The Study Of Immune Function Related Genes And Sex Linked Nucleic Acid Marker Of Chinese Shrimp (Fenneropenaeus Chinensis)

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.