而且可能参与幼虫从表皮摄取营养的过程。利用MmeCB特异性抑制剂（CA074Me）处理饥饿幼虫，发现其生长受到明显抑制，验证了MmeCB参与幼虫表皮营养代谢的推论。利用免疫组织化学技术，研究了MmeCB蛋白在文蛤幼虫中的时空分布，发现其在L2期幼虫表面和胃部有阳性信号，而在L3期，MmeCB在幼虫面盘基部有强烈的表达，提示MmeCB在文蛤幼虫中不仅起到营养的作用，而且可能和幼虫附着变态有关。利用CA074Me分别处理文蛤胚胎和变态期幼虫，发现抑制MmeCB的活性对胚胎发育和幼虫变态都有显著影响。研究结果提示MmeCB在幼虫发育各阶段均具有重要的生物学功能。根据caspase在不同物种中的保守区域设计简并引物，在文蛤幼虫中扩增到cDNA序列片段；检测有活性的caspase在文蛤幼虫各发育时期的分布部位，发现其在L1至L3期幼虫中都有分布，说明整个幼虫形态变化过程中都有caspase的参与；细胞凋亡检测结果显示，幼虫主要发生细胞凋亡的部位在L3期幼虫的面盘，即变态过程中要退化的器官，说明细胞凋亡可能是文蛤幼虫变态过程中面盘退化的主要机制；用caspase特异性抑制剂处理变态前幼虫，发现幼虫变态率下降，初步验证了caspase在文蛤幼虫变态过程中的作用。通过对上述三种基因的研究，分别探讨了文蛤幼虫发育阶段中的几个主要事件（L1期到L2期的贝壳形成、L2到L3期的幼虫营养摄食及L3到L4期的附着变态）相关的基因及其功能，为研究贝类生长发育调控的分子机理提供了新的线索。
其他摘要	Bivalve culture is one of most widely practiced mariculture.in China. Seed rearing is a key phase in large scale bivalve culture industry. The developmental status of early larvae would greatly affect the metamorphosis rate, the later growth and survival of bivalve. Investigation of molecular mechanism in larval development is thus important for production practice. Clam Meretrix meretrix is distributed along the coastal and estuarine areas in eastern Asia. It is an important commercial clam widely cultured in the coast of China. In this paper, we studied M. meretrix ferritin (MmeFer), cathepsin B (MmeCB) and caspase genes, which are involved in clam larval shell formation, nutrition, metabolism and apoptosis, respectively. We have cloned the three genes, investigated the temporal and spatial expression profile both at gene and protein level in trochophore (L1), D-veliger (L2), pediveliger (L3) and postlarvae (L4). The potential roles of these proteins were analyzed with specific inhibitors during larval development. Firstly, embryos were found developed into trochophore-like larvae with no shell if cultured at gastrula stage in artificial seawater without iron. Shell-like structures were formed only in the presence of iron. The larvae which had been transferred at L1 stage into ASW developed normal shell. This indicated that iron and iron associated protein are important for larval shell formation. The EST sequence which is homologous with ferritin, which is a principal iron metabolic protein, was selected from the M. meretrix cDNA library. The full-length of ferritin subunit cDNA (MmeFer) was cloned by RACE. The results of real-time PCR revealed that the MmeFer mRNA expression changed before and after the larval shell formation. The result of whole mount in situ hybridization showed that MmeFer was located at the position of shell initiation in trochophore stage, indicating MmeFer plays a role in shell initiation in M. meretrix. The full-length of M. meretrix cathepsin B (MmeCB) cDNA was cloned with 3’ and 5’ RACE. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. From L2 to L4 stages, MmeCB mRNA was detected in the digestive tract, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in L2 indicating that it is associated with another pathway of nutrient metabolism. Cathepsin B specific inhibitor (CA074Me) was applied to study the function of MmeCB in the growth of unfed larvae. Larval growth was found influenced which support that MmeCB may participate in nutrient metabolism in epidermis. MmeCB protein expression was detected by immunocytochemistry with specific antibody during larval development. In L3 larvae, MmeCB expression can be detected not only in the digestive tract and epidermis near the shell, but also at the bottom of velum, showing MmeCB might play a role in velum degradation during larval metamorphosis. The potential role of MmeCB in hatching and metamorphosis was studied with CA074Me. The results showed that inhibiting the activity of MmeCB can affect the embryo development and larval metamorphosis, suggesting the multifunction of MmeCB during M. meretrix larval development. Degenerate primers were designed according to the conserved sequence of caspase. A cDNA segment was isolated from M. meretrix larvae. The results of caspase in situ detection in larval developmental stages revealed that there were activated caspase from L1 to L3, indicating caspase is involved in the whole developmental process. The main apoptosis area was found in velum at L3 before metamorphosis, indicating the degradation of velum might be the process of apoptosis. The larvae before metamorphosis were treated by caspase specific inhibitor to analyze the role of caspase in this process. The results indicated that caspase have function metamorphosis. In conclusion, the three genes (MmeFer, MmeCB and caspase) associated with larval shell formation from L1 to L2 stage, larval nutrient metabolism from L2 to L3, and metamorphosis from L3 to L4, have been characterized and analyzed respectively. The investigation will be helpful for understanding the molecular mechanism in bivalve larval development.
页数	128
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1105
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	王晓梅. 文蛤（Meretrix meretrix）幼虫生长发育相关基因的克隆和功能分析[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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