生物技术通报 ›› 2022, Vol. 38 ›› Issue (10): 164-172.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0055

• 研究报告 • 上一篇    下一篇

杧果钾通道基因MiSPIK的克隆、表达与功能分析

韩蕾1,2(), 李俊林1,2, 高爱平3, 黄建峰3, 李建召1(), 宋志忠1,3,4()   

  1. 1.鲁东大学农林工程研究院 山东省高校作物高产抗逆分子模块育种重点实验室,烟台 264025
    2.中国热带农业科学院作物品种资源研究所,海口 571100
    3.南京林业大学林学院,南京 210037
    4.剑桥大学植物系,剑桥,英国 CB2 3EA
  • 收稿日期:2022-01-11 出版日期:2022-10-26 发布日期:2022-11-11
  • 作者简介:韩蕾,女,博士,副教授,研究方向:植物养分高效利用及耐逆机理;E-mail:hanlei1610@qq.com
  • 基金资助:
    国家重点研究计划子课题(2019YFD1000504);国家自然科学基金项目(31501743);国家自然科学基金项目(31601819);山东省农业良种工程项目(2019LZGC009)

Cloning,Expression and Functional Analysis of Potassium Channel Gene MiSPIK in Mangifera indica

HAN Lei1,2(), LI Jun-lin1,2, GAO Ai-ping3, HUANG Jian-feng3, LI Jian-zhao1(), SONG Zhi-zhong1,3,4()   

  1. 1. The Engineering Research Institute of Agriculture and Forestry,Ludong University/Key Laboratory of Molecular Module-based Breeding of High Yield and Abiotic Resistant Plants in Universities of Shandong,Yantai 264025
    2. Tropical Crops Genetic Resources Institute,Chinese Academy of Tropical Agricultural Sciences,Haikou 571100
    3. College of Forestry,Nanjing Forestry University,Nanjing 210037
    4. Department of Plant Science,University of Cambridge,Cambridge CB2 3EA,UK
  • Received:2022-01-11 Published:2022-10-26 Online:2022-11-11

摘要:

Shaker类型钾通道在植物生长发育中起重要作用,其在果树中的生物学功能依然未知。克隆并揭示杧果SPIK钾通道基因的功能,为研究热带果树Shaker类型钾通道的生物学功能提供基因资源和理论基础。以杧果品种‘桂热82’为材料,利用RACE-PCR技术克隆Shaker类型钾离子通道基因MiSPIK(GenBank ID:OM179914),利用生物信息学手段分析该基因及编码蛋白的序列特征,通过实时荧光定量PCR(RT-qPCR)分析该基因的组织特异性表达特征,及其在转录水平对缺钾、高钾、低温、NaCl和PEG处理等5种非生物胁迫的响应情况,并创制MiSPIK超表达转基因拟南芥株系。结果显示,MiSPIK蛋白的分子量为90 006.83 kD,等电点(pI)为7.59,含有6个跨膜结构,属于不稳定的亲水蛋白。杧果MiSPIK与梨PbrSPIK和拟南芥AtSPIK的氨基酸序列一致性高达59.46%,三者紧密聚为一类,遗传距离最近。在转录水平上,MiSPIK在杧果花粉中特异性表达,并在一年生嫁接苗根部受缺钾或NaCl处理的抑制均显著降低,受高钾、PEG或低温胁迫诱导而显著增强。MiSPIK在拟南芥中表达后增强了转基因株系的钾素富集能力,并促进转基因植株提早抽薹和开花。杧果MiSPIK是花粉中特异表达的Shaker类型钾通道基因,其表达水平易受多种非生物胁迫的调控,可能在杧果钾素营养高效与利用中发挥作用。

关键词: 杧果, Shaker类型钾离子通道, SPIK, 花粉, 异源超表达

Abstract:

Shaker-type potassium(K+)channels play important roles in plant growth and development. However,their biological function in fruit crops is still unknown. We focused on the cloning and functional verification of SPIK channel gene in mango(Mangifera indica),which provide gene resources and theoretical foundation for the biological function analysis of Shaker type K+ channels in tropical fruit plants. The Shaker type K+ channel gene MiSPIK(GenBank ID:OM179914)was isolated by RACE-PCR technology from ‘Guire 82’ mango. The sequence characteristics of this gene and its coding protein were analyzed via bioinformatics. The tissue-specific expression characteristics and its responses to 5 abiotic stresses,including K+ deficiency,high K+ stress,low temperature,NaCl and PEG treatment,were further detected in the transcriptional level via qRT-PCR. MiSPIK-overexpressed transgenic Arabidopsis line was created. Results showed that MiSPIK protein had a molecular weight of 90 006.83 kD with the isoelectric point(pI)of 7.59,which possessed 6 transmembrane domains and belonged to unstable hydrophilic protein. The amino acid sequence identity was 59.46% among MiSPIK,pear PbrSPIK,and Arabidopsis AtSPIK,and phylogenetic tree analysis demonstrated that these three SPIK homologs were tightly clustered together and had the closest genetic distance. In the transcriptional level,MiSPIK was specifically expressed in mango pollens,and its expression in the roots of annual grafted seedlings significantly reduced by K+ deficiency or NaCl treatment,and significantly enhanced by high K+ stress,PEG or low temperature treatment. In addition,over-expression of MiSPIK in Arabidopsis significantly enhanced the K+ accumulation status and induced earlier bolting and flowering of transgenic plants. MiSPIK was a pollen-specifically expressed Shaker type K+ channel gene in mango,whose expression was prone to be regulated by distinct abiotic stresses,and may take part in the high efficient utilization of K+ nutrition in mango.

Key words: Mangifera indica, Shaker type K+ channel, SPIK, pollen, heterogenous expression