Zeaxanthin is a derivative of β-carotene found in nature and it is the most curcial portion of the macula and retina,helping protect the macular region of the eye from harmful form of light can cause photoxidative damage to the eye.The biosynthesis of zeaxanthin is regulated by a series if enzymes,β-carotene hydroxylase(HYB) is the rate-limiting enzyme during the synthesis.To investigate the changes of Dschyb expression and content of zeaxanthin in Dunaliella salina under some stress,the full-length cDNA of chyb (GenBank accession No.JN118489) had been obtained from D.salina based on RACE technology,and four factors influencing Dschyb and zeaxanthin were analyzed.The cDNA was comprised of 1 433 bp containing a 969 bp open reading fragment,which encoded a polypeptide of 322 amio acids with a predicted molecular mass of 35.5 kDa and theoretical pI of 9.01.It had four conserved histidine residue motifs and was homologous with Volvox carteri f.nagariensis (64％) and Chlamydomonas reinhardtii(58％).Sequence analysis showed that D.salina CHYB had four transmembrane domains and chloroplast transit peptide,which further proved that β-carotene hydroxylase was located in the thylakoid membrane,no signal peptide was predicted in DsCHYB.Phylogenetic tree demostrated CHYB in D.salina and CHYBs from other green algae like Volvox carteri f.nagariensis and Chlamydomonas reinhardtii were grouped into one clade.Upon the observation of chyb regulation expression,the Dschyb expression level had little fluctuation when treated with intensive light at the treatment groups of 6 and 12 h,but with a significant rise from 24 h (P＜0.01),and at the peak of 48 h (P＜0.01).When darked-cultured cells were exposed to high light,sodium acetate and ferrous sulfate,the Dschyb transcription obviously increased within the initial 6 h (P＜0.01),but then declined after 12 h of the treatment.When D.sallina was exposed to glucose,the expression level of Dschyb reached a peak after 1.5 h.In contrast to the control group,there was no change observed in the group of 6 h.Most strikingly,glucose induction had not been observed after treatment with RNA polymerase inhibitor actinomycin D,indicating that actinomycin D might have a strong inhibitory effect on glucose.Dschyb mRNA was expressed significantly less than that in the control group (P＜0.01),especially in groups of 1.5 and 3 h.Zeaxanthin was identified according to their chromatograms on HPLC and UV-vis absorption spectra.The retention time of the standard was 26.742 min while the sample was 29.008 min.The content of zeaxanthin increased due to sodium acetate,ferrous sulfate,high light and glucose treatment.The content of zeaxanthin had increased 16％ (P＜0.05),28％(P＜0.05) and 53％(P＜0.01) compared with control group,respectively.These results support that high light and glucose play critical roles in Dschyb expression and provide a helpful message for production of zeaxanthin.%玉米黄质(3,3'-二羟基-β-胡萝卜素)是自然界中常见的一种β-胡萝卜素衍生物,是黄斑和视网膜中最关键的部分.玉米黄质是通过一系列酶作用合成,其中β-胡萝卜素羟化酶(chyb)是关键限速酶,为了研究盐生杜氏藻(Dunaliella salina)中不同胁迫下β-胡萝卜素羟化酶基因的表达及玉米黄质含量的变化.本研究采用RACE方法首次从盐生杜氏藻中扩增出β-胡萝卜素羟化酶基因(Dschyb),并分析强光、葡萄糖等因素对DSchyb表达及玉米黄质含量的影响.结果表明,该基因全长1 433 bp(GenBank登录号:JN118489),包含一个969bp的开放阅读框,编码322个氨基酸,氨基酸的分子量为35.51kD,等电点(PI)为9.01.DsCHYB包含有4个保守的组氨酸基序,与团藻及莱茵衣藻的同源性分别是64％和58％.DsCHYB具有4个跨膜结构及叶绿体导肽,进一步证明该酶定位于叶绿体类囊膜上.系统进化树分析标明,DsCHYB与其他绿藻如团藻(Volvox carteri f.Nagariensis)、莱茵衣藻(Chlamydomonas reinhardtii)的CHYB共处一个进化支,亲缘关系很近.Dschyb基因的表达调控研究显示,在经强光刺激24 h后,Dschyb 基因表达显著上调(P＜0.01),在48 h表达最高(P＜0.01).在经乙酸钠、硫酸亚铁和强光共同处理6h时,Dschyb表达急剧上升(P＜0.01),处理12h后下降;在葡萄糖处理1.5h后,其表达达到最高,6h后表达与对照组无显著性差异;添加放线菌素D后,Dschyb的表达相对对照降低,表明放线菌素D可能对葡萄糖诱导Dschyb表达具有抑制作用.高效液相色谱测定其玉米黄质含量,钠铁、强光及葡萄糖处理均能提高盐生杜氏藻的玉米黄质的含量,其玉米黄质含量比对照组分别增长16％(P＜0.05),28％(P＜0.05)和53％(P＜0.01).本研究结果为玉米黄质合成调控及其在藻类中功能研究提供理论指导.
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