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作物学报 ›› 2025, Vol. 51 ›› Issue (6): 1489-1500.doi: 10.3724/SP.J.1006.2025.44191

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

花生磷脂酰肌醇转运蛋白基因AhSFH的克隆及其响应黄曲霉菌侵染的表达特征分析

郭腾达1,2(), 崔梦杰2,*(), 陈琳杰2, 韩锁义1,2, 郭敬坤1,2, 吴晨迪2, 付留洋1, 黄冰艳2, 董文召2, 张新友1,2,*()   

  1. 1郑州大学生命科学学院, 河南郑州 450001
    2河南省作物分子育种研究院/农业农村部黄淮海油料作物重点实验室/河南省油料作物遗传改良重点实验室, 河南郑州 450002
  • 收稿日期:2024-11-19 接受日期:2025-03-26 出版日期:2025-06-12 网络出版日期:2025-04-07
  • 通讯作者: *张新友, E-mail: haasxinyou@163.com;崔梦杰, E-mail: cui2015104035@163.com
  • 作者简介:E-mail: gtd8586@163.com
  • 基金资助:
    本研究由国家自然科学基金青年科学基金项目(32301851);河南省农业科学院优秀青年基金项目(2024YQ03);河南省科技攻关项目(242102110308)

Cloning and expression analysis of the phosphatidylinositol transfer protein AhSFH gene in peanuts responsive to Aspergillus flavus infection

GUO Teng-Da1,2(), CUI Meng-Jie2,*(), CHEN Lin-Jie2, HAN Suo-Yi1,2, GUO Jing-Kun1,2, WU Chen-Di2, FU Liu-Yang1, HUANG Bing-Yan2, DONG Wen-Zhao2, ZHANG Xin-You1,2,*()   

  1. 1School of Life Sciences, Zhengzhou University, Zhengzhou 450001, Henan, China
    2Henan Academy of Crop Molecular Breeding / Key Laboratory of Oil Crops in Huang-Huai-Hai Plains, Ministry of Agriculture and Rural Affairs / Henan Provincial Key Laboratory for Oil Crops Improvement, Zhengzhou 450002, Henan, China
  • Received:2024-11-19 Accepted:2025-03-26 Published:2025-06-12 Published online:2025-04-07
  • Contact: *E-mail: haasxinyou@163.com;E-mail: cui2015104035@163.com
  • Supported by:
    the Youth Fund Project of National Natural Science Foundation of China(32301851);the Excellent Youth Fund Project of Henan Academy of Agricultural Sciences(2024YQ03);the Science and Technology Research Project of Henan Province(242102110308)

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摘要： 磷脂酰肌醇转运蛋白(phosphatidylinositol transfer protein, PITP)是真核生物细胞内膜系统间转运磷脂酰肌醇和磷脂酰胆碱单体的一类蛋白质, 其参与植物生长发育、信号转导、逆境胁迫响应等多种重要的生命过程。花生中响应黄曲霉侵染的PITP基因尚未见报道。本研究以高抗黄曲霉侵染花生种质J11为材料, 利用RT-PCR技术克隆获得花生磷脂酰肌醇转运蛋白类基因, 并通过生物信息学分析、实时荧光定量PCR (RT-qPCR)技术、亚细胞定位等对其进行分子特征鉴定和功能预测。结果显示, 该基因编码区序列长度为1836 bp, 编码一个分子式为C3114H4938N880O943S35、包含611个氨基酸、分子量为70.91 kD、等电点为7.84的不稳定亲水性蛋白。该蛋白无信号肽和跨膜结构域, 含有典型的Sec14和Nodulin结构域, 属于植物PITP家族的SFH亚族, 将其命名为AhSFH, 与大豆和蓖麻SFH蛋白有较近的亲缘关系。亚细胞定位结果显示, AhSFH蛋白主要定位于细胞质中。启动子作用元件分析发现, AhSFH基因启动子区包含大量的光响应、激素和胁迫响应元件。转录组和RT-qPCR分析发现, 在黄曲霉侵染初期(T2~T3), AhSFH基因在抗性材料中的表达水平急剧上升, 且显著高于高感材料。蛋白互作预测分析显示, AhSFH蛋白与多个转移酶相关家族蛋白具有关联性。本研究表明, 花生AhSFH基因可强烈响应黄曲霉的侵染, 可能在抗性花生材料抵抗黄曲霉侵染中发挥正调控作用。

关键词: 花生, 磷脂酰肌醇转运蛋白, AhSFH, 黄曲霉, 表达分析

Abstract:

