發(fā)布時間：2018-07-31 05:12

【摘要】：以獨行菜(Lepidium apetalum)為材料,通過分析獨行菜轉(zhuǎn)錄組數(shù)據(jù),設(shè)計特異性引物,克隆了獨行菜肉桂酸-4-羥化酶(cinnamate-4-hydroxylase,C4H)基因的cDNA序列,命名為LaC4H,Gen Bank登錄號為KX064050,并進行生物信息學分析,原核表達、純化,組織特異性分析和誘導(dǎo)表達分析。結(jié)果表明:(1)LaC4H基因開放閱讀框(ORF)長1 518 bp,編碼505個氨基酸,其蛋白質(zhì)分子質(zhì)量為57.73 k D。(2)生物信息學分析顯示La C4H蛋白包含細胞色素P450的保守基序和5個特征性底物結(jié)合位點,是細胞色素P450超家族成員,系統(tǒng)進化分析顯示LaC4H蛋白與擬南芥等十字花科植物C4H蛋白同源性較高。(3)通過構(gòu)建原核表達載體pET-32a-LaC4H在大腸桿菌BL21(DE3)菌株中成功表達LaC4H重組蛋白,利用Ni~(2+)親和色譜得到了純化的LaC4H重組蛋白。(4)熒光定量PCR結(jié)果表明LaC4H基因在莖中表達量最高,葉和花中次之,根中表達量最低。經(jīng)茉莉酸甲酯(methyl jasmonate,MeJA)誘導(dǎo)后,葉中LaC4H表達量明顯上升,誘導(dǎo)后48 h達到最高值,葉中LaC4H的表達量與黃酮含量之間呈正相關(guān)。這為進一步研究LaC4H基因在獨行菜黃酮類化合物生物合成途徑中的功能奠定基礎(chǔ)。
[Abstract]:The cDNA sequence of cinnamate-4-hydroxylaseC4H gene was cloned by analyzing the transcriptome data and designing a specific primer. The cDNA sequence was named LaC4HG Bank accession number was KX064050, and bioinformatics analysis was carried out. Prokaryotic expression, purification, tissue specific analysis and induced expression analysis. The results showed that: (1) LaC4H gene open reading frame (ORF) was 1 518 BP long, encoding 505 amino acids, and its protein molecular weight was 57.73 k D. (2. Bioinformatics analysis showed that La C4H protein contained the conserved motif of cytochrome P450 and five characteristic substrate binding sites. LaC4H protein is a member of cytochrome P450 superfamily. Phylogenetic analysis shows that LaC4H protein has high homology with C4H protein in Arabidopsis and other cruciferous plants. (3) LaC4H recombinant protein was successfully expressed in Escherichia coli BL21 (DE3) strain by construction of prokaryotic expression vector pET-32a-LaC4H. The purified LaC4H recombinant protein was obtained by Ni ~ (2) affinity chromatography. (4) the results of fluorescence quantitative PCR showed that the expression of LaC4H gene was the highest in stem, second in leaves and flowers, and the lowest in root. After being induced by methyl jasmonate, the expression of LaC4H in leaves increased significantly and reached the highest value 48 h after induction. There was a positive correlation between the expression of LaC4H and the content of flavonoids in leaves. This lays a foundation for the further study of the function of LaC4H gene in the biosynthesis pathway of flavonoids.
【作者單位】： 河南中醫(yī)藥大學藥學院;呼吸疾病診療與新藥研發(fā)河南省協(xié)同創(chuàng)新中心;
【基金】：國家重點基礎(chǔ)研究發(fā)展計劃“973計劃”項目(2013CB531802) 河南省科技攻關(guān)計劃(162102310468) 河南中醫(yī)學院博士科研基金(BSJJ2011-07)
【分類號】：R284
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本文編號：2154519