Abstract：Carotene hydroxylases are key enzymes that catalyze the synthesis of carotenoids in metabolic pathway of carotenoids. It consists of the β-carotenoid hydroxylase (chyb) and the cytochrome P450 (cyp97) family. In order to investigate the bioinformatic characteristics of the chyb and the cyp97 gene family and the expression of chyb and cyp97 under different stress conditions in Dunaliella viridis, cDNA sequences of chyb1, chyb2 and cyp97c of Dunaliella viridis were obtained from the transcriptome data. Bioinformatic analysis showed that the proteins encoded by chyb1, chyb2 and cyp97c genes of D. viridis were hydrophilic proteins, of which CHYB1 and CHYB2 had four transmembrane structures respectively, and CYP97C protein had no cross-membrane structure. Multiple sequence alignment showed that both CHYB1 and CHYB2 contained conservative histidine residues (HXXXXH and HXXHH) while CYP97C contained a heme site (FXXGXRXCXG) and a conservative site (EXXR). Phylogenetic tree analysis revealed that the chyb1 and chyb2, belonging to chyb gene family, while cyp97c was a member of cyp97 gene family and most closely related to the Chlamydomonas reinhardtii. Moreover, qRT-PCR results revealed that all of the 3 tested genes were up-regulated (P<0.01) during high light stress. Besides the content of lutein, zeaxanthin, and the active oxygen level in D. viridis increased significantly, indicated that carotenoid hydroxylase gene family was involved in high light stress and regulated reactive oxygen level to protect algal from oxygen stress. In addition, excess salt or low temperature stress or plant growth regulators (e.g. methyl jasmonate, acetylsalicylic acid, arachidonic acid, ammonium cerous sulfate) could not induce these two family genes simultaneously, revealed that different family genes had different preference to stress response. The present study initially revealed the metabolic control mechanism of carotenoid of D. viridis, which provides excellent genetic resources for increasing the content of lutein and zeaxanthin, and a new sight for improving plant stress tolerance.
[1] 陈俊粤, 龚一富, 马颖瑞, 等. 2017. 绿色杜氏藻异戊烯基焦磷酸异构酶基因(DvIPI)的生物信息学与诱导表达分析[J]. 生命科学研究, 21(3): 201-207. (Chen J Y, Gong Y F, Ma Y R, et al.2017. Bioinformatics and inducible expression analysis of the heterogeneous enzyme gene (DVIPI) in Dunaliella viridis[J]. Life Science Research, 21(3): 201-207.) [2] 丁丽莺. 2013. 包合技术应用于万寿菊制剂初探[D]. 硕士学位论文, 南京师范大学, 导师: 任勇, pp. 23-25. (Ding L Y.2013. Preliminary study on application of encapsulation technology in Tagetes erecta[D]. Thesis for M.S., Nanjing Normal University, Supervisor: Ren Y, pp. 23-25.) [3] 郭玉朋. 2014. 植物光呼吸途径研究进展[J]. 草业学报, 23(4): 322-329. (Guo Y P.2014. Research progress of plant light respiration pathway[J]. Acta Prataculturae Sinica, 23(4): 322-329.) [4] 贺丽虹, 赵淑娟, 胡之璧. 2008. 植物细胞色素P450基因与功能研究进展[J]. 药物生物技术, 15(2): 142-147. (He L H, Zhao S J, Hu Z B.2008. Research progress of plant cytochrome P450 gene and function[J]. Chinese Journal of Pharmaceutical Biotechnology, 15(2): 142-147.) [5] 侯召丽. 2010. 八氢番茄红素合成酶基因转化绿藻的初步研究[D]. 硕士学位论文, 南开大学, 导师: 陈德富, pp. 2-3. (Hou S L.2010. A preliminary study on the transformation of green algae by the gene of the lycopene synthase[D]. Thesis for M.S., Nankai University, Supervisor: Chen D F, pp. 2-3.) [6] 雷波, 卢坤, 丁福章, 等. 2012. 