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) fr...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 .展开更多
目的:探讨慢性浅表性胃炎不同证型与胃黏膜水通道蛋白3、4基因表达的相关性.方法:胃镜下取胃体上部黏膜,液氮罐保存,荧光定量PCR法检测胃黏膜AQP3、AQP4的基因表达.结果:脾胃湿热证组AQP3、AQP4高于胃阴不足证组和正常人组(4.5980±...目的:探讨慢性浅表性胃炎不同证型与胃黏膜水通道蛋白3、4基因表达的相关性.方法:胃镜下取胃体上部黏膜,液氮罐保存,荧光定量PCR法检测胃黏膜AQP3、AQP4的基因表达.结果:脾胃湿热证组AQP3、AQP4高于胃阴不足证组和正常人组(4.5980±0.8234 vs 3.4362±0.3450,3.8495±0.5072,7.7062±0.6859 vs 6.800±0.5544,7.0384±0.6706;P<0.051,脾虚湿困证组、寒湿困脾证组AQP3均高于胃阴不足证组(4.5158±0.5603,4.8083±0.8419 vs 3.4362±0.3450,P<0.05).脾胃湿热证组、脾虚湿困证组、寒湿困脾证组3组间AQP3、AQP4比较没有明显差异(P>0.05).胃阴不足证组和正常人组间AQP3、AQP4比较也没有明显差异(P>0.05).结论:慢性胃炎中医证型不同,胃黏膜AQP3、AQP4基因表达不同,胃黏膜AQP3、AQP4可能成为脾虚湿困、寒湿困脾、脾胃湿热、胃阴不足等病证的发生机制之一.展开更多
文摘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 .
文摘目的:探讨慢性浅表性胃炎不同证型与胃黏膜水通道蛋白3、4基因表达的相关性.方法:胃镜下取胃体上部黏膜,液氮罐保存,荧光定量PCR法检测胃黏膜AQP3、AQP4的基因表达.结果:脾胃湿热证组AQP3、AQP4高于胃阴不足证组和正常人组(4.5980±0.8234 vs 3.4362±0.3450,3.8495±0.5072,7.7062±0.6859 vs 6.800±0.5544,7.0384±0.6706;P<0.051,脾虚湿困证组、寒湿困脾证组AQP3均高于胃阴不足证组(4.5158±0.5603,4.8083±0.8419 vs 3.4362±0.3450,P<0.05).脾胃湿热证组、脾虚湿困证组、寒湿困脾证组3组间AQP3、AQP4比较没有明显差异(P>0.05).胃阴不足证组和正常人组间AQP3、AQP4比较也没有明显差异(P>0.05).结论:慢性胃炎中医证型不同,胃黏膜AQP3、AQP4基因表达不同,胃黏膜AQP3、AQP4可能成为脾虚湿困、寒湿困脾、脾胃湿热、胃阴不足等病证的发生机制之一.
