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高劲松

作品数:1 被引量:15H指数:1
供职机构:中山大学达安基因诊断中心更多>>
发文基金:中国科学院知识创新工程重要方向项目广东省科技创新百项工程项目国家自然科学基金更多>>
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近江牡蛎Hsc70蛋白基因cDNA片段的克隆及Southern杂交和RT-PCR分析被引量:15
2003年
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
张其中吴信忠高劲松潘金培
关键词:近江牡蛎CDNA片段克隆SOUTHERN杂交RT-PCR分析
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