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作物杂志,2022, 第1期: 77–83 doi: 10.16035/j.issn.1001-7283.2022.01.011

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

苦荞FtC4H基因克隆与生物信息学分析

尹桂芳1(), 段迎2, 杨晓琳2, 蔡苏云2, 王艳青1, 卢文洁1, 孙道旺1, 贺润丽2(), 王莉花1()   

  1. 1云南省农业科学院生物技术与种质资源研究所/云南省农业生物技术重点实验室/农业农村部西南作物基因资源与种质创制重点实验室,650205,云南昆明
    2山西中医药大学中药与食品工程学院,030619,山西太原
  • 收稿日期:2021-01-18 修回日期:2021-07-19 出版日期:2022-02-15 发布日期:2022-02-16
  • 通讯作者: 贺润丽,王莉花
  • 作者简介:尹桂芳,主要从事荞麦特异性状分子标记与育种研究,E-mail： 1434651675@qq.com
  • 基金资助:
    国家自然科学基金地区科学基金(31460379);财政部和农业农村部：国家现代农业产业技术体系(CARS-07-C-2);山西省重点研发计划项目(201803D221012-6);2019年中医药公共卫生服务补助专项“全国中药资源普查项目”(财社[2019]68号)

Cloning and Bioinformatics Analysis of FtC4H Gene from Tartary Buckwheat

Yin Guifang1(), Duan Ying2, Yang Xiaolin2, Cai Suyun2, Wang Yanqing1, Lu Wenjie1, Sun Daowang1, He Runli2(), Wang Lihua1()   

  1. 1Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences/ Yunnan Provincial Key Laboratory of Agricultural Biotechnology/Key Laboratory of Southwestern Crop Gene Resources and Germplasm Innovation, Ministry of Agriculture and Rural Affairs, Kunming 650205, Yunnan, China
    2College of Traditional Chinese Medicine and Food Engineering, Shanxi University of Chinese Medicine, Taiyuan 030619, Shanxi, China
  • Received:2021-01-18 Revised:2021-07-19 Online:2022-02-15 Published:2022-02-16
  • Contact: He Runli,Wang Lihua

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摘要：

克隆苦荞苯丙烷类物质代谢途径中的关键酶肉桂酸-4-羟基化酶基因（FtC4H）,为进一步研究其功能奠定基础。以云荞1号和小米荞为材料,提取不同发育期果壳RNA,利用RT-PCR法克隆苦荞FtC4H基因,运用生物信息学分析FtC4H蛋白的特征,构建FtC4H蛋白系统进化树,分析其基因表达。结果表明,克隆的FtC4H基因序列包含1299bp完整的cDNA开放阅读框,编码432个氨基酸,为亲水性不稳定碱性蛋白,具有P450超家族保守域,不具有跨膜结构域,有丰富的二级结构,三级结构预测显示FtC4H与6vby.1.A的序列相似度高。系统进化分析结果表明,本研究克隆的FtC4H与已报道的苦荞其他C4H基因不同。qRT-PCR结果表明,FtC4H在小米荞的花和叶中相对表达量显著高于云荞1号。

关键词: 苦荞, RT-PCR克隆, 肉桂酸-4-羟基化酶, 生物信息学分析, 实时荧光定量PCR

Abstract:

In this paper, the key enzyme gene cinnamic acid-4-hydroxylase (FtC4H) in the benzene propanes metabolic pathway of tartary buckwheat was cloned, laying a foundation for further research on its function. The RNA of Yunqiao 1 and Xiaomiqiao husks at different developmental stages was extracted, and the FtC4H gene from tartary buckwheat was cloned by RT-PCR, and the characteristics of FtC4H protein were analyzed by bioinformatics. The phylogenetic tree of FtC4H protein was constructed and analyzed FtC4H gene expression. The results showed that the cloned gene sequence had complete cDNA open reading frame of 1299bp, encoding 432 amino acids. FtC4H was a hydrophilic and unstable basic protein with P450 superfamily conserved domain and without transmembrane domain; The secondary structure of FtC4H was complex; the prediction of tertiary structure showed a high similarity with 6vby.1.A sequence. The results of phylogenetic analysis showed that the FtC4H cloned in this study was different from other C4H genes of tartary buckwheat reported. qRT-PCR showed that the relative expression level of FtC4H in the flowers and leaves of Xiaomiqiao were significant higher than that of Yunqiao 1.

Key words: Tartary buckwheat, RT-PCR cloning, Cinnamic acid-4-hydroxylase, Bioinformatics analysis, qRT-PCR

表1

引物序列及用途

用途
Application		 引物名称
Primer name		 引物序列（5'-3'）
Primer sequence (5'-3')
RT-PCR FtC4H-F GTAATCTCAAAGCTCCGCGG
FtC4H-R ACACATAACAGCGACGATGAC
实时荧光定量PCR H3-F AAGAAGTCCCACAGATACCGC
qRT-PCR H3-R AGCCTCCTGAAGAGCTAGCAC
FtC4H-F GGTCAGTCGAGTGGGCATTA
FtC4H-R TTGGCGTCGTTGAGGTTC

图1

FtC4H克隆片段的PCR电泳图 M：SM0331 DNA Marker;1：云荞1号;2：小米荞

图2

FtC4H的核苷酸序列及氨基酸序列

图3

苦荞FtC4H蛋白保守结构域分析

图4

苦荞FtC4H蛋白质二级结构预测 蓝色：α-螺旋;红色：延伸链;绿色：β-折叠;紫色：无规则卷曲

图5

苦荞FtC4H蛋白质三级结构预测

图6

FtC4H与已克隆苦荞C4H基因的多序列比对

图7

FtC4H与已克隆苦荞C4H蛋白的多序列比对 蓝色框为脯氨酸富含区;红色框为血红素结合域;蓝色线为底物结合位点基序;绿色线为E-R-R三联体

图8

FtC4H与其他植物来源C4H基于蛋白质序列的系统进化树

图9

FtC4H基因在苦荞不同器官的相对表达量 “*”和“**”分别表示在0.05和0.01水平差异显著

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