中国农业科学 ›› 2024, Vol. 57 ›› Issue (22): 4416-4430.doi: 10.3864/j.issn.0578-1752.2024.22.003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

谷子SiCIPK21的克隆及功能分析

杜艳伟1(), 阎晓光1(), 赵晋锋1(), 贾苏卿2, 王高鸿1, 余爱丽1, 张鹏1   

  1. 1 山西农业大学谷子研究所,山西长治 046011
    2 山西农业大学玉米研究所,山西忻州 034000
  • 收稿日期:2024-05-12 接受日期:2024-07-12 出版日期:2024-11-16 发布日期:2024-11-22
  • 通信作者:
    阎晓光,E-mail:
    赵晋锋,E-mail:
  • 联系方式: 杜艳伟,E-mail:yanweidu@163.com。
  • 基金资助:
    山西省基础研究计划(202103021223153); 山西省基础研究计划(202103021224163); 国家重要特色物种谷子育种联合攻关项目(gzlg-01-03); 山西农业大学科技创新提升工程(CXGC202440)

Cloning and Functional Verification of SiCIPK21 Gene in Foxtail Millet

DU YanWei1(), YAN XiaoGuang1(), ZHAO JinFeng1(), JIA SuQing2, WANG GaoHong1, YU AiLi1, ZHANG Peng1   

  1. 1 Millet Research Institute, Shanxi Agricultural University, Changzhi 046011, Shanxi
    2 Maize Research Institute, Shanxi Agricultural University, Xinzhou 034000, Shanxi
  • Received:2024-05-12 Accepted:2024-07-12 Published:2024-11-16 Online:2024-11-22

摘要:

【目的】Ca2+-CBL-CIPK信号系统在植物响应非生物逆境中具有重要功能。克隆谷子SiCIPK21,并研究其抗逆功能,为谷子抗逆分子育种提供关键候选基因和理论依据。【方法】利用生物信息学分析SiCIPK21启动子区域的顺式作用元件,预测其蛋白与拟南芥AtCBL的互作关系。通过PCR技术克隆SiCIPK21,构建融合表达载体在烟草中瞬时表达,确定亚细胞定位。从谷子品种豫谷1号叶片中特异性扩增SiCIPK21的部分片段,构建重组载体VIGS-pTRV2-SiCIPK21,以谷子八氢番茄红素脱氢酶(SiPDS)为指示基因,选取二叶期的谷子幼苗,通过子叶注射进行侵染,利用病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术研究盐(250 mmol·L-1 NaCl)胁迫下SiCIPK21在谷子苗期的作用。在拟南芥中过表达SiCIPK21获得T3代转基因株系。在不同浓度NaCl(150/175 mmol·L-1)、甘露醇(300/400 mmol·L-1)和ABA(0.25/0.5 μmol·L-1)处理下对萌发期的表型进行分析,同时,对苗期的耐盐和耐旱表型进行分析。【结果】亚细胞定位显示,SiCIPK21位于细胞核。SiCIPK21可能与拟南芥AtCBL2、AtCBL3、AtCBL4、AtCBL9和AtCBL10互作。SiCIPK21启动子区域含有逆境应答元件,暗示SiCIPK21可能参与谷子的逆境应答。VIGS基因沉默试验表明,沉默谷子SiCIPK21植株对盐胁迫的敏感性增加。通过遗传转化获得3个独立的T3代拟南芥过表达株系(2#、3#和6#)。在不同浓度NaCl(150/175 mmol·L-1)、甘露醇(300/400 mmol·L-1)和ABA(0.25/0.5 μmol·L-1)条件下的萌发率、萌发速度、绿色子叶展开率、根系长度和鲜重均显著高于野生型植株(WT);在拟南芥苗期,过表达株系的成活率和叶绿素含量显著提高,对盐胁迫和干旱的耐受性增强。【结论】SiCIPK21是植物响应盐和干旱胁迫的正调控因子,SiCIPK21可作为提高谷子抗逆分子育种的候选基因。

关键词: 谷子, SiCIPK21, 非生物胁迫, 表型分析, 基因沉默

Abstract:

【Objective】The Ca2+-CBL-CIPK signaling pathway has important functions in plant response to abiotic stresses. By cloning the SiCIPK21 gene and studying its function under stress conditions, we provide a key candidate gene and theoretical basis for molecular breeding of foxtail millet with stress tolerance.【Method】Bioinformatics was used to analyze the cis-acting elements in the promoter region of this gene and predict the interactions between this protein and AtCBLs in Arabidopsis thaliana. SiCIPK21 was cloned by PCR, and a fusion expression vector was constructed for transient expression in tobacco to determine the subcellular localization. foxtail millet cv. Yugu 1 was used as material, and specifically amplified part of the SiCIPK21 gene fragment from Yugu 1 leaves, and recombinant vector VIGS-pTRV2-SiCIPK21 was constructed, using the phytoene desaturase gene (SiPDS) as the indicator gene, and seedlings of foxtail millet at the two-leaf stage were selected and infiltrated by cotyledon injection to investigate the role of SiCIPK21 under salt stress (250 mmol·L-1 NaCl) by using virus-induced gene silencing (VIGS) technology. T3 generation transgenic lines were obtained by overexpressing SiCIPK21 in Arabidopsis thaliana. Phenotypes at germination were analyzed under different concentrations of NaCl (150/175 mmol·L-1), mannitol (300/400 mmol·L-1) and ABA (0.25/0.5 μmol·L-1) treatments, and salt and drought tolerant phenotypes at seedling stage were also analyzed.【Result】Subcellular localization revealed that SiCIPK21 was located in the nucleus. The protein SiCIPK21 might interact with AtCBL2, AtCBL3, AtCBL4, AtCBL9, and AtCBL10 in Arabidopsis thaliana. The promoter region of SiCIPK21 contained adverse response elements, suggesting that SiCIPK21 may participate in the adverse responses. The VIGS gene silencing demonstrated that SiCIPK21-silenced foxtail millet plants had increased sensitivity to salt stress than the control plants. Three independent T3 generation Arabidopsis thaliana overexpression lines (2#, 3# and 6#) were obtained by genetic transformation. Overexpression lines showed significantly higher germination rate, germination speed, green cotyledon unfolding rate, root length and fresh weight than the wild-type plants (WT) at different concentrations of NaCl (150/175 mmol·L-1), mannitol (300/400 mmol·L-1) and ABA (0.25/0.5 μmol·L-1). Moreover, phenotypic analysis of salt and drought tolerance in Arabidopsis seedlings showed that overexpression lines had significantly higher survival rates and chlorophyll contents than WT.【Conclusion】SiCIPK21 is a positive regulator of plant response to salt and drought stresses, which makes it a candidate gene for improving stress tolerance by molecular breeding in foxtail millet.

Key words: foxtail millet, SiCIPK21, abiotic stress, phenotype analysis, gene silencing