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题名: 近江牡蛎Hsc70蛋白基因cDNA片段的克隆及Southern杂交和RT-PCR分析
作者: 张其中 ; 吴信忠 ; 高劲松 ; 潘金培
关键词: 近江牡蛎 ; Hsc70蛋白基因 ; cDNA片段 ; 克隆 ; Southern杂交 ; RT-PCR分析
刊名: 动物学报
发表日期: 2003-01-01
卷: 49, 期:5, 页:708-712
部门归属: [1]中国科学院南海海洋研究所,广州510301 [2]中山大学达安基因诊断中心,广州510089
项目归属: 海洋生物
摘要: 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 3612 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 30s. 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-^32pdCTP 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 investi-gate 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 .
内容类型: 期刊论文
URI标识: http://ir.scsio.ac.cn/handle/344004/7431
Appears in Collections:广东省海洋药物重点实验室_期刊论文

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Recommended Citation:
张其中;吴信忠; 高劲松;潘金培.近江牡蛎Hsc70蛋白基因cDNA片段的克隆及Southern杂交和RT-PCR分析,动物学报,2003,49(5):708-712
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文件名: 近江牡蛎Hsc70蛋白基因cDNA片段的克隆及Southern杂交和RT-PCR分析.pdf
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