中间锦鸡儿<i>CiMYB15</i>基因正调控拟南芥黄酮代谢 <sup>*</sup>

doi:10.13523/j.cb.20181002

中国生物工程杂志

2018, Vol. 38

Issue (10): 8-19 DOI: 10.13523/j.cb.20181002

研究报告

中间锦鸡儿CiMYB15基因正调控拟南芥黄酮代谢 ^*

柴文娟¹,杨杞^1,^2,³,李国婧^1,^2,³,王瑞刚^1,^2,^3,^**(

)

1 内蒙古农业大学生命科学学院呼和浩特 010018
2 内蒙古自治区植物逆境生理与分子生物学重点实验室呼和浩特 010018
3 内蒙古自治区抗逆植物遗传资源利用与分子改良科技创新团队呼和浩特 010018

CiMYB15 from Caragana Intermedia Positively Regulates Flavonoids Metabolism of Arabidopsis

Wen-juan CHAI¹,Qi YANG^1,^2,³,Guo-jing LI^1,^2,³,Rui-gang WANG^1,^2,^3,^**(

)

1 College of Life Sciences,Inner Mongolia Agricultural University,Hohhot 010018,China
2 Inner Mongolia Key Laboratory of Plant Stress Physiology and Molecular Biology,Hohhot 010018,China
3 Inner Mongolia Scientific Innovation Team of Genetic Resource Utilization and Molecular improvement of Stress Resistant Plants, Hohhot 010018, China

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摘要：

目的: R2R3-MYB类转录因子参与调控植物初生和次生代谢。方法: 从中间锦鸡儿(Caragana intermedia)干旱转录组数据库中搜索并克隆了一个R2R3-MYB基因,命名为CiMYB15(GenBank登录号MH678649);将CiMYB15基因编码区转入野生型拟南芥中,利用分光光度法测定了野生型和转基因拟南芥中总黄酮含量,并用qRT-PCR检测了转基因植物中AtCHS基因的表达情况。同时采用染色体步移法克隆了CiMYB15基因的启动子序列。结果表明:(1)CiMYB15基因gDNA长度为1 960 bp,包含三个外显子(134、131和521 bp)和两个内含子(281和893 bp);开放阅读框长度为786 bp,编码262个氨基酸。(2)克隆得到1 580 bp的启动子序列,序列中主要包含损伤诱导元件G-box和P-box、盐诱导作用元件GT1-motif、参与干旱诱导的反应元件MBS,以及真菌侵害应答元件BOX-W1、植物-病原菌互作元件EIER;此外,还包含调节黄酮合成基因的MYB转录因子的结合位点。(3)CiMYB15基因的表达受到紫外胁迫的诱导。(4)CiMYB15基因过表达株系的总黄酮含量高于野生型。(5)过表达植物中AtCHS基因的表达量亦高于野生型。以上结果说明,CiMYB15基因正调控拟南芥黄酮代谢。

关键词： 中间锦鸡儿; 总黄酮; CiMYB15基因; 启动子; 查尔酮合酶基因

Abstract:

Objective: R2R3-MYB transcription factors regulate primary and secondary metabolism in plants.Methods: A R2R3-MYB encoding sequence, characterized and cloned from drought treated transcriptome of Caragana intermedia, was named as CiMYB15. The gene was transferred into Arabidopsis thaliana, and the total flavonoids contents of transgenic lines and wild type were measured by spectrophotometric method, and the expression of AtCHS in transgenic plants was analyzed by qRT-PCR. A 1 580bp fragment of CiMYB15 promoter was isolated by genome walking. The results revealed that: (1) the length of CiMYB15 gDNA was 1 960bp, it’s consisted of three exons (134, 131 and 521 bp) and two introns (281 and 893 bp). The open reading frame (ORF) encodes a polypeptide of 262 amino acids. (2)The main cis-elements of CiMYB15 promoter include abiotic stress responded elements (G-box, P-box, GT1-motif and MBS), biotic stress responded elements (BOX-W1 and EIER), and MYB binding sites of flavonoids synthase regulatory genes. (3) The expression of CiMYB15 was induced by UV-B. (4) Total flavonoids contents of CiMYB15 overexpression plants were higher than that of wild type Arabidopsis. (5) Furthermore, the expression level of AtCHS was increased in transgenic Arabidopsis. In brief, CiMYB15 positively regulated the flavonoids metabolism.

Key words: Caragana intermedia Total flavonoids CiMYB15 Promoter Chalcone synthase (CHS)

收稿日期: 2018-07-07 出版日期: 2018-11-09

ZTFLH:

Q785

基金资助: * 内蒙古自治区科技创新引导项目(KCBJ2018012);内蒙古自治区科技创新团队(201503004)

通讯作者: 王瑞刚 E-mail: ruigangwang@126.com

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引用本文:

柴文娟,杨杞,李国婧,王瑞刚. 中间锦鸡儿CiMYB15基因正调控拟南芥黄酮代谢 ^*[J]. 中国生物工程杂志, 2018, 38(10): 8-19.

Wen-juan CHAI,Qi YANG,Guo-jing LI,Rui-gang WANG. CiMYB15 from Caragana Intermedia Positively Regulates Flavonoids Metabolism of Arabidopsis. China Biotechnology, 2018, 38(10): 8-19.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20181002 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I10/8

表1 CiMYB15基因克隆与功能分析引物信息

图1 CiMYB15基因克隆的电泳图

图2 CiMYB15的cDNA、gDNA及推导的氨基酸序列

图3 CiMYB15和其它植物MYB蛋白序列比较

图4 CiMYB15与其它植物系统进化分析

图5 CiMYB15基因启动子扩增结果

表2 CiMYB15基因启动子分析预测

图6 qRT-PCR检测UV-B处理下CiMYB15基因的表达水平

图7 pCanG-CiMYB15融表达载体的构建及转基因株系的鉴定

图8 野生型和CiMYB15转基因株系中的总黄酮含量

图9 野生型和CiMYB15转基因株系中黄酮代谢相关基因的检测

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