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作物学报 ›› 2023, Vol. 49 ›› Issue (10): 2654-2664.doi: 10.3724/SP.J.1006.2023.24280

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

割手密转录因子SsWRKY1提高甘蔗品种抗旱能力的功能鉴定

沈庆庆1(), 王天菊2, 王俊刚3, 张树珍3, 赵雪婷1, 何丽莲1(), 李富生1,4()   

  1. 1云南农业大学农学与生物技术学院, 云南昆明 650201
    2楚雄师范学院资源环境与化学学院, 云南楚雄 675000
    3中国热带农业科学院热带生物技术研究所, 海南海口 571101
    4云南农业大学云南省作物生产与智慧农业重点实验室, 云南昆明 650201
  • 收稿日期:2022-12-20 接受日期:2023-04-17 出版日期:2023-10-12 网络出版日期:2023-04-26
  • 通讯作者: 李富生, E-mail: lfs810@sina.com; 何丽莲, E-mail: helilian905@sohu.com
  • 作者简介:E-mail: wsqq2022@163.com
  • 基金资助:
    国家自然科学基金项目(31960451);国家重点研发计划项目(2018YFD1000503);云南省作物生产与智慧农业重点实验室专项(202105AG070007);云南省重大科技专项(202202AE090021);云南省教育厅科学研究基金项目(2020Y203)

Functional identification of Saccharum spontaneum transcription factor SsWRKY1 to improve drought tolerance in sugarcane

SHEN Qing-Qing1(), WANG Tian-Ju2, WANG Jun-Gang3, ZHANG Shu-Zhen3, ZHAO Xue-Ting1, HE Li-Lian1(), LI Fu-Sheng1,4()   

  1. 1College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, Yunnan, China
    2College of Resources Environment and Chemistry, Chuxiong Normal University, Chuxiong 675000, Yunnan, China
    3Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, Hainan, China
    4Key Laboratory for Crop Production and Smart Agriculture of Yunnan Province, Yunnan Agricultural University, Kunming 650201, Yunnan, China
  • Received:2022-12-20 Accepted:2023-04-17 Published:2023-10-12 Published online:2023-04-26
  • Contact: E-mail: lfs810@sina.com; E-mail: helilian905@sohu.com
  • Supported by:
    National Natural Science Foundation of China(31960451);National Key Research and Development Program of China(2018YFD1000503);Special Projects of the Key Laboratory for Crop Production and Smart Agriculture of Yunnan Province(202105AG070007);Major Science and Technology Projects in Yunnan Province(202202AE090021);Scientific Research Fund Project of Department of Education Yunnan Province(2020Y203)

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摘要：

SsWRKY1是甘蔗属野生种割手密WRKY家族成员基因, 开展SsWRKY1基因的功能分析, 为研究SsWRKY1参与干旱调控的分子机制提供有价值的信息。本研究利用农杆菌介导转化技术获得SsWRKY1过表达株系和RNAi干扰株系, PEG干旱胁迫处理发现过表达株系受干旱胁迫伤害程度明显轻于非转基因植株, 而干扰表达株系则表现严重的干旱胁迫伤害; 过表达株系脯氨酸(Pro)含量显著增加, 丙二醛(MDA)含量显著下降, 超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)等抗氧化酶活性出现不同程度的提高, 且胁迫响应基因MAPK级联信号传递基因MAPK、ABA生物合成基因NCED、脯氨酸生物合成基因P5CS、以及ROS清除系统基因SOD、POD、CAT的表达水平明显升高, 说明过表达SsWRKY1能够提高甘蔗应对干旱胁迫的能力。本研究表明, SsWRKY1基因通过激活抗氧化系统和调节胁迫响应基因的方式增强了甘蔗对干旱的抗性, 可作为甘蔗抗性遗传改良的重要基因资源。

关键词: WRKY转录因子, 转基因甘蔗, 抗旱性, 功能鉴定

Abstract:

