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作物学报 ›› 2025, Vol. 51 ›› Issue (1): 58-67.doi: 10.3724/SP.J.1006.2025.44092

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

脂质转移蛋白SiLTP1基因参与谷子耐盐响应初探

孟凡花1, 刘敏1(), 沈傲1,2, 刘炜1,*()   

  1. 1山东省农业科学院农作物种质资源研究所, 山东济南 250100
    2上海应用技术大学生态技术与工程学院, 上海 201418
  • 收稿日期:2024-06-06 接受日期:2024-09-18 出版日期:2025-01-12 网络出版日期:2024-10-10
  • 通讯作者: *刘炜, E-mail: wheiliu@163.com
  • 作者简介:刘敏, E-mail: liumin20210101@163.com
    **同等贡献
  • 基金资助:
    山东省农业良种工程项目(2021LZGC006);山东省重点研发计划项目(2021LZGC025);国家自然科学基金项目(32201736);国家自然科学基金项目(32171955);山东省农业科学院农业科技创新工程项目(CXGC2023F13)

Preliminary investigation of the SiLTP1: a lipid transfer protein gene involved in the salt tolerance of foxtail millet

MENG Fan-Hua1, LIU Min1(), SHEN Ao1,2, LIU Wei1,*()   

  1. 1Institute of Crop Germplasm Resources, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China
    2School of Ecological Technology & Engineering, Shanghai Institute of Technology, Shanghai 201418, China
  • Received:2024-06-06 Accepted:2024-09-18 Published:2025-01-12 Published online:2024-10-10
  • Contact: *E-mail: wheiliu@163.com
  • About author:**Contributed equally to this work
  • Supported by:
    Agricultural Fine Seed Project of Shandong Province(2021LZGC006);Key Research and Development Project of Shandong Province(2021LZGC025);National Natural Science Foundation of China(32201736);National Natural Science Foundation of China(32171955);Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences(CXGC2023F13)

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摘要：

土壤盐渍化严重制约农业生产发展, 通过提高植物耐盐性进行盐碱地综合利用, 对于保障粮食安全, 提升农业生产效率具有重要意义。脂质转移蛋白是一类在高等植物中广泛存在的小分子蛋白质, 能够参与植株生长、信号转导以及多种生物和非生物胁迫过程。前期本课题组克隆到一个谷子中编码脂质转移蛋白的基因SiLTP1, 本研究通过构建其原核表达载体和植物双元过表达载体, 获得了该基因的原核表达蛋白及同源转化过表达阳性植株, 经进一步筛选获得4个纯合株系。体外耐盐性试验显示, SiLTP1的原核表达蛋白具有一定耐盐性; 转基因植株苗期耐盐生理指标测定显示, SiLTP1过表达植株在遭受盐胁迫时, 植株体内MDA积累减少, 抗氧化酶含量升高, 可积累更少的过氧化氢, 从而降低胁迫对植株的氧化损伤, 显示基因过表达植株具有更好的耐盐性。本研究结果初步揭示, SiLTP1可正向调控谷子的耐盐性, 其在谷子耐盐抗逆方面具有潜在功能, 为耐盐谷子品种改良及新品种培育提供了理论基础和候选基因资源。

关键词: 脂质转移蛋白, SiLTP1, 盐胁迫, 谷子, 耐盐响应

Abstract:

Soil salinization poses a significant barrier to agricultural productivity. Enhancing the salt tolerance of crops is crucial for the effective utilization of saline-alkali land, thereby ensuring food security and improving agricultural efficiency. Lipid transfer proteins (LTPs) are a class of small, widely distributed proteins in higher plants, playing crucial roles in plant growth, signal transduction, and responses to various biotic and abiotic stresses. In a previous study, a lipid transfer protein gene, designated SiLTP1, was cloned from foxtail millet. In this study, we constructed both a prokaryotic expression vector and a plant binary overexpression vector for SiLTP1. The prokaryotically expressed protein and four homozygous transgenic lines were successfully obtained and characterized. In vitro salt tolerance assays revealed that the SiLTP1 protein exhibited a notable degree of salt tolerance. Physiological measurements of the transgenic seedlings indicated reduced oxidative damage, as evidenced by lower malondialdehyde (MDA) accumulation, increased superoxide dismutase (SOD) activity, and reduced hydrogen peroxide (H2O2) levels under salt stress, demonstrating enhanced salt tolerance. These findings suggest that SiLTP1 plays a positive role in regulating salt tolerance in foxtail millet, potentially by mitigating oxidative stress. This study provides a theoretical foundation and valuable genetic resources for the future development and breeding of salt-tolerant foxtail millet varieties.

Key words: lipid transfer proteins, SiLTP1, salt stress, foxtail millet, salt tolerance response

图1

SiLTP1基因的原核蛋白表达 A: 菌液PCR验证; M: DNA marker; 1~6: pET32a-SiLTP1阳性菌液。B: SDS-PAGE电泳; M: protein maker; 1~4: pET32a- SiLTP1分别经0.05 mmol L-1、0.20 mmol L-1、0.50 mmol L-1和1.00 mmol L-1 IPTG诱导5 h表达的总蛋白; 5~6: pET32a空载体表达的总蛋白; 7: 未经载体转化的植株总蛋白。图中32 kD条带为pET32a-SiLTP1表达的融合蛋白, 21 kD条带为载体表达的标签蛋白。"

图2

斑点法检测pET32a-SiLTP1菌株的耐盐性 重组菌株(pET32a-SiLTP1)和对照菌株(pET32a)在0 mmol L-1、100 mmol L-1、200 mmol L-1、300 mmol L-1、500 mmol L-1和700 mmol L-1不同NaCl浓度的LB固体培养基上的生长情况。"

图3

重组菌株pET32a-SiLTP1 (蓝色)和对照菌株pET32a (橙色)在含200 mmol L-1 NaCl的LB液体培养基中的生长曲线 * 表示在0.05概率水平差异显著。"

图4

SiLTP1过表达株系的筛选鉴定 A: SiLTP1过表达植株的潮霉素筛选。B: SiLTP1过表达植株的PCR鉴定; M: DNA marker; 1~4: T3代过表达株系; 5: 阴性对照Ci846。C: SiLTP1基因在对照Ci846和过表达株系OE1~OE4中的相对表达量。不同小写字母表示在0.05概率水平差异显著。"

图5

盐胁迫下对照及SiLTP1过表达植株的表型 A: 0 mmol L-1和100 mmol L-1 NaCl处理7 d后谷子各株系表型。B: 0 mmol L-1和100 mmol L-1 NaCl处理7 d后谷子各株系的根长和苗长统计。不同小写字母表示在0.05概率水平差异显著。"

图6

盐处理后Ci846及SiLTP1过表达株系的生理指标测定及DAB染色 不同小写字母表示在0.05概率水平差异显著。"

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