本文選題：油用牡丹　切入點(diǎn)：脂肪酸脫氫酶　出處：《中國(guó)農(nóng)業(yè)科學(xué)》2017年10期

【摘要】：【目的】ω-3脂肪酸脫氫酶(ω-3 FAD)為植物脂肪酸生物合成途徑中的關(guān)鍵酶,通過(guò)對(duì)油用牡丹‘鳳丹’ω-3 FAD基因結(jié)構(gòu)特征及其在不同組織中的表達(dá)模式進(jìn)行分析,為研究FAD3在油用牡丹‘鳳丹’脂肪酸形成過(guò)程中的調(diào)控提供理論基礎(chǔ)�！痉椒ā坎捎肦ACE和RT-PCR的方法克隆油用牡丹‘鳳丹’ω-3 FAD基因,用Vector NTI Advance 11軟件進(jìn)行序列分析,BLAST進(jìn)行同源性比較,并用MEGA7.0的鄰接法(neighbor-joining,NJ)構(gòu)建系統(tǒng)進(jìn)化樹(shù),利用在線(xiàn)蛋白質(zhì)分析工具Ex PASy預(yù)測(cè)FAD3蛋白的基本參數(shù)和特性,利用Phyre2預(yù)測(cè)蛋白質(zhì)二級(jí)和三級(jí)結(jié)構(gòu),并通過(guò)實(shí)時(shí)熒光定量PCR檢測(cè)該基因在油用牡丹‘鳳丹’不同發(fā)育期的種子以及不同組織中的特異性表達(dá)情況。【結(jié)果】獲得油用牡丹‘鳳丹’脂肪酸脫氫酶FAD3,命名為FAD3(Gen Bank登錄號(hào):KX906966)。序列分析表明該基因c DNA序列全長(zhǎng)1 723 bp,其中,開(kāi)放閱讀框1 308 bp,編碼435個(gè)氨基酸,3′端非編碼區(qū)長(zhǎng)287 bp,5′末端非編碼區(qū)長(zhǎng)99 bp,預(yù)測(cè)成熟蛋白分子量為49.9 k D,理論等電點(diǎn)(p I)為7.42,N端無(wú)信號(hào)肽,脂肪系數(shù)為83.08,不穩(wěn)定指數(shù)35.67,總水平親水性為-0.222。經(jīng)FAD3蛋白的二級(jí)結(jié)構(gòu)預(yù)測(cè),FAD3以α-螺旋、隨機(jī)卷曲為主,其次是延伸鏈、β-轉(zhuǎn)角含量較少;多序列比對(duì)結(jié)果表明,油用牡丹FAD3氨基酸序列含有2個(gè)保守結(jié)構(gòu)域,系統(tǒng)發(fā)育分析結(jié)果顯示‘鳳丹’與芍藥處于同一分支,其親緣關(guān)系最近。TMHMM和Target P亞細(xì)胞定位分析得知,FAD3蛋白有3個(gè)跨膜區(qū)域,可能定位于內(nèi)質(zhì)網(wǎng)中發(fā)揮功能。組織特異性結(jié)果分析表明,FAD3在‘鳳丹’的根、莖、葉、花瓣、雌蕊、雄蕊、種子中均有表達(dá),其中,在葉中表達(dá)量最高,雌蕊次之,在雄蕊中表達(dá)量最低;不同時(shí)期種子中,10 d表達(dá)量最高,20 d次之,在60 d中表達(dá)量最低�！窘Y(jié)論】成功從油用牡丹‘鳳丹’中克隆獲得FAD3全長(zhǎng)c DNA序列,其在‘鳳丹’不同組織中呈現(xiàn)出多種表達(dá)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 偽 -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.
【作者單位】： 重慶師范大學(xué)生命科學(xué)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金(31171588) 重慶市林業(yè)重點(diǎn)科技攻關(guān)項(xiàng)目(渝林科研2015-3) 重慶市社會(huì)民生科技創(chuàng)新專(zhuān)項(xiàng)(cstc2016shmszx80051) 重慶師范大學(xué)基金(14XLB015)
【分類(lèi)號(hào)】：Q943.2;S565.9
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本文編號(hào)：1687866