编码缺刻缘绿藻乙酰辅酶A羧化酶BCCP亚基的基因克隆与表达分析
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上海海洋大学水产与生命学院,上海海洋大学水产与生命学院

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国家自然科学基金(31172389);国家海洋局可再生能源专项(SHME2011SW02);上海高校海洋科学一流学科建设项目


Cloning and transcription analysis of genes coding for BCCP subunit of acetyl-CoA carboxylase in Myrmecia incisa
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College of Fisheries and Life Science,Shanghai Ocean University,College of Fisheries and Life Science,Shanghai Ocean University

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    摘要:

    为了探讨在氮饥饿对缺刻缘绿藻花生四烯酸(ArA)合成与积累的影响,通过cDNA末端快速扩增(RACE)和设计简并引物进行反转录(RT)-PCR扩增,克隆了编码该藻异质型乙酰辅酶A羧化酶(ACCase)的2个生物素羧基载体蛋白(BCCP)的基因序列。其中MiBCCP1基因的cDNA序列长1 267 bp,包含的5’-非翻译区(UTR)长44 bp,3’-UTR长524 bp,开放阅读框(ORF)长699 bp,编码232个氨基酸并含有45个氨基酸序列的叶绿体定位信号肽;预测成熟蛋白分子量约为20 ku。MiBCCP2基因的ORF长789 bp,编码263个氨基酸并含有49个氨基酸的叶绿体定位信号肽,推测成熟蛋白分子量约为22 ku,但其羧基端缺乏生物素酰基化位点。邻接法(NJ)构建的聚类图显示它们分属于2个不同的分支(靴带值为100)。采用半定量反转录(RT)-PCR技术分析这2个基因的表达量,结果显示,在氮饥饿光照培养条件下,它们的相对转录量都先短暂升高然后持续下调,从而表明缺刻缘绿藻脂肪酸的从头合成能力有下降的趋势。结合该藻的ArA含量在该培养条件下明显增加的结果,推测胞质中同质型ACCase对缺刻缘绿藻ArA合成与积累起着更重要的作用。

    Abstract:

    Acetyl-CoA carboxylase(ACCase)catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA,which is a key and rate-limited step in the biosynthesis of fatty acids.In order to investigate its effect on the arachidonic acid(ArA)in Myrmecia incisa grown under nitrogen starvation,two genes coding for biotin carboxyl carrier protein(BCCP)which was one subunit of the heteromeric ACCase complex,were cloned by using Rapid Amplification Complementary DNA ends(RACE)method and reverse transcription(RT)-PCR with designed degenerated primers.The full-length cDNA sequence of MiBCCP1 was composed of 1 267 bp,comprising a 44-bp 5'-untranslated region(UTR)and a 524-bp 3'-UTR with a typical poly A tail.The open reading frame(ORF)was 699 bp in length encoding a 232-amino-acid protein.After restriction of a 45-amino-acid chloroplast signal peptide,the putative molecular weight of mature MiBCCP1 was about 20 ku.The coding sequence of MiBCCP2 gene was also cloned and it was 789 bp in length encoding a 263-amino-acid protein with a 49-amino-acid chloroplast signal peptide,too.The putative molecular weight of mature MiBCCP2 was approximately 22 ku but it lacked a characteristic biotinylation domain at the C-terminus.Neighbor-joining(NJ)dendrogram showed that these two cloned genes belonged to BCCP1 and BCCP2 clades,which was supported by the bootstrip value of 100.Semi-quantitative reverse transcription(RT)-PCR result showed that during the stress course of nitrogen starvation,the relative transcription of both MiBCCP1 and MiBCCP2 genes increased temporally possibly due to shock,and subsequently declined to the end of this experiment,suggesting that M.incisa had a trend to a declined ability of fatty acid biosynthesis de novo.Taking the significantly increased ArA level in this microalga during this culture course together,it was supposed that the homomeric ACCase in the cytosol playsed a more important role than the heteromeric one in modulating biosynthesis and accumulation of ArA in M.incisa under the nitrogen starvation stress.

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李燕,周志刚.编码缺刻缘绿藻乙酰辅酶A羧化酶BCCP亚基的基因克隆与表达分析[J].水产学报,2014,38(6):794~802

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  • 收稿日期:2014-02-05
  • 最后修改日期:2014-04-08
  • 录用日期:2014-05-19
  • 在线发布日期: 2014-06-16
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