原壳小球藻番茄红素ε环化酶基因的克隆和分析
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摘要
叶黄素(lutein)是含氧类胡萝卜素,即类叶黄素(xanthophylls)中的一种,是一种性能优异的抗氧化剂。由于富含叶黄素,小球藻(Chlorella)作为一种极具潜力的叶黄素源而备受关注。与其它叶黄素源相比,小球藻具有生长迅速、可进行异养培养、含游离叶黄素等优势。目前关于小球藻叶黄素的研究中,主要采用培养条件的优化来提高叶黄素产量,少见采用代谢途径改造的办法来实现这一目标。本论文把叶黄素代谢过程中关键分支点编码番茄红素ε环化酶(lycopene epsilon cyclase)的LCYE基因作为研究的对象,通过对原壳小球藻(Chlorella protothecoides CS-41)LCYE基因的克隆和特征分析,为进一步揭示番茄红素ε环化酶的特性和叶黄素合成的分子代谢机制奠定基础。研究工作
主要从以下几个方面开展:
1、通过cDNA末端快速扩增(RACE)和RT-PCR对C. protothecoides CS-41核基因组上的LCYE基因进行扩增,得到2107 bp的cDNA序列(GenBank登录号:FJ752528),序列分析显示,其开放阅读框(Open reading frame, ORF)的长度为1731 bp,编码576个氨基酸。
2、通过LCYE基因对应的氨基酸序列的生物信息学分析,发现它与高等植物和其它藻类的LCYE有相似性,相似性最高的是莱茵衣藻(Chlamydomonas reinhardtii XM001696477.1),达到67%,其中,第48～459个氨基酸残基为一个典型的番茄红素环化酶蛋白(Lycopene cyclase protein)结构域(pfam05834),第261～284个氨基酸残基为一个典型的环化酶保守的模体,还发现其具有环化酶所特有的保守功能结构域,包括N端的双核苷酸结合位点、以及预测跨膜区域等。同时,进化树分析结果表明,藻类群中小球藻和衣藻的亲缘关系比较近。
3、采用Taqman荧光定量PCR和高效液相色谱法(HPLC)的分别对不同培养条件下原壳小球藻的LCYE基因的表达水平和叶黄素产量进行定量分析。实验结果显示,在不同的培养条件下,LCYE基因的表达水平和叶黄素产量有一定的相关性。
Lutein is one of xanthophylls which are a group of compounds that are oxygenated derivatives of carotenoids. Natural lutein demonstrates high performance in anti-oxidation effect. Chlorella is noted for its high cellular lutein content, which makes it a potential resource for lutein production. Comparing to other lutein resources, Chlorella has many advantages such as the high specific growth rate and the ability of growing heterotrophically. Most researches focused on the optimization of cultivation conditions to improve lutein productivity; however, few researches used metabolic engineering technology to achieve this aim. The objective of this study is to clone the full-length cDNA of LCYE gene from Chlorella protothecoides CS-41 and to preliminarily understand the regulatory expression mechanism of LCYE at the molecular level by bioinformatics analysis. The following aspects were included in this study.
1、A full-length cDNA (GenBank Accession No.FJ752528)of LCYE was cloned from Chlorella protothecoides CS-41 by RACE (rapid-amplification of cDNA ends) and RT-PCR, which is a 2107-bp sequence. It was found that there was a 1731-bp open reading frame by ORF finder, and that this gene encodes a putative LCYE from Chlorella protothecoides CS-41.
2、Homology studies showed that the deduced amino acid sequence of LCYE gene had a significant similarity with the corresponding sequences of other green algae and higher plants. It shared the highest sequence similarity, up to 67%, with the LCYE of Chlamydomonas reinhardtii. It was predicted that one typical lycopene cyclase protein domain (Pfam05834) was located between the 48th -459th amino acid. Several typical motifs for all cyclases were found by the alignment analysis of the LCYE amino acid sequences: one Dinucleotide-binding site at the N-terminus, one Cyclase motif 1, and one Predicted TM helix at the C-terminus. The anaysis of phylogenetic tree showed the closest relationship between Chlorella protothecoides CS-41 and Chlamydomonas reinhardtii.
