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作物学报 ›› 2024, Vol. 50 ›› Issue (9): 2157-2166.doi: 10.3724/SP.J.1006.2024.44018

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

花生干旱诱导型启动子AhMYB44-11-Pro的克隆与功能分析

刘永惠(), 沈一, 沈悦, 梁满, 沙琴, 张旭尧, 陈志德*()   

  1. 江苏省农业科学院经济作物研究所, 江苏南京 210014
  • 收稿日期:2024-01-29 接受日期:2024-05-21 出版日期:2024-09-12 网络出版日期:2024-06-12
  • 通讯作者: *陈志德, E-mail: chen701865@aliyun.com
  • 作者简介:E-mail: yonghuiliu_126@126.com
  • 基金资助:
    财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-13);江苏省种业振兴揭榜挂帅项目(JBGS [2021] 062)

Cloning and functional analysis of drought-inducible promoter AhMYB44-11- Pro in peanut (Arachis hypogaea L.)

LIU Yong-Hui(), SHEN Yi, SHEN Yue, LIANG Man, SHA Qin, ZHANG Xu-Yao, CHEN Zhi-De*()   

  1. Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China
  • Received:2024-01-29 Accepted:2024-05-21 Published:2024-09-12 Published online:2024-06-12
  • Contact: *E-mail: chen701865@aliyun.com
  • Supported by:
    China Agriculture Research System of MOF and MARA(CARS-13);Open Competition Project for Seed Industry of Jiangsu Province(JBGS [2021] 062)

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摘要：

干旱是严重威胁我国花生产量与品质的主要环境因素之一。为阐明干旱胁迫响应基因AhMYB44-11的调控机制, 揭示其启动子的功能, 本研究从花生基因组中扩增获得该基因的启动子序列AhMYB44-11-Pro, 分别构建全长及5°端部分缺失启动子的GUS融合表达载体, 利用农杆菌介导的遗传转化体系, 分析启动子的活性和表达模式。结果显示, 相比于同源基因AhMYB44-01的启动子, AhMYB44-11-Pro具有更多的MBS、Myb-binding sit等响应干旱胁迫的顺式作用元件; 同时其拟南芥转基因阳性植株经脱水处理后GUS染色加深, GUS酶活性显著增加; 表明启动子的活性受干旱胁迫诱导表达, 证实AhMYB44-11-Pro是一个干旱诱导型启动子。此外, AhMYB44-11-Pro含有种子胚乳特异表达元件和赤霉素响应元件, GUS染色显示在角果发育过程中其活性呈上调趋势, 由此推测AhMYB44-11可能在种子发育尤其是干物质积累过程中发挥重要作用。本研究结果将为全面解析AhMYB44的生物学功能奠定基础, 也为作物抗旱遗传改良提供参考依据。

关键词: 花生, AhMYB44, 启动子, 干旱胁迫, GUS活性

Abstract:

Drought is a significantly environmental factor that poses a serious threat to peanut yield and quality in China. Our study aims to elucidate the regulatory mechanism of the drought stress response gene AhMYB44-11 and uncover the function of its promoter. We isolated the promoter sequence AhMYB44-11-Pro from the peanut genome and constructed recombinant expression vectors comprising the full-length promoter and several 5'-terminal deletion promoters, each fused with the GUS reporter gene. These vectors were then introduced into plant tissue via Agrobacterium rhizogenes-mediated transformation to assess promoter activity and expression patterns. The results revealed that AhMYB44-11-Pro contains a higher number of drought-responsive cis-regulatory elements, including MBS and Myb-binding sites, compared to the promoter of its homologous gene AhMYB44-01. Furthermore, dehydration treatment significantly enhanced GUS staining and activity in AhMYB44-11-Pro transgenic Arabidopsis, indicating that the promoter’s activity is upregulated under drought stress. These findings confirm that AhMYB44-11-Pro functions as a drought-inducible promoter. Additionally, AhMYB44-11-Pro contains a seed endosperm-specific expression element and gibberellin response elements, demonstrating an increased expression trend during pod development, as evidenced by GUS histochemical staining. This suggests that AhMYB44-11 may play a crucial role in seed development, particularly in dry matter accumulation. This research lays the groundwork for a comprehensive analysis of the biological functions of AhMYB44 and provides a valuable reference for genetically enhancing crop drought resistance.

Key words: peanut, AhMYB44, promoter, drought stress, GUS activity

表1

启动子序列克隆及载体构建引物"

用途
Function		 引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
基因克隆Gene cloning Pro0-F GGCGTGCGGTCTTCTGGGGT
Pro0-R GCTACGATGCCGCTATCCAC
全长以及缺失序列载体构建
Vectors construction of full-length and several deletion sequences		 Pro1-F GTCGACCTGCAGGCATGCAAGCTTGTTCTTGTAACAACTTGAGGC
Pro2-F GTCGACCTGCAGGCATGCAAGCTTAAGTTTGATAGTAAGGGTG
Pro3-F GTCGACCTGCAGGCATGCAAGCTTGTTGATTGGATTCTTTGC
Pro4-F GTCGACCTGCAGGCATGCAAGCTTATGGTCTTGTCTTCGTTA
Pro5-F GTCGACCTGCAGGCATGCAAGCTTCAGAGCAGGGATGACTTG
Pro6-F GTCGACCTGCAGGCATGCAAGCTTATCGTAACGCCCTACCTA
Pro (1-6)-R GAAATTTACCCTCAGATCTACCATGGAGTGCTTAGTTTCTTTTGTTTTG

图1

AhMYB44-01/11启动子的顺式作用元件分布示意图"

图2

AhMYB44-11启动子克隆及其5°缺失启动子片段的扩增电泳图 M: DL2000 marker; P0: 含有全长启动子序列的长片段; P1~P6: 启动子全长及5°端不同程度截短的片段。"

图3

AhMYB44-11-Pro及截短片段的GUS融合表达重组载体结构示意图 P1~P6: 启动子全长及5°端不同程度截短的片段。"

图4

AhMYB44-11-Pro系列转基因烟草GUS染色 P1~P6: 分别转化不同长度的AhMYB44-11-Pro系列表达载体的转基因烟草叶片。"

图5

AhMYB44-11-Pro系列转基因烟草GUS活性检测 P1~P6: 分别转化不同长度的AhMYB44-11-Pro系列表达载体的转基因烟草叶片。小写字母代表P < 0.05, 大写字母代表P < 0.01。"

图6

AhMYB44-11-Pro的转基因拟南芥不同生长阶段的GUS染色 A: 萌发2 d的幼苗; B: 萌发3 d的幼苗; C: 萌发7 d的幼苗; D: 茎的横切面; E: 花序; F: 茎节; G: 角果。"

图7

AhMYB44-11-Pro的转基因拟南芥干旱胁迫诱导表达分析 A: 半定量分析转基因株系Pro-21和Pro-27中AhMYB44-11-Pro的转录水平; 35S: 转化含CaMV35S启动子的空载体阳性植株; Actin2: 半定量内参基因。B和C: 转基因拟南芥幼苗在正常生长条件和脱水胁迫处理下的GUS染色和活性定量检测, **代表P < 0.01。"

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