Haematococcus pluvialis is a freshwater planktonic single-cell microalgae. It will accumulate high amount of carotenoids under unfavorable environmental conditions. As one of carotenoids, lycopene is an important intermediate in the carotenoid biosynthesis pathway. Lycopene β-cyclase (LycB) is the key enzyme that catalyzes the circularization of lycopene to form β-carotene. In this study, we constructed a p1301-BS-RNAi vector using lycB from Dunaliella salina as the interference sequence with kanamycin and atrazine resistance marker, and then transformed it into H. pluvialis by electroporation. Sixteen independent transgenic lines were obtained after resistance selection, genome PCR, and RT-PCR analyses. Seven well-grown lines were selected to determine the contents of carotenoids by HPLC analysis after inducing by high light. The lycopene content in these lines was increased by 99.4% while the β-carotene content was decreased by 48.4%, indicating that the interference by heterogenous lycB could inhibit the conversion of lycopene into β-carotene. The amount of increase in lycopene was only 5% of the amount of decrease in β-carotene, indicating that 95% of the decreased β-carotene was converted into other metabolites. Therefore, in order to largely increase the lycopene content in H. pluvialis, it is necessary to coordinatively regulate other metabolic pathways.
[1] 陈兴才, 黄伟光, 欧阳琴. 雨生红球藻培养及虾青素累积条件探讨. 中国食品学报, 2006, 6(4): 41-45.
[2] Grünewald K, Hirschberg J, Hagen C. Ketocarotenoid biosynthesis outside of plastids in unicellular green alga Haematococcus pluvialis. J Biol Chem, 2001, 276(8): 6023-6029.
[3] Drago? N, Bercea V, Bica A, Drug? B, Nicoar? A, Coman C. Astaxanthin production from a new strain of Haematococcus pluvialis grown in batch culture. Ann RSCB, 2010, XV(2): 353-361.
[4] Collins AM, Jones HDT, Han DX, Hu Q, Beechem TE, Timlin JA. Carotenoid distribution in living cells of Haematococcus pluvialis (Chlorophyceae). PLoS One, 2011, 6(9): e24302.
[5] Rosati C, Aquilani R, Dharmapuri S, Pallara P, Marusic C, Tavazza R, Bouvier F, Camara B, Giuliano G. Metabolic engineering of beta-carotene and lycopene content in tomato fruit. Plant J, 2000, 24(3): 413-419.
[6] 万群, 张兴国, 宋明. 果实特异性RNAi介导的Lcy基因沉默来增加番茄中番茄红素的含量. 生物工程学报, 2007, 23(3): 429-433.
[7] 牟春林, 陈喜文, 侯召丽, 王绘砖, 陈德富, 辛乃宏, 郭连城, 张俊杰. 工业生产β-胡萝卜素杜氏藻的分离及种属鉴定. 盐业与化工, 2009, 38(4): 25-30.
[8] 王绘砖, 陈喜文, 郝晓华, 陈德富. 利用雨生红球藻表达系统高通量筛选活力提高的阿特拉津氯水解酶突变子. 生物工程学报, 2011, 27(4): 620-628.
[9] 刘健晖, 李爱芬. 雨生红球藻在不同培养基的生长比较. 生态科学, 2006, 25(2): 113-115, 121.
[10] de Souza ML, Wackett LP, Boundy-Mills KL, Mandel-baum RT, Sadowsky MJ. Cloning, characterization, and expression of a gene region from Pseudomonas sp. strain ADP involved in the dechlorination of atrazine. Appl Environ Microbiol, 1995, 61(9): 3373-3378.
[11] Farré G, Bai C, Twyman RM, Capell T, Christou P, Zhu CF. Nutritious crops producing multiple carotenoids--a meta-bolic balancing act. Trends Plant Sci, 2011, 16(10): 532-540.
[12] Kelkel M, Schumacher M, Dicato M, Diederich M. Antioxidant and anti-proliferative properties of lycopene. Free Radic Res, 2011, 45(8): 925-940.
[13] Ronen G, Carmel-Goren L, Zamir D, Hirschberq J. An alternative pathway to β-carotene formation in plant chromoplasts discovered by map-based cloning of beta and old-gold color mutations in tomato. Proc Natl Acad Sci USA, 2000, 97(20): 11102-11107.
[14] 张学成, 孟振, 时艳侠, 王培磊. 光照、温度和营养盐对三株盐生杜氏藻生长和色素积累的影响. 中国海洋大学学报(自然科学版), 2006, 36(5): 754-762.
[15] 晏露, 姜建国, 刘冠楠, 陈倩, 朱跃辉. 盐藻番茄红素β-环化酶全长cDNA的克隆及结构分析. 现代食品科技, 2010, 26(3): 218-221.
[16] Steinbrenner J, Linden H. Light induction of carotenoid biosynthesis genes in the green alga Haematococcus pluvialis: regulation by photosynthetic redox control. Plant Mol Biol, 2003, 52(2): 343-356.
[17] Diretto G, Tavazza R, Welsch R, Pizzichini D, Mourgues F, Papacchioli V, Beyer P, Giuliano G. Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase. BMC Plant Biol, 2006, 6: 13.
[18] Pecker I, Gabbay R, Cunningham FX Jr, Hirschberg J. Cloning and characterization of the cDNA for lycopene β-cyclase from tomato reveals decrease in its expression during fruit ripening. Plant Mol Biol, 1996, 30(4): 807-819.
[19] Cunningham FX Jr, Pogson B, Sun Z, McDonald KA, DellaPenna D, Gantt E. Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation. Plant Cell, 1996, 8(9): 1613-1626.
[20] Huguency P, Badillo A, Chen HC, Klein A, Hirschberg J, Camara B, Kuntz M. Metabolism of cyclic carotenoids: a model for the alteration of this biosynthetic pathway in Capsicum annuum chromoplasts. Plant J, 1995, 8(3): 417-424.
[21] Bouvier F, d'Harlingue A, Camara B. Molecular analysis of carotenoid cyclase inhibition. Arch Biochem Biophys, 1997, 346(1): 53-64.
[22] Bouvier F, D'harlingue A, Backhaus RA, Kumagai MH, Camara B. Identification of neoxanthin synthase as a carotenoid cyclase paralog. Eur J Biochem, 2000, 267(21): 6346-6352.
[23] Hirsc
