| 宋康华,洪克前,侯晓婉,谷 会,姚全胜.菠萝Ac4CL5基因的克隆及表达分析[J].中国南方果树,2025,54(2): |
| 菠萝Ac4CL5基因的克隆及表达分析 |
| Cloning and expression analysis of Ac4CL5 gene in pineapple fruit |
| 投稿时间：2024-02-05 修订日期：2024-03-28 |
| DOI： |
| 中文关键词: 菠萝 4-香豆酸辅酶A连接酶 (4CL) 生物信息 原核表达 转录调控 |
| 英文关键词:Pineapple 4-coumaric acid: coenzyme A ligase (4CL) Bioinformation Prokaryotic expression Regulation |
| 基金项目:海南省自然科学( No. 321QN0921 ),中央级公益性科研院所基本科研业务费专项 ( No. S1630062023005；No. 1630062022006S)。 |
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| 中文摘要: |
| 【目的】了解Ac4CL5基因在菠萝果肉酚类物质合成中的作用以及调控机制,为通过该基因遗传改良菠萝果实品质和抗逆性奠定基础。【方法】以‘巴厘’菠萝果肉为材料 ,克隆菠萝4-香豆酸辅酶A连接酶(4-coumaric acid: coenzyme A ligase, 4CL)Ac4CL5基因,并进行生物信息学分析,同时对Ac4CL5基因进行原核表达分析和上游调控因子的筛选。【结果】Ac4CL5编码区全长1659 bp,比基因组获得序列少92 bp,推测是品种差异造成的；生物信息学分析表明,Ac4CL5具有4CL基因家族典型的保守结构域,亚细胞定位预测显示Ac4CL5定位于过氧化物酶体；进化分析结果表明,菠萝 Ac4CL5属于Class I家族 Type I 类,推测Ac4CL5参与菠萝果实木质素的生物合成；利用原核表达系统成功诱导出可溶性Ac4CL5目的蛋白,对纯化蛋白的酶活性和最适催化底物分析表明,Ac4CL5的最适催化底物为香豆酸,咖啡酸次之。利用Ac4CL5基因启动子进行酵母单杂交筛库发现,溴结合域蛋白GTE9)和C2H2型锌指蛋白可能是调控Ac4CL5表达的上游调控因子。【结论】Ac4CL5蛋白主要以香豆酸和咖啡酸为底物,其超量表达与菠萝黑心病发生中该类酚类物质的大量积累密切相关。Ac4CL5可能受到溴结合域蛋白GTE9和C2H2型锌指蛋白转录因子的调控。 |
| 英文摘要: |
| [Objective] To understand the role of the pineapple 4-coumaric acid: coenzyme A ligase 5 (Ac4CL5) gene in the phenolics synthesis of pineapple fruit and lay the foundation for improving the quality of pineapple fruit genetically. [Method] Ac4CL5 gene was cloned from ‘Comte de Paris’ pineapple flesh. At the same time, prokaryotic expression analysis and upstream regulatory factor screening of the Ac4CL5 gene were carried out to gain a deeper understanding of the role and regulatory mechanism of Ac4CL5 gene in the synthesis of phenolic compounds in pineapple pulp. [Result]The full length coding region of Ac4CL5 was 1659 bp, and 92 bp less than the sequence obtained from genome website, which was presumably due to differences in variety; Bioinformatics analysis showed that conserved domains of 4CL were found in Ac4CL5; Subcellular localization prediction showed that Ac4CL5 was localized in peroxisomes; Evolutionary analysis results indicated that Ac4CL5 was belong to the type I of Class I and was involved in the lignin biosynthesis; The soluble target protein was successfully induced through the prokaryotic expression system. Analysis of the enzyme activity with different |
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