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作物学报 ›› 2025, Vol. 51 ›› Issue (11): 2971-2982.doi: 10.3724/SP.J.1006.2025.54025

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

MeERF6-MePAP2模块响应低温胁迫的分子机制研究

艾力1,2(), 李梦桃1,2, 卢发宝1,2, 刘晓晨1,2, 麦伟涛1,2, 周新成2,3,*(), 陈新2,3,*()   

  1. 1 海南大学热带农林学院, 海南海口 570228
    2 热带作物生物育种全国重点实验室 / 中国热带农业科学院热带生物技术研究所, 海南海口 571101
    3 中国热带农业科学院三亚研究院, 海南三亚 572025
  • 收稿日期:2025-02-21 接受日期:2025-07-09 出版日期:2025-11-12 网络出版日期:2025-07-14
  • 通讯作者: *周新成, E-mail: zhouxincheng@itbb.org.cn; 陈新, E-mail: chenxin@itbb.org.cn
  • 作者简介:E-mail: 18395321365@qq.com
  • 基金资助:
    热带作物生物育种全国重点实验室创新团队科研项目(NKLTCBCXTD07);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-11-HNCX)

Molecular mechanism of the MeERF6-MePAP2 module in response to low-temperature stress

AI Li1,2(), LI Meng-Tao1,2, LU Fa-Bao1,2, LIU Xiao-Chen1,2, MAI Wei-Tao1,2, ZHOU Xin-Cheng2,3,*(), CHEN Xin2,3,*()   

  1. 1 College of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, Hainan, China
    2 National Key Laboratory of Tropical Crop Biological Breeding / Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, Hainan, China
    3 Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, Sanya 572025, Hainan, China
  • Received:2025-02-21 Accepted:2025-07-09 Published:2025-11-12 Published online:2025-07-14
  • Contact: *E-mail: zhouxincheng@itbb.org.cn; E-mail: chenxin@itbb.org.cn
  • Supported by:
    National Key Laboratory of Tropical Crop Biotechnology Innovation Team Research Project(NKLTCBCXTD07);China Agriculture Research System of MOF and MARA(CARS-11-HNCX)

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摘要：

以木薯品种SC124的自交一代分离群体为研究对象, 通过集团分离分析法(BSA)和基因组重测序, 定位并克隆了MYB类转录因子基因MePAP2。研究发现, MePAP2启动子区域存在一段84 bp的插入缺失, 该插入缺失与紫色叶柄和绿色叶柄性状的分离显著相关, 并与植株在低温环境下的生长势密切相关。进一步以MePAP2启动子为诱饵利用酵母单杂交试验, 筛选到AP2/ERF家族转录因子基因MeERF6, 并通过双荧光素酶试验和活体成像技术确定MeERF6能够直接结合MePAP2启动子并正向调控其转录活性。MeERF6在低温胁迫下的响应时间早于MePAP2, 且其在叶柄组织中的转录活性显著高于其他部位。本研究揭示了MeERF6-MePAP2模块在木薯低温胁迫响应中的分子调控机制, 为培育耐低温木薯新品种提供了重要的理论依据和基因资源。

关键词: 木薯, 低温胁迫, MePAP2, MeERF6, 酵母单杂

Abstract:

Using a segregating population derived from the self-pollination of the cassava variety SC124, we employed Bulked Segregant Analysis (BSA) combined with genome re-sequencing to map and clone the MYB-family transcription factor MePAP2. Genetic analysis revealed an 84 bp insertion-deletion (InDel) in the promoter region of MePAP2, which was significantly associated with the segregation of the purple/green petiole trait and correlated with plant growth vigor under low-temperature conditions. A yeast one-hybrid assay using the MePAP2 promoter as bait identified the AP2/ERF-family transcription factor MeERF6. Dual-luciferase reporter assays and in vivo imaging demonstrated that MeERF6 directly binds to the MePAP2 promoter and positively regulates its transcription. Notably, MeERF6 responded to low-temperature stress earlier than MePAP2, and its transcriptional activity was significantly higher in petiole tissue compared to other organs. Collectively, these findings elucidate a hierarchical regulatory pathway involving the MeERF6-MePAP2 module in cassava’s response to low-temperature stress, providing valuable genetic targets for the molecular breeding of cold-tolerant cassava varieties.

