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作物学报 ›› 2023, Vol. 49 ›› Issue (6): 1573-1583.doi: 10.3724/SP.J.1006.2023.21028

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甜荞FeSGT1基因克隆及抗旱功能解析

朱旭东1(), 杨兰锋1, 陈媛媛1, 侯泽豪1,2, 罗旖柔1, 熊泽浩1, 方正武1,*()   

  1. 1长江大学农学院/主要粮食作物产业化湖北省协同创新中心, 湖北荆州 434025
    2中国农业科学院作物科学研究所, 北京 100081
  • 收稿日期:2022-04-18 接受日期:2022-09-05 出版日期:2023-06-12 网络出版日期:2022-09-15
  • 通讯作者: *方正武, E-mail: fangzhengwu88@163.com
  • 作者简介:E-mail: 202071604@yangtzeu.edu.cn
  • 基金资助:
    国家自然科学基金项目(31671755)

Biological functional analysis of common buckwheat (Fagopyrum esculentum) FeSGT1 gene in enhancing drought stress resistance

ZHU Xu-Dong1(), YANG Lan-Feng1, CHEN Yuan-Yuan1, HOU Ze-Hao1,2, LUO Yi-Rou1, XIONG Ze-Hao1, FANG Zheng-Wu1,*()   

  1. 1College of Agriculture, Yangtze University/Hubei Center for Collaborative Innovation of Grain Industry, Jingzhou 434025, Hubei, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2022-04-18 Accepted:2022-09-05 Published:2023-06-12 Published online:2022-09-15
  • Contact: *E-mail: fangzhengwu88@163.com
  • Supported by:
    National Natural Science Foundation of China(31671755)

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摘要：

SGT1 (suppressor of the G2 allele of skp1)作为skp1-4的抑制因子, 在植物的非生物胁迫响应中具有重要作用。根据甜荞干旱胁迫下的转录组学和蛋白质组学分析, 我们克隆出一个与甜荞耐旱性状相关的候选基因FeSGT1。生物信息学分析表明FeSGT1包含一个1086 bp开放阅读框(ORF), 编码361个氨基酸, 具有3个(TPR、CS和SGS)保守结构域。进化分析表明, FeSGT1与藜麦CqSGT1 (XP_021726759.1)、甜菜BvSGT1 (XP_010671588.1)和菠菜SoSGT1 (XP_021839743.1)亲缘关系较近。亚细胞定位初步显示FeSGT1蛋白定位于细胞膜上。qRT-PCR分析发现FeSGT1在干旱胁迫24 h内表达呈现上调趋势。在盐、低温(4℃)胁迫和ABA处理下, FeSGT1基因表达在12 h达到高峰, 24 h后开始下降。在拟南芥中过表达FeSGT1基因发现, 在干旱和盐胁迫下, 转基因植株的发芽率、根长、鲜重和存活率显著提高, 丙二醛(MDA)和过氧化氢(H2O2)含量明显降低, 而过氧化氢酶(CAT)活性显著升高。过表达FeSGT1显著增强了转基因拟南芥植株的耐旱和耐盐能力, 为深入研究FeSGT1基因调控甜荞抗旱分子机制奠定了基础。

关键词: 甜荞, FeSGT1, 转基因拟南芥, 抗旱性

Abstract:

SGT1 (Suppressor of the G2 Allele of skP1) is an inhibitor of skp1-4, which plays an important role in the abiotic stress response of plants. Based on the early transcriptomics and proteinomics analyses of common buckwheat under drought stress, a FeSGT1 gene was screened and cloned, which contained a 1086 bp open reading frame encoding 361 amino acids and 3 domains including TPR, CS, and SGS. Homologous protein comparison showed that FeSGT1 was closely related to CqSGT1 (XP_021726759.1), BvSGT1 (XP_010671588.1), and SoSGT1 (XP_021839743.1). Besides, FeSGT1 gene encoded membrane localization protein. The relative expression levels revealed that FeSGT1 tended to be up-regulated within 24 hours of drought stress. The expression of FeSGT1 gene peaked at 12 hours and began to decline after 24 hours under salt, low temperature (4℃), and ABA treatments. Overexpression of FeSGT1 gene in transgenic Arabidopsis not only conferred drought and salt tolerance, but also significantly increased root length, fresh weight, and survival rate compared with the wild type (WT) plant, accompanied by the elevated activities of catalase (CAT), the lowered malonaldehyde (MDA) and H2O2 contents, thus allowing plants to better adapt to adverse environments. Our results provided information in the exploring of the molecular regulation mechanism responding to drought tolerance in common buckwheat.

