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作物学报 ›› 2023, Vol. 49 ›› Issue (1): 36-45.doi: 10.3724/SP.J.1006.2023.24006

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

马铃薯StMAPKK4基因表达分析及互作蛋白筛选与鉴定

濮雪1(), 王凯彤1, 张宁1,2,*(), 司怀军1,2   

  1. 1甘肃农业大学生命科学技术学院, 甘肃兰州 730070
    2甘肃省干旱生境作物学省部共建国家重点实验室, 甘肃兰州 730070
  • 收稿日期:2022-01-04 接受日期:2022-05-05 出版日期:2023-01-12 网络出版日期:2022-05-23
  • 通讯作者: 张宁
  • 作者简介:E-mail: 824153833@qq.com
  • 基金资助:
    国家自然科学基金项目(31960444);甘肃省干旱生境作物学重点实验室——省部共建国家重点实验室培育基地主任基金课题(GSCS-2019-Z03)

Relative expression analysis of StMAPKK4 gene and screening and identification of its interacting proteins in potato (Solanum tuberosum L.)

PU Xue1(), WANG Kai-Tong1, ZHANG Ning1,2,*(), SI Huai-Jun1,2   

  1. 1College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, Gansu, China
    2State Key Laboratory of Aridland Crop Science, Gansu Agricultural University, Lanzhou 730070, Gansu, China
  • Received:2022-01-04 Accepted:2022-05-05 Published:2023-01-12 Published online:2022-05-23
  • Contact: ZHANG Ning
  • Supported by:
    National Natural Science Foundation of China(31960444);Gansu Provincial Key Laboratory of Aridland Crop Science, Gansu Agricultural University(GSCS-2019-Z03)

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摘要：

MAPKKs是促丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)级联反应主要成员之一, 位于该级联反应通路中间, 对信号传递起着收集和发散的关键作用。有研究表明马铃薯StMAPKK4基因响应干旱胁迫, 为进一步探究该基因的生物学功能, 本研究对StMAPKK4基因进行了生物信息学分析。结果表明, StMAPKK4与科民茄亲缘关系最近。其含有蛋白激酶家族的Protein kinase domain (PF00069)结构域, 位置定位于64~302 aa之间。StMAPKK4含有多个激素(茉莉酸甲酯、乙烯、脱落酸、ABA)及胁迫相关响应元件。qRT-PCR分析结果表明, StMAPKK4基因在马铃薯茎中表达量最高, 且在干旱及盐处理下均上调表达。亚细胞定位表明该基因定位于细胞膜上。利用酵母双杂交方法筛选出8个与StMAPKK4相互作用的蛋白, 并通过回转试验验证了其互作的真实性。对互作蛋白进行Blast比对结果显示, StMAPKK4与多酚氧化酶、藻蓝蛋白、天冬氨酸转氨酶、渗透素、磷酸盐转运蛋白等多种蛋白互作, 初步判断StMAPKK4可能参与光合作用响应机制、植物体低温、干旱及盐胁迫等一系列非生物胁迫、促进根系对磷元素的吸收等生理生化过程。

关键词: 马铃薯, StMAPKK4, 生物信息学, qRT-PCR, 亚细胞定位, 酵母双杂交

Abstract:

MAPKKs, one of the main members of mitogen-activated protein kinase (MAPK) cascade, are located in the middle of the cascade pathway and play a key role in signal transduction. Studies have shown that potato StMAPKK4 gene responds to drought stress. To further explore the biological function of StMAPKK4 gene, bioinformatics analysis was conducted in the study. The results showed that StMAPKK4 was most closely related to Solanum commersonii. It contained the protein kinase domain (PF00069) of the protein kinase family, which located between 64 aa and 302 aa. StMAPKK4 contained multiple hormones (methyl jasmonate, ethylene, and abscisic acid) and stress-related response elements. The qRT-PCR analysis revealed that StMAPKK4 gene expression was the highest in potato stem. The relative expression levels were up-regulated under drought and salt treatments. Subcellular localization indicated that StMAPKK4 was localized on the cell membrane. Furthermore, eight proteins interacting with StMAPKK4 were screened by yeast two-hybrid method, and their interaction was verified by rotation experiment. Blast comparison of the interacting proteins indicated that StMAPKK4 interacted with polyphenol oxidase, phycocyanin, aspartate aminotransferase, osmotin, phosphate transporter, and other proteins. It was preliminarily concluded that StMAPKK4 may be involved in the response mechanism of photosynthesis, a series of abiotic stresses such as low temperature, drought, and salt stresses in plants, and promote the uptake of phosphorus in roots.

