油用牡丹脂肪酸脱氢酶基因FAD3的克隆与表达分析

发布时间：2018-03-30 22:11

  本文选题：油用牡丹　切入点：脂肪酸脱氢酶　出处：《中国农业科学》2017年10期

【摘要】：【目的】ω-3脂肪酸脱氢酶(ω-3 FAD)为植物脂肪酸生物合成途径中的关键酶,通过对油用牡丹‘凤丹’ω-3 FAD基因结构特征及其在不同组织中的表达模式进行分析,为研究FAD3在油用牡丹‘凤丹’脂肪酸形成过程中的调控提供理论基础。【方法】采用RACE和RT-PCR的方法克隆油用牡丹‘凤丹’ω-3 FAD基因,用Vector NTI Advance 11软件进行序列分析,BLAST进行同源性比较,并用MEGA7.0的邻接法(neighbor-joining,NJ)构建系统进化树,利用在线蛋白质分析工具Ex PASy预测FAD3蛋白的基本参数和特性,利用Phyre2预测蛋白质二级和三级结构,并通过实时荧光定量PCR检测该基因在油用牡丹‘凤丹’不同发育期的种子以及不同组织中的特异性表达情况。【结果】获得油用牡丹‘凤丹’脂肪酸脱氢酶FAD3,命名为FAD3(Gen Bank登录号:KX906966)。序列分析表明该基因c DNA序列全长1 723 bp,其中,开放阅读框1 308 bp,编码435个氨基酸,3′端非编码区长287 bp,5′末端非编码区长99 bp,预测成熟蛋白分子量为49.9 k D,理论等电点(p I)为7.42,N端无信号肽,脂肪系数为83.08,不稳定指数35.67,总水平亲水性为-0.222。经FAD3蛋白的二级结构预测,FAD3以α-螺旋、随机卷曲为主,其次是延伸链、β-转角含量较少;多序列比对结果表明,油用牡丹FAD3氨基酸序列含有2个保守结构域,系统发育分析结果显示‘凤丹’与芍药处于同一分支,其亲缘关系最近。TMHMM和Target P亚细胞定位分析得知,FAD3蛋白有3个跨膜区域,可能定位于内质网中发挥功能。组织特异性结果分析表明,FAD3在‘凤丹’的根、茎、叶、花瓣、雌蕊、雄蕊、种子中均有表达,其中,在叶中表达量最高,雌蕊次之,在雄蕊中表达量最低;不同时期种子中,10 d表达量最高,20 d次之,在60 d中表达量最低。【结论】成功从油用牡丹‘凤丹’中克隆获得FAD3全长c DNA序列,其在‘凤丹’不同组织中呈现出多种表达模式。
[Abstract]:[objective] 蠅 -3 fatty acid dehydrogenase (蠅 -3 FAD) is a key enzyme in the biosynthesis pathway of plant fatty acids. The structural characteristics of 蠅 -3 fatty acid dehydrogenase (FAD) gene and its expression patterns in different tissues were analyzed.In order to study the regulation of FAD3 in fatty acid formation of oil peony 'Fengdan'. [methods] RACE and RT-PCR were used to clone the gene of 'Fengdan' 蠅 -3 FAD.Vector NTI Advance 11 software was used to analyze the sequence and compare the homology of blast. The phylogenetic tree was constructed by using the adjacent method of MEGA7.0. The basic parameters and properties of FAD3 protein were predicted by the on-line protein analysis tool Ex PASy.Using Phyre2 to predict the secondary and tertiary structures of proteins,The specific expression of the gene in the seeds and tissues of oil peony 'Fengdan' was detected by real-time fluorescence quantitative PCR. [results] Fatty acid dehydrogenase (FAD3) of oil used peony 'Fengdan' was obtained.The name is FAD3(Gen Bank login number: KX906966.Sequence analysis showed that the c DNA sequence of the gene was 1 723 BP, in which,Open reading frame 1 308 BP, encoding 435 amino acid 3 '-terminal noncoding region 287 BP 5' -terminal non-coding region 99 BP, predicted molecular weight of mature protein is 49.9 KD, theoretical isoelectric point I) is 7.42 N terminal signal free peptide.The fat coefficient was 83.08, the instability index was 35.67, and the total hydrophilicity was -0.222.The secondary structure of FAD3 protein predicted that FAD3 consisted of 伪 -helix, random crimp, extension chain and less 尾 -rotation angle. The results of multiple sequence alignment showed that the amino acid sequence of FAD3 contained two conserved domains.Phylogenetic analysis showed that 'Fengdan' was in the same branch of Paeonia lactiflora, and its close relationship. TMHMM and Target P subcellular localization analysis showed that there were three transmembrane regions of FAD3 protein, which may play a role in the endoplasmic reticulum.The results of tissue specific analysis showed that FAD3 was expressed in roots, stems, leaves, petals, pistil, stamens and seeds of 'Fengdan'. The highest expression was found in leaves, followed by pistil, and the lowest in stamens.The highest expression level was 10 days in seeds of different stages, followed by the lowest expression in 60 days. [conclusion] the full-length FAD3 c DNA sequence was successfully cloned from oil peony 'Fengdan'.There are many expression patterns in different tissues of Fengdan.
【作者单位】： 重庆师范大学生命科学学院;
【基金】：国家自然科学基金(31171588) 重庆市林业重点科技攻关项目(渝林科研2015-3) 重庆市社会民生科技创新专项(cstc2016shmszx80051) 重庆师范大学基金(14XLB015)
【分类号】：Q943.2;S565.9
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本文编号：1687866