Key words: cassava, low-temperature stress, MePAP2, MeERF6, yeast one-hybrid

表1

本研究所用引物"

引物名称
Primer name		 上游引物
Forward primer (5′-3′)		 下游引物
Reverse primer (5′-3′)
MePAP2-PCR
MePAP2-pAbAi		 CAAGTTGGAGGGTCTTAGCTTGT
CTTGAATTCGAGCTCGGTACCACCCCTAACCATTTGCTTTCAT		 GATGTCCAAGTTGGGTGGGTAT
AGCACATGCCTCGAGGTCGACTCCACTCCACACACTGCCTATTT
MeERF6-pGreen II62-sk CGCTCTAGAACTAGTGGATCCATGGCGGTAGAAACTGTAGCA GATAAGCTTGATATCGAATTCTCAGTTTGTCTTTGTCCGTTTGC
MePAP2-pGreen II0800 CTATAGGGCGAATTGGGTACCACC
CCTAACCATTTGCTTTCATC		 AAGCTTATCGATACCGTCGACTCCACTCCACACACTGCCTATTT
qMeActin TCTTCTCAACTGAGGAGCTGCT CCTTCGTCTGGACCTTGCTG
qMeERF6 ATAAGAGACCCTAACCGGAAAG GCGTCATCTGTTACATCAGTATC

附图1

紫色叶柄与绿色叶柄植株的生长势"

图1

SC124自交一代的叶柄颜色、生长势和关键位点的插入缺失分子标记鉴定 A: SC124自交一代叶柄出现紫色与绿色分离。B: SC124自交一代心叶出现紫色与绿色分离。C: 紫色叶柄和绿色叶柄植株的平均茎粗, 误差线上小写字母代表5%显著水平下的差异性。D: 紫色叶柄和绿色叶柄植株的平均株高, 误差线上小写字母代表5%显著水平下的差异性。E: 紫色叶柄和绿色叶柄启动子区域序列差异比较, 红色框中表示紫色叶柄与绿色叶柄的差异序列。F: 根据紫色叶柄启动子区域进行引物设计示意图。G: 利用PCR技术验证紫色叶柄、绿色叶柄启动子区域差异位点。"

附表1

SC124自交分离群体株高及茎粗田间性状调查表"

