马铃薯StMAPKK4基因表达分析及互作蛋白筛选与鉴定

doi:10.3724/SP.J.1006.2023.24006

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

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)

摘要/Abstract

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

濮雪, 王凯彤, 张宁, 司怀军. 马铃薯StMAPKK4基因表达分析及互作蛋白筛选与鉴定[J]. 作物学报, 2023, 49(1): 36-45.

PU Xue, WANG Kai-Tong, ZHANG Ning, SI Huai-Jun. Relative expression analysis of StMAPKK4 gene and screening and identification of its interacting proteins in potato (Solanum tuberosum L.)[J]. Acta Agronomica Sinica, 2023, 49(1): 36-45.

图/表 12

表1

表2

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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

表3

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表4

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引物名称 Primer name	引物序列 Primer sequence (5'-3')
pCEGFP-F	acgggggacgagctcggtaccATGGCCTTAGTTCGTGATCGC	Kpn I
pCEGFP-F	gcccttgctcaccatgtcgacGGTGGATTTCAAATCGATACTCTGT	Sal I
pGBKT7-F	tcagaggaggacctgCATATGTACAATCCTTCCCCCTTTTAAC	Nde I
pGBKT7-R	ctagttatgcggccgCTGCAGTCCCAGTTTTTAGCTTGCTT	Pst I

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

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

马铃薯StMAPKK4基因表达分析及互作蛋白筛选与鉴定

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

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