Bioinformatics Analysis and Expression Study of DHDDS of Dunaliella viridis

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.015

Abstract

Abstract: Dunaliella viridis is a unicellular green alga producing valuable secondary metabolite carotenoids, and dehydrodolichyl diphosphate synthase(DHDDS)is an enzyme in the synthesis pathway of carotenoids. It was to investigate the correlation between expression of DHDDS gene in D. viridis treated with different concentrations of MeJA and the content of carotenoids. Total RNA was extracted from D. viridis and the full length of DHDDS was acquired by de-novo transcriptome sequencing, then the sequence was analyzed by bioinformatics website and software to predict the protein structure. Transcriptional levels of D. viridis treated with different concentrations of MeJA were examined by Real-time quantitative PCR(RT-qPCR). The total length of gene DHDDS was 2 211 bp, containing a 1 740 bp open reading frame(ORF)that encoded 579 amino acids. Theoretical isoelectric point of DHDDS protein was 7.63. Relative molecular mass was 62 472.7 Da. Bioinformatics prediction revealed that there was no signal peptide and transmembrane domain in DHDDS protein, and it was located in cytoplasmic matrix. Sequence alignment showed that DHDDS of D. viridis shared the highest homology of 59% with that of Chlorella variabilis. The RT-qPCR showed that the expression of DHDDS gene in D. viridis treated by 100 mol/L of MeJA was the highest with significant difference：at this concentration, the content of carotenoids was higher than other groups. There was certain correlation between the expression of DHDDS gene and the content of carotenoids. Conclusively, the DHDDS of D. viridis is an enzyme located in cytoplasmic matrix, relating to synthesis pathway of carotenoids. Within certain concentration, MeJA in lower concentration shows promoting effect on the expression, and inhibiting in higher concentration, there is a correlation between the expression and the content of carotenoids.

Key words: Dunaliella viridis, DHDDS, carotenoids contents, MeJA

Pan Yifang, Gong Yifu, Yu Kai, Zhu Shuaiqi, Wang Heyu. Bioinformatics Analysis and Expression Study of DHDDS of Dunaliella viridis[J]. Biotechnology Bulletin, 2015, 31(7): 101-108.

