日本沼虾Bax基因克隆及其在低氧胁迫过程中的作用
Q785%S966.12; 为探究细胞凋亡相关基因Bax(B-cell lymphoma-2 associated X protein)在日本沼虾低氧胁迫过程中所发挥的作用,实验采用cDNA末端快速扩增(RACE)PCR技术获得日本沼虾细胞凋亡相关基因Bax的cDNA全长序列,采用半定量RT-PCR与实时荧光定量PCR(qPCR)分析其在日本沼虾不同组织、不同低氧阶段的表达情况,同时采用Western blot与免疫组化分析了低氧下日本沼虾Bax蛋白的表达与定位.日本沼虾Bax基因cDNA全长2 287 bp(NCBI 登录号:MZ823353),包括 5'非编码区(UTR)为 42...
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| Published in | 水产学报 Vol. 48; no. 8; pp. 65 - 78 |
|---|---|
| Main Authors | , , , , |
| Format | Journal Article |
| Language | Chinese |
| Published |
上海海洋大学,农业农村部淡水水产种质资源重点实验室,上海 201306%上海海洋大学,农业农村部淡水水产种质资源重点实验室,上海 201306
01.08.2024
上海海洋大学,水产种质资源发掘与利用教育部重点实验室,上海 201306 |
| Subjects | |
| Online Access | Get full text |
| ISSN | 1000-0615 |
| DOI | 10.11964/jfc.20220513497 |
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| Abstract | Q785%S966.12; 为探究细胞凋亡相关基因Bax(B-cell lymphoma-2 associated X protein)在日本沼虾低氧胁迫过程中所发挥的作用,实验采用cDNA末端快速扩增(RACE)PCR技术获得日本沼虾细胞凋亡相关基因Bax的cDNA全长序列,采用半定量RT-PCR与实时荧光定量PCR(qPCR)分析其在日本沼虾不同组织、不同低氧阶段的表达情况,同时采用Western blot与免疫组化分析了低氧下日本沼虾Bax蛋白的表达与定位.日本沼虾Bax基因cDNA全长2 287 bp(NCBI 登录号:MZ823353),包括 5'非编码区(UTR)为 42 bp,3'UTR 为 814 bp,开放阅读框(ORF)1 431 bp,编码476个氨基酸.通过软件和生物信息网站对其序列进行分析,氨基酸相似度比对显示,日本沼虾细胞凋亡基因Bax富含高度保守的BH1、BH2及BH3结构域.系统进化树分析显示,日本沼虾Bax基因与斑节对虾等甲壳动物Bax聚为一支,具有最近的亲缘关系.RT-PCR结果表明,日本沼虾Bax基因在肝胰腺中表达量最高,在脑中表达量最低.在低氧胁迫1～96h时,日本沼虾鳃和肝胰腺组织Bax基因表达量均显著高于对照组,这与日本沼虾Bax蛋白表达丰度基本相似.通过构建原核表达载体获得体外重组蛋白Bax,并将纯化重组蛋白免疫兔子获得抗血清.免疫组化结果显示,鳃和肝胰腺Bax蛋白阳性信号主要定位在鳃上皮细胞和肝细胞中.最后,采用流式细胞仪分析表明,日本沼虾血细胞凋亡率在低氧胁迫96h时显著高于对照组,这与日本沼虾Bax基因转录水平和蛋白表达丰度相吻合.研究表明,日本沼虾Bax基因在日本沼虾不同组织应答低氧胁迫分子过程中均具有促凋亡作用.本研究可为探明日本沼虾不耐低氧的分子机制提供理论参考. |
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| AbstractList | Q785%S966.12; 为探究细胞凋亡相关基因Bax(B-cell lymphoma-2 associated X protein)在日本沼虾低氧胁迫过程中所发挥的作用,实验采用cDNA末端快速扩增(RACE)PCR技术获得日本沼虾细胞凋亡相关基因Bax的cDNA全长序列,采用半定量RT-PCR与实时荧光定量PCR(qPCR)分析其在日本沼虾不同组织、不同低氧阶段的表达情况,同时采用Western blot与免疫组化分析了低氧下日本沼虾Bax蛋白的表达与定位.日本沼虾Bax基因cDNA全长2 287 bp(NCBI 登录号:MZ823353),包括 5'非编码区(UTR)为 42 bp,3'UTR 为 814 bp,开放阅读框(ORF)1 431 bp,编码476个氨基酸.通过软件和生物信息网站对其序列进行分析,氨基酸相似度比对显示,日本沼虾细胞凋亡基因Bax富含高度保守的BH1、BH2及BH3结构域.系统进化树分析显示,日本沼虾Bax基因与斑节对虾等甲壳动物Bax聚为一支,具有最近的亲缘关系.RT-PCR结果表明,日本沼虾Bax基因在肝胰腺中表达量最高,在脑中表达量最低.在低氧胁迫1～96h时,日本沼虾鳃和肝胰腺组织Bax基因表达量均显著高于对照组,这与日本沼虾Bax蛋白表达丰度基本相似.通过构建原核表达载体获得体外重组蛋白Bax,并将纯化重组蛋白免疫兔子获得抗血清.免疫组化结果显示,鳃和肝胰腺Bax蛋白阳性信号主要定位在鳃上皮细胞和肝细胞中.最后,采用流式细胞仪分析表明,日本沼虾血细胞凋亡率在低氧胁迫96h时显著高于对照组,这与日本沼虾Bax基因转录水平和蛋白表达丰度相吻合.研究表明,日本沼虾Bax基因在日本沼虾不同组织应答低氧胁迫分子过程中均具有促凋亡作用.本研究可为探明日本沼虾不耐低氧的分子机制提供理论参考. |
| Abstract_FL | Hypoxia phenomenon in pond water is a common and prominent stress factor in aquaculture,which will severely restrict the behavior,growth,reproduction and other aspects of aquaculture animals,and even lead to large-scale death.Bax is a pro-apoptotic protein in the Bcl-2 protein family,although most studies have investig-ated the function of Bax in many vertebrates,little information to date was observed in the crustaceans.The full-length cDNA sequence of pro-apoptotic Bax gene was obtained by RACE(rapid-amplification