导　　师： 许强华

学科专业： I0803

授予学位： 硕士

作　　者： ;

机构地区： 上海海洋大学

摘　　要： 三疣梭子蟹,隶属于甲壳纲、十足目、梭子蟹科,梭子蟹属,具有肉质鲜美、营养丰富、产量高等特点,是中国沿海的重要经济蟹类。它是一种广盐性蟹类,对盐度有一定的适应范围,不同的盐度条件将影响三疣梭子蟹种群的分布和洄游。由于过度捕捞,梭子蟹自然资源正日益减少。近年来,我国沿海已广泛开展了三疣梭子蟹的人工养殖,它已经成为我国最重要的养殖海产蟹。盐度是三疣梭子蟹人工养殖过程中的一个重要的因素。关于广盐性蟹类的渗透调控机理已有部分研究,在分子水平上的研究较少。从分子水平上弄清三疣梭子蟹的渗透调控机理对三疣梭子蟹种质资源的改良和人工养殖具有重要意义。筛选获得渗透调节相关基因是其必要的前提。 本研究以三疣梭子蟹鳃组织为材料,构建其全长cDNA文库。从该文库随机挑选5012个单克隆进行测序,共获得高质量的ESTs序列4433个,平均长度为748bp。经过CAP3组装后获得2426个假定转录本/(重叠群238个,单拷贝2188个/)。每个重叠群中EST的数量在2～641个,平均为10.34个。所有转录本经BlastX在NCBI的非冗余蛋白库进行比较分析发现,有858条ESTs可以找到其同源序列/(E-value<10?4/)。 最初的注释结果表明:1.7/%的ESTs为蛋白酶,5.2/%的ESTs为核糖体蛋白,6.9/%的ESTs为线粒体转录子。相似的序列用物种进行分类,我们发现:果蝇/(20.05/%/),小鼠/(8.51/%/),大鼠/(8.04/%/),人/(7.58/%/),爪蟾/(3.96/%/)和斑马鱼/(3.96/%/)。转录子的注释结果显示,在4433个EST中有629个单拷贝,占所以EST的14.2/%;通过Gene Ontology的生物功能分类发现有292个单拷贝序列可能与盐度调控有关,占整个单拷贝的12/%。这些候选基因大致可进一步划分为7个功能类别:抗氧化活性/(5.4/%/),转运蛋白/(15.8/%/),胁迫相关蛋白/(11.0/%/),信号转导/(13.7/%/),蛋白合成/(22.4/%/),转录/(13.7/%/)以及新陈代谢与能量代谢/(17.5/%/)。 利用T3,T7通用引物,通过PCR扩增获得2426个uni基因。纯化后的PCR扩增产物送至博奥生物有限公司,用于cDNA芯片制备。每个PCR产物在芯片上重复三次。分别提取10‰、25‰、40‰三组样本各3只蟹的总RNA,分别命名为:G-10、G-25与G-40。我们一共杂交4张芯片:将G-10与G-25杂交,并且进行cy3与cy5荧光交换;另外,将G-40与G-25杂交,并且进行cy3与cy5荧光交换,从而分别获得低盐//高盐与对照条件下差异表达的基因。 通过cDNA芯片杂交我们一共筛选获得了417个显著差异表达的基因。利用cluster3.0来分析来进行不同盐度条件下,差异表达基因的聚类分析,结果显示:这些差异表达的基因可以分成8个cluster,每个Cluster具有不同的基因表达模式。统计发现:低盐条件下/(10‰养殖水体/),158个基因的表达量呈现显著上调,54个基因的表达量呈显著下调;高盐条件下/(40‰养殖水体/),123个基因的表达量显著上调,222个基因的表达量显著下调。 从中挑取用于芯片杂交的14个基因,利用实时荧光定量RT-PCR技术,来检测不同盐度情况下/(10‰、25‰、40‰/),该基因的表达水平,并与芯片杂交的实验结果相对比,以此来验证芯片杂交实验结果。结果表明:12个基因检测结果基本与芯片杂交结果保持一致。 The swimming crab, Portunus trituberculatus /(Crustacea: Decapoda: Portunidae: Portunus/) is a commercially important species, widely distributed along the coast of China. It is a euryhaline crab species and can survive in wide-range salinity conditions, but different water salinity conditions might influence its distribution and migration rout. The swimming crab natural resources were declinied significantly due to overfishing in the past several decades. The artificial propagation of this species was promoted along the coastal waters, and now the swimming crab became the most important farmed marine crab in China. Salinity is an important factor in the crab artificial propagation. There were lots of literatures regarding on the osmotic regulation physiological mechanisms of euryhaline crab so far, while the researches on molecular mechanism was quite few. To clarify the molecular mechanism of osmotic regulation has great significance on the improvement of germplasm resources and artificial propagation of P.trituberculatus. The screening of osmo-regulation related genes is the necessary prerequisite of the study on the mechanism of osmotic regulation. In our study, full-length cDNA library was constructed from gills of the swimming crab, Portunus trituberculatus. 5012 clones randomly picked out from cDNA library for sequencing, resulted in 4433 high quality ESTs with the average length of 748bp. All the ESTs were assembled using CAP3 software,producing 2426 potential transcripts /(238 contigs and 2188 singlets/). The number of ESTs per contig is between 2 to 641, with an average of 10.34 ESTs. The length of contigs is between 293 and 1356bp, with an average length of 796bp. All transcripts were compared to non-redundant protein database of NCBI using BLASTx, and homologous sequences can be found for 858 ESTs/(E-value<10?4/). The