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作物学报 ›› 2023, Vol. 49 ›› Issue (5): 1272-1281.doi: 10.3724/SP.J.1006.2023.24070

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

CBL互作蛋白激酶GmCIPK10增强大豆耐盐性

李慧1,2(), 路依萍1, 汪小凯1, 王璐瑶1, 邱婷婷1, 张雪婷1, 黄海燕1, 崔晓玉1,*()   

  1. 1临沂大学农林科学学院, 中国山东临沂 276000
    2菲律宾克里斯汀大学国际学院, 菲律宾 1004
  • 收稿日期:2022-03-29 接受日期:2022-07-21 出版日期:2023-05-12 网络出版日期:2022-08-18
  • 通讯作者: *崔晓玉, E-mail: cuixiaoyu@lyu.edu.cn
  • 作者简介:E-mail: lihuiqau@163.com
  • 基金资助:
    国家自然科学基金项目(32001459);山东省自然科学基金项目(ZR2020QC123)

GmCIPK10, a CBL-interacting protein kinase promotes salt tolerance in soybean

LI Hui1,2(), LU Yi-Ping1, WANG Xiao-Kai1, WANG Lu-Yao1, QIU Ting-Ting1, ZHANG Xue-Ting1, HUANG Hai-Yan1, CUI Xiao-Yu1,*()   

  1. 1College of Agriculture and Forestry Sciences, Linyi University, Linyi 276000, Shandong, China
    2Center for International Education, Philippine Christian University, 1004, the Philippines
  • Received:2022-03-29 Accepted:2022-07-21 Published:2023-05-12 Published online:2022-08-18
  • Contact: *E-mail: cuixiaoyu@lyu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32001459);Natural Science Foundation of Shandong Province(ZR2020QC123)

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摘要：

盐胁迫严重威胁大豆产量和品质。类钙调磷酸酶B亚基互作蛋白激酶(CIPKs)在植物应对环境胁迫过程中发挥重要作用。但是, 目前对大豆CIPKs的生物学功能知之甚少。本研究从大豆基因组克隆到GmCIPK10。生物信息学分析结果表明, GmCIPK10属于不含内含子型CIPKs, 包含一个丝氨酸(Ser)/苏氨酸(Thr)蛋白激酶结构域和NAF/FISL基序。表达模式分析结果表明, 在盐(NaCl)、甲基紫精(MV)和过氧化氢(H2O2)处理下, GmCIPK10的转录水平升高。在拟南芥和大豆毛状根中过表达GmCIPK10能够提高转基因植株的抗盐性。进一步的生理指标测定发现, 在盐胁迫下, 过表达GmCIPK10能够降低转基因植株中丙二醛(MDA)和H2O2积累, 增强抗氧化酶活性以及降低钠离子(Na+)/钾离子(K+)比值。此外, qRT-PCR分析发现GmCIPK10促进抗氧化和耐盐相关基因表达响应盐胁迫。酵母双杂交、Pull-down和双分子荧光互补试验结果证明GmCIPK10与钙离子(Ca2+)感应器GmCBL4相互作用。这些结果为解析CBL-CIPK信号通路在大豆盐胁迫应答的作用提供了参考。

关键词: GmCIPK10, 耐盐性, ROS清除, Na+/K+稳态, 大豆

Abstract:

Salt stress seriously restricts the yield and quality of soybean. Calcineurin B subunit-interacting protein kinases (CIPKs) play a vital role in response to environmental stresses in plant. However, few study is known about the biological function of soybean CIPKs. In this study, GmCIPK10 was cloned from soybean genome. Bioinformatics analysis exhibited that GmCIPK10 encoded an intron-poor type CIPKs with a serine (Ser)/threonine (Thr) protein kinase domain and a NAF/FISL motif. The relative expression pattern levels showed that the transcript level of GmCIPK10 was increased under NaCl, MV, and H2O2 treatments. Overexpression of GmCIPK10 in Arabidopsis and soybean hairy roots improved salt tolerance of transgenic plants. Further physiological indicator assays demonstrated that GmCIPK10 overexpression could decrease the accumulation of MDA and H2O2, enhance the activity of antioxidant enzymes, and reduce sodium (Na+)/potassium (K+) in transgenic plants under salt stress. In addition, qRT-PCR illustrated that GmCIPK10 promoted the relative expression level of antioxidant- and salt tolerance-related gene in salt stress. The yeast two-hybrid, pull-down, and bimolecular fluorescence complementation experiments confirmed that GmCIPK10 interacts with the Ca2+ sensor GmCBL4. These results provide a reference for investigating the role of the CBL-CIPK signaling pathway in response to salt stress in soybean.

