小麦转录因子基因TaWRKY33的耐盐性分析

doi:10.3864/j.issn.0578-1752.2018.24.001

Abstract

Abstract:

【Objective】 WRKY transcription factors are one of the largest families of transcriptional regulators in plants which functions in the regulation of various physiological programs, including pathogen defense, growth, development and abiotic stresses. Wheat transcription factor TaWRKY33 enhanced drought and heat tolerance in transgenic Arabidopsis. To further investigate its function and stress response mechanism, this article studied its salt tolerance and screened wheat cDNA library to obtain its putative interacting proteins by yeast two-hybrid system. Meanwhile, dual luciferase system was used to detect transcriptional activity of TaWRKY33 transcription factors.【Method】 TaWRKY33 was tested under salt stress using quantitative real-time PCR (qRT-PCR) based on SYBR Green I technology. The coding sequence of TaWRKY33 was cloned into pBI121 driven by CaMV35S promoter. The construct was transformed mediated by Agrobacterium into Arabidopsis plants (Col-0) to obtain transgenic lines. Meanwhile, pWMB110- TaWRKY33 binary vector was used to create over expressed wheat lines. Homozygous T₃seeds of Arabidopsis transgenic lines and T₂ wheat overexpression lines were used for salt tolerance analysis. The wheat cDNA was used as the template for amplifying the TaWRKY33 coding sequence, and the bait plasmid pGBKT7- TaWRKY33 was constructed. We transformed the recombinant plasmid and cDNA library into yeast cell AH109. We screened positive clones via SD/-Trp/-Leu/-His/-Ade and SD/Raf/Gal/X-α-gal plate. Predicted clones were sequenced and analyzed by BLAST. The protoplasts of wheat were prepared, and the reporters and effector plasmids were transformed by transient expression experiments, and the relative fluorescence values were calculated to illustrate transcription activity of transcription factors. 【Result】 qRT-PCR analysis showed that TaWRKY33 was induced by salt. The transient expression experiment of double luciferase showed that TaWRKY33 could activate the luciferase activity in wheat cells. From the perspective of functional analysis, formed longer roots compared with wild type plants, the fresh weight of overexpressing Arabidopsis was significantly different from that of wild type. Importantly, from the perspective of fresh weight, relative electrical conductivity and Na ⁺ content in salt treatment showed that wheat with overexpression of TaWRKY33 had better salt tolerance than control. Through preliminary analysis, the candidate proteins screened by yeast two-hybrid system showed influence on signal transduction and immune process, which demonstrates that TaWRKY33 plays an important role in stress signal transduction and gene transcription regulation in plants.【Conclusion】 Salt-inducible TaWRKY33 improved salt tolerance in transgenic Arabidopsis and wheat and it has potential transcriptional activation activity in cells; TaWRKY33 might function via interacting with a diverse array of protein partners.

Key words: Triticum aestivum, WRKY transcription factor, yeast two-hybrid system, protein interaction, salt tolerance

ZHANG HuiYuan,LIU YongWei,YANG JunFeng,ZHANG ShuangXi,YU TaiFei,CHEN Jun,CHEN Ming,ZHOU YongBin,MA YouZhi,XU ZhaoShi,FU JinDong. Identification and Analysis of Salt Tolerance of Wheat Transcription Factor TaWRKY33 Protein[J].Scientia Agricultura Sinica, 2018, 51(24): 4591-4602.

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Table 1

BLAST analysis and predicted functions of candidate genes"

TaWRKY33的互作蛋白 The proteins interacting with TaWRKY33	GenBank登录号 GenBank accession No.	功能 Functions
烯醇化酶Enolase 乙醇酸氧化酶Glycolate oxidase	KC342469 AAB82143	糖酵解过程Glycolytic process 光呼吸反应中的关键酶,能氧化乙醇酸形成乙醛酸 One of the key enzymes in photorespiration where it oxidizes glycolate to glyoxylate
1,5-二磷酸核酮糖羧化酶/加氧酶小亚基 Chloroplast ribulose-1,5-bisphosphate carboxylase/ oxygenase small subunit (rbcS) gene	KT288199	主要在固定CO₂中对D-核酮糖二磷酸羧化以及对戊糖底物的氧化裂解 To catalyze the primary CO₂ fixation step in the reductive pentose phosphate pathway
叶绿素a/b结合蛋白 Chlorophyll a/b-binding protein	AAT81763	在光合作用中利用光能同化二氧化碳 To utilizie light energy to assimilate carbon dioxide in photosynthesis
ATP合成酶γ亚基ATP synthase gamma subunit	ADC33136	与其他亚基组成ATP合成酶 To form the ATP synthase with other subunits
半胱氨酸蛋白酶抑制剂 Cysteine proteinase inhibitor	AY062608	抑制蛋白氨基酸肽链内切酶的水解作用,维持细胞蛋白的构象 Protect cells from inappropriate endogenous or external proteolysis and may regulate intra-or extracellular protein breakdown
泛素结合酶2E2	XM_003568254	参与蛋白的降解途径和DNA的修复途径 To be involved in the ubiquitin-mediated protein degradation pathway and the DNA repair pathway
电压依赖性阴离子通道VDAC3	X82148	电压门控离子通道活性Vo ltage-gated anion channel activity
铝胁迫基因AIP	ACV44213	受到铝、干旱以及高盐胁迫上调表达 Up-regulated by the imposition of aluminum, high-salt, and drought stress
WD40重复蛋白WD40 尿酸氧化酶Urate oxidase 未知蛋白Unknown protein	NM_124800 DQ365938	控制植物表皮特征,并且能通过与其他蛋白互作来调控下游基因的表达 Controls plant epidermal traits, and the combination and interaction of these regulatory proteins determine the set of downstream genes to be expressed 参与尿酸合成第一步 This protein is involved in step 1 of the subpathway that synthesizes (S)-allantoin from urate 小麦染色体3B基因组片段 Triticum aestivum chromosome 3B, genomic scaffold

Table 1

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Identification and Analysis of Salt Tolerance of Wheat Transcription Factor TaWRKY33 Protein

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