Cloning,Southern blotting and RT-PCR analysis of a cDNA fragment encoding of 70 kDa heat shock cognate protein (Hsc70) from the oyster (Crassostrea ariakensis)
ZHANG Qi Zhong ①② WU Xin Zhong ①** GAO Jin Song ③ PAN Jin Pei ① (① South China Sea Institute of Oceanology, The Chinese Academy of Sciences, Guangzhou 510301, China) (② College of Life Science, Southwest China Normal University, Chongqing 400715, China) (③ Da'An Gene Diagnostic Center of Sun Yat Sen University, Guangzhou 510089, China )
In order to elucidate the molecular mechanisms of the oyster (Crassostrea ariakensis) against adverse stimulating factors, we cloned and sequenced a partial cDNA encoding a 70 kDa heat shock cognate protein (Hsc70) from the oyster. The live oysters were obtained from Chengcun, Yangxi County, Guangdong Province, China. Various tissues, including mantle, gills, adductor muscle, heart and blood cells, were respectively collected from 5 untreated live oysters or treated ones at 36℃ for 1 5 hours, and immediately frozen in liquid nitrogen except for the blood cells which were suspended with Trizol Reagent after centrifugation ( 12 000 r/min for 30 s) and stored at -20℃. Total RNA was isolated using Trizol Reagent according to the manufacture's instructions. The first strand cDNA was synthesized using reverse transcriptase Superscript Ⅱ according to the manufacture's instructions. The primers were designed from a conserved region of C. gigas Hsc70 cDNA sequence (GeneBank accession No. AF144646). The polymerase chain reaction (PCR) was performed for 30 cycles with denaturation at 94℃ for 30 s, annealing at 49℃ for 40 s, and elongation at 72℃ for 30 s. The product was cloned to pGEM T easy vector and sequenced. It is 509 base pairs (bp) and possesses 94% identity with the cDNA encoding C. gigas Hsc70 using Blastn. This homology was strongly confirmed by amino acid sequence comparison using the Blastx (99%). The 509 bp fragment was labeled with α 32 pdCTP and a random primer DNA labeling kit and employed as a probe to perform Southern blotting, the result demonstrated that the cDNA came from a partial mRNA transcript of C. ariakensis genomic DNA gene. The polymerase chain reaction (PCR) was carried out to investigate the expression of Hsc70, Using the cDNAs of several tissues, such as gills (heat shocked), mantle, adductor muscle (heat shocked), heart, blood cells (one sample with heat shock for 1 5 hours at 36℃ and another without any stimulus). The PCR results revealed that Hsc70 transcripts could be detected in all the tissues analyzed and greatly increased in the tissues with heat shock. The results showed that the Hsc70 is ubiquitously and constitutively expressed but can be stimulated by heat shock. All the facts above firmly established that the cloned cDNA fragment was a part of the cDNA encoding a Hsc70 protein in the oyster C. ariakensis .
【Fund】： 国家自然科学基金(No .3 9970 5 81);; 国家自然科学基金农业倾斜项目(No .3 0 170 741);; 中国科学院知识创新工程重要方向项目(KSCX2 SW 3 0 2 8);; 广东省科技创新百项工程资助项目(No .99B0 62 0 1G)~~
【CateGory Index】： Q78
【CateGory Index】： Q78