Key words: GmCIPK10, salt tolerance, ROS scavenging, Na+/K+ homeostasis, soybean

表1

本研究所使用的引物"

引物
Primer ID		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
AtActin GAAATCACAGCACTTGCACC TGGAATGTGCTGAGGGAAGC
GmTubulin GAGGCAAGATGAGCACCAAG ACGGAACATTTCCTGAATGGAG
GmCIPK10 CAAGCCTGTTTTTGCATCTGAAT GATAGTGTGTTTGGATCCCAGC
GmCAT1 AGCTAGCGCAAAGGGTTTCT AAGGTTTCAGGGCTACCACG
GmPOD21 CCGTTTCGTGGGTCAGAAATCT- CCGACGCCTGCTCCGACACTA
GmZAT10 AAGCCTTCTCCTCTTACCAAGCA TCGACGCCGA ACTCGTTGT
GmMYB118 ATCATACTGTTCGGAGTCAC CAGACACTGTAGAGACCTTGTT
GmLEA5 CCGATGTATCGGTAAGAGT AGGCTTTTGA ACCATCTC
GmNHX1 GTCGGGGCACACTTCACTAA GGATGCTGCTTGGACGATGA
GmSALT3 AAGCAGGTGCTTAACGACGA CAAATCTGTTAGCCGCGACG
GmSOS1 ATCGGCTGGGAAAGATTGGG CACCAGGGCCAGCTAGTAAG

图1

GmCIPK10的蛋白结构和进化关系分析"

图2

GmCIPK10响应盐胁迫和氧化胁迫的表达模式 以GmTubulin为内参基因, 每组3棵, 3次生物学重复。MV: 甲基紫精。*: P < 0.05。"

图3

过表达GmCIPK10提高转基因拟南芥的耐盐性 A和B: GmCIPK10-OE和WT拟南芥植株经盐胁迫(75 mmol L-1 NaCl)处理10 d后的表型和主根长; C: GmCIPK10基因表达量; D: MDA含量; E: H2O2含量; 每组4棵, 3次生物学重复; WT: 野生型; OE: 过表达。*: P < 0.05。"

图4

过表达GmCIPK10提高转基因大豆发状根的耐盐性 A和B: GmCIPK10-OE和VC大豆植株的表型经盐胁迫(200 mmol L-1 NaCl)处理10 d后的表型和成活率; C: GmCIPK10基因表达量; GmCIPK10-OE和VC大豆植株经盐胁迫处理7 d后的; D: MDA含量; E: H2O2 含量; F: CAT活性; G: POD活性; H: K+离子含量; I: Na+离子含量; J: Na+/K+比值; 每组10 棵, 3次生物学重复; VC: 载体对照; OE: 过表达; FW: 鲜重; DW: 干重。*: P < 0.05。"

图5

GmCIPK10调节盐胁迫和氧化胁迫相关基因表达响应盐胁迫 每组3棵, 3次生物学重复。VC: 载体对照; OE: 过表达。*: P < 0.05。"

图6

GmCBL4与GmCIPK10互作 A: 酵母双杂交检测GmCBL4和GmCIPK10互作; B: Pull-down检测GmCBL4和GmCIPK10互作; C: BiFC检测GmCBL4和GmCIPK10互作。BiFC: 双分子荧光互补; YFP: 黄色荧光蛋白。标尺为12 μm。"

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