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作物学报 ›› 2024, Vol. 50 ›› Issue (12): 3155-3164.doi: 10.3724/SP.J.1006.2024.44054

• 研究简报 • 上一篇    

花生转录因子基因AhWRI1的克隆及表达分析

殷祥贞1(), 赵健鑫2, 郝翠翠2, 潘丽娟1, 陈娜1, 许静1, 姜骁1, 赵旭红1, 王恩琪2, 曹欢2, 禹山林1, 迟晓元1,*()   

  1. 1山东省花生研究所, 山东青岛 266100
    2青岛科技大学海洋科学与生物工程学院, 山东青岛 266042
  • 收稿日期:2024-03-29 接受日期:2024-08-15 出版日期:2024-12-12 网络出版日期:2024-09-03
  • 通讯作者: *迟晓元, E-mail: chi000@126.com
  • 作者简介:E-mail: yinxiangzhen1985@163.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);山东省农业科学院农业科技创新工程项目(CXGC2023F20);山东省农业科学院农业科技创新工程项目(CXGC2024F20);广东省重点领域研发计划项目(2020B020219003);新疆维吾尔自治区重大科技专项(2022A02008-3);泰山学者工程专项, 山东省重点研发计划(农业良种工程)项目(2022LZGC007);山东省自然科学基金项目(ZR2021QC172);山东省自然科学基金项目(ZR2023QC146);山东省重点研发计划(乡村振兴科技创新提振行动计划)项目(2022TZXD0031)

Cloning and expression analysis of transcription factor AhWRI1s in peanut

YIN Xiang-Zhen1(), ZHAO Jian-Xin2, HAO Cui-Cui2, PAN Li-Juan1, CHEN Na1, XU Jing1, JIANG Xiao1, ZHAO Xu-Hong1, WANG En-Qi2, CAO Huan2, YU Shan-Lin1, CHI Xiao-Yuan1,*()   

  1. 1Shandong Peanut Research Institute, Qingdao 266100, Shandong, China
    2College of Marine Science and Biological Engineering, Qingdao University of Science and Technology, Qingdao 266042, Shandong, China
  • Received:2024-03-29 Accepted:2024-08-15 Published:2024-12-12 Published online:2024-09-03
  • Contact: *E-mail: chi000@126.com
  • Supported by:
    China Agriculture Research System of MOF and MARA(CARS-13);Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences(CXGC2023F20);Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences(CXGC2024F20);Key-Area Research and Development Program of Guangdong Province(2020B020219003);Major Scientific and Technological Project in Xinjiang(2022A02008-3);Taishan Scholar Project Funding, the Research and Development Program of Shandong Province (the Improved Variety Engineering Project)(2022LZGC007);Natural Science Foundation of Shandong Province(ZR2021QC172);Natural Science Foundation of Shandong Province(ZR2023QC146);Key Research and Development Plan of Shandong Province (Action Plan to Boost Scientific and Technological Innovation in Rural Revitalization)(2022TZXD0031)

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摘要：

花生是世界范围内广泛栽培的油料和经济作物之一, 由于其高油脂和高蛋白质含量, 已成为人们主要的油脂和蛋白质来源。随着世界对植物油需求的不断增加, 改良花生脂肪酸组成和提高油脂含量成为花生育种工作的重要内容。转录调控因子可调控油脂合成相关代谢途径中一系列基因的表达, 显著影响油脂合成和代谢。本研究从花生品种花育33号的叶片中克隆得到2个转录因子基因AhWRI1-1和AhWRI1-2, AhWRI1-1的ORF为1101 bp, 编码366个氨基酸; AhWRI1-2的ORF为1128 bp, 编码375个氨基酸。生物信息学分析发现, AhWRI1-1和AhWRI1-2均含有2个AP2/EREBP结构域。利用qRT-PCR检测AhWRI1-1和AhWRI1-2在不同组织中的表达模式发现, AhWRI1-1在种子中的表达量最高, 可能参与调节脂肪酸合成和油脂积累; AhWRI1-2在下胚轴中的表达量最高, 可能参与下胚轴的发育。此外, AhWRI1-1和AhWRI1-2对非生物胁迫的响应存在差异, 表明AhWRI1-1和AhWRI1-2非生物胁迫中的作用也存在差异。通过在酵母中的转录激活试验验证, AhWRI1-1和AhWRI1-2均具有转录激活活性。本研究为以后对AhWRI1-1和AhWRI1-2的功能进行深入研究奠定了基础。

