榅桲果实CAD基因的克隆、序列分析及表达  

Cloning, Sequence Analysis and Expression of CAD Gene in Quince (Cydonia oblonga Mill) Fruit

作　　者：车玉红[1] 杨波[2] 郭春苗[2] 木巴热克·阿尤普 吴津蓉[1] 杜鹃[1] CHE Yuhong;YANG Bo;GUO Chunmiao;Mubareke Ayoupu;WU Jinrun;DU Juan(Xinjiang Agricultural Professional Technology College,Changji Xinjiang 831100,China;Institute of Horticultural Crops,Xinjiang Academy of Agricultural Sciences,Urumqi 830091,China)

机构地区：[1]新疆农业职业技术学院,新疆昌吉831100 [2]新疆农业科学院园艺作物研究所,乌鲁木齐830091

出　　处：《新疆农业科学》2020年第5期814-821,共8页Xinjiang Agricultural Sciences

基　　金：新疆维吾尔自治区自然科学基金(2018D01A45);自治区农牧科技骨干人才培养项目。

摘　　要：【目的】研究榅桲果实木质化与CAD基因的关系。【方法】基于GenBank报道的近缘物种CAD基因cDNA序列,应用Primer Premier 5.0软件设计PCR扩增引物。提取榅桲总RNA,经反转录后合成cDNA,应用RT-PCR方法成功扩增出CAD基因片段并克隆到pMD18-T载体。通过DNAstar软件进行同源序列比对,ClustalX结合MEGA4.1软件构建系统进化树,采用Protparam在线程序分析蛋白质的理化性质,用DNAstar的Protean程序预测二级结构。通过间苯二酚染色鉴定果肉发育时期木质素的积累程度,并利用RT-qPCR对CAD基因在发育期时期的表达规律进行检测。【结果】榅桲CAD基因其开放阅读框(ORF)序列为1 071 bp,编码356个氨基酸,与其他物种序列同源性最高达96%,进化关系上与苹果较近。在果实6个发育时期,木质素积累逐渐降低,而CAD基因表达与果实木质化程度紧密相关,随着木质素合成趋缓呈现一个逐步下调的趋势。【结论】CAD基因是调控榅桲果实木质化的关键基因。【Objective】 To investigate the relationship between the lignization and CAD genes in Quince Frui.【Method】Based on the cDNA sequence of cinnamyl alcohol dehydrogenase(CAD) gene reported by GenBank, PCR primers were designed by primer premier 5.0. The total RNA was extracted and the cDNA was synthesized by reverse transcription. The CAD gene fragment was successfully amplified by RT-PCR and cloned into pMD 18-T vector. Through the homologous sequence alignment of DNAStar software, clustalx combined with mega 4.1 software, the phylogenetic tree was built and the physicochemical properties of proteins were analyzed by protparam online program, and the secondary structure was predicted by protean program of dnastar. Finally, the accumulation of lignin in pulp during pulp development was identified by resorcinol staining, and the expression of CAD gene during development was detected by RT-qPCR. 【Result】 The results showed that the open reading frame(ORF) sequence of quince CAD gene was 1,071 bp, encoding 356 amino acids, and the homology with other species was as high as 96%, which was close to apple in evolutionary relationship. During the six stages of fruit development, the accumulation of lignin decreased gradually, while the expression of CAD gene was closely related to the degree of lignin fruit, and showed a trend of decreasing step by step with the slow synthesis.【Conclusion】CAD gene is the key gene to regulate the lignification of Quince Fruit.

关 键 词：榅桲 CAD基因 克隆 序列分析 表达 

分 类 号：S661.9[农业科学—果树学]