Molecular cloning of clottable protein (CP) of the white shrimp Litopenaeus vannamei and its transcription under extrinsic influences

• Main Authors: Chiang, Pei-Chi, 姜佩琪
• Other Authors: Cheng, Winton
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/69445790873863584126

碩士 === 國立屏東科技大學 === 水產養殖系 === 95 === Blood coagulation is an essential immune mechanism for aquatic animals with an open circulatory system such as crustaceans. This research is aimed to comprehend the molecular characteristics of the clottable protein in white shrimp, Litopenaeus vannamei, through molecular cloning of its cDNA and tissues expression, profiles. The expression of CP transcripts under different temperature, salinity and ammonia and post-Vibrio alginolyticusc injection were also investgated. CP cDNA was obtained from the heart of the white shrimp by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA using oligonucleotide primers based on the CP sequence of tiger shrimp, Penaeus monodon, and freshwater crayfish, Pacifastacus leniusculus. The full-length cDNAs of CP consisted of 5495 bp open reading frame encode 1666 amino acids (aa) with predicted include a 14-aa signal peptide, molecular mass of the mature protein was 187.917 kDa with an estimated pI of 5.27. Two putative integrin binding motif (cell adhesion site), RGD (Arg-Gly-Asp) and three N-glycosylation site were conserved in CP of white shrimp. Sequence comparison showed that CP deduced aa of L. vannamei had overall similarities of 85% and 37% to that of P. monodon and P. leniusculus respectively. Shrimp CP was found to exist in haemocyte, heart, gill, muscle, hepatopancreans, lymph organ and intestine by RT-PCR and real-time PCR. The clotting time of V. alginolyticus injection shrimps are significant longer than un-injection ones at 12 and 24 hours. At 22 and 34 ℃ water temperature for 2 days, the clotting time is significant longer than 28 ℃ treatment, moreover, after 7 days, it is significant shorter at 22 ℃treatment and significant longer at 34 ℃compared to 28 ℃. The clotting time in salinity 35 and 45 ‰ treatment for 2 and 7 days is significant longer than other treatments. When white shrimps expose to 5 and 10 mg l-1 ammonia-N for 7 days, the clotting time is significant longer than 1 mg l-1 and control groups. In V. alginolyticus challenge tests, shrimp CP expression in haemocyte, hepatopancrans and muscle were no significantly, heart CP was sharply increased post 24 h injection, but gill CP and intestine CP expression were down-regulation post 6 and 3 h, respectively, and return to initial levels at 24 and 24 h, respectively. Shrimp were exposed to 22 oC and 34 oC water for 2 days, hemocyte CP of shrimp at 34 oC was higher than shrimp at 22 oC, but both of them were higher than control shrimp at 28 oC. Heart CP and hepatopancreans CP were significantly raised when shrimp to be placed in 34 oC wate for 2 days, but return to initial levels after 7 days. Gill CP expression was significantly decreased after 2 days exposure to 34 oC water, and significantly increased after 7 days. The increment of CP expression was detected in muscle and intestine of shrimp at 34 oC for 2 days, but the graduals decrease of CP expression was detected in muscle of shrimp at 34 oC for 7 days. In salinity trials, heamocyte CP expression was lower than others treatments at 45 ‰ for 2 day, but return to initial levels post 7 days exposure to 45 ‰. Heart CP was down-regulation at 5 ‰ salt water for 2 days, but return to initial levels after 7 days. Shrimp at 15 ‰ salt water for 2 day, its gill CP expression was significantly increased, and a recovery was detected at 7 days. The expression of hepatopancreans CP was increase significantly at 35 and 45‰ for 7 days, and no significant different expression of CP was measured in muscle and intestine. Heamocyte CP expression was significantly increased after exposure to 1, 5 and 10 mg l-1 ammonia-N for 7 days, but no significantly different was found in the tissues of heart, gill and intestine. Up-regulation of CP was detected in shrimp at 1, 5 and 10 mg l-1 ammonia-N for 2 days when compared with control group. Muscle CP of shrimp at 10 mg l-1 ammonia-N for 2 days was increased significantly, and similar up-regulation was also to be found in muscle CP of shrimp at 5 and 10 mg l-1 ammonia-N for 7 days.