油茶长链脂肪酰基CoA合成酶基因1(CoLACS1)分子特征与表达分析
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国家林业公益性行业科研专项重大项目(201404702);湖南省自然科学基金项目(2016JJ5031)


Molecular Characterization and Expression Analysis of long chain fatty acid acyl-CoA synthetase gene 1 (CoLACS1) from Camellia oleifera
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    摘要:

    长链脂肪酰基CoA合成酶(LACS)在脂肪酸的合成与分解代谢中起着重要作用。以油茶(Camellia oleifera)国家审定品种‘华硕’种仁转录组数据为基础,根据LACS 基因Unigene序列设计引物,分离克隆了油茶一条LACS1基因全长cDNA 序列,命名为CoLACS1(GeneBank登录号:KJ960228),基因全长2114bp,开放阅读框2088bp,编码695个氨基酸;生物信息学分析显示CoLACS1具有三个B1ock,从分子特征可判断CoLACS1属于LACS家族;氨基酸同源比对显示与其它物种的LACS氨基酸序列具有较高的相似性,其中与拟南芥LACS7相似性78%,与麻风树、大豆、毛果杨等物种LACS6(peroxisomal)相似性可达80%以上;对CoLACS1进行原核表达分析,构建的pET30a- CoLACS1载体成功转化到BL21( DE3) 中经1 mmol/L IPTG诱导表达,菌液检测获得预测的目的蛋白(分子量约为76 kD);分析转录组数据中CoLACS1的Unigene序列RTKM值和对CoLACS1实施实时荧光定量PCR,结果表明CoLACS1在油茶‘华硕’种子发育各时期平稳表达,表达丰度变化不大,荧光定量结果变化规律与转录组数据分析一致;同时分析‘华硕’种仁不同时期含油率和脂肪酸成分变化,双变量统计分析发现CoLACS1表达模式与油茶油脂积累规律呈显著相关性。本研究为进一步研究油茶油脂积累与代谢的基因调控提供理论依据。

    Abstract:

    long chain fatty acid acyl-CoA synthetase(LACS)plays an important role in fatty acid anabolism an catabolism. Based on seeds transcriptome Unigene sequence of Camellia oleifera named ‘Huashuo’(‘HS’), cDNA with complete sequence 2114 bp encoded LACS was isolated and cloned from Camellia oleifera seed, designated as CoLACS1 (GeneBank accession number:KJ960228). CoLACS1 had an open reading frame with 2088bp encoding 695 amino acids. sequence analysis demonstrated that the CoLACS1 possessed three blocks, the typical molecular characteristics of LACS, and belonged to LACS family. Phylogenetic analysis showed CoLACS1 is highly conserved and had the similarity( 78 %) with AtLACS7 and the similarity( over 80 % )with LACS6(peroxisomal)of Jatropha curcas, glycine max and Populus trichocarpa et al. The expression vector of pET30a-CoLACS1 was transformed to BL21 ( DE3) bacteria successfully,which was induced by 1 mmol/L IPTG to succeed in expressing the recombinant protein with molecular weight about 76 kD. The gene expression changes by qPCR,which were similar to those of RPKM in transcriptome data,indicate that the CoLACS1 expresses stably in different developmental stage of Camellia oleifera seeds and there was striking relationship between the gene expression of CoLACS1 and the accumulating of lipid in Camellia oleifera seed. The studies provide a theoretical basis for the futher reseach of gene regulating in lipid accumulating and metabolism of Camellia oleifera.

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江南,谭晓风,张琳,等.油茶长链脂肪酰基CoA合成酶基因1(CoLACS1)分子特征与表达分析[J].植物遗传资源学报,2017,18(1):139-147.

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  • 收稿日期:2016-03-17
  • 最后修改日期:2016-05-26
  • 录用日期:2016-07-01
  • 在线发布日期: 2017-01-17
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