木薯<i>MebZIP2</i>基因克隆及其功能分析

陈甘露; 颜彦; 孟宪伟; 付莉莉; 邱先进; 丁泽红; 胡伟

木薯MebZIP2基因克隆及其功能分析

陈甘露^{1, 2,},
颜彦²,
孟宪伟²,
付莉莉²,
邱先进^1, ,,
丁泽红^{2, 3, ,},
胡伟^{2, 3}

1.
长江大学农学院，湖北　荆州　434025
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三亚研究院，海南　三亚　572025

基金项目: 国家自然科学基金项目（32260486）；三亚崖州湾科技城科技专项（SCKJ-JYRC-2023-68）；中国热带农业科学院基本科研业务费专项（1630052023004）；海南省重点研发计划（ZDYF2022XDNY259）

详细信息

作者简介:
陈甘露（2000 — ），女，硕士研究生，主要从事木薯分子生物学研究，E-mail：cgl15355126069@163.com

通讯作者:
邱先进（1984 — ），男，副教授，主要从事作物遗传育种研究，E-mail：xjqiu216@yangtzeu.edu.cn

丁泽红（1982 — ），男，研究员，主要从事木薯抗逆和品质遗传改良研究，E-mail：dingzehong@itbb.org.cn

中图分类号: S533
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- 被引次数: 0
出版历程
- 收稿日期: 2023-10-12
- 修回日期: 2023-11-15
- 网络出版日期: 2024-03-28

Gene Cloning and Functional Analysis of MebZIP2 in Cassava

CHEN Ganlu^{1, 2
,},
YAN Yan²,
MENG Xianwei²,
FU Lili²,
QIU Xianjin^{1
, ,},
DING Zehong^{2, 3
, ,},
HU Wei^{2, 3}

1.
College of Agriculture, Yangtze University, Jingzhou, Hubei　434025, China
2.
Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou, Hainan　571101, China
3.
Sanya Research Institute of Chinese Academy of Tropical Agricultural Sciences, Sanya, Hainan　572025, China

摘要

摘要: 目的 bZIP转录因子在植物生长发育、淀粉合成和非生物胁迫等方面发挥重要作用。克隆木薯MebZIP2基因并进行亚细胞定位，基因表达分析和酵母单杂交分析，为进一步研究MebZIP2基因的功能提供参考。方法从木薯SC205中扩增MebZIP2基因编码区（CDS）序列，对其进行生物信息学分析，并构建pNC-Green-SubN融合表达载体，通过农杆菌介导转染烟草表皮细胞，观察荧光信号以确定MebZIP2蛋白的亚细胞定位情况。在木薯不同组织和块根不同发育阶段分析MebZIP2基因表达特征，利用酵母单杂交研究其与淀粉合成基因启动子的互作情况。结果 MebZIP2基因CDS序列长度为465 bp，编码154个氨基酸，蛋白分子量为17 891.35 Da，理论等电点为5.23，属于不稳定亲水性蛋白。MebZIP2含有bZIP保守结构域，其氨基酸序列与橡胶树bZIP蛋白的相似性最高，为74.22%。MebZIP2基因启动子区域含有光响应元件，赤霉素、水杨酸和茉莉酸等激素响应元件，以及胚乳表达和胁迫响应元件。MebZIP2蛋白亚细胞定位于细胞核和细胞膜中。MebZIP2基因在根尖分生组织、须根、茎和块根发育前期的表达量较高。MebZIP2与淀粉合成基因MeAPL5a、MeGBSS1和MeISA1共表达，而且MebZIP2蛋白可以与它们的启动子互作。结论 MebZIP2基因属于bZIP基因家族成员，其表达具有组织特异性和块根发育阶段性，MebZIP2蛋白可以与淀粉合成基因MeAPL5a、MeGBSS1和MeISA1的启动子互作，可能参与了木薯块根发育和淀粉合成。
- 木薯 /
- bZIP转录因子 /
- MebZIP2 /
- 基因克隆 /
- 功能分析
Abstract: : Objective bZIP transcription factors play an important role in plant growth and development, starch synthesis, and abiotic stress. Cloning of cassava MebZIP2 and analysis of its subcellular localization, gene expression, and yeast one-hybrid can provide useful references to further study the function of MebZIP2. Method The coding sequence (CDS) of MebZIP2 was amplified from cassava SC205 and then used to perform bioinformatics analysis. A pNC-Green-SubN fusion expression vector was constructed and transferred into tobacco epidermal cells by Agrobacterium-mediated method, and fluorescence signals were observed to determine subcellular localization of the MebZIP2 protein. The expression profiles of MebZIP2 were analyzed in different tissues and at different development stages of cassava storage root. The interactions between MebZIP2 and the promoters of starch synthesis genes were studied by yeast one-hybrid. Result The CDS sequence of MebZIP2 was 465 bp in length and encoded 154 amino acids. The molecular weight was 17891.35 Da, the theoretical isoelectric point was 5.23, and MebZIP2 was an unstable hydrophilic protein. MebZIP2 contained a bZIP-conserved domain and showed the highest sequence similarity (74.22%) to a bZIP protein from the Hevea brasiliensis. The promoter region of MebZIP2 contained light-responsive elements, hormone (such as gibberellin, salicylic acid, and jasmonic acid) responsive elements, as well as endosperm expression and stress-responsive elements. MebZIP2 protein was localized both in the cell membrane and nucleus. MebZIP2 showed high expression levels in root apical meristems, fibrous roots, stems, and at early developmental stages of storage roots. MebZIP2 was co-expressed with several starch synthesis genes such as MeAPL5a, MeGBSS1, and MeISA1, and MebZIP2 could interact with their promoters. Conclusion MebZIP2 belonged to the bZIP gene family, and its expression showed tissue-specificity and root developmental stage-specificity. MebZIP2 protein could interact with the promoters of starch synthesis genes (MeAPL5a, MeGBSS1 and MeISA1), and it might be involved in storage root development and starch synthesis in cassava.
- Cassava /
- bZIP transcription factor /
- MebZIP2 /
- gene cloning /
- functional analysis

HTML全文

图 1 MebZIP2基因扩增产物的电泳图

M：DL2000 DNA标记；1：MebZIP2基因。

Figure 1. The electrophoresis of PCR amplification of MebZIP2 gene

M: DL2000 DNA Marker; 1: MebZIP2 gene.