烟草橙蛋白基因的克隆与生物信息学分析[J]. 烟草农学, 297(4): 60-66. (Lei B, Lu K, Ding F Z, et al.2012. Cloning and bioinformatics analysis of tobacco orange protein gene[J]. Tobacco Agronomy, 297(4): 60-66.) [7] 刘佳, 全雪丽, 姜明亮, 等. 2016. 低温胁迫对人参皂苷生物合成途径基因家族表达特性的影响研究[J]. 中草药, 47(11): 1956-1961. (Liu J, Quan X L, Jiang M L, et al.2016. Effects of low temperature stress on the expression characteristics of gene family of ginsenoside biosynthesis pathway[J]. Chinese Traditional and Herbal Drugs, 47(11): 1956-1961.) [8] 吕明珠. 2012. 水稻类胡萝卜素羟化酶CYP97A4参与Iutein生物合成与强光破坏防御[D]. 博士学位论文, 中国科学院, 导师: 林鸿宣, pp. 7-8. (Lv M Z.2012. Rice carotenoids CYP97A4 involved in iutein biosynthesis and strong light damage defense[D]. Thesis for M.S., Chinese Academy of Sciences, Supervisor: Lin H X, pp. 7-8.) [9] 焦芳婵, 曾建敏, 吴兴富, 等. 2015. 烟草β-胡萝卜素羟化酶基因的特征分析[J]. 分子植物育种, 13(8): 1831-1837. (Jiao F S, Zeng J M, Wu X F, et al.2015. Characteristic analysis of tobacco β-carotene hydroxylase gene[J]. Molecular Plant Breeding. 13(8): 1831-1837.) [10] 施蕴渝, 吴季辉. 2008. 核磁共振波谱研究蛋白质三维结构及功能[J]. 中国科学技术大学学报, 38(8): 941-949. (Shi Y Y, Wu J H.2008. NMR spectroscopy studies the three-dimensional structure and function of proteins[J]. Journal of China University of Science and Technology, 38(8): 941-949.) [11] 孙化雨, 陈颖, 赵韩生, 等. 2015. 毛竹β-胡萝卜素羟化酶基因的分子特征及其功能[J]. 林业科学, 51(10): 53-59. (Sun H Y, Chen Y, Zhao H S, et al.2015. Molecular characteristics and function of β-carotene hydroxylase gene in Phyllostachys pubescens[J]. Scientia Silvae Sinicae, 51(10): 53-59.) [12] 王冬梅, 王玮蔚, 孙雪, 等. 2014. 一株杜氏藻的分子鉴定与耐盐特性[J]. 植物生理学报, 50(3): 315-323. (Wang D M, Wang W W, Sun X, et al.2014. Molecular identification and salt-tolerant characteristics of a Dunaliella salina[J]. Plant Physiology Journal, 50(3): 315-323.) [13] 王加龙. 2006. OsCH2和OsHSP82基因过表达载体构建和水稻遗传转化[D]. 硕士学位论文, 湖南农业大学, 导师: 陈信波, pp. 7-8. (Wang J L.2006. Construction of OsCH2 and OsHSP82 gene expression vectors and rice genetic transformation[D]. Thesis for M.S., Hunan Agricultural University, Supervisor: Chen X B, pp. 7-8.) [14] 王丽丽. 2009. 诱导子对雨生红球藻虾青素含量的影响及其分子机理的初步研究[D]. 硕士学位论文, 宁波大学, 导师: 龚一富, pp. 34-45. (Wang L L.2009. Preliminary study on the effect of inducer on the content of astaxanthin and its molecular mechanism in Haematococcus plutialis[D]. Thesis for M.S., Ningbo University, Supervisor: Gong Y F, pp. 34-45.) [15] 王培磊. 2005. 胁迫因子对杜氏藻生长及其β-胡萝卜素积累的影响研究[D].博士学位论文, 中国海洋大学, 导师: 张学成, pp. 13-21. (Wang P L.2005. Effects of stress factors on growth and β-carotene accumulation of Dunaliella viridis[D]. Thesis for M.S., Ocean University of China, Supervisor: Zhang X C, pp. 13-21.) [16] 解敏敏, 龚达平, 李凤霞, 等. 2013. 烟草细胞色素P450的基因组学分析[J]. 遗传, 35(3): 379-387. (Xie M M,Gong D P, Li F X, et al.2013. Genomic analysis of tobacco cytochrome P450[J]. Hereditas (Beijing), 35(3): 379-387.) [17] 许峰, 龚一富, 刘林, 等. 2015. 转盐生杜氏藻β-胡萝卜素羟化酶基因(chyb)烟草的获得及耐盐性鉴定[J]. 农业生物技术学报, 23(9): 1149-1156. (Xu F, Gong Y F, Liu L, et al.2015. Acquisition and salt-tolerance evaluation of transgenic tobacco (Nicotiana tabacum)with β-carotene hydroxylase gene (chyb) from Dunaliela viridis[J]. Journal of Agricultural Biotechnology, 23(9): 1149-1156. [18] 俞凯, 龚一富, 朱帅旗, 等. 2017. 不同外源诱导子对三角褐指藻lcyb基因转录及其岩藻黄素含量的影响[J]. 