孟衡玲 张薇 卢丙越 苏一兰 薛春丽

摘要：【目的】克隆铁皮石斛中查尔酮合酶（CHS）基因，分析其基本生物学信息及组织表达特异性，为进一步研究CHS基因在铁皮石斛中的表达调控机理及其在铁皮石斛中的功能打下理论基础。【方法】采用铁皮石斛转录组序列与NCBI同源序列进行比对，根据保守区段设计引物克隆铁皮石斛CHS基因的cDNA全长，采用实时荧光定量PCR定量表达分析不同生长年限、不同组织部位中CHS基因的表达水平。【结果】铁皮石斛CHS基因cDNA编码区1188 bp，编码395个氨基酸，GenBank登录号KT783451，分子量为43.2 kD，理论等电点6.05。铁皮石斛GHS氨基酸序列与同属植物金钗石斛（Dendrobium nobile）的CHS氨基酸序列同源性最高，达99%，与非同科植物欧洲大叶杨（Populus trichocarpa）、巨桉（Eucalyptus grandis）和紫玉兰（Magnolia liliiflora）等植物的同源性在83%左右。该基因编码的蛋白质属非分泌蛋白，定位于细胞质基质中，为非跨膜结构的亲水性不稳定蛋白。CHS基因在1年生铁皮石斛叶片中的表达量最高，随着植株年龄的增长，叶片中CHS基因的表达量降低，茎中的表达量升高。【结论】铁皮石斛CHS基因早期主要参与激素运输和器官形态建成，后期主要参与类黄酮物质合成。

关键词： 铁皮石斛；查尔酮合成酶；基因克隆；實时荧光定量PCR

中图分类号： S567.239；Q785 文献标志码：A 文章编号：2095-1191（2016）12-2015-05

Abstract：【Objective】The present study cloned chalcone synthase（CHS） gene and analyzed its basic biological information and tissue-specific expression in Dendrobium officinale， in order to lay a theoretical basis for further research on the function and expression regulation mechanism of CHS gene in D. officinale. 【Method】Comparing conserved sequence with NCBI homologous sequence， real-time fluorescence quantitative PCR was adopted to analyze CHS gene expression in different tissues at different growth years based on cDNA full length of design-primer-cloned CHS gene in conserved domain. 【Result】The coding region of CHS gene in D. officinale was 1188 bp encoding 395 amino acids， molecular weight was 43.2 kD， theoretical isoelectric point 6.05， GenBank accession number KT783451. In terms of amino acid sequence，CHS in D. officinale showed the highest homology（99%） with that in Dendrobium nobile， its same generic plant. The homology between CHS sequence in D. officinale and its non-family plants Populus trichocarpa， Eucalyptus grandis and Magnolia liliiflora was around 83%. The encoded protein of CHS gene belonged to non-secreted protein which was located in cytoplasmic matrix. It was hydrophilic unstable protein with non-transmembrane structure. The expression level of CHS gene was the highest in one-year leaves of D. officinale. As growth year increased， the expression level of CHS gene decreased in leaves， but increased in stems. 【Conclusion】CHS gene in D. officinale is mainly involved in auxin transport and organ morphogenesis at early stage and participats in flavonoid biosynthesis at late stage.

Key words： Dendrobium officinale； chalcone synthase； gene cloning； real-time fluorescence quantitative PCR

0 引言

【研究意义】黄酮二糖碳苷类化合物是自然界中抗氧化作用最强的天然化合物之一，具有抑制活性氧和清除自由基的作用，在铁皮石斛（Dendrobium officinale）的茎、叶和花中均有分布且含量丰富（周桂芬和吕圭源，2012）。查尔酮合成酶（Chalcone synthase，CHS）是植物次生代谢途径中黄酮类物质合成的第一个关键酶，参与类黄酮合成（康亚兰等，2014；……