木薯<i>MeERF1.2</i>基因克隆及表达分析

曾坚; 吴伟姗; 谢彩虹; 沈梓欣; 叶晓雪; 颜彦; 李冰; 陈志晟; 彭红元; 胡伟; 曾力旺

doi:10.19303/j.issn.1008-0384.2023.04.003

木薯MeERF1.2基因克隆及表达分析

doi: 10.19303/j.issn.1008-0384.2023.04.003

曾坚^1,,
吴伟姗¹,
谢彩虹¹,
沈梓欣¹,
叶晓雪²,
颜彦^{2, 3},
李冰⁴,
陈志晟²,
彭红元¹,
胡伟^{2, 3},
曾力旺^{2, 3, ,}

1.
韶关学院广东省粤北食药资源利用与保护重点实验室/英东生物与农业学院，广东　韶关　512005
2.
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科技信息研究所，海南　海口　571101
4.
平江县第一中学，湖南　岳阳　414500

基金项目: 广东省基础与应用基础研究基金项目（2021A1515011236、 2023A1515010336）；中央级公益性科研院所基本科研业务费专项（1630052022027）；海南省自然科学基金面上项目（320MS098、 322MS128）；韶关学院重点项目（SZ2022KJ05）；广东省普通高校重点领域专项（2022ZDZX4047）；国家自然科学基金项目（31901537）；广东省教育厅重大项目（2017KZDXM077）；校级大学生创新创业训练计划项目（Sycxcy2022089）

详细信息

作者简介:
曾坚（1987−），男，博士，副教授，研究方向：植物基因功能研究（E-mail：zengjian@sgu.edu.cn）

通讯作者:
曾力旺（1987−），女，硕士，助理研究员，研究方向：植物基因分子生物学（E-mail：zengliwang@163.com）

中图分类号: S533
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- 被引次数: 0
出版历程
- 收稿日期: 2022-06-09
- 录用日期: 2022-06-09
- 修回日期: 2022-11-15
- 网络出版日期: 2023-03-28
- 刊出日期: 2023-04-28

Cloning and Expression of MeERF1.2 in Cassava

ZENG Jian^1
,,
WU Weishan¹,
XIE Caihong¹,
SHEN Zixin¹,
YE Xiaoxue²,
YAN Yan^{2, 3},
LI Bing⁴,
CHEN Zhisheng²,
PENG Hongyuan¹,
HU Wei^{2, 3},
ZENG Liwang^{2, 3
, ,}

1.
Guangdong Provincial Key Laboratory of Utilization and Conservation of Food and Medicinal Resources in Northern Region/Henry Fok College of Biology and Agriculture, Shaoguan University, Shaoguan, Guangdong 　512005, China
2.
Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science,　Haikou, Hainan 571101, China
3.
Institute of Scientific and Technical Information, Chinese Academy of Tropical Agricultural Science, Haikou, Hainan 　571101, China
4.
The First Middle School of Pingjiang Country, Yueyang, Hunan　414500, China

摘要

摘要: 目的乙烯响应因子（Ethylene response factor，ERF）是乙烯信号转导通路的重要成员，克隆并分析其在木薯块根采后生理性变质（Post-harvest physiological deterioration，PPD）过程中的表达情况，能为进一步研究乙烯信号在木薯PPD过程中的功能提供参考。方法以木薯栽培品种华南8号（SC8）为材料，采用RT-PCR技术克隆木薯MeERF1.2基因，对其进行相关生物信息学分析，如遗传进化关系、结构域、蛋白质结构预测、理化性质等。对MeERF1.2基因在细胞中的亚细胞定位进行确认，并用qRT-PCR技术分析MeERF1.2基因在木薯块根PPD过程中的表达水平。结果克隆得到的MeERF1.2基因全长为660 bp，编码的氨基酸残基数为219，分子量和等电点分别为25.04 kD和5.61，含有AP2家族结构域，和橡胶HbERF1B-like的亲缘关系最近，序列相似性达到88.74%。MeERF1.2基因定位于细胞核。和对照0 h相比，MeERF1.2基因的表达量在木薯块根的采后过程中表现为显著上升趋势，即MeERF1.2基因的表达受到PPD过程的诱导。结论克隆得到的MeERF1.2基因包含ERF基因家族的保守结构域，属于ERF基因家族；MeERF1.2基因的表达在采后过程中受到诱导，可能参与了木薯块根的PPD过程，为后续进一步分析乙烯信号转导通路在PPD过程中的作用奠定了基础。
- 木薯 /
- 乙烯 /
- ERF /
- 活性氧 /
- 采后生理性变质
Abstract: Objective Critical factors in the ethylene signal transduction pathway involving the post-harvest physiological deterioration (PPD) of cassava were investigated. Method One of the possible ethylene response factors (ERFs) in Manihot esculenta cv.SC8, MeERF1.2, was cloned using RT-PCR to analyze the expressions during PPD. Physicochemical properties, conserved domain, genetic evolutionary relationship, and protein structure of the gene were studied with bioinformatics tools. Distribution of MeERF1.2 in the plant cells was verified by online software and subcellular localization. Result MeERF1.2 had an ORF of 660 bp encoded 188 amino acids with a molecular weight of 25.04 kD and a pI of 5.61. The protein contained an AP2 domain showing a high sequence similarity of 88.74% with the HbERF1B-like gene. It was localized in the nucleus and significantly upregulated during PPD from 0 to 6, 12, and 48 h displaying an apparent induction trend in the process. Conclusion MeERF1.2 was one of the ERFs induced in cassava tubers during PPD and played a vital role in the signal transduction pathway of the plant.
- Cassava /
- ethylene /
- ERF /
- ROS /
- post-harvest physiological deterioration