3、Taqman real-time PCR and HPLC (high performance liquid chromatography) were respectively used to determine the expression level of LCYE gene and the lutein content of different cultures, and it was indicated that the expression of LCYE gene correlated with lutein content in different cultivation conditions.
主要从以下几个方面开展:
1、通过cDNA末端快速扩增(RACE)和RT-PCR对C. protothecoides CS-41核基因组上的LCYE基因进行扩增,得到2107 bp的cDNA序列(GenBank登录号:FJ752528),序列分析显示,其开放阅读框(Open reading frame, ORF)的长度为1731 bp,编码576个氨基酸。
2、通过LCYE基因对应的氨基酸序列的生物信息学分析,发现它与高等植物和其它藻类的LCYE有相似性,相似性最高的是莱茵衣藻(Chlamydomonas reinhardtii XM001696477.1),达到67%,其中,第48～459个氨基酸残基为一个典型的番茄红素环化酶蛋白(Lycopene cyclase protein)结构域(pfam05834),第261～284个氨基酸残基为一个典型的环化酶保守的模体,还发现其具有环化酶所特有的保守功能结构域,包括N端的双核苷酸结合位点、以及预测跨膜区域等。同时,进化树分析结果表明,藻类群中小球藻和衣藻的亲缘关系比较近。
3、采用Taqman荧光定量PCR和高效液相色谱法(HPLC)的分别对不同培养条件下原壳小球藻的LCYE基因的表达水平和叶黄素产量进行定量分析。实验结果显示,在不同的培养条件下,LCYE基因的表达水平和叶黄素产量有一定的相关性。
Lutein is one of xanthophylls which are a group of compounds that are oxygenated derivatives of carotenoids. Natural lutein demonstrates high performance in anti-oxidation effect. Chlorella is noted for its high cellular lutein content, which makes it a potential resource for lutein production. Comparing to other lutein resources, Chlorella has many advantages such as the high specific growth rate and the ability of growing heterotrophically. Most researches focused on the optimization of cultivation conditions to improve lutein productivity; however, few researches used metabolic engineering technology to achieve this aim. The objective of this study is to clone the full-length cDNA of LCYE gene from Chlorella protothecoides CS-41 and to preliminarily understand the regulatory expression mechanism of LCYE at the molecular level by bioinformatics analysis. The following aspects were included in this study.
1、A full-length cDNA (GenBank Accession No.FJ752528)of LCYE was cloned from Chlorella protothecoides CS-41 by RACE (rapid-amplification of cDNA ends) and RT-PCR, which is a 2107-bp sequence. It was found that there was a 1731-bp open reading frame by ORF finder, and that this gene encodes a putative LCYE from Chlorella protothecoides CS-41.
2、Homology studies showed that the deduced amino acid sequence of LCYE gene had a significant similarity with the corresponding sequences of other green algae and higher plants. It shared the highest sequence similarity, up to 67%, with the LCYE of Chlamydomonas reinhardtii. It was predicted that one typical lycopene cyclase protein domain (Pfam05834) was located between the 48th -459th amino acid. Several typical motifs for all cyclases were found by the alignment analysis of the LCYE amino acid sequences: one Dinucleotide-binding site at the N-terminus, one Cyclase motif 1, and one Predicted TM helix at the C-terminus. The anaysis of phylogenetic tree showed the closest relationship between Chlorella protothecoides CS-41 and Chlamydomonas reinhardtii.
3、Taqman real-time PCR and HPLC (high performance liquid chromatography) were respectively used to determine the expression level of LCYE gene and the lutein content of different cultures, and it was indicated that the expression of LCYE gene correlated with lutein content in different cultivation conditions.
引文
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