References

[1]姬翔. 杜氏盐藻(Dunaliella salina)14-3-3蛋白cDNA全长的克隆与序列分析[D]. 郑州：郑州大学, 2006.
[2]张翅. 杜氏盐藻(Dunaliella salina)cDNA文库构建及表达序列标签分析[D]. 武汉：华中科技大学, 2009.
[3] 伍先绍, 贺稚非, 龚霄. 杜氏盐藻及其在功能食品中的应用[J]. 中国食品添加剂, 2008(2)：127-130.
[4]刘红涛, 冯书营, 陈涛, 等. 杜氏盐藻分子生物学最新进展及展望[J]. 中国生物工程杂志, 2007, 27(10)：113-118.
[5]冯书营, 刘红涛, 李杰, 等. 杜氏盐藻基因工程研究现状及应用前景[J]. 中国生物工程杂志, 2007, 27(2)：108-112.
[6]Sakaihara T, Honda A, Tateyama S, et al. Subcellular fractionation of polyprenyl diphosphate synthase activities responsible for the syntheses of polyprenols and dolichols in spinach leaves[J]. J Biachem, 2000, 128(6)：1073-1078.
[7] Mueller MJ, Brodschelm W, Spannagl E, et al. Signaling in the elicitation process is mediated through the octadecanoid pathway leading to jasmonic acid[J]. Proc Natl Acad Sci USA, 1993, 90：7490.
[8]Tamogami S, Rakwal R, Kodama O. Phytoalexin production elicited by exogenously applied jasmonic acid in rice leaves(Oryza sativa L.)is under the control of cytokinins and ascorbic acid[J]. FEBS Lett, 1997, 412：61.
[9]余龙江, 朱敏, 刘幸福, 等. 茉莉酸甲酯对中国红豆杉胚性细胞紫杉醇生物合成的影响[J]. 武汉植物学研究, 1999, 17(4)：371-374.
[10]Qi FH, Zhan YG, Jing TZ. A review on elicitors and their regulation on secondary metabolites in plant cell culture[J]. Nat Prod Res Dev, 2008, 20：568-573.
[11]马君兰, 赵越. 外源茉莉酸甲酯(MeJA)对大豆异黄酮合成途径的影响[J]. 东北农业大学学报, 2011, 42(5)：14-18.
[12]廖巧. 甲基茉莉酸诱导青蒿素积累的机理研究[D]. 重庆：西南大学, 2012.
[13]桂连友. 外源茉莉酸甲酯对茶树抗虫作用的诱导及其机理研究[D]. 杭州：浙江大学, 2004.
[14]施江. 外源茉莉酸甲酯诱导对茶树鲜叶次生代谢产物的影响[D]. 北京：中国农业科学院, 2014.
[15]王鑫威, 王丽丽, 龚一富, 等. 茉莉酸甲酯对雨生红球藻虾青素含量和dxs基因表达的影响[J]. 水产学报, 2011, 12：1822-1828.
[16]章丽, 龚一富, 刘晓丹, 等. 外源MeJA胁迫对盐生杜氏藻生理生化特性的影响[J]. 生物学杂志, 2013, 3：38-42, 47.
[17]Núria C, Montserrat A, Oriol F, et al. Characterization of dehydrod-olichyl diphosphate synthase of Arabidopsis thaliana, a key enzyme in dolichol biosynthesis[J]. FEBS Letters, 2000(477)：170-174.
[18]Alexandrov NN, Brover VV, Freidin S, et al. Insights into corn genes derived from large-scale cDNA sequencing[J]. Plant Mol Biol, 2009, 69(1-2)：179-194.
[19]Hu MC, Gong HY, Lin GH, et al. XBP-1, a key regulator of unfolded protein response, activates transcription of IGF1 and Akt phosphorylation in zebrafish embryonic cell line[J]. Biochem Biophys Res Commun, 2007, 359(3)：778-783.
[20]吕军, 张颖, 冯立芹, 等. 生物信息学工具BLAST的使用简介[J]. 内蒙古大学学报：自然科学版, 2003, 34(2)：179-187.
[21]Emanuelesson O, Nielsen H, Brunak S, et al. Predicting subcellular localization of proteins based on their N-terminaal amino acid sequence[J]. J Mol Biol, 2000, 300(4)：1005-1016.
[22]Emanuelesson O, Nielsen H, von Heijne G. Chloro P, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites[J]. Protein Science, 1999, 8(5)：978-984.
[23]Bendsten JD, Nielsen H, Heijne GV, et al. Prediction of signal peptides：SignalP3. 0[J]. J Mol Biol, 2004, 340：783-795.
[24]Gasteiger E, Hoogland C, Gattiker A, et al. Protein identification and analysis tools on the ExPAsy Aerver. In：John M, Walker ed. The Proteomics Protocols Handbook[M]. New Jersey：Humana Press, 2005：571-607.
[25]Higgins D, Thompson J, Gibson T, et al. CLUSTAL W：improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gapenalties and weight matrix choice[J]. Nucleic Acides Res, 1999, 22：4673-4680.
[26]Guex N, Peitsch MC. SWISS-MODEL and the Swiss-PdbViewer：An environment for comparative protein modeling[J]. Electrophoresis, 1997, 18：2714-2723.
[27]Geourjon C, Deleage G. SOPMA：Significant improvement in protein secondary structure prediction by consensus prediction from multiple alinments[J]. Comput Appl Biosci, 1995, 11(6)：681-684.
[28]Stephan Z, Julia D, Rong W, et al. Whole-exome sequencing links a variantin DHDDS to retinitis pigmentosa[J]. The American Journal of Human Genetics, 2011, 88：201-206.
[29]李嵘, 王喆之. 植物萜类合成酶3-羟基-3-甲基戊二酰辅酶A还原酶的生物信息学分析[J]. 广西植物, 2006, 26(5)：464-473.
[30]崔大方. 植物分类学[M]. 第3版. 北京：中国农业出版社, 2010：85-255.
[31]Schroeder JI, Allen GJ, Hugouvieux V, et al. Guard cell signal transduction[J]. Annual Review of Plant Biology, 2001, 52(1)：627-658.
[32]Yukimune Y, Tabata H, Higashi Y. Methyl jasmonate-induced overproduction of paclitaxel and baccatin III in taxus cell suspension cultures[J]. Nat Biotechnol, 1996, 14(9)：1129-1132.
[33]朱颖, 王丽丽, 柴芸彬, 龚一富. 茉莉酸甲酯对杜氏盐藻β-胡萝卜素含量的影响[J]. 宁波大学学报：理工版, 2010, 1：13-17.
[34]Choi D, Bostock RM, Avdiushko S, et al. Lipid-derived signals that discriminate wound and pathogen responsive isopernoid pathways in plants：Methyl jasmonate and the fungal elicitor arachidonic acid induce different 3-hydroxy-3-methylglutaryl-coenzyme A reductase genes and antimicrobial isoprenoids in Solanum tuberosum L. [J]. Proc Natl Acad Sci, 1994, 91(6)：2329-2333.