SsWRKY1 is a member gene of the WRKY family in Saccharum spontaneum. The functional analysis provides valuable information for studying the molecular mechanism of SsWRKY1 involved in drought regulation. In this study, SsWRKY1 overexpression lines and RNAi interference expression lines were obtained by Agrobacterium-mediated transformation technology. The results showed that overexpression lines were significantly less damaged by drought stress than non-transgenic plants, while the interference expression lines showed severe drought stress damage with PEG to simulate drought stress. The content of proline in the overexpression lines increased significantly, the content of malondialdehyde decreased significantly, and the activities of antioxidant enzymes such as superoxide dismutase, peroxidase, and catalase increased to varying degrees. The relative expression levels of MAPK Cascade signal transduction gene MAPK, ABA biosynthesis gene NCED, proline biosynthesis gene P5CS, ROS scavenging system gene SOD, POD, and CAT were significantly increased, indicating that SsWRKY1 overexpression could improve the ability of sugarcane to cope with drought stress. In conclusion, SsWRKY1 enhanced the drought resistance of sugarcane by activating the antioxidant system and regulating stress response genes and could be used as an important gene resource for genetic improvement of sugarcane resistance.

Key words: WRKY transcription factors, transgenic sugarcane, drought tolerance, functional identification

图1

SsWRKY1过表达和RNAi干扰表达载体图谱 A: SsWRKY1过表达载体。B: SsWRKY1 RNAi干扰表达载体。"

表1

胁迫响应基因qRT-PCR分析的引物"

引物名称
Primer name		 引物序列
Primer sequences (5'-3')
SOD-F
SOD-R		 GTGAAGGCTGTTGCTGTGCTT
CGGTTCTCATCTTCTGGTGCT
POD-F
POD-R		 AATCGGAGAAGTCCTCGTCCCATAG
TTCTACCACAACAACCTCGCCAAG
CAT-F
CAT-R		 CATCCCACTCAACTACAGGCACATG
TCACACCACAAGTAGGCTTCCAATG
P5CS-F
P5CS-R		 TTTGGATTGGGTGCTGAGGTTGG
ATCCTTGTCACCGTTCACCACTTG
MAPK-F
MAPK-R		 GGTCTTCCTCTCCTCCGTCGTC
TCCTCCGCTCATCGTCTTCTTCC
NCED-F
NCED-R		 CTTCCACGGCACCTTCATCACTG
GGCTCCTCTGTTCCTCCTCCAAG

图2

SsWRKY1过表达和RNAi干扰表达载体分子检测 A: 过表达载体酶切鉴定。B: RNAi干扰表达载体菌液检测。"

图3

农杆菌介导的甘蔗遗传转化 A: 胚性愈伤组织。B: 愈伤组织开始分化成苗。C: Basta第1次抗性筛选。D: Basta第2次抗性筛选。E: 抗性植株的生根及炼苗。F: 转基因植株移栽。"

图4

SsWRKY1转基因甘蔗的分子检测 A: 目的基因PCR检测。M: DNA marker DL2000, +: 阳性对照, -: 阴性对照, 1~10: 转基因甘蔗。B: Bar基因试纸检测。-: 阴性对照, 1~5: 转基因甘蔗。C: 过表达株系中目的基因qRT-PCR检测, WT代表未转化甘蔗植株, OE4、OE5、OE8代表不同的过表达株系。D: 干扰表达株系中目的基因qRT-PCR检测, WT代表未转化甘蔗植株, R1、R2、R5代表不同的RNAi干扰表达株系。APRT基因的表达为内参, 差异显著性分析采用t测验方法, *P < 0.05、**P < 0.01。"

图5

SsWRKY1过表达株系的抗旱性鉴定 A: 干旱胁迫条件下过表达株系的表型变化, WT代表未转化甘蔗植株, OE4、OE5、OE8代表不同的过表达株系。差异显著性分析采用t测验方法, *P < 0.05、**P < 0.01。"

图6

SsWRKY1干涉表达株系的抗旱性鉴定 A: 干旱胁迫条件下干涉表达株系的表型变化, WT代表未转化甘蔗植株, R1、R2、R5代表不同的RNAi干扰表达株系。差异显著性分析采用t测验方法, *P < 0.05、**P < 0.01。"

图7

SsWRKY1过表达株系中干旱胁迫响应基因的表达谱分析 WT代表未转化甘蔗植株, OE4、OE5、OE8代表不同的过表达株系。差异显著性分析采用t测验方法, *P < 0.05、**P < 0.01。"

图8

SsWRKY1干扰表达株系中干旱胁迫响应基因的表达谱分析 WT代表未转化甘蔗植株, R1、R2、R5代表不同的RNAi干扰表达株系。差异显著性分析采用t测验方法, *P < 0.05、**P < 0.01。"

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