results of primary annotation indicated that protease /(1.7/%/), ribosomal protein /(5.2/%/) and mitochondrium /(6.9/%/). Similar sequences were classified by species, we found that Drosophila melanogaster /(20.05/%/), Mus musculus /(8.51/%/), Rattus norvegicus /(8.04/%/), Homo sapiens /(7.58/%/), Xenopus aevis /(3.96/%/) and Danio rerio /(3.96/%/). The functional annotation of the P. trituberculatus transcripts resulted in identification of about 292 unique sequences /(629 ESTs/) involved in salinity adaptation, accounted for about 12.0/% /(292//2426/) of total unique sequences and 14.2/% /(629//4433/) of the total ESTs. Some of them were identified according to GO classifications; others were determined based on the published literature on the genes related to salinity adaptation. These candidate genes were further grouped into at least seven function categories: reactive oxygen scavengers /(5.4/%/), transporter protein /(15.8/%/), stress protein /(11.0/%/), signaling transduction /(13.7/%/), protein synthesis and destination /(22.4/%/), transcription /(13.7/%/), and metabolism and energy /(17.5/%/). By using T3 and T7 primers, 2426 uni-genes were amplified by PCR and the purified PCR amplification products were used for preparation of cDNA array /(CapitalBio Corporation/). Each product was repeated three times on the array. Total RNA Were extracted from three groups of samples /(10‰, 25‰, 40‰/) and named as G-10, G-25 and G-40 respectively. Total RNA was used to prepare the fluorescent dye labeled cDNA using the linear mRNA amplification procedures were performed by CapitalBio Corporation. Then the RNA samples of treatment and control were labeled with either fluorochromes cyanine-3 /(Cy3/)-dCTP or cyanine-5 /(Cy5/)-dCTP. Analyses were performed twice per sample, using a dye-swapping experiment /(technical replicate/) in which cDNA from the control /(G-25/) was labeled with Cy3 and cDNA from low salinity /(G-10/) or high salinity /(G-40/) challenge was labeled with Cy5. In the second analysis, control cDNA was labeled with Cy5 and cDNA from salinity challenge was labeled with Cy3. This dye reversal helps to minimize error due to fluor-associated bias. Therefore, we could obtain differentially expressed genes between low-salinity//high salility and control conditions separately. We obtained a total of 417 significantly differentially expressed genes by cDNA microarray screening. The differentially expressed genes were grouped using hierarchical cluster analysis, and eight different clusters were defined. Each Cluster has different gene expression profile.The response to low salinity /(10‰/) was a dramatic up-regulation of 158 genes and a down regulation of 54 genes. And the response to high salinity /(40‰/) was a dramatic up-regulation of 123 genes and down regulation of 222 genes. Interestingly, 5 genes showed concomitant up-regulation whereas 10 down-regulation in both low and high salinity challenges. 130 genes showed opposite direction of regulation responding to the two different salinity challenges. The expression levels of 14 genes were analyzed by real time quantitative RT-PCR at different salinity conditions /(10‰, 25‰, 40‰/) to demonstrate the results of array hybridization. The results indicated that 12 of 14 genes were consistent with array analyzed.

关 键 词： 三疣梭子蟹 盐度调控 文库 基因芯片 实时荧光定量

分 类 号： [TS2 S96]

领　　域： [轻工技术与工程] [农业科学] [农业科学]