株系编号
Strain number		 绿色叶柄Green petiole 株系编号
Strain number		 紫色叶柄Purple petiole
株高
Plant height (cm)		 茎粗
Stem diameter (cm)		 株高
Plant height (cm)		 茎粗
Stem diameter (cm)
AS0570 170 2.6 AS0581 128 1.5
Lb-25 140 1.5 AS0590 112 1.5
AS0565 150 2.0 AS0589 126 1.6
Lb-26 146 1.7 AS0587 167 1.9
AS0547 105 1.3 AS0585 189 1.7
AS0546 115 1.5 AS0583 152 1.9
AS0545 162 2.1 AS0554 174 1.7
Lb-32 93 1.6 AS0557 140 2.4
AS0560 108 1.0 Zb-13 180 2.0
AS0550 83 1.5 AS0568 177 2.0
AS0258 82 1.5 AS0535 110 1.5
AS0265 130 2.7 AS0540 150 2.0
AS0278 166 2.2 AS0138 180 2.1
AS0300 90 1.5 AS0505 130 1.9
Lb-35 153 1.9 AS0533 141 2.0
Lb-41 80 1.0 AS0192 127 1.7
Zb-22 189 2.2 AS0751 166 2.4
AS0161 92 1.3 AS002 115 1.9
AS0261 140 1.5 AS0768 190 2.0
AS0299 146 1.7 AS0549 114 1.6
AS0760 180 2.3 AS0543 187 2.2
AS0295 93 1.1 AS0060 165 1.9
AS0197 130 1.9 AS0553 112 1.6
AS0584 130 1.5 AS0566 155 2.0
AS0688 75 1.0 Zb-34 175 2.1
AS0694 140 1.5 AS0563 200 2.0
AS0576 125 1.7 AS0558 146 1.7
AS0201 176 1.9 AS0561 186 2.4
AS0136 164 2.0 AS0562 162 2.1
AS0249 160 1.7 AS0426 133 1.4
AS0245 124 1.7 AS0255 172 1.8
AS0458 86 1.2 SC0274 100 2.1
AS0242 75 0.9 Zb-48 118 1.3
AS0019 118 1.3 AS0092 168 2.1
AS0230 107 1.3 AS0209 121 1.8
AS0752 82 1.6 AS022 155 2.1
AS0188 100 1.0 AS0190 100 1.7
AS0687 90 1.1 AS0216 125 1.8
AS0059 140 1.9 AS0549 160 1.8
AS0062 57 0.7 AS0681 140 1.7
AS0342 73 0.9 Zb-46 127 1.6
AS0224 43 0.7 AS0104 160 2.7
AS0276 120 1.8 AS0053 197 2.5
AS0273 87 2.0 AS0514 168 2.4
AS0287 122 1.6 AS0080 167 1.8
AS0286 133 1.9 Zb-27 138 1.6
AS0269 100 1.6 AS0532 124 1.5
Lb-15 180 2.3 Zb-29 120 1.3
Lb-37 170 1.9 AS0081 70 1.3

图2

MePAP2特性分析 A: MePAP2与10种代表性促进花青素合成的植物MYB转录因子的蛋白序列系统发育分析。其他物种的登录号为: AtPAP1 (NP_176057.1)、AtPAP2 (NP_176813.1)、AtMYB113 (NP_176811.1)、FaMYB10 (USN17647.1)、MdMYB9 (NP_001280749.1)、VvMYBA1 (XP_010664911.1)、VvMYB5b (NP_001267854.1)、VvMYB114 (XP_034707784.1)、MdMYB11 (NP_001280958.1)。B: MePAP2与其他物种同源蛋白的序列比对。灰色方框代表R2结构域, 黑色方框代表R3结构域, 红色框表示bHLH相互作用基序。"

图3

MeERF6的筛选与鉴定 A: 绿色叶柄、紫色叶柄酵母单杂交AbA背景浓度筛选。B: 绿色叶柄、紫色叶柄酵母菌落划线纯化鉴定。C: 使用保守结构域在线预测网站Smart对MeERF6保守结构域进行预测。D: MeERF6蛋白在烟草中的亚细胞定位, 标尺为50 μm。"

图4

MeERF6和MePAP2响应低温处理的表达谱 A: MePAP2在4℃低温处理下柄、茎、叶3个不同组织部位的转录活性分析, 误差线上小写字母代表5%显著水平下的差异性。B: MeERF6在4℃低温处理下柄、茎、叶3个不同组织部位的转录活性分析, 误差线上小写字母代表5%显著水平下的差异性。"

图5

酵母单杂、双荧光素酶测定、活体成像3种技术验证启动子与转录因子互作 A: 酵母单杂交点对点试验验证MeERF6与MePAP2pro在酵母体内存在相互作用。B: 活体成像(LCA)试验验证MeERF6与MePAP2绿色叶柄启动子区在烟草体内存在互作, MePAP2pro + MeERF6为试验组。C: 活体成像(LCA)试验验证MeERF6与MePAP2紫色叶柄启动子区在烟草体内存在互作, MePAP2pro + MeERF6为试验组。D: 双荧光素酶测定明确MeERF6对MePAP2绿色叶柄与紫色叶柄启动子区的调控方式, MePAP2pro + MeERF6为试验组, 试验数据取3次重复平均值, 误差线上小写字母代表5%显著水平下的差异性。"

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