目的 分離珍稀瀕危藥用植物鐵皮石斛咖啡酰輔酶A氧甲基轉(zhuǎn)移酶（caffeoyl CoA O-methyltransferase，CCoAOMT）基因（DoOMT）并進(jìn)行生物信息學(xué)和表達(dá)模式分析。方法 采用RT-PCR和RACE技術(shù)獲基因cDNA全長；利用生物信息學(xué)軟件預(yù)測蛋白的理化性質(zhì)、結(jié)構(gòu)域和三維建模等分子特性；用DNASTAR 6.0和MEGA 4.0分別進(jìn)行氨基酸多序列比對和進(jìn)化關(guān)系分析；借助實(shí)時(shí)定量PCR檢測基因表達(dá)模式。結(jié)果 分離到DoOMT（GenBank注冊號KF876839），cDNA全長1 005 bp，編碼一條由239個(gè)氨基酸組成的多肽，相對分子質(zhì)量為2.708×104，等電點(diǎn)5.03；DoOMT蛋白不含跨膜域和信號肽，具有氧甲基轉(zhuǎn)移酶family 3、甲基轉(zhuǎn)移酶的保守結(jié)構(gòu)域（13～238、31～238）和8個(gè)保守基序；DoOMT與植物CCoAOMTs蛋白一致性為49.4%～78.7%，所在分支隸屬于CCoAOMTs分子進(jìn)化的1b類群，與單子葉植物香草親緣關(guān)系最近；DoOMT基因轉(zhuǎn)錄本在石斛根、莖、葉器官中為組成型表達(dá)，莖中相對表達(dá)量較高，為葉中的4.562倍，根和葉中無顯著差異。結(jié)論 鐵皮石斛咖啡酰輔酶A氧甲基轉(zhuǎn)移酶基因DoOMT的分子特征為進(jìn)一步研究其在鐵皮石斛次生代謝和生長發(fā)育過程中的作用奠定基礎(chǔ)。

Objective To isolate the caffeoyl CoA O-methyltransferase (CCoAOMT) gene (DoOMT) in a rare endangered medicinal orchid species Dendrobium officinale, and to carry out the bioinformatics and expression mode analysis. Methods RT-PCR and RACE technologies were used to obtain the full length cDNA of DoOMT gene. The characteristics of physiochemical properties, conserved domains, and three dimensional structure of the deduced DoOMT protein were determined using a series of bioinformatic tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 6.0 and MEGA 4.0 softwares, respectively. Real time quantitative PCR was used for gene expression analysis. Results The full length cDNA of DoOMT was 1 005 bp in length and encoded a 239-aa protein with a molecular weight of 27 080 and an isoelectric point of 5.03; The deduced DoOMT protein, without transmembrane or signal peptide residues, contained the oxygen methyltransferase family 3, methyltransferase conserved domains (13—238, 31—238), and eight conserved signature sequences. DoOMT had high identities (49.4%—78.7%) with CCoAOMTs proteins from various plants; DoOMT belonged to the 1b subgroup of the CCoAOMT evolutionary tree, and was closely related to the monocot Vanilla planifolia. DoOMT transcripts were constitutively expressed in the leaves, stems, and roots. The transcription level of DoOMT was markedly higher than that of leaves with 4.562 fold, whereas the transcript amount showed no significant difference in leaves and roots. Conclusion Molecular characterization of DoOMT will be useful for further functional determination of the gene involving in the secondary metabolism along with growth and development in D. officinale.

國家自然科學(xué)基金資助（31070300, 31101608）；陜西省青年科技新星項(xiàng)目（2012KJXX-44）；陜西省教育廳專項(xiàng)科研計(jì)劃項(xiàng)目（2013JK0829）