紫花苜蓿MsWRKY42的分离、鉴定及其对非生物胁迫的响应

doi:10.3864/j.issn.0578-1752.2020.17.004

Abstract

Abstract:

【Objective】The WRKY gene family in plants encodes a large group of transcription factors (TFs) that play essential roles in diverse stress responses, developmental and physiological processes. It laid a foundation for further research on the role of WRKY transcription factor in alfalfa stress-resistant molecular regulation by analyzing the role of MsWRKY42 transcription factor in alfalfa. 【Method】MsWRKY42 sequence was obtained by homologous alignment and sequence characteristics of it were analyzed by online bioinformatics tools. The phylogenetic tree of MsWRKY42 and Arabidopsis WRKY genes was constructed by MEGA-X. The putative cis-elements in promoter region of MsWRKY42 was analyzed by PlantCARE. The real-time quantitative PCR (qRT-PCR) was used to analyze the expression pattern of MsWRKY42 in different organs and its response to abiotic including NaCl (0.3 mol·L^-1), PEG(15%), 4℃, 40℃, low phosphorus and ABA (0.1×10^-3mol·L^-1). The fusion expression vector of pCAMBIA1300- WRKY-GFP was constructed and delivered into Nicotiana benthamiana by Agrobacterium mediated method to determine the subcellular localization of WRKY protein. The yeast one-hybrid technique was used to analyze the binding activity of MsWRKY42 and the cis-acting element W-box.【Result】The gene contained a 1 692 bp open reading frame encoding 563 amino acids. Multiple sequence alignment and phylogenetic tree analysis results indicated that the protein is a member of the IIb sub-group WRKY family and contains a WRKY conserved domain and a C₂H₂ zinc finger motif. It has the highest similarity to AtWRKY42 in the Arabidopsis family, therefore, named as MsWRKY42. A variety of cis-acting elements were identified in the promoter region of MsWRKY42, including regulatory elements such as stress response, hormone response, and diurnal regulation. Real-Time PCR analysis showed that MsWRKY42 had the highest expression in roots and leaves. The expression of MsWRKY42 was up-regulated by NaCl, PEG, low temperature, high temperature, low phosphorus and ABA treatments. Subcellular localization indicated that MsWRKY42 was mainly located on the nucleus in plant cells. The results of the binding activity analysis showed that MsWRKY42 was able to specifically bind to W-box.【Conclusion】MsWRKY42 is a typical transcription factor, which can specifically bind to cis-acting elements, and the protein is localized in the nucleus. The gene was expressed in different tissues of alfalfa, and was induced by NaCl, PEG, high temperature, low phosphorus and ABA treatment. It is speculated that the MsWRKY42 gene may play a role in response to multiple stress defense responses as a WRKY transcription factor.

Key words: alfalfa, WRKY transcription factor, abiotic stress, subcellular localization, binding activity analysis

LIU JiaoJiao,WANG XueMin,MA Lin,CUI MiaoMiao,CAO XiaoYu,ZHAO Wei. Isolation, Identification, and Response to Abiotic Stress of MsWRKY42 Gene from Medicago sativa L.[J].Scientia Agricultura Sinica, 2020, 53(17): 3455-3466.

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Figures/Tables 10

Table 1

Promoter prediction by PlantCARE"

顺式作用元件 Cis-elements	序列 Sequence (5′-3′)	功能 Function	数量 No.
CGTCA-motif	CGTCA	参与MeJA反应的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness	2
GARE-motif	TCTGTTG	赤霉素反应元件 Gibberellin-responsive element	1
I-box	GTATAAGGCC	部分光响应元件 Part of a light responsive element	1
Box 4	ATTAAT	参与光响应的保守 DNA 模块的部分元件 Part of a conserved DNA module involved in light responsiveness	5
HD-Zip 3	GTAAT(G/C)ATTAC	蛋白质结合位点 Protein binding site	1
circadian	CAAAGATATC	参与昼夜节律顺行调控元件 Cis-acting regulatory element involved in circadian control	1
TCCC-motif	TCTCCCT	部分光响应元件 Part of a light responsive element	1
TGACG-motif	TGACG	参与MeJA反应的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness	2
3-AF3 binding site	CACTATCTAAC	保守DNA模块阵列的一部分（CMA3） Part of a conserved DNA module array (CMA3)	1
TATC-box	TATCCCA	参与赤霉素反应的顺式作用元件 Cis-acting element involved in gibberellin-responsiveness	1
Sp1	GGGCGG	光响应元件 Light responsive element	1
TC-rich repeats	ATTCTCTAAC	参与防御和胁迫反应的顺式作用元件 Cis-acting element involved in defense and stress responsiveness	1
GCN4_motif	TGAGTCA	参与胚乳表达的顺式调控元件 Cis-regulatory element involved in endosperm expression	1
CAAT-box	CAAAT	启动子和增强子区域中常见的顺式作用元件 Common cis-acting element in promoter and enhancer regions	16
G-Box	CACGTG	参与光响应顺式作用元件 Cis-acting regulatory element involved in light responsiveness	5
GATA-motif	GATAGGG	部分光响应元件 Part of a light responsive element	1
O2-site	GATGA(C/T)(A/G)TG(A/G)	参与玉米醇溶蛋白代谢调节的顺式作用调节元件 Cis-acting regulatory element involved in zein metabolism regulation	1
GT1-motif	GGTTAAT	光响应元件 Light responsive element	2
TATA-box	TATA	在转录起始的-30 附近的核心启动子元件 Core promoter element around -30 of transcription start	41
ARE	AAACCA	厌氧诱导必需元件 Cis-acting regulatory element essential for the anaerobic induction	2
GC-motif	CCCCCG	参与缺氧特异性诱导的类似增强子元件 Enhancer-like element involved in anoxic specific inducibility	1
TCA-element	CCATCTTTTT	参与水杨酸响应的顺式作用元件 Cis-acting element involved in salicylic acid responsiveness	1
TCT-motif	TCTTAC	部分光响应元件 Part of a light responsive element	2
ATCT-motif	AATCTAATCC	部分参与光响应的保守DNA区域 Part of a conserved DNA module involved in light responsiveness	2
ABRE	CACGTG	参与脱落酸反应的顺式作用元件 Cis-acting element involved in the abscisic acid responsiveness	5

Table 1

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Isolation, Identification, and Response to Abiotic Stress of MsWRKY42 Gene from Medicago sativa L.

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