【作者】 王兵；

【导师】 相建海；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2003， 博士

【摘要】 因为中国对虾比较显著的性别二态性可以给生产带来巨大利益,性控研究一直是国际同行研究的热点与难点,对对虾性别相关核酸片段的研究不但具有广阔的应用价值,而且还可以为进一步的研究提供理论依据和指导; 由于对对虾抗病机制了解的甚少,对虾养殖业因病害大规模爆发而损失惨重,因而免疫基因的研究有助于弄清对虾的抗病机理,为对虾病害的预防和控制奠定基础。本研究运用差异显示技术,筛选了中国对虾性别相关核酸片段; 利用cDNA文库和消减文库、EST大规模测序、基因芯片和生物信息学等技术手段,对中国对虾免疫相关基因进行研究,获得了一些重要进展,结果如下: 1.通过双随机引物差异显示技术,筛选到了一条中国对虾性别相关核酸片段,该片段大小为±500bp。在PCR反应条件相同的情况下,该片段表现为在雌性个体的扩增产物要多于雄性个体。将该片段克隆后进行了测序,并根据测序结果设计了特异性PCR扩增引物。该片段的差异在DNA和RNA中都可以检测出,可以作为探针为性别控制技术提供快速检测手段。2.构建了中国对虾头胸部组织的cDNA文库并首次对中国对虾EST进行大规模的测序,共获得EST 10446条,拼接后得到3120条无冗余基因片段。在中国对虾EST数据的分析中,通过功能注释,共获得了1373条已知基因,其余1747条unique基因为未知基因。从EST数据中挖掘出微卫星324个,约占总EST数目的3%,并对中国对虾的新陈代谢途径、基因功能分类等重要生物学信息进行了分析。3.构建了中国对虾WSSV感染前后的正、反向抑制性消减杂交cDNA文库(正向库容为3.7e×10~4,反向文库为1.2e×10~4)。获得了相当数量的分别在正常和感染虾体内特异表达的基因,为抗WSSV基因的筛选奠定了基础。4.利用已知序列的EST和对虾消减文库中的克隆,构建了中国对虾表达谱芯片。该芯片共有待研究cDNA片段3114个,其中1578个克隆来自EST数据中的unique基因,1536个克隆来自消减文库(正、反向文库各768个)。5.利用cDNA芯片杂交技术,对WSSV感染后6小时组织和濒死对虾组织进行了研究,筛选与抗WSSV病毒相关的基因。经数次实验结果叠加后,来自更多还原

【Abstract】 Sex control research of this species is one of most concerned one by scientists because the sex dimorphism of Chinese shrimp can provide great benefit for shrimp culture industries. The investigating of sex-linked markers not only provides help to the culture industry but also facilitate the research of the shrimp sex determination. Outbreak of the WSSV disease makes very serious lost every year. The poor knowledge of shrimp immune system block the prevent and research of WSSV disease. So the immune function related genes are very important to the further research of shrimp diseases. In this study, technologies of DDRT, cDNA library, subtracted suppressed hybridized cDNA libraries, large scale sequencing of ESTs, gene chip and bioinformatics were used. Some important progresses were acquired: 1. Through the DDRT technology, one sex linked DNA sequence (±500bp) was cloned. This fragment had high expression level in the female than the male under the same PCR conditions. The expression pattern of this DNA fragment indicates that the sex determination of the Chinese shrimp is heterogametic. The DNA fragment was cloned and sequenced. Special primers were designed. Problem discovered in the experiment can be used as reference in the further work. The specific primers of the sex linked DNA fragment can service as quick sex detection method for the Chinese shrimp. 2.cDNA library of the cephalothorax of Chinese shrimp was constructed and sequenced, 10446 ESTs were obtained and aligned into 3120 unique genes. Those unique genes were analyzed by Blast against NCBI database, a total of 1373 contigs/singletons were known genes, and the remaining 1747 ESTs were putative novel genes. To analyze the gene expression patterns of Fenneropenaeus chinesis, ESTs matching known genes were categorized into different functional groups according to the categorized KEGG GO TREE. Total numbers of 324 SSRs were found in this ESTs database by motif search. Metabolism pathway and function classification of the unique genes were also surveyed. All the information above will provide useful information for the future research. 3.Reverse and forward cDNA suppressed subtraction hybridization libraries of WSSV infected and normal tissues were constructed. The forward library had a titer of 3.7e x104 and the reverse library had a titer of 1.2ex104. 4. A total of 3114 clones from ESTs and SSH libraries of Chinese shrimp were selected to spot on to the microarray, 1578 from ESTs and 1536 from SSH libraries (768 each). 5.The chips were used to test the different gene expression of 6 hrs post WSSV challenge and moribund shrimp of WSSV naturally infected. Total numbers of 109 clones had different expression after WSSV infection, 51 of them were unique genes of ESTs, and 58 of them were from clones of SSH libraries. Twenty-six of the 51 clones were known genes (22 up-regulated, 4 down-regulated). The 58 clones from SSH libraries were sequenced and blasted with NCBI databases, 46 clones are known genes (27 up-regulated, 19 down-regulated). Some expressive rules were figured out: · We found that most clones, which expressed in high level at 6 hrs post WSSV challenge are immune related; Most of the clones that expressed in high level in the tissues of moribund shrimp are related to the WSSV infection. · The detection of WSSV invasion cooperative genes may help us to know the control and infection system of the WSSV, such as larking RNA binding protein, ubiquin and EF-1 alpha. The housekeeping genes were also found terminated in the moribund tissues. · The anti-disease related genes, such as HSPs, trehalose and aldo-keto reductases etc, may play very important role in the process of anti-WSSV disease for the shrimp. And their expression pattern was firstly found, which is helpful to understand the immune systems of the Chinese shrimps. · We also found the antibacterial peptides, peritrophin like protein, etc. may have close relationship with the WSSV disease infection, and WSSV may block the expression of the cuticle proteins of the shrimp. The fact that many different expression genes were found in this experiment means the microarray technology could be very helpful in the research of shrimp defense system.更多还原