关键词: 花生, AP2/EREBP转录因子, 非生物胁迫, 基因表达分析, 转录激活

Abstract:

Peanut is one of the widely cultivated oil and economic crops worldwide and has become a major source of oil and protein for humans due to its high oil and protein content. With the increasing global demand for vegetable oil, improving the fatty acid composition and increasing the lipid content of peanut seeds has become a top priority in peanut breeding. Transcriptional regulators can modulate the expression of a series of genes in metabolic pathways related to lipid synthesis, significantly affecting lipid synthesis and metabolism. In this study, two transcription factors, AhWRI1-1 and AhWRI1-2, were cloned from the leaves of Huayu 33. The ORF of AhWRI1-1 was 1101 bp, encoding 366 amino acids, and the ORF of AhWRI1-2 was 1128 bp, encoding 375 amino acids. Bioinformatics analysis revealed that both AhWRI1-1 and AhWRI1-2 contained two AP2/EREBP conserved domains. The expression patterns of AhWRI1-1 and AhWRI1-2 in different tissues were detected by qRT-PCR. The results showed that AhWRI1-1 had the highest expression in seeds, suggesting its involvement in the regulation of fatty acid synthesis and oil accumulation, while AhWRI1-2 had the highest expression in hypocotyls, indicating its role in hypocotyl development. Additionally, the differences in the responses of AhWRI1-1 and AhWRI1-2 to abiotic stresses suggested that these transcription factors may play different roles under such conditions. Transcriptional activation experiments in yeast showed that both AhWRI1-1 and AhWRI1-2 possess transcriptional activation activities. This study lays the foundation for future in-depth functional studies of AhWRI1-1 and AhWRI1-2.

Key words: peanut, AP2/EREBP transcription factor, abiotic stress, gene expression analysis, transcriptional activation

表1

PCR引物"

引物名称
Primer name		 序列
Sequence (5′-3′)
AhWRI1-1-F ATGAAGAGGTCTCCTAATTCCTCT
AhWRI1-1-R TTAATAATTGGCATAGACATGCATATC
AhWRI1-2-F ATGGAAATGATGACAAAGAAA
AhWRI1-2-R TCACAGATAAGGGAGATTGC
qAhWRI1-1-F TGCCGAATTATCCGAGTT
qAhWRI1-1-R CAGCCATACACATAAGATTGAT
qAhWRI1-2-F TGAAGAGGAGTTGGATGTC
qAhWRI1-2-R TGTAGTGTTGGTAGTGGAAT

图1

AhWRI1-1 (a)和AhWRI1-2基因(b)的核苷酸与氨基酸序列"

图2

花生与其他植物WRI1蛋白的氨基酸序列比对 WRI1蛋白的2个AP2/EREBP结构域用横线标出。"

图3

AhWRI1-1和AhWRI1-2的基因结构"

图4

植物WRI1系统发育树"

图5

AhWRI1-1和AhWRI1-2基因在花生不同组织和种子不同发育时期的表达分析 a: AhWRI1-1在不同组织中的表达分析; b: AhWRI1-2在不同组织中的表达分析; c: AhWRI1-1在种子不同发育时期的表达分析; d: AhWRI1-1在种子不同发育时期的表达分析。将Actin11为内参基因。在a、b、c和d中, 分别将AhWRI1-1在种子中、AhWRI1-2在下胚轴中、AhWRI1-1在48 d的种子中、AhWRI1-2在12 d的种子中的表达量设置为1, 即分别将它们作为参考点。"

图6

AhWRI1-1 (a)和AhWRI1-2 (b)在非生物胁迫和激素处理条件下的表达分析 Actin11为内参基因, 分别将AhWRI1-1和AhWRI1-2在0 h的表达量设置为1, 即分别将它们作为参考点。ABA: 脱落酸; GA: 赤霉素; ET: 乙烯利; JA: 茉莉酸; SA: 水杨酸。"

图7

AhWRI1-1和AhWRI1-2转录激活活性分析"

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