Key words: Common buckwheat (Fagopyrum esculentum), FeSGT1, transgenic Arabidopsis, drought resistance

表1

引物序列汇总"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Purpose
FeSGT1-F ATGGCGTCCGATCTCGAAAAG ORF扩增
FeSGT1-R TTAGTATTCCCATTTCTTCACCTC Amplification of ORF
Actin-QF ACCTTGCTGGACGTGACCTTAC 荧光定量内参基因
Actin-QR CCATCAGGAAGCTCATAGTTC Reference gene of qRT-PCR
FeSGT1-QF ATCACGCTGAACTCTTCGCA 荧光定量PCR
FeSGT1-QR TGCGTTCATCACACTCACGA Quantitative real time-PCR
pH-FeSGT1-F CTCGAGATGGCCGGCCCTGATATTTTG 亚细胞定位
pH-FeSGT1-R CCTAGGATCCTCTTGGCTCTCATC Subcellular Localization
pBI-FeSGT1-F CTCGAGATGGCCGGCCCTGATATTTTG 转基因拟南芥
pBI-FeSGT1-R CCCGGGCTAATCCTCTTGGCTCTCATC Transgenic Arabidopsis
p121-SGT1-F TTTGGAGAGGACCTCGACCT 转基因拟南芥鉴定
p121-SGT1-R TTAGTATTCCCATTTCTTCACCTC Identification of transgenic Arabidopsis

图1

甜荞FeSGT1的鉴定和结构分析 A: FeSGT1与其他植物SGT1蛋白的多重比对; B: FeSGT1蛋白系统进化树分析; C: FeSGT1蛋白三级结构分析。"

图2

FeSGT1的亚细胞定位 FeSGT1蛋白在烟草叶片中的亚细胞定位。绿色表示GFP信号, 红色表示叶绿体自身荧光。"

图3

甜荞FeSGT1 基因在不同非生物胁迫下的表达分析 *、**分别表示在0.05和0.01概率水平差异显著。数据表示为平均值±SD (n = 3)"

图4

FeSGT1转基因拟南芥干旱处理表型分析 A: 干旱胁迫下WT和OE株系的种子萌发分析。B: 干旱胁迫下WT和OE株系的发芽率; 计算后5 d的发芽率。C: 干旱胁迫下WT和OE株系的根长测定。D: 干旱胁迫下WT和OE株系的总根长和鲜重; *表示在0.05概率水平差异显著。"

图5

FeSGT1基因的异位表达增强了转基因拟南芥的抗旱性 A: 干旱胁迫下3周龄WT和OE株系的表型分析。B: 复水7 d后WT和OE株系在干旱条件下的存活率。C~E: 对照和干旱处理下WT和OE株系的MDA (C) H2O2 (D)含量和CAT (E)活性。*、**分别表示在0.05 和0.01概率水平差异显著。"

图6

盐处理下FeSGT1转基因植株表型分析 A: 盐胁迫下WT和OE株系的种子萌发分析。B: 盐胁迫下WT和OE株系的发芽率; 计算后5 d的发芽率。C: 盐胁迫下WT和OE株系的根长测定。D: 盐胁迫下WT和OE株系的总根长和鲜重; *表示在0.05概率水平差异显著。"

图7

FeSGT1基因的异位表达增强了转基因拟南芥的耐盐性 A:盐胁迫下3周龄WT和OE株系的表型分析。B: WT和OE株系在盐胁迫下的存活率。C~E: 对照和盐处理下WT和OE株系的MDA (C)、H2O2 (D)含量和CAT (E)活性。*表示在0.05概率水平差异显著。"

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