农业生物技术学报, 25(12): 2009-2017. (Yu K, Gong Y F, Zhu S Q, et al.2017. Effects of different exogenous elicitors on lcyb gene transcription and fucoxanthin content in Phaeodactylum tricornutum[J]. Journal of Agricultural Biotechnology, 25(12): 2009-2017.) [19] 于晓娜. 2014. 两株绿藻类胡萝卜素羟化酶基因的克隆和表达分析[D]. 硕士学位论文, 南京农业大学, 导师: 秦松. pp. 14-15. (Yu X N.2014. Gene cloning and expression analysis of carotenoid hydroxylase from two green algae[D]. Thesis for M.S., Nanjing Normal University, Supervisor: Qin S, pp. 14-15.) [20] 章丽, 龚一富, 刘晓丹, 等. 2013. 盐生杜氏藻β-胡萝卜素羟化酶基因(chyb)的克隆及表达分析[J]. 农业生物技术学报, 21(8): 920-930. (Zhang L, Gong Y F, Liu X D, et al.2013. Cloning and expression analysis of β-carotene hydroxylase gene (chyb) from Dunaliella salina[J]. Journal of Agricultural Biotechnology, 21(8): 920-930. [21] 钟兰, 王凯, 谭军. 2002. 籼稻细胞色素P450超基因家族成员及其EST证据[J]. 中国科学, 32(6): 500-504. (Zhong L, Wang K, Tan J.2002. P450 family members and est evidences of indica rice cytochrome-gene[J]. Science China, 32(6): 500-504.) [22] 周丽, 粱新乐, 励建荣. 2003. 类胡萝卜素抗氧化作用研究进展[J]. 食品研究与开发, 24(2): 21-23. (Zhou L, Liang X Y, Li J R.2003. Research progress in antioxidant action of carotenoids[J]. Food Research and Development, 24(2): 21-23.) [23] 朱帅旗, 章丽, 俞凯, 等. 2017. 绿色杜氏藻不同β-胡萝卜素羟化酶基因家族胁迫应答模式研究[J]. 遗传, 39(2): 156-165. (Zhu S Q, Zhang L, Yu K, et al.2017. Study on the response pattern of different β-carotene hydroxylase gene family stress responses in Dunaliella viridis[J]. Hereditas (Beijing), 39(2): 156-165.) [24] Alscher R G, Donahue J L, Cramer C L.1997. Reactive oxygen species and antioxidants: Relationships in green cells[J]. Physiologia Plantarum, 100(2): 224-233. [25] Ben-Amotz A, Gressel J, Avron M.2007. Massive accumulation of phytoene induced by norflurazon in Dunaliella bardawil (chlorophyceae) prevents recovery from photoinhibition[J]. Journal of Phycology, 23(2): 176-181. [26] Ben-Amotz A, Shaish A, Avron M.1989. Mode of action of the massively accumulated β-Carotene of Dunaliella bardawil in protecting the alga against damage by excess irradiation[J]. Plant Physiology, 91(3): 1040-1043. [27] Boussiba S.2000. Carotenogenesis in the green alga Haematococcus pluvialis: Cellular physiology and stress response[J]. Physiologia Plantarum, 108(2): 111-117. [28] Bouvier F, Keller Y, D'Harlingue A, et al.1998. Xanthophyll biosynthesis: Molecular and functional characterization of carotenoid hydroxylases from pepper fruits (Capsicum annuum L.)[J]. Biochimica et Biophysica Acta, 1391(3): 320-328. [29] Butnariu M, Rodino S, Petrache P, et al.2014. Determination and quantification of maize zeaxanthin stability[J]. Digest Journal of Nanomaterials and Biostructures, 2(9): 745-755. [30] Galpaz N, Wang Q, Menda N, et al.2008. Abscisic acid deficiency in the tomato mutant high-pigment 3 leading to increased plastid number and higher fruit lycopene content[J]. Plant Journal, 53(5): 717-730. [31] Gao Y Y, Yang M C, Wang C H.2013. Nutrient deprivation enhances lipid content in marine microalgae[J]. Bioresource Technology, 147(30): 484-491. [32] Gill R W, Sanseau P.2000. Rapid in silico cloning of genes using expressed sequence tags (ESTs)[J]. Biotechnology Annual Review, 5(00): 25-44. [33] Gong Y F, Liao Z H, Guo B H, et al.2006. Molecular cloning and expression profile analysis of Ginkgo biloba dxs gene encoding 