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首页 > 过刊浏览>2021年第28卷第2期 >123-135. DOI:10.12264/JFSC2020-0245
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缺刻缘绿藻CDP-乙醇胺:1,2-二酰甘油乙醇胺磷酸转移酶的基因特征与功能鉴定
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焦建璐(1994–),女,硕士研究生,研究方向为藻类生物技术.

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国家自然科学基金项目(31772821)； 国家“双一流”水产学科项目.


Characterization and identification of a CDP-ethanolamine: 1, 2-diacylglycerol ethanolamine phosphotransferase gene from the microalga (Myrmecia incisa)
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    摘要:

    运用反转录 PCR 与 5'-及 3'-末端快速扩增技术, 从缺刻缘绿藻(Myrmecia incisa Reisigl) H4301 中克隆到胞苷二磷酸(CDP)-乙醇胺: 二酰基甘油乙醇胺磷酸转移酶(EPT)的基因 MiEPT。其 cDNA 序列全长 1914 bp, 其中, 5'-非翻译区(UTR)长 217 bp, 3'-UTR 长 533 bp, 开放阅读框(ORF)长 1164 bp, 编码由 387 个氨基酸组成的蛋白。以缺刻缘绿藻基因组 DNA 为模板扩增到该基因的 DNA 序列, 长为 2853 bp。这两条序列的比对结果显示, MiEPT 含有 6 个内含子, 它们将其 ORF 分隔成 7 个外显子。经预测, MiEPT 为含有 8 个跨膜区的膜整合蛋白。多序列比结果显示, MiEPT 具有 CDP-醇磷脂酰转移酶基序。基于不同物种 EPT 的氨基酸序列所构建的邻接聚类结果表明, MiEPT 与已知功能的莱茵衣藻(Chlamydomonas reinhardtii)EPT 聚类在一起, 由此推测, MiEPT 可以催化磷脂酰乙醇胺(PE)的生物合成。为鉴定 MiEPT 的基因功能, 利用表达载体 pET-23a 实现了在大肠杆菌中重组表达 MiEPT 的目的。使用超离心技术, 获得部分纯化含重组 MiEPT 的组分。体外酶促反应的产物经薄层层析分析显示, 重组表达的 MiEPT 具有 EPT 和胆碱磷酸转移酶(CPT)的活性。经棒状薄层色谱的定量分析, 可知 MiEPT 的 EPT 酶活性是 CPT 的 10.3倍, 从而表明, MiEPT 在缺刻缘绿藻中能同时参与 PE 和磷脂酰胆碱的生物合成, 但更倾向利用 CDP-乙醇胺生产 PE。

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焦建璐,封兵,毕燕会,周志刚.缺刻缘绿藻CDP-乙醇胺:1,2-二酰甘油乙醇胺磷酸转移酶的基因特征与功能鉴定[J].中国水产科学,2021,28(2):123-135
JIAO Jianlu, FENG Bing, BI Yanhui, ZHOU Zhigang. Characterization and identification of a CDP-ethanolamine: 1, 2-diacylglycerol ethanolamine phosphotransferase gene from the microalga (Myrmecia incisa)[J]. Journal of Fishery Sciences of China,2021,28(2):123-135

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