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
旨在探讨热应激对H9c2心肌细胞构成型HSP70(constitutive or cognate HSPs,Hsc70)出入核及细胞凋亡的影响。以42℃作为热应激模型温度,通过转染Hsc70siRNA抑制Hsc70表达,Western blot检测细胞质和细胞核内Hsc70表达,ELISA检测细胞培养液...旨在探讨热应激对H9c2心肌细胞构成型HSP70(constitutive or cognate HSPs,Hsc70)出入核及细胞凋亡的影响。以42℃作为热应激模型温度,通过转染Hsc70siRNA抑制Hsc70表达,Western blot检测细胞质和细胞核内Hsc70表达,ELISA检测细胞培养液LDH浓度,Annexin V-FITC/PI双染法检测细胞凋亡。结果表明,正常H9c2心肌细胞质Hsc70表达量较高,细胞核中表达量很低,细胞质和细胞核Hsp72表达量非常低。热应激后细胞质Hsc70表达量无显著差异,而细胞核Hsc70热应激30和100min后极显著升高(P<0.01),热应激240min后开始降低;细胞质和细胞核Hsp72热应激后显著升高(P<0.05或P<0.01)。Hsc70抑制表达后,细胞质Hsc70水平显著降低,热应激后Hsc70入核明显减少,但仍然有入核现象;Hsc70抑制表达对细胞质和细胞核Hsp72表达无显著影响。与热应激组相比,热应激+Hsc70siRNA组LDH表达量呈升高趋势,热应激100min两组出现显著差异(P<0.05);Hsc70抑制表达后H9c2细胞在热应激后更容易发生凋亡,而且在热应激30和100min内,两组之间存在显著差异(P<0.05)。结果提示,热应激可诱使Hsc70出入细胞核,Hsc70抑制表达后热应激诱导Hsc70入核显著降低,细胞损伤加重,细胞凋亡升高。展开更多
组成型热休克蛋白70-4(heat shock protein 70 cognate 4,HSC70-4)是HSP70家族的重要成员,对蛋白质的正确折叠与转运有着重要意义。本研究以中华蜜蜂转录组数据中获得的HSC70-4基因序列为基础,通过对中华蜜蜂不同发育阶段、不同组织以...组成型热休克蛋白70-4(heat shock protein 70 cognate 4,HSC70-4)是HSP70家族的重要成员,对蛋白质的正确折叠与转运有着重要意义。本研究以中华蜜蜂转录组数据中获得的HSC70-4基因序列为基础,通过对中华蜜蜂不同发育阶段、不同组织以及不同低温胁迫下的HSC70-4 m RNA表达量进行测定,以期为揭示该基因在中华蜜蜂生长发育和耐寒抗冻过程中的生理功能提供理论依据。结果显示,中华蜜蜂HSC70-4基因包含1923 bp的开放阅读框,编码641个氨基酸,蛋白分子量为70.4 k Da。中华蜜蜂HSC70-4氨基酸序列中包含3个HSP70家族的标签序列,其N端含有HSC70家族的GGXP四肽结构标志,C端包含EEVD结构。与膜翅目其它昆虫的氨基酸序列一致性在94%以上,具有较高的保守性。中华蜜蜂HSC70-4在成虫期的表达量显著高于幼虫期和蛹期(P<0.01),从幼虫期至10日龄成虫期呈逐渐上升趋势,但在15日龄至30日龄间呈现波动起伏。HSC70-4在中华蜜蜂不同组织中的表达存在显著差异(P<0.01),且在胸部高度表达,在足中中度表达,在其余组织中低度表达。中华蜜蜂HSC70-4的表达受低温胁迫的诱导,在低温胁迫2 h时其表达量最低,4 h时表达量最高。本研究结果表明中华蜜蜂HSC70-4在中华蜜蜂的生长发育过程中应对低温胁迫时发挥生理功能。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 .
文摘组成型热休克蛋白70-4(heat shock protein 70 cognate 4,HSC70-4)是HSP70家族的重要成员,对蛋白质的正确折叠与转运有着重要意义。本研究以中华蜜蜂转录组数据中获得的HSC70-4基因序列为基础,通过对中华蜜蜂不同发育阶段、不同组织以及不同低温胁迫下的HSC70-4 m RNA表达量进行测定,以期为揭示该基因在中华蜜蜂生长发育和耐寒抗冻过程中的生理功能提供理论依据。结果显示,中华蜜蜂HSC70-4基因包含1923 bp的开放阅读框,编码641个氨基酸,蛋白分子量为70.4 k Da。中华蜜蜂HSC70-4氨基酸序列中包含3个HSP70家族的标签序列,其N端含有HSC70家族的GGXP四肽结构标志,C端包含EEVD结构。与膜翅目其它昆虫的氨基酸序列一致性在94%以上,具有较高的保守性。中华蜜蜂HSC70-4在成虫期的表达量显著高于幼虫期和蛹期(P<0.01),从幼虫期至10日龄成虫期呈逐渐上升趋势,但在15日龄至30日龄间呈现波动起伏。HSC70-4在中华蜜蜂不同组织中的表达存在显著差异(P<0.01),且在胸部高度表达,在足中中度表达,在其余组织中低度表达。中华蜜蜂HSC70-4的表达受低温胁迫的诱导,在低温胁迫2 h时其表达量最低,4 h时表达量最高。本研究结果表明中华蜜蜂HSC70-4在中华蜜蜂的生长发育过程中应对低温胁迫时发挥生理功能。