of cDNA ends)PCR technology to explore the role of Bax(B-cell lymphoma-2 associated X protein)in the oriental river prawn Macrobrachium nipponense under hypoxic stress.RT-PCR was used to analyze the distribution of Bax gene in dif-ferent tissues of M.nipponense.Meanwhile,real-time quantitative PCR(qPCR)and Western blot were used to detect the expression of Bax gene in different hypoxic stress stages.Bax recombinant protein and antiserum were prepared,and the localization of Bax protein was analyzed by immunohistochemistry.The present results showed that the full-length cDNA of Bax gene was 2 287 bp(NCBI accession no MZ823353),including 42 bp of 5'non-coding region(UTR),814 bp of 3'UTR,1 431 bp of open reading frame(ORF)encoding 476 amino acids.Sequence analysis showed that Bax was rich in highly conserved BH1,BH2 and BH3 domains.Phylogenetic tree analysis showed that Bax gene of M.nipponense was closely related to the Bax gene of Penaeus monodon.RT-PCR results showed that Bax mRNA expression was the highest in the hepatopancreas and the lowest in the brain.qPCR results indicated that Bax expression level in the gill and hepatopancreas tissues of M.nipponense was signi-ficantly higher than that of the control group under hypoxia 1-96 h.Western blot analysis also confirmed that Bax protein expression level was basically similar to gene transcription level.The recombinant Bax protein was obtained by constructing prokaryotic expression vector in vitro,which were immunized rabbits to obtain anti-serum.Immunohistochemical results showed that the positive signal of Bax protein in the gill and hepatopancreas were mainly located in the gill epithelial cells and hepatocytes.Finally,flow cytometry analysis showed that the apoptosis rate of haemocytes in hypoxia 96 h group significantly higher than those in the control group,which was consistent with the expression pattern of Bax protein expression abundance and gene transcription level.These res-ults suggest that Bax gene can promote apoptosis in different tissues of M.nipponense in response to hypoxic stress.The present study has provided theoretical reference for exploring the molecular mechanism of hypoxia sensitivity in M.nipponense. |
| Author | 薛程 赵倩倩 孙盛明 郑诚 孙西超 |
| AuthorAffiliation | 上海海洋大学,农业农村部淡水水产种质资源重点实验室,上海 201306%上海海洋大学,农业农村部淡水水产种质资源重点实验室,上海 201306;上海海洋大学,水产种质资源发掘与利用教育部重点实验室,上海 201306 |
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| Author_FL | ZHAO Qianqian ZHENG Cheng SUN Xichao SUN Shengming XUE Cheng |
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| DocumentTitle_FL | Cloning of Bax gene in Macrobrachium nipponense and its role in hypoxia stress |
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| Keywords | 细胞凋亡 低氧 hypoxic 原核表达 Bax apoptosis 日本沼虾 Macrobrachium nipponense prokaryotic expression |
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| Title | 日本沼虾Bax基因克隆及其在低氧胁迫过程中的作用 |
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