绿色杜氏藻DvGGPS生物信息学及转录差异研究

香叶酰香叶酰焦磷酸合成酶(GGPS)能够催化焦磷酸异戊烯酯(IPP)与二甲基丙烯焦磷酸(DMAPP)反应形成二萜化合物通用前体香叶酰基香叶酰焦磷酸(GGPP)。为深入了解GGPS蛋白在类胡萝卜素合成途径中的作用,通过Illumina Hi Seq TM 2000高通量测序获得Dv GGPS基因c DNA全长序列,对GGPS基因序列进行生物信息学分析,并研究花生四烯酸(AA)、乙酰水杨酸(ASA)和硫酸铈铵(ACS)对Dv GGPS基因转录水平的影响及绿色杜氏藻类胡萝卜素含量变化。生物信息学分析结果表明,Dv GGPS基因c DNA全长2 229 bp,含1 041 bp开放阅读框,编码346个氨基酸。GGPS蛋白质序列理论等电点(p I)为5.82,相对分子质量为37.66 k Da,该蛋白为疏水性蛋白,无信号肽和跨膜区域。二级结构预测显示该蛋白分子中α-螺旋结构最多,占57.23%。同源比对结果表明,绿色杜氏藻GGPS蛋白与雨生红球藻亲缘关系最近。qRT-PCR结果表明,62.5 mg·L~(-1)AA、50 mg·L~(-1)ASA和0.8 mg·L~(-1)ACS处理的Dv GGPS基因转录水平达到最高,此时类胡萝卜素含量也均有提高,说明绿色杜氏藻类胡萝卜素的生物学合成可能是通过诱导Dv GGPS基因表达实现的,Dv GGPS基因在类胡萝卜素生物合成中起关键作用。Dv GGPS基因的克隆及表达调控研究为今后探明类胡萝卜素积累的分子机理提供了重要参考,也为进一步通过代谢工程手段提高绿色杜氏藻类胡萝卜素含量奠定了理论基础。

Bioinformatics Analysis and Transcription Difference of Geranylgeranyl Pyrophosphate Synthase (GGPS) in Dunaliella viridis

Geranylgeranyl pyrophosphate synthase (GGPS) can catalyze isoprenyl pyrophosphate with dimethylallyl diphosphate to form diterpeniods precursor.To understand more about the role of GGPS in the carotenoid biosynthesis,the full length cDNA of GGPS gene from Dunaliella viridis was sequenced by HiSeqTM Illumina 2000 and analyzed by using different bioinformatics software.Aacetylsalicylic acid (ASA),ammonium cerous sulfate (ACS) and arachidonic acid (AA) were used to induce the expression of GGPS gene and carotenoid production in D.Viridis.Bioinformatic prediction of GGPS gene revealed that it has a full length of 2 229 bp,containing an open reading frame of 1 041 bp,encoding 346 amino acids.The theoretical isoelectric point of GGPS protein was 5.82,with the relative molecular mass of 37.66 kDa.GGPS protein was hydrophobic,and had neither signal peptide nor transmembrane domain.The secondary structure analysis illustrated that approximately 57.23％ of GGPS was helical in structure.Phylogenetic tree results showed that the amino acid sequence of GGPS protein of D.virdis was similar to Haematococcus pluvialis.The RT-qPCR showed that the expression of GGPS gene in D.viridis treated at 62.5 mg·L-1 AA,50 mg· L-1 ASA,0.8 mg·L-1ACS was the highest,and significant increase in the content of carotenoids was also observed.The results showed that the carotenoid biosynthesis was achieved by the induction of GGPS gene.This paper would not only provide reference for molecular mechanism of carotenoid biosynthesis study,but also lay the theoretical basis for improving content of carotenoid though metabolic engineering strategies in D.virdis.