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图 2 MebZIP2与其他物种中同源氨基酸序列的比对结果

Figure 2. Alignment of MebZIP2 and its homology amino acid sequences

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图 3 MebZIP2与其他物种中同源的bZIP蛋白的系统发育进化树

Figure 3. Phylogenetic tree of MebZIP2 and its homology amino acid sequences

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图 4 MebZIP2基因启动子中顺式元件分析

Figure 4. Cis-element analysis of MebZIP2 promoter

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图 5 MebZIP2蛋白的亚细胞定位

Figure 5. Subcellular localization of MebZIP2 protein

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图 6 MebZIP2基因表达分析

（A）MebZIP2在木薯不同组织中的表达分析。（B）MebZIP2在木薯块根不同发育阶段的表达分析。S1～S7表示木薯种植后第100、140、180、220、260、300、340天。不同字母表示均值之间显著差异（邓肯多重检验，P ＜ 0.05）。

Figure 6. MebZIP2 gene expression analysis

(A) Expression analysis of MebZIP2 in different tissues of cassava. (B) Expression analysis of MebZIP2 at different developmental stages of cassava storage root. S1～S7 indicate the 100th, 140th, 180th, 220th, 260th, 300th, 340th day after cassava planting. Different letters indicate significant differences between means (Duncan multiple test, P ＜ 0.05).

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图 7 MebZIP2与淀粉合成基因启动子的互作

A. MebZIP2与淀粉合成基因MeAPL5a、MeGBSS1、MeISA1在木薯块根不同发育阶段的表达趋势。S1～S7表示木薯种植后第100、140、180、220、260、300、340天。B. MebZIP2与淀粉合成基因MeAPL5a、MeGBSS1、MeISA1启动子的酵母单杂交结果。DDO：SD/-Trp-Leu培养基；TDO：不含3-AT的SD/-Trp/-Leu/-His培养基；TDO/5 mmol·L⁻¹ 3-AT：含5 mmol·L⁻¹ 3-AT的SD/-Trp/-Leu/-His培养基。

Figure 7. Interaction of MebZIP2 with promoters of starch biosynthesis genes

A. Expression analysis of MebZIP2 and starch biosynthesis genes (MeAPL5a, MeGBSS1 and MeISA1) at different developmental stages of cassava storage roots. S1～S7 indicate the 100th, 140th, 180th, 220th, 260th, 300th, 340th day after cassava planting. B. Yeast one-hybrid results between MebZIP2 and the promoters of MeAPL5a, MeGBSS1, and MeISA1, respectively. DDO: SD/-Trp-Leo medium; TDO: SD/-Trp/-Leu/-His medium without 3-AT; TDO/5 mmol·L⁻¹ 3-AT: SD/-Trp/-Leu/-His medium with 5 mmol·L⁻¹ 3-AT.

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表 1 供试引物序列

Table 1. The primers used in this work

引物名称 Primer name	引物序列 Primer sequence	引物用途 Primer usage
MebZIP2-F	5'-GCCATGGAGGCCAGTGAATTCATGGTCTCTTCTAGTGGGGCAT-3′	基因克隆
MebZIP2-R	5′-CAGCTCGAGCTCGATGGATCCGAACAGGGCCATGTCCATG-3′	基因克隆
pNC-MebZIP2-F	5′-AGTGGTCTCTGTCCAGTCCTATGGTCTCTTCTAGTGGGGCAT-3′	亚细胞定位
pNC-MebZIP2-R	5′-GGTCTCAGCAGACCACAAGTGAACAGGGCCATGTCCATG-3′	亚细胞定位
qRTMebZIP2-F	5′-GGATCAGCTCGAGAAAGAAAAC-3′	荧光定量PCR
qRTMebZIP2-R	5′-TTAGGCCATTGCTGAAATTCAC-3′	荧光定量PCR
Actin-F	5′-TGATGAGTCTGGTCCATCCA-3′	内参基因
Actin-R	5′-CCTCCTACGACCCAATCTCA-3′	内参基因
MeAPL5a-F	5′-GACTCACTATAGGGCGAATTCGGAAGTGAGCAATAAAAGTAGAAATG-3′	启动子克隆
MeAPL5a-R	5′-ATAATGCCAGGAATTACTAGTAGCAGCTGAATGTGGAGTGTTT-3′	启动子克隆
MeGBSSI-F	5′-GACTCACTATAGGGCGAATTCGAAATGTAGGACCCTGGCGA-3′	启动子克隆
MeGBSSI-R	5′-ATAATGCCAGGAATTACTAGTCTCTATGCGGAAACAATTAAATCTG-3′	启动子克隆
MeISA1-F	5′-GACTCACTATAGGGCGAATTCCTTGAAAATGAAAAAGATTAAAGAGAAA-3′	启动子克隆
MeISA1-R	5′-ATAATGCCAGGAATTACTAGTCGTTGGTATTACTTGGTGATAATCAC-3′	启动子克隆

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