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石蕾， 梁成伟*， 郝翠翠， 张伟
（青岛科技大学 化工学院， 山东 青岛 266042）
摘要： 在缺氮、高光条件下，雨生红球藻（Haematococcus pluvialis）体由生长状态变为休眠状态，并形成厚壁孢子，同时在细胞内产生大量虾青素。4-羟基-3-甲基-2-(E)-丁烯基-4-磷酸还原酶（HDR）是甲基赤藓糖磷酸(MEP)途径中的最后一个酶，催化4-羟基-3-甲基-(2E)-丁烯基-4-磷酸生成异戊烯基焦磷酸(IPP)，而IPP合成途径是叶绿素和类胡萝卜素合成的上游途径。本研究根据雨生红球藻的转录组中获得的HDR基因的部分序列设计引物，通过RACE的方法获得雨生红球藻的HDR基因全长序列，命名为HpHDR。生物信息学分析结果表明：HpHDR长1421 bp，编码434个氨基酸，属于LYTB蛋白质家族，其氨基酸的相似性与其它绿藻植物超过90%。通过实时定量RT-PCR方法分析了在缺氮、高光及高光缺氮胁迫条件下雨生红球藻中HDR基因表达调控模式，结果显示：在3种诱导条件下，HDR基因均存在转录上调，但是表达模式不同。在缺氮（-N）条件下，HDR基因在22 d表达量最大，为正常条件下的18.0倍；在高光(HL)条件下，HDR基因在第7 d的最大表达量为正常条件下的22.3倍；在高光-缺氮（HL-N）条件下，HDR基因在第2 d达到最大表达量，为正常条件下的54.4倍。研究结果表明HDR基因的表达量与雨生红球藻在缺氮、高光、高光缺氮条件下色素合成量之间存在正相关性。
关键词： 雨生红球藻； 异戊烯焦磷酸（IPP）； HDR； 基因表达
中图分类号： R 931.711文献标志码： A
引用格式：石蕾， 梁成伟， 郝翠翠，等.雨生红球藻4-羟基-3-甲基-2-(E)-丁烯基-4-磷酸还原酶基因(HDR)的克隆及表达分析\[J\].青岛科技大学学报（自然科学版），2017，38（4）：32-39.
SHI Lei, LIANG Chengwei, HAO Cuicui, et al.Cloning and expression analysis of 4-Hydroxy-3-methyl-2-(E)-butenyl-4-diphosphatereductase gene (HDR) from Haematococcus pluvialis\[J\].Journal of Qingdao University of Science and Technology（Natural Science Edition),2017,38(4)：32-39.
Cloning and Expression Analysis of 4-Hydroxy-3-methyl-2-(E)-butenyl-4-diphosphatereductase Gene (HDR) from Haematococcus Pluvialis
SHI Lei, LIANG Chengwei, HAO Cuicui, ZHANG Wei
(College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao 266042, China)
Abstract: Haematococcus pluvialis is a kind of single-celled algae.When green cells encounter stress conditions such as nitrogen deficiency, high light intensity, the alga rapidly differentiates from a vegetative stage into a resting stage, forming aplanospores which accu-mulate large amounts of astaxanthin. Isopentenyl diphosphate(IPP) biosynthesis pathway is upstream of carotenoid and chlorophyll synthesis pathway. 4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphatereductase (HDR) is the terminal-acting enzyme in the plastid MEP pathway which provides isoprenoids. In the study, we designed primers according the partial sequence of HDR gene sequence from Haematococcus pluvialis and obtained the full length HDR gene through the method of RACE, named HpHDR. Bioinformatics analysis indicated that the length of open reading flame (ORF) in HpHDR gene is 1421 bp,encoding 434 amino acids. HpHDR belonged to the LYTB family and have more than 90% similarity with HDRs from other plants. The expression patterns of HDR were analyzed by qtRT-PCR. The results showed that HDR gene all up-regulated under the inducement of N starvation (-N), high light (HL) and high light with nritron free (HL-N). Under the condition of nitrogen deficiency, HDR showed significantly up-regulated expression level, with 16.3-fold higher than that of control respectively. Under the condition of HL, HDR gene obviously up-regulated at the transcript level with 22.3- fold higher than control, respectively. Under the condition of HL-N, the transcript levels of HDR, changed most significantly, with 54.4-fold higher than control, respectively. The correlation between the pigments content and the amount of HDR gene expression under stress conditions were also analyzed in this paper.
Key words: Haematococcus pluvialis； IPP； HDR； gene expression
收稿日期： 2016-02-14
基金项目： 国家自然科学基金项目（31000135）.
作者简介： 石蕾（1993—），女，硕士研究生. *通信联系人.
文章编号： 1672-6987（2017）04-0032-08； DOI： 10.16351/j.1672-6987.2017.04.005
