| 荸荠查尔酮异构酶基因的克隆与表达分析 |
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| 引用本文： | 陈夏鑫,陈振林,帅 良,段振华,商飞飞,宋慕波.荸荠查尔酮异构酶基因的克隆与表达分析[J].保鲜与加工,2020,20(4):30-36. |
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| 作者姓名： | 陈夏鑫 陈振林 帅 良 段振华 商飞飞 宋慕波 |
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| 作者单位： | 1. 大连工业大学食品学院，辽宁 大连 116034；2. 贺州学院食品科学与工程技术研究院，广西 贺州 542899 |
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| 基金项目： | 国家自然科学基金（31801607）；国家重点研发计划(2018YFD0901003)；广西自然科学基金项目（2020GXNSFAA259087,2017GXNSFAA198082）；广西特聘专家专项经费共同资助(厅发[2016]21号)；贺州市创新驱动发展专项(贺科创PT1907006) |
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| 摘 要： | 查尔酮异构酶(CHI)是植物黄酮合成途径的关键酶,为了研究鲜切荸荠贮藏过程中表面黄化的机理,利用RACE技术对荸荠CHI基因编码区cDNA全长进行克隆,并分析其在不同组织和鲜切荸荠贮藏过程中的表达。结果表明,荸荠CHI基因(CwCHI)cDNA序列长度为1 003 bp(GeneBank:MG719238),含有一个744 bp的最大开放阅读框,其DNA包含3个内含子和4个外显子。Cw CHI可编码247个氨基酸,经预测其分子量为26.410 kD,理论等电点为4.89。Cw CHI蛋白的二级结构以α-螺旋为主,占38.06%。通过多重比对发现,CwCHI具有该家族特有的保守结构域,决定底物偏好性的第201位氨基酸为Ser,与其他Ⅰ型CHI蛋白相同。系统进化分析表明CwCHI与油棕榈和菠萝的CHI蛋白亲缘关系较近。荧光定量PCR分析表明,CwCHI基因在荸荠皮中的表达量最高,而在荸荠肉中的表达最低。鲜切荸荠黄化过程中CHI活性和CwCHI表达量快速上升,水杨酸处理抑制了CHI活性的上升和CwCHI的表达。
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| 关 键 词： | 荸荠 查尔酮异构酶 基因克隆 表达分析 |
Cloning and Expression Analysis of CHI Gene in Chinese Water Chestnut |
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| Abstract: | Chalcone isomerase(CHI) is a key enzyme in biosynthesis pathway of flavonoids. To study the mechanism of surface yellowing in fresh cut Chinese water chestnut (CWC) during storage, the full-length cDNA of CWC CHI gene was cloned, and the expression of CHI gene in different tissues and in fresh cut CWC during storage was analyzed. The result indicated that, a 1 003 bp full-length cDNA of CWC gene (CwCHI) with an intact maximum open reading frame of 744 bp was obtained, and the DNA contained 3 introns and 4 exons. The CwCHI encoded 247 amino acids, and the predicted molecular weight of CwCHI was 26.410 kD, pI was 4.89. Alpha helix was the main part(38.06%) in secondary structure of CwCHI protein. The multiple sequence alignment showed that CwCHI had the characteristic conservative domains of the CHI family, and same as other type I CHI proteins, the amino acid residue 201 of CwCHI protein was Ser, which influences substrate preference. Phylogenetic tree analysis showed that CwCHI was very closely related to CHI protein of Elaeis oleifera and Ananas comosus. The analysis of Q-PCR showed that the expression level of CwCHI was the highest in peel and the lowest in pulp. The expression level of CwCHI and CHI activity increased dramatically during surface yellowing of fresh cut CWC. However, salicylic acid treatment significant inhibited the increase of CHI activity and the expression of CwCHI. |
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| Keywords: | Chinese water chestnut chalcone isomerase gene cloning expression analysis |
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