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

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摘要番茄红素β-环化酶(lycopene beta-cyclase, LCYB)能催化番茄红素形成β-胡萝卜素及其氧化物，是处于植物类胡萝卜素合成途径中的一个分支点。为研究三角褐指藻(Phaeodactylum tricornutum)在不同外源诱导下lcyb基因的表达情况及其对岩藻黄素积累的影响，本研究通过从头测序(de novo测序)获得三角褐指藻lcyb基因cDNA全长并对其进行生物信息学分析，进一步对经过不同浓度的茉莉酸甲酯(methyl jasmonate, MeJA)、乙酰水杨酸(acetylsalicylic acid, ASA)、花生四烯酸(arachidonic acid, AA)和硫酸铈铵(ammonium cerous sulfate, ACS)处理的三角褐指藻lcyb基因表达进行分析，并对其岩藻黄素含量进行测定。结果表明，本研究通过转录组获得的三角褐指藻lcyb基因序列与NCBI上已知序列(GenBank No. XM002176576)相同，其cDNA全长为2 060 bp，含1个长度为1 980 bp的开放阅读框(open reading fragment, ORF)，编码659个氨基酸，该蛋白在N端含有1个双核苷酸结合位点，在C端含有1个螺旋跨膜区域，存在叶绿体导肽和信号肽，定位于叶绿体类囊膜上。系统发育进化树显示，三角褐指藻等硅藻共处1个进化支，亲缘关系较近。诱导转录结果表明，50 μmol/L MeJA、10 mg/L ASA、0.1 mg/L AA和0.4 mg/L ACS处理后的三角褐指藻lcyb基因在本实验范围内转录表达水平最高，且三角褐指藻中的岩藻黄素含量随着lcyb基因转录量的增加而增加，并呈正相关，说明该酶在岩藻黄素合成过程中起到重要作用，诱导子能增强该基因转录从而达到提高岩藻黄素含量的目的。本研究为岩藻黄素的合成调控提供理论指导，也为进一步通过代谢工程手段提高三角褐指藻中的岩藻黄素含量提供参考。

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关键词 ：三角褐指藻, 番茄红素 β-环化酶(LCYB), 岩藻黄素, 生物信息学

Abstract：Lycopene beta-cyclase (LCYB), which catalyzes the formation of β-carotene and its oxides, is an enzyme act as branching point of the plant carotenoid synthesis pathway. This study investigated the effect of elicitors on the expression of lcyb gene and content of fucoidin in Phaeodactylum tricornutum. Firstly, the full-length cDNA of lcyb had been obtained from P. tricornutum, and bioinformatics analysis was carried out. Then, aethyl jasmonate (MeJA), aacetylsalicylic acid (ASA)，arachidonic acid (AA) and ammonium cerous sulfate (ACS) were used to induce the expression of Ptlcyb gene and the content of fucoxanthin in P. tricornutum. The results showed that the lcyb gene of Phaeodactylum tricornutum obtained by de novo sequencing was same as the sequence on NCBI(GenBank No. XM002176576). The full length of lcyb gene was comprised by 2 060 bp, contanting a 1 980 bp open reading fragment(ORF), which encoded a polypeptide of 659 amio acids. Sequence analysis showed that P. tricornutum LCYB had a dinucleotides-biding site at the N-terminus and a Predicted TM helix at the C-terminus. In addition, signal peptide and the presence of chloroplast peptide han been found, which further proved that LCYB was located in the thylakoid membrane. Phylogenetic tree demostrated that diatom such as P. tricornutum coexisted in an evolutionary branch, which was closely related. Upon the observation of Ptlcyb regulation expression, the expression level of Ptlcyb had significant rise when the concentration was 50 μmol/L MeJA, 10 mg/L ASA, 0.1 mg /L AA and 0. 4 mg/L ACS. At the same time, the content of fucoxanthin in P. tricornutum was significantly higher than that in the control group (P ＜0.01). The variation trend of fucoxanthin content and Ptlcyb expression were basically the same, which showed that Ptlcyb played an important role in the synthesis of fucoxanthin. This study provide theoretical guidance for the regulation of fucoxanthin synthesis and also provide a reference for further improving the content of fucoxanthin by means of metabolic engineering in P. tricornutum.

Key words： Phaeodactylum tricornutum Lycopene β-cyclase (LCYB) fucoxanthin Bioinformatics

收稿日期: 2017-03-24 出版日期: 2017-12-10

基金资助:国家星火计划项目

通讯作者: 龚一富 E-mail: gongyifu@163.com

引用本文:

俞凯龚一富朱帅旗刘浩王何瑜. 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]. , 2017, 25(12): 2009-2017.

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http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2017/V25/I12/2009

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