绿色杜氏藻类胡萝卜素羟化酶基因家族分子特征及胁迫表达分析
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摘要
类胡萝卜素羟化酶(Carotenoid hydroxylase)是类胡萝卜素代谢途径中的关键酶,可催化多种类胡萝卜素的合成。类胡萝卜素羟化酶包括亚铁血红素依赖型β-胡萝卜素羟化酶(β-carotenoid hydroxylase, CHYB)和细胞色素P450家族类胡萝卜素羟化酶(CYP97)。为探究chyb基因家族和cyp97基因家族在绿色杜氏藻(Dunaliella viridis)中的生物信息学特征及在不同胁迫条件下基因的表达情况,本研究通过绿色杜氏藻转录组数据,获得chyb1、chyb2和cyp97c基因cDNA序列,对其进行生物信息学分析,并通过荧光定量PCR探究其在不同非生物胁迫下的基因表达水平。生物信息学结果表明,这3条基因编码的蛋白质均为亲水性蛋白,其中CHYB1蛋白和CHYB2蛋白各有4个跨膜结构,CYP97C蛋白无跨膜结构。氨基酸多重序列比对结果显示,CHYB1和CHYB2各含有2种保守的组氨酸残基HXXXXH和HXXHH,CYP97C蛋白含有1个血红素位点FXXGXRXCXG和1个谷氨酸(E)和精氨酸(R)完全保守的螺旋K区保守位点EXXR。系统进化树分析显示,绿色杜氏藻cyp97c属于cyp97基因家族,莱茵衣藻(Chlamydomonas reinhardtii)与绿色杜氏藻的亲缘关系最近。qRT-PCR结果表明,在强光胁迫下绿色杜氏藻chyb1、chyb2和cyp97c基因的表达量均有极显著上升(P<0.01),且藻体内叶黄素和玉米黄素含量显著提高,活性氧水平明显上升,表明类胡萝卜素羟化酶基因家族参与高光胁迫并受到活性氧调控进而保护藻体免受氧胁迫的伤害;此外,高盐、低温及甲基茉莉酸(methyl jasmonate, MeJA)、乙酰水杨酸(acetylsalicylic acid, ASA)、花生四烯酸(arachidonic acid, AA)、硫酸铈铵(ammonium cerous sulfate, ACS)等植物生长调节物质处理均不能同时诱导这两个家族基因,表明不同家族基因对胁迫应答具有不同的偏好性。本研究初步揭示绿色杜氏藻类胡萝卜素代谢调控机理,将为提高叶黄素和玉米黄素含量提供优良的基因资源,同时为植物抗逆提供新思路。
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 cyp97 c of Dunaliella viridis were obtained from the transcriptome data. Bioinformatic analysis showed that the proteins encoded by chyb1, chyb2 and cyp97 c genes of D. viridis were hydrophilic proteins, of which CHYB1 and CHYB2 had four transmembrane structures respectively, and CYP97 C protein had no cross-membrane structure. Multiple sequence alignment showed that both CHYB1 and CHYB2 contained conservative histidine residues(HXXXXH and HXXHH) while CYP97 C 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 cyp97 c was a member of cyp97 gene family and most closely related to the Chlamydomonas reinhardtii. Moreover, q RT-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.
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 cyp97 c of Dunaliella viridis were obtained from the transcriptome data. Bioinformatic analysis showed that the proteins encoded by chyb1, chyb2 and cyp97 c genes of D. viridis were hydrophilic proteins, of which CHYB1 and CHYB2 had four transmembrane structures respectively, and CYP97 C protein had no cross-membrane structure. Multiple sequence alignment showed that both CHYB1 and CHYB2 contained conservative histidine residues(HXXXXH and HXXHH) while CYP97 C 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 cyp97 c was a member of cyp97 gene family and most closely related to the Chlamydomonas reinhardtii. Moreover, q RT-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.
引文
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