本文選題：金釵石斛 + -羥基--甲基戊二酰輔酶A合酶��； 參考：《中草藥》2017年12期

【摘要】：目的克隆金釵石斛Dendrobium nobile中3-羥基-3-甲基戊二酰輔酶A合酶(3-hydroxy-3-methylglutaryl-CoA synthase,HMGS)基因DnHMGS,并進(jìn)行生物信息學(xué)和表達(dá)分析。方法采用反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)、cDNA末端快速擴(kuò)增(RACE)技術(shù)獲得DnHMGS基因cDNA全長;生物信息學(xué)分析編碼蛋白的理化特性、結(jié)構(gòu)域等特征;用DNASTAR、MEGA軟件分別進(jìn)行氨基酸多序列比對和進(jìn)化樹構(gòu)建分析;借助實(shí)時(shí)熒光定量PCR(qRT-PCR)技術(shù)檢測基因組織表達(dá)模式。結(jié)果 DnHMGS基因全長為1 816 bp(GenBank注冊號KX789180),編碼一條由474個(gè)氨基酸組成的多肽,相對分子質(zhì)量為52 458.47,等電點(diǎn)5.98。DnHMGS蛋白具有植物HMGS酶的典型結(jié)構(gòu)域和活性中心,與鳳梨、稻、玉米等單子葉植物親緣關(guān)系較近。DnHMGS基因具有組織表達(dá)特異性,接菌前,DnHMGS轉(zhuǎn)錄本在金釵石斛葉中的表達(dá)量最高,為根、莖中的2倍以上。但接菌后DnHMGS基因表達(dá)情況轉(zhuǎn)變?yōu)榍o葉根。結(jié)論首次從金釵石斛中克隆得到HMGS基因的全長cDNA,該基因的分子鑒定為進(jìn)一步揭示該基因在金釵石斛萜類物質(zhì)合成代謝途徑中的作用及菌根真菌影響石斛堿生物合成的調(diào)控機(jī)制奠定了基礎(chǔ)。
[Abstract]:Objective to clone the 3-hydroxy-3-methylglutaryl-CoA synthase (3-hydroxy-3-methylglutaryl-CoA synthase-HMGS) gene from Dendrobium nobile of Dendrobium nobile and analyze its bioinformatics and expression. Methods the full length of DnHMGS gene cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA terminal of RT-PCRN, and the physicochemical properties and domain of the encoded protein were analyzed by bioinformatics. The amino acid multi-sequence alignment and phylogenetic tree construction were analyzed by DNASTARGA-MEGA software, and the expression patterns of gene tissues were detected by real-time quantitative PCR qRT-PCRR technique. Results the total length of DnHMGS gene was 1 816 bp(GenBank registration number KX789180, encoding a polypeptide composed of 474 amino acids with relative molecular weight of 52 458.47. The isoelectric point 5.98.DnHMGS protein had the typical domain and active center of plant HMGS enzyme, and was similar to pineapple and rice. The expression of DnHMGS gene in maize and other monocotyledonous plants was higher than that in the roots and stems of DnHMGS. The expression of DnHMGS transcripts was the highest in the leaves of Dendrobium nobile before inoculation with DnHMGS, which was more than 2 times of that in the roots and stems of Dendrobium nobile. But the expression of DnHMGS gene changed to stem and leaf root after inoculation. Conclusion the full-length HMGS gene was cloned from Dendrobium nobile for the first time. The molecular identification of the gene is intended to further reveal the role of the gene in the pathway of synthesis and metabolism of terpenoids and the effect of mycorrhizal fungi on alkaloid biosynthesis of Dendrobium nobile. The control mechanism has laid the foundation.
【作者單位】： 中國醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院藥用植物研究所;
【基金】：國家自然科學(xué)基金資助項(xiàng)目(31170314,81473331)
【分類號】：S567.239

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