Key words: potato, StMAPKK4 gene, bioinformatics, qRT-PCR, subcellular localization, yeast two-hybrid

表1

引物名称及序列"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 限制性内切酶
Restriction endonuclease
pCEGFP-F acgggggacgagctcggtaccATGGCCTTAGTTCGTGATCGC Kpn I
pCEGFP-F gcccttgctcaccatgtcgacGGTGGATTTCAAATCGATACTCTGT Sal I
pGBKT7-F tcagaggaggacctgCATATGTACAATCCTTCCCCCTTTTAAC Nde I
pGBKT7-R ctagttatgcggccgCTGCAGTCCCAGTTTTTAGCTTGCTT Pst I

表2

引物名称及序列"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')
Ef1a-F CAAGGATGACCCAGCCAAG
Ef1a-R TTCCTTACCTGAACGCCTGT
StMAPKK4-F TCAACTCTACCGCCAACACC
StMAPKK4-R GCACAAGTCCCGACGTATGA

图1

马铃薯StMAPKK4基因与其他9个物种同源基因发生树"

图2

马铃薯StMAPKK4蛋白保守结构域分析"

图3

马铃薯StMAPKK4蛋白质磷酸化位点分析"

表3

马铃薯StMAPKK4基因启动子区顺式作用元件分析"

顺式作用件
Cis-acting elements		 核心序列
Core sequences		 功能注释
Function annotation		 数量
Number
CGTCA-motif CGTCA 茉莉酸甲酯响应元件
Cis-acting regulatory element involved in the MeJA-responsiveness		 1
TGACG-motif TGACG 茉莉酸甲酯响应元件
Cis-acting regulatory element involved in the MeJA-responsiveness		 1
GATA-motif AAGGATAAGG 部分光响应元件
Part of a module for light response		 1
TCT-motif TCTTAC 部分光响应元件
Part of a module for light response		 1
顺式作用件
Cis-acting elements		 核心序列
Core sequences		 功能注释
Function annotation		 数量
Number
GT1-motif GGTTAAT 参与光反应顺式作用元件
Light responsive element		 2
ERE ATTTTAAA 乙烯响应元件
Ethylene responsive element		 5
ARE AAACCA 厌氧诱导的顺式调节元件
Cis-acting regulatory element essential for the anaerobic induction		 1
ABRE CACGTG ABA响应元件
Cis-acting element involved in the abscisic acid responsiveness		 6
AT1-motif AATTATTTTTTATT 部分光响应元件
Part of a module for light response		 1
AE-box AGAAACAA 部分光响应元件
Part of a module for light response		 3
STRE AGGGG 脱落酸、干旱响应调控元件
Cis-acting and drought element involved in abscisic acid response		 2
MYC CATGTG 逆境胁迫相关顺式调控元件
Stress related cis-regulatory elements		 6
TATA-box TATATAAA 转录起始位点−30的核心元件
Core promoter element around −30 of transcription start		 7
CAAT-box CAAT 启动子增强子区顺式作用元件
Common cis-acting element in promoter and enhancer regions		 33
W-box TTGACC 可诱导性抗性基因响应元件
Inducible resistance gene responsive element		 1
MYB TAACCA 干旱诱导相关元件
MYB binding site involved in drought-inducibility		 13
TATA-box TATA 转录起始位点−30的核心元件
Core promoter element around −30 of transcription start		 71
AT-rich sequence TAAAATACT 富含AT的DNA结合蛋白结合位点
AT-rich DNA binding protein binding sites		 1
G-box CACGTG 参与光反应的顺式作用元件
Light responsive element		 8
Box 4 ATTAAT 参与光反应的保守DNA模块部分
Part of a conserved DNA module involved in light responsiveness		 1
I-box GTATAAGGCC 部分光响应元件
Part of a module for light response		 1

图4

马铃薯StMAPKK4基因的组织特异性表达分析 柱上不同小写字母表示差异显著(P < 0.05)。"

图5

干旱和盐胁迫下StMAPKK4基因表达量分析 柱上不同小写字母表示差异显著(P < 0.05)。"

图6

马铃薯StMAPKK4亚细胞定位 pCEGFP: 空白对照(无StMAPKK4基因的pCEGFP空载体); pCEGFP-StMAPKK4: pCEGFP-StMAPKK4融合蛋白; 黄色、橙色、红色、蓝色箭头分别表示细胞核、细胞质、细胞膜和叶绿体。"

图7

酵母双杂交诱饵载体pGBKT7-StMAPKK4的毒性检测与自激活活性检测"

表4

StMAPKK4互作蛋白信息"

克隆
No.		 基因编号
Gene ID		 注释
Gene annotation		 基因全长
Full length of genes (bp)		 阳性克隆次数
Number of positive clones
1 XM_006347021.2 多酚氧化酶Polyphenol oxidase 2096 13
2 XM_006367901.2 ARF-GTPase活化因子ARF-GTPase activator 753 7
3 XM_006342026.2 藻蓝蛋白Plastocyanin 1483 7
4 XM_006366150.2 天冬氨酸转氨酶Aspartate aminotransferase 1739 5
5 XM_006367956.2 渗透素Osmotin 921 15
6 NM_001287896.1 磷酸盐转运蛋白Phosphate transporter 1567 10
7 XM_006358242.2 水解酶, 水解O-糖基化合物
Hydrolase, hydrolyzing O-glycosyl compounds		 3017 6
8 XM_006355206.2 核糖体蛋白S10p/S20e家族蛋白
Ribosomal protein S10p/S20e family protein		 676 5

图8

StMAPKK4互作蛋白的酵母双杂交验证 各菌落上方的序号为表4中互作蛋白的编号。"

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