Studies On Relevant Genes Involved In Immune Response To WSSV Challenge In Chinese Shrimp Fenneropenaeus Chinensis

White spot syndrome is a highly contagious viral disease caused by white spot syndrome virus (WSSV), which resulted in severe economic losses to the shrimp farming industry since its first report in Taiwan in 1993. Until present, there are still no effective ways to cure or completely prevent the prevalence of WSSV. After years of farming practices and WSSV challenge experiments, it showed that there were some anti WSSV infection genetic mechanism, but the specific mechanism needs further explanation. So relevant genes involved in immune response to WSSV challenge in Chinese shrimp Fenneropenaeus chinensis were studied to provide data for the prevention and control of diseases caused by WSSV. In this study, the Chinese shrimp new variety "Huanghai No.2" which showed better resistance to WSSV in farming practice was used as specific material. Genes involved in immune response to WSSV challenge were screened by DDRT-PCR. The five genes, chitin binding protein (FcCBP),β-1,3-glucan binding protein (FcβGBP-HDL), transcription repressor (FcTR), C-type lectin (FcCTL) and glutamine synthetase (FcGS) which might play important roles in the response to WSSV challenge were cloned. Their characteristics, possible functions and expression patterns were also studied. The results are summarized as follows:1. Forty-six positive differential fragments were recovered by DDRT-PCR. Then sequences of the positive differential fragments were determined and blasted in GenBank. Most of them were found to encode proteins related to oxidative phosphorylation, protein synthesis, glycolysis and signal transduction.2. The FcCBP gene which encoded a protein with a chitin-binding domain type 2 in the C-terminal domain was cloned from F. chinensis by using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of FcCBP showed 34% identity with Nasonia vitripennis chitin-binding protein. The genomic DN A of FcCBP was composed of five exons and four GT-AG introns. Quantitative realtime PCR analysis revealed the presence of FcCBP transcripts in all experiment tissues with the maximum level in intestines. The expression of FcCBP mRNA in hepatopancreas was up-reguleted sharply at 0-6 h post WSSV challenge, then down-regulated. The expression in gills was up-regulated at 0-12 h, then drop back. The results showed that, FcCBP was emergency induced in response to WSSV challenge and might prevent the WSSV further challenge at the the first line of defense.3. The full length cDNA of FcβGBP-HDL cloned in this study was of 6713 bp encoding a polypeptide with an estimated molecular mass of 240.7 kDa which was the largestβGBP molecular mass reported, one RGD motif and two glucanase-like motifs without catalysis activity. The deduced amino acid sequence of FcβGBP-HDL showed 88% identity with Litopenaeus vannameiβGBP-HDL. There were many binding sites involved in the regulation of immune gene's transcriptions in other arthropods, such as two GATA-1, three AP-1 and one c-Ets-1. Quantitative realtime PCR analysis reveals the presence of FcβGBP-HDL transcripts in all experiment tissues with the maximum level in muscles. The expression of FcβGBP-HDL mRNA in hepatopancreas was down-regulated sharply at 0～6 h post WSSV challenge and then up-regulated, while in gills was up-reguleted sharply at 0～6 h post WSSV challenge and then drop back. It was speculated that FcβGBP-HDL was involved in shrimp immune responses as a glucan receptor, and promotes the defense response as an induced sensitive gene.4. The FcTR gene encoding protein of 354 amino acids with four ZnF_C2H2 domains and two SFP1 domains in the C-terminal domain was the first cloned transcriptional repressor gene from F. chinensis. The deduced amino acid sequence of FcTR showed 68% identity with Branchiostoma floridae transcription repressor. The genomic DNA of FcTR was composed of four exons and three GT-AG introns. Quantitative realtime PCR analysis revealed the presence of FcTR transcripts in all experiment tissues with the maximum level in intestines. The expression of FcTR mRNA in hepatopancreas was down-reguleted sharply at 0～6 h post WSSV challenge and then up-regulated, while in gills was up-regulated sharply at 0～6 h post WSSV challenge with the climax at 24 h and then down-regulated. The results suggested that FcTR might close some genes related to WSSV challenge to prevent the WSSV further challenge.5. A novel C-type lectin which showed only 34% identify with C-type lectin reported and encoded a polypeptide with one carbohydrate-recognition domain in the C-terminal domain is cloned from F. chinensis. The full length genomic DNA of FcCTL was composed of five exons and four GT-AG introns, and there were three CT type microsatellites in intron3 and CA type and GT type microsatellites in intron4. Unlike other C-type lectins only transcripted in hemocytes or hepatopancreas, FcCTL was transcripted in all experiment tissues in F. chinensis WSSV unchallenge. The expression of FcCTL mRNA in hepatopancreas was up-reguleted at 0-24 h post WSSV challenge with the climax at 24 h and then drop back, while in gills was up-regulated at 0-6 h with the climax at 6 h, then down-regulated. The results suggested FcCTL might play more important role than other lectins and could resist the invasion of pathogens in broader tissues.6. FcGS was the first glutamine synthetase gene cloned from F. chinensis. The full length cDNA of FcGS was of 1444 bp encoding a polypeptide of 366 amino acids with one GS beta-Grasp domain in the N-terminal domain and one GS catalysis domain in the C-terminal domain. The deduced amino acid sequence of FcGS showed 87% identity with Panulirus argus glutamine synthetase. The full length genomic DNA of FcGS was composed of two exons and one GT-AG intron. Quantitative Realtime PCR analysis revealed the presence of FcGS transcripts in all experiment tissues with the maximum level in muscles. The expression of FcGS mRNA in hepatopancreas was up-regulated at 0-48 h post WSSV challenge with the climax at 48 h, in gilles was up-reguleted at 0-6 h post WSSV challenge and then down-regulated. The results indicated that FcGS might be involved in the immune responses to WSSV challenge.These results suggested that the five genes cloned from F. chinensis in this study had domains related to immune response, and modified their expression corresponding to various tissues post WSSV challenge. So they might be a part of the genes involved in immune response to WSSV challenge. It was also found in this study that the trends of expression modification post WSSV challenge among the five genes were amazing similar:All of them were upregulated sharply in gills at 0-6 h post WSSV challenge and then down-regulated, and up-regulated in hepatopancreas as a whole. Thus, both gills and hepatopancreas might be the main tissues, but played different roles in immune response to WSSV challenge:gills was emergency indeuced as the first line of defence, however hepatopancreas was delayed induced as the main tissue to produce humoral immune molecules. The further studies were needed to clarify the immune response mechanism of these genes to WSSV challenge in F. chinensis.