HTML全文

图 1 MeERF1.2基因扩增结果

M：DNA Marker；1：样品; 2：阴性对照。

Figure 1. PCR amplification of MeERF1.2

M: DNA Marker; 1: sample; 2: negative control.

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图 2 结构域分析

Figure 2. Analysis of conserved domain

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图 3 MeERF1.2蛋白序列的同源性比对

Figure 3. Homologous alignment of MeERF1.2 sequences

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图 4 ERF基因的系统进化树

Figure 4. Phylogenetic tree of ERF genes

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图 5 MeERF1.2蛋白亚细胞定位分析

Figure 5. Subcellular localization of MeERF1.2 protein

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图 6 MeERF1.2在不同组织中的表达量

L：叶，M：中脉，S：茎，P：叶柄，LB：侧芽，OES：分化胚组织，FR：须根，SR：根，RAM：根顶端分生组织。不同小写字母表明在Duncan’s多重比较中差异显著（P＜0.05）

Figure 6. Expressions of MeERF1.2 in tissues

L: leaf; M: midvein; S: stem; P: petiole; LB: lateral bud; OES: organized embryogenic structure; FR: fibrous root; SR: storage root; RAM: root apical meristem. Data with different letters indicate significant differences based on Duncan's multiple range tests (P＜0.05).

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图 7 MeERF1.2基因在PPD中的表达变化

A MeERF1.2基因在采后腐烂过程中转录组测序表达量；B: MeERF1.2基因在采后腐烂过程中实时定量PCR检测结果；*和**分别代表与0 h差异显著（P＜0.05）和极显著（P＜0.01）。

Figure 7. Expressions of MeERF1.2 during PPD

A: expression of MeERF1.2 during cassava PPD shown by transcriptome sequencing; B: expression of MeERF1.2 during cassava PPD shown by qRT-PCR; * and ** represent significantly different from control (0 h) at P＜0.05 and P＜0.01, respectively.

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表 1 扩增引物序列

Table 1. Primers and their sequences

引物名称 Primer	引物序列 Primer sequence	引物用途 Primer usage
MeERF1.2-F	5′-ATGGATTCCTCCATCTTTCATTCT-3′	基因克隆
MeERF1.2-R	5′-CCAAGGTCTTATAGCATTCTCAGAT-3′	基因克隆
BiMeERF1.2-F	5′-AGTGGTCTCTGTCCAGTCCTATGGATTCCTCCATCTTT-3′	亚细胞定位
BiMeERF1.2-R	5′-GGTCTCAGCAGACCACAAGTCCAAGGTCTTATAGCATT-3′	亚细胞定位
qMeERF1.2-F	5′-GAGCTGGGGCTGTACTCAAT-3′	荧光定量PCR
qMeERF1.2-R	5′-CAGGTGAGCACCCTTCTTCT-3′	荧光定量PCR
MeEF1-F	5′-TGAACCACCCTGGTCAGATTGGAA-3′	内参基因
MeEF1-R	5′-AACTTGGGCTCCTTCTCAAGCTCT-3′	内参基因

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