斑节对虾UBE2H基因克隆及其表达
【目的】克隆斑节对虾（Penaeusmonodon）泛素结合酶E2 H基因（UBE2H），了解其组织特异性表达情况，为揭示UBE2H在斑节对虾卵巢发育过程的作用提供依据。【方法】PCR扩增斑节对虾UBE2H基因的开放阅读框，利用实时荧光定量PCR检测UBE2H基因在斑节对虾各组织及不同发育期斑节对虾肝胰腺和卵巢中的表达情况；并构建pET32a-UBE2H原核表达重组质粒，进行诱导表达及蛋白纯化。【结果】克隆获得的斑节对虾UBE2H基因全长1010 bp （GenBank登录号KU870456），其中，5＇非编码区77 bp，3＇非编码区378 bp，开放阅读框555 bp（编码184个氨基酸）...
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| Published in | 南方农业学报 Vol. 47; no. 11; pp. 1958 - 1965 |
|---|---|
| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
中国水产科学研究院 南海水产研究所/农业部南海渔业资源开发利用重点实验室，广州 510300
2016
华南农业大学 动物科学学院，广州 510642%中国水产科学研究院 南海水产研究所/农业部南海渔业资源开发利用重点实验室,广州,510300%华南农业大学 动物科学学院,广州,510642 |
| Subjects | |
| Online Access | Get full text |
| ISSN | 2095-1191 |
| DOI | 10.3969/jissn.2095-1191.2016.11.1958 |
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| Abstract | 【目的】克隆斑节对虾（Penaeusmonodon）泛素结合酶E2 H基因（UBE2H），了解其组织特异性表达情况，为揭示UBE2H在斑节对虾卵巢发育过程的作用提供依据。【方法】PCR扩增斑节对虾UBE2H基因的开放阅读框，利用实时荧光定量PCR检测UBE2H基因在斑节对虾各组织及不同发育期斑节对虾肝胰腺和卵巢中的表达情况；并构建pET32a-UBE2H原核表达重组质粒，进行诱导表达及蛋白纯化。【结果】克隆获得的斑节对虾UBE2H基因全长1010 bp （GenBank登录号KU870456），其中，5＇非编码区77 bp，3＇非编码区378 bp，开放阅读框555 bp（编码184个氨基酸）。斑节对虾UBE2H蛋白等电点（pI）4.88，分子量20.85 kD。斑节对虾UBE2H氨基酸序列与其他物种相似度很高，为78.00%-83.00%。实时荧光定量PCR检测结果显示，UBE2H基因在斑节对虾淋巴组织中表达量最高，其次为鳃；在斑节对虾不同卵巢发育期肝胰腺和卵巢中的表达量均呈先上升后下降的变化趋势，但其峰值出现时间存在差异，肝胰腺的最高表达量出现在卵巢III期，卵巢的最高表达量出现在卵巢II期。经原核表达获得的斑节对虾UBE2H融合蛋白约39.00 kD，纯化后的蛋白浓度为2.92μg/μL。【结论】斑节对虾UBE2H参与了其卵母细胞发育和肝胰腺中卵黄蛋白的转运过程，与斑节对虾的卵巢发育密切相关。 |
|---|---|
| AbstractList | S945.47; 【目的】克隆斑节对虾（Penaeusmonodon）泛素结合酶E2 H基因（UBE2H），了解其组织特异性表达情况，为揭示UBE2H在斑节对虾卵巢发育过程的作用提供依据。【方法】PCR扩增斑节对虾UBE2H基因的开放阅读框，利用实时荧光定量PCR检测UBE2H基因在斑节对虾各组织及不同发育期斑节对虾肝胰腺和卵巢中的表达情况；并构建pET32a-UBE2H原核表达重组质粒，进行诱导表达及蛋白纯化。【结果】克隆获得的斑节对虾UBE2H基因全长1010 bp （GenBank登录号KU870456），其中，5'非编码区77 bp，3'非编码区378 bp，开放阅读框555 bp（编码184个氨基酸）。斑节对虾UBE2H蛋白等电点（pI）4.88，分子量20.85 kD。斑节对虾UBE2H氨基酸序列与其他物种相似度很高，为78.00%~83.00%。实时荧光定量PCR检测结果显示，UBE2H基因在斑节对虾淋巴组织中表达量最高，其次为鳃；在斑节对虾不同卵巢发育期肝胰腺和卵巢中的表达量均呈先上升后下降的变化趋势，但其峰值出现时间存在差异，肝胰腺的最高表达量出现在卵巢III期，卵巢的最高表达量出现在卵巢II期。经原核表达获得的斑节对虾UBE2H融合蛋白约39.00 kD，纯化后的蛋白浓度为2.92μg/μL。【结论】斑节对虾UBE2H参与了其卵母细胞发育和肝胰腺中卵黄蛋白的转运过程，与斑节对虾的卵巢发育密切相关。 【目的】克隆斑节对虾（Penaeusmonodon）泛素结合酶E2 H基因（UBE2H），了解其组织特异性表达情况，为揭示UBE2H在斑节对虾卵巢发育过程的作用提供依据。【方法】PCR扩增斑节对虾UBE2H基因的开放阅读框，利用实时荧光定量PCR检测UBE2H基因在斑节对虾各组织及不同发育期斑节对虾肝胰腺和卵巢中的表达情况；并构建pET32a-UBE2H原核表达重组质粒，进行诱导表达及蛋白纯化。【结果】克隆获得的斑节对虾UBE2H基因全长1010 bp （GenBank登录号KU870456），其中，5＇非编码区77 bp，3＇非编码区378 bp，开放阅读框555 bp（编码184个氨基酸）。斑节对虾UBE2H蛋白等电点（pI）4.88，分子量20.85 kD。斑节对虾UBE2H氨基酸序列与其他物种相似度很高，为78.00%-83.00%。实时荧光定量PCR检测结果显示，UBE2H基因在斑节对虾淋巴组织中表达量最高，其次为鳃；在斑节对虾不同卵巢发育期肝胰腺和卵巢中的表达量均呈先上升后下降的变化趋势，但其峰值出现时间存在差异，肝胰腺的最高表达量出现在卵巢III期，卵巢的最高表达量出现在卵巢II期。经原核表达获得的斑节对虾UBE2H融合蛋白约39.00 kD，纯化后的蛋白浓度为2.92μg/μL。【结论】斑节对虾UBE2H参与了其卵母细胞发育和肝胰腺中卵黄蛋白的转运过程，与斑节对虾的卵巢发育密切相关。 |
| Abstract_FL | Objective]The present study was conducted to clone ubiquitin-conjugating enzyme E2 H gene(UBE2H) of Penaeus monodon and explore its tissue specific expression, in order to provide references for revealing ovary develop-ment of P. monodon. [Method]Open reading frame of UBE2H gene in P. monodon was amplified by PCR, and UBE2H gene expressions in different tissues, and in hepatopancreas and ovary of P. monodon at different ovary developmental stages were detected using real-time quantitative PCR. pET32a-UBE2H prokaryotic expression recombinant plasmid was con-structed, and