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Cloning and Expression Analysis of Scallop HSP22 Gene and Its Association with Heat Stress Resistance

Author: ZhangLei
Tutor: YangGuanPin;SongLinSheng
School: Ocean University of China
Course: Marine biology
Keywords: Chlamys farreri Argopecten irradians heat shock protein 22 heavy metal Vibrio anguillarum heat stress real-time PCR single nucleotide polymorphism
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 218
Quote: 5
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Abstract


Mollusc culture is a traditional marine aquaculture industry in China. It plays an important role in the economic development of the costal provinces. But since 1990s, scallop aquaculture in China has been experiencing a large-scale mortality, and the culture industry suffered from a major great loss. The current large-scale mortality problem may result from the interaction of many different factors, such as foreign phathogens, environmental factors, and mollusc immunity. Many researches have been carried out focused on the pathogens isolation and diagnosis, culture environment improvement, and culture technique optimization, and a lot of progress has been achieved. In the present study, large scale EST sequencing method together with RACE technique was used to isolate and clone two full length cDNA of heat shock protein 22 from two species of scallop, Chlamys farreri and Argopecten irradians. The expression patterns of AiHSP22 mRNA in different tissues and in haemocytes of scallops exposed to Cd2+, Pb2+ or Cu2+ were investigated by real-time quantitative RT-PCR. And using the same method, the expression patterns of CfHSP22 mRNA in different tissues and in haemocytes of scallops challenged by Vibrio anguillarum were investigated. In addition, the nucleotide sequence polymorphisms in extron region of HSP22 gene from Zhikong scallop Chlamys farreri (designated as CfHSP22) were investigated to explore their association with susceptibility/resistance to heat stress. The results are as follows.The full-length cDNA of Argopecten irradians HSP22 (designated as AiHSP22) was 1112 bp, consisting of a 5-terminal untranslated region (UTR) of 51 bp, a 3′UTR of 473 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 588 bp encoding a polypeptide of 195 amino acids with the estimated molecular mass of 22.71 kDa and a predicted isoelectric point of 8.44. Analysis of the protein domain features indicated that the deduced amino-acid sequence of AiHSP22 contained anα-crystallin domain in its C-terminal half which was a hallmark of the subfamily of sHSP. The deduced amino acid sequence of AiHSP22 showed high similarity to previously identified HSP22s. The expression patterns of AiHSP22 mRNA in different tissues and in haemocytes of scallops exposed to Cd2+, Pb2+ or Cu2+ were investigated by real-time quantitative RT-PCR. The mRNA of AiHSP22 was constitutively expressed in all examined tissues, including haemocyte, muscle, kidney, gonad, gill and heart. The expression level in heart and muscle was higher than that in other tissues. The mRNA level of AiHSP22 in haemocytes was up-regulated after a ten-day exposure of scallops to Cu2+, Pb2+ and Cd2+. However, the expression of AiHSP22 did not increase linearly along with the rise of heavy metal concentration. The result also indicated that AiHSP22 was the least sensitive to Cd2+, compared with Cu2+ and Pb2+. The sensitive response of AiHSP22 to Cu2+, Pb2+ and Cd2+ stress indicated that it could be developed as an indicator of exposure to heavy metals for the pollution monitoring programs in aquatic environment.The full-length cDNA of Chlamys farreri HSP22 (designated as CfHSP22) was of 1279 bp, consisting of a 5-terminal untranslated region (UTR) of 122 bp, a 3’UTR of 581 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 576 bp encoding a polypeptide of 191 amino acids with the putative molecular mass of 22.21 kDa and the predicted isoelectric point of 9.69. There was anα-crystallin domain, hallmark of the sHSP subfamily, in the C-terminus of deduced amino-acid sequence of CfHSP22. The deduced amino acid sequence of CfHSP22 showed high similarity to previously identified HSP22s. The expression patterns of CfHSP22 mRNA in different tissues and in haemocytes of scallops challenged by Vibrio anguillarum were investigated by real-time quantitative RT-PCR. The mRNA of CfHSP22 was constitutively expressed in all examined tissues including haemocyte, muscle, kidney, gonad, gill, heart and hepatopancreas, and the expression level in hepatopancreas was higher than that in other tissues. The relative expression level of CfHSP22 transcript was up-regulated and reached maximal level at 12 h after bacterial injection, and then dropped progressively to the original level at 48 h post challenge. The results suggested that the involvement of CfHSP22 in responses against bacterial infection and further highlighted its functional importance in the immune system of Chlamys farreri.One site of single nucleotide polymorphism (SNP) was identified in the extron region of CfHSP22, +94C/A SNP was selected to analyze its distribution in the susceptible and resistant stocks, which was identified according to the survival time after 30℃heat stress. Using Bi-PASA PCR, three genotypes were found at each site, which were C/C, C/A and C/A at locus +94, respectively. The frequency of +94A/A and +94C/A genotype in the resistant stock were significantly higher than those in the susceptible stock (P<0.05), indicating a significant association with the resistance of Zhikong scallop to heat stress. To confirm the presumption, another independent challenge experiment was performed, in which the cumulative mortality of scallops with +94C/C genotype was extremely significant higher than those with +94A/A genotype (P<0.01), and +94C/C genotype was significantly higher than those with +94C/A genotype (P<0.05), those further validate the association between +94C/C、+94C/A genotypes and the resistance of Zhikong scallop to heat stress. These results suggested that the +94C/A could be a potential marker applied in future selection of Zhikong scallop with enhanced resistance to heat stress.

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