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

Abstract

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

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.

Figures/Tables 12

Table 1

Table 2

Fig. 1

Fig. 2

Fig. 3

Table 3

Cis-acting elements in the promoter region of StMAPKK4 gene in potato"

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

Table 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Table 4

Fig. 8

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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

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

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