1-deoxy-D-xylulose 5-phosphate synthase, the first committed enzyme of the 2-C-met-hyl-D-erythritol 4-phosphate pathway[J]. Planta Medica, 72(4): 329-335. [34] Grünewald K, Hirschberg J.2001. Hagen C. Ketocarotenoid biosynthesis outside of plastids in the unicellular green alga Haematococcus pluvialis[J]. Journal of Biological Chemistry, 276(8): 6023-6029. [35] Hu Q, Miyashita H, Iwasaki I, et al.1998. A photosystem I reaction center driven by chlorophyll d in oxygenic photosynthesis[J]. Proceedings of the National Academy of Sciences of the USA, 95(22): 13319-13323. [36] Kim J, Smith J J, Tian L, et al.2009. The evolution and function of carotenoid hydroxylases in Arabidopsis[J]. Plant&Cell Physiology, 50(3): 463-479. [37] Krinsky N I.2009. Carotenoid protection against oxidation: Pure and applied chemistry[J]. Annals of Internal Medicine, 52(1): 806-18. [38] Oren A.2005. A hundred years of dunaliella research: 1905-2005[J]. Saline Systems, 1(1): 1-14. [39] Ruizsola M A, Rodríguezvillalón A, Rodríguezconcepción M.2014. Lightsensitive phytochrome interacting factors (PIFs) are not required to regulate phytoene synthase gene expression in the root[J]. Plant Signaling & Behavior, 9(8): 353-372. [40] Saeed B, Das M, Khurana P.2015. Overexpression of ring-carotene hydroxylase1 (BCH1) in Indian mulberry, Morus indica, cv. K2, confers tolerance against UV, high temperature and high irradiance stress induced oxidative damage[J]. Plant Cell Tissue & Organ Culture, 120(3): 1003-1014. [41] Stickforth P, Steiger S, Hess W R, et al.2003. A novel type of lycopene ε-cyclase in the marine cyanobacterium, Prochlorococcus marinus MED4[J]. Archives of Microbiology, 179(6): 409-415. [42] Tao N G, Juan X U, Cheng Y J, et al.2005. Isolation and characterization of copia-like retrotransposons from 12 sweet orange (Citrus sinensis, Osbeck) cultivars[J]. Journal of Botany, 47(12): 1507-1515. [43] Tian L, Dellapenna D.2001. Characterization of a second carotenoid β-hydroxylase gene from Arabidopsis and its relationship to the LUT1 locus[J]. Plant Molecular Biology, 47(3): 379-388. [44] Tian L, Dellapenna D.2004. Progress in understanding the origin and functions of carotenoid hydroxylases in plants[J]. Archives of Biochemistry & Biophysics, 430(1): 22-29. [45] Tian L, Musetti V, Kim J, et al.2004. The Arabidopsis LUT1 locus encodes a member of the cytochrome p450 family that is required for carotenoid epsilon-ring hydroxylation activity[J]. Proceedings of the National Academy of Sciences of the USA, 101(1): 402-417. [46] Yang D G, Qin D L, Li Z, et al.2016. Physiological effects of exogenous salicylic acid on maize seedlings under low temperature stress[J]. Maize Science, 21(4): 122-129. [47] Yoshii Y.2006. Diversity and evolution of photosynthetic antenna systems in green plants[J]. Phycological Research, 54(3): 220-229. [48] Zhang L, Ma G, Kato M, et al.2012. Regulation of carotenoid accumulation and the expression of carotenoid metabolic genes in citrus juice sacs in vitro[J]. Journal of Experimental Botany, 63(2): 871-886. [49] Zhou J, Gong Y F, Ma Y R, et al.2017, Bioinformatics analysis and transcription difference of geranylgeranyl pyrophosphate synthase (GGPS) in Dunaliella viridis[J]. Journal of Nuclear Agricultural Sciences, 31(4): 635-642.
