文冠果XsWRI1基因克隆、转录活性及组织特异性表达分析

doi:10.12302/j.issn.1000-2006.202309001

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南京林业大学学报（自然科学版） ›› 2025, Vol. 49 ›› Issue (2): 23-30.doi: 10.12302/j.issn.1000-2006.202309001

• 专题报道：推进乡村全面振兴视域下的多功能油用树种文冠果研究（执行主编尹佟明李维林） • 上一篇下一篇

文冠果XsWRI1基因克隆、转录活性及组织特异性表达分析

张薇(), 李麟坤, 梁重钧, 王利兵^*()

中国林业科学研究院林业研究所,林木遗传育种全国重点实验室,北京 100091

收稿日期:2023-09-01 接受日期:2023-11-29 出版日期:2025-03-30 发布日期:2025-03-28
通讯作者: *王利兵(wlibing@caf.ac.cn),研究员。
作者简介:
张薇(w.zhang35@caf.ac.cn),博士生。
基金资助:
青年拔尖人才支持计划项目

Cloning, transcriptional activation, and tissue expression analysis of XsWRI1 from Xanthoceras sorbifolium

ZHANG Wei(), LI Linkun, LIANG Chongjun, WANG Libing^*()

State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China

Received:2023-09-01 Accepted:2023-11-29 Online:2025-03-30 Published:2025-03-28

摘要/Abstract

摘要：

【目的】表征文冠果(Xanthoceras sorbifolium)中WRINKLED1(WRI1)转录因子的序列同一性、转录激活活性和功能域,为探究其在种子油生物合成中的调控作用提供参考。【方法】利用cDNA末端快速扩增(RACE)技术从成熟文冠果胚乳组织中克隆XsWRI1全长cDNA序列,利用生物信息学工具分析蛋白质序列特性。构建pGBKT7-XsWRI1载体,并将其转化到Y2HGold酵母感受态验证转录激活活性。通过实时荧光定量PCR技术(qRT-PCR)对根、茎、叶、花瓣、雄蕊和发育中的种仁组织特异性表达模式进行定量分析。【结果】XsWRI1基因(GenBank登录号:OR500287)全长1 688 bp,编码414个氨基酸,为亲水性不稳定蛋白。酵母活性检测证实XsWRI1具有较强的转录激活活性。组织特异性表达分析显示,XsWRI1的表达在发育胚乳中占主导地位,营养器官(根、茎、叶)和其他生殖器官(花瓣、雄蕊)中的表达水平可以忽略不计。结构预测确定了2个保守的AP2/EREBP DNA结合结构域(残基76-148和177-238)和一个核定位信号。【结论】本研究阐明XsWRI1在文冠果中的分子特征和组织特异性调控作用,突出了其在脂质生物合成途径中的潜在作用。这些发现为有针对性的基因操作,以增强木本油料作物的种子油积累奠定了基础。

关键词: 文冠果, XsWRI1, 基因克隆, 转录激活, 组织特异性表达, 种子油生物合成

Abstract:

【Objective】This study aimed to characterize the sequence identity, transcriptional activation potential, and functional domains of the WRINKLED1 (WRI1) transcription factor in Xanthoceras sorbifolium (yellowhorn), providing insights into its regulatory role in seed oil biosynthesis. 【Method】The full-length cDNA of XsWRI1 was cloned from endosperm tissues of mature yellowhorn using rapid amplification of cDNA ends (RACE). Bioinformatics tools were employed to analyze protein sequence properties. A recombinant pGBKT7-XsWRI1 vector was constructed and transformed into Y2HGold yeast cells to assess transcriptional activation activity. Tissue-specific expression patterns were quantified via quantitative real-time PCR (qRT-PCR) across the root, stem, leaf, petal, stamen and developing embryo tissues. 【Result】The XsWRI1 gene (GenBank accession: OR500287) spans 1 688 bp, encoding a hydrophilic and unstable protein of 414 amino acids. Yeast activity testing confirmed strong transcriptional activation activity of XsWRI1. Tissue-specific expression analysis revealed predominant XsWRI1 expression in developing embryos, with negligible levels in vegetative organs (root, stem, leaf) and reproductive structures (petal, stamen). Structural prediction identified two conserved AP2/EREBP DNA-binding domains (residues 76-148 and 177-238) and a nuclear localization signal. 【Conclusion】This study elucidates the molecular characteristics and tissue-specific regulatory role of XsWRI1 in yellowhorn, highlighting its potential function in lipid biosynthesis pathways. These findings establish a foundation for targeted genetic manipulation to enhance seed oil accumulation in woody oil crops.

Key words: Xanthoceras sorbifolium, XsWRI1, gene cloning, transcriptional activity, tissue-specific expression, seed oil biosynthesis

中图分类号:

S727.32

张薇,李麟坤,梁重钧,等. 文冠果XsWRI1基因克隆、转录活性及组织特异性表达分析[J]. 南京林业大学学报（自然科学版）, 2025, 49(2): 23-30.

ZHANG Wei, LI Linkun, LIANG Chongjun, WANG Libing. Cloning, transcriptional activation, and tissue expression analysis of XsWRI1 from Xanthoceras sorbifolium[J].Journal of Nanjing Forestry University (Natural Science Edition), 2025, 49(2): 23-30.DOI: 10.12302/j.issn.1000-2006.202309001.

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引物名称 primer name	引物序列(5'—3') primer sequences 5'-3'	引物用途 primer purpose
3'race outer	GAATAACTCCGTTCTCAGTGC	3'race
3'race inner	GATGCGGGATTTGAGGAGAACATAG
5'race outer	TGTCTCTGTTTTGAGTTTTTCTG	5'race
5'race inner	AGTAACATCAGATAAAGCACAAG
XsWRI1-F	TTTACTACACATTCTGTCTTATT	ORF扩增 ORF amplification
XsWRI1-R	AACAACAAACAACATCAACAA
BD-WRI1-F	aggaggacctgcatatggcc ATGAAGAGGTCTTCTACTAC	pGBKT7载体构建 the pGBKT7 vector construction
BD-WRI1-R	cccgggaattcggcctccat TCAAACAGAATAGATATTAC
qRT WRI1-F	ATAACATACAGAACAAGAAGGGAAG	荧光定量 fluorescence quantitative
qRT WRI1-R	CAGCAAGATCATAAGTACGAGCA
qRT Tip41-F	AAGATGAATTGGCTGATAACGGA	荧光定量内参 fluorescence quantitative internal reference
qRT Tip41-R	AGCTCTCACGAAGAATGACTGGG

SapBase编号	相对表达量relative expression
SapBase编号	44 d	54 d	68 d	81 d
EVM0012075	127.87	112.62	52.33	6.32
EVM0007314	0.33	0.09	0	0
EVM0014879	0	0.02	0	0

文冠果XsWRI1基因克隆、转录活性及组织特异性表达分析

Cloning, transcriptional activation, and tissue expression analysis of XsWRI1 from Xanthoceras sorbifolium

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