Phosphatidylinositol transfer proteins (PITPs) are a class of proteins responsible for transporting phosphatidylinositol and phosphatidylcholine monomers across the inner membrane systems of eukaryotic cells. They play essential roles in plant growth and development, signal transduction, stress responses, and other vital biological processes. To date, the involvement of PITP genes in peanut (Arachis hypogaea) responses to Aspergillus flavus infection has not been reported. In this study, the PITP gene was cloned from the highly resistant peanut variety “J11” using RT-PCR, and its molecular characterization and functional prediction were analyzed through bioinformatics, RT-qPCR, and subcellular localization studies. The results showed that the gene’s coding region is 1836 bp in length, encoding an unstable hydrophilic protein with the molecular formula C3114H4938N880O943S35. The protein consists of 611 amino acids, with a molecular weight of 70.91 kD and an isoelectric point of 7.84. It lacks signal peptide and transmembrane domains but contains typical Sec14 and Nodulin domains. It belongs to the SFH subfamily of the plant PITP family and is closely related to soybean and ricinus SFH proteins. Subcellular localization analysis indicated that the AhSFH protein is primarily localized in the cytoplasm. Promoter cis-acting element analysis revealed that the AhSFH promoter contains large number of light-, hormone-, and stress-responsive elements. Transcriptome and RT-qPCR analyses showed that AhSFH expression increased sharply in resistant materials during the early stages of A. flavus infection (T2-T3), surpassing the expression levels observed in highly susceptible materials. Additionally, protein interaction prediction suggested that AhSFH is associated with several transferase-related family proteins. These findings indicate that the AhSFH gene in peanuts plays a crucial role in responding to A. flavus infection and may function as a positive regulator in enhancing peanut resistance to this pathogen.

Key words: peanut, phosphatidylinositol transport protein, AhSFH, Aspergillus flavus, expression analysis

表1

引物序列"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Function
AhSFH-F/R ATGTCAGGCTCTGAAGGACAATG&TCAACAGAATAGCCGCCTCTG 基因克隆Gene cloning
qAhSFH-F/R TGCGCATCACCACAATAGAT&GTCATGGCAGCCAAAAGATT 基因表达分析
Analysis of gene expression
sAhSFH-F/R AACACGGGGGACTTTGCAACatgtcaggctctgaaggacaatgtagtc& TCCTCGCCCTTGCTCACCATagatcctcctccagatcctcctc 亚细胞定位Subcellular localization

图1

AhSFH基因的克隆及鉴定分析 A: AhSFH基因的染色体定位。B: AhSFH基因的凝胶电泳图; M: DL2000 marker; 1: AhSFH扩增产物。C: AhSFH基因的结构分析。D: AhSFH蛋白的跨膜结构预测。E: AhSFH蛋白的亲疏水性预测; 数值小于0表示亲水, 大于0表示疏水。F: AhSFH基因的ORF及其编码氨基酸序列。"

表2

AhSFH蛋白的理化性质"

基因
Gene		 基因位置
Gene location		 编码长度
Encoding length (bp)		 蛋白质分子质量
Protein molecular weight (kD)		 等电点
Isoelectric point		 跨膜结构
Transmembrane structures		 不稳定指数
Instability index
AhSFH (arahy.E2N38F.1) Chr.19: 267015-272130 1836 70.91 7.84 0 53.72

图2

AhSFH蛋白的二级结构(A)和三级结构(B) 蓝色h: α-螺旋; 绿色t: β-折叠; 橙色c: 无规则卷曲; 红色e: 延伸链。"

图3

AhSFH蛋白与其他植物磷脂酰肌醇转运蛋白的系统进化树分析 A: AhSFH蛋白与其他植物同源蛋白序列比对分析。B: 植物PITP蛋白家族系统进化分析。C: 植物PITP蛋白家族成员保守结构分析。"

图4

AhSFH-GFP蛋白在烟草叶片表皮细胞中的亚细胞定位 GFP: 绿色荧光信号; mKATE-NLS: 细胞核指示蛋白红色荧光信号; Chlorophyll: 叶绿体自发荧光信号; Bright field: 明视野; Merged: 叠加图。标尺为20 μm。"

表3

AhSFH启动子顺式作用元件分析"

元件 Motif 数量 Number 功能 Function
CAAT-box 21 核心启动子元件 Core promoter element
TATA-box 60 核心启动子元件 Core promoter element
ATCT-motif 1 光反应 Light responsiveness
Box 4 2 光反应 Light responsiveness
G-box 1 光反应 Light responsiveness
GATA-motif 1 光反应 Light responsiveness
GARE-motif 1 赤霉素响应元件 Gibberellin-responsive element
HD-Zip 3 1 蛋白结合位点 Protein binding site
LTR 1 低温反应 Low-temperature responsiveness
ARE 1 厌氧诱导 Anaerobic induction
AT-rich element 1 富含AT的DNA结合蛋白结合位点 Binding site of AT-rich DNA binding protein
TCA-element 1 水杨酸响应 Salicylic acid responsiveness
TGA-element 1 生长素响应 Auxin-responsive element

图5

AhSFH启动子区的激素响应元件和胁迫响应元件分析 实线框与虚线框分别代表激素和胁迫响应元件。"

图6

AhSFH在黄曲霉接种后不同时间的表达 A: 基于转录组数据的表达分析。B和C: 基于荧光定量PCR的表达分析。**: 在0.01水平差异显著。"

图7

AhSFH蛋白的相互作用网络分析"

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