induction expression and protein purification was carried out. [Result]The cloned UBE2H gene(GenBank accession No. KU870456)was1010bpinlength,including5'UTRof77bpand3'UTRof378bpandanopenreading frame(ORF) of 555 bp(encoding 184 amino acids). Isoelectric point(pI) of UBE2H protein was 4.88, molecular weight was 20.85 kD. Amino acid sequence of UBE2H protein was highly similar to those of other species, the similarity reached 78.00%-83.00%. Real-time quantitative PCR results indicated that expression quantity of UBE2H gene was the highest in lymphoid tissue in P. monodon, and followed by gill. The expression quantities of UBE2H gene in hepatopan-creas and ovary at different ovary developmental stages both went through an up-down variation, but peak times were dif-ferent. The largest expression quantity of UBE2H gene in hepatopancreas occurred at ovary developmental stageⅢ, whereas the largest expression quantity in ovary occurred at ovary developmental stageⅡ. Fusion protein UBE2H obtained through prokaryotic expression was 39.00 kD, protein concentration after purification was 2.92 μg/μL. [Conclusion]UBE2H gene of P. monodon participate in oocyte development and transport of yolk protein in hepatopancreas, and is closely related to ovary development of P. monodon. |
| Author | 唐蕾 傅明骏 刘文生 黄建华 周发林 江世贵 |
| AuthorAffiliation | 中国水产科学研究院南海水产研究所／农业部南海渔业资源开发利用重点实验室,广州510300 华南农业大学动物科学学院,广州510642 |
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| Author_FL | JIANG Shi-gui HUANG Jian-hua ZHOU Fa-lin FU Ming-jun LIU Wen-sheng TANG Lei |
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| Keywords | 克隆 UBE2H基因 原核表达 实时荧光定量PCR 斑节对虾 UBE2H gene clone Penaeus monodon prokaryotic expression real-time quantitative PCR |
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| Notes | 45-1381/S Objective]The present study was conducted to clone ubiquitin-conjugating enzyme E2 H gene（UBE2H） of Penaeus monodon and explore its tissue specific expression, in order to provide references for revealing ovary develop-ment of P. monodon. [Method]Open reading frame of UBE2H gene in P. monodon was amplified by PCR, and UBE2H gene expressions in different tissues, and in hepatopancreas and ovary of P. monodon at different ovary developmental stages were detected using real-time quantitative PCR. pET32a-UBE2H prokaryotic expression recombinant plasmid was con-structed, and induction expression and protein purification was carried out. [Result]The cloned UBE2H gene（GenBank accession No. KU870456）was1010bpinlength,including5＇UTRof77bpand3＇UTRof378bpandanopenreading frame（ORF） of 555 bp（encoding 184 amino acids）. Isoelectric point（pI） of UBE2H protein was 4.88, molecular weight was 20.85 kD. Amino acid sequence of UBE2H protein was highly similar to those of other species, the similarity reached 78.00%-83. |
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| Snippet | 【目的】克隆斑节对虾（Penaeusmonodon）泛素结合酶E2 H基因（UBE2H），了解其组织特异性表达情况，为揭示UBE2H在斑节对虾卵巢发育过程的作用提供依据。【方法】PCR扩增斑节对... S945.47; 【目的】克隆斑节对虾（Penaeusmonodon）泛素结合酶E2 H基因（UBE2H），了解其组织特异性表达情况，为揭示UBE2H在斑节对虾卵巢发育过程的作用提供依据。【方法】PCR扩... |
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| SubjectTerms | UBE2H基因 克隆 原核表达 实时荧光定量PCR 斑节对虾 |
| Title | 斑节对虾UBE2H基因克隆及其表达 |
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