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

中国农业科学 ›› 2018, Vol. 51 ›› Issue (24): 4591-4602.doi: 10.3864/j.issn.0578-1752.2018.24.001

• 作物遗传育种·种质资源·分子遗传学 • 下一篇

小麦转录因子基因TaWRKY33的耐盐性分析

张惠媛¹(),刘永伟²(),杨军峰³,张双喜⁴,于太飞¹,陈隽¹,陈明¹,周永斌¹,马有志¹,徐兆师¹(),付金东¹()

1 中国农业科学院作物科学研究所/国家农作物基因资源与基因改良重大科学工程/农业部麦类生物学与遗传育种重点实验室,北京 100081
2 河北省农林科学院遗传生理研究所/河北省植物转基因中心,石家庄 050051
3 河北旺丰种业有限公司,河北邢台 054900
4 宁夏农林科学院农作物研究所,宁夏永宁 750105

收稿日期:2018-06-27 接受日期:2018-09-12 出版日期:2018-12-16 发布日期:2018-12-16
基金资助:
转基因生物新品种培育重大专项(2018ZX0800909B);宁夏自然科学基金(NZ17126);河北省现代农业科技创新工程项目(494-0402- JBN-C7GQ)

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

ZHANG HuiYuan¹(),LIU YongWei²(),YANG JunFeng³,ZHANG ShuangXi⁴,YU TaiFei¹,CHEN Jun¹,CHEN Ming¹,ZHOU YongBin¹,MA YouZhi¹,XU ZhaoShi¹(),FU JinDong¹()

1 Institute of Crop Science, Chinese Academy of Agricultural Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement/Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture, Beijing 100081
2 Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences/Plant Genetic Engineering Center of Hebei Province, Shijiazhuang 050051
3 Hebei Wangfeng Seed Industry Co., Ltd. Xingtai 054900, Hebei
4 Institute of Crop Science, Ningxia Academy of Agriculture and Forestry Sciences, Yongning 750105, Ningxia

Received:2018-06-27 Accepted:2018-09-12 Online:2018-12-16 Published:2018-12-16

摘要/Abstract

摘要：

目的 WRKY转录因子是植物转录调节因子家族之一,参与调控植物病原菌的防御、生长发育和抗逆应答等多种生理过程。小麦WRKY转录因子基因TaWRKY33可以提高转基因拟南芥对干旱和高温的抗性。通过分析TaWRKY33的耐盐性,检测TaWRKY33转录因子的转录活性,深入分析转录因子基因TaWRKY33的功能,以解析其耐盐调控机制。方法以盐胁迫处理的小麦cDNA为模板,利用实时荧光定量PCR检测TaWRKY33在盐胁迫条件下的表达模式;将TaWRKY33的CDS序列插入带有CaMV35S启动子的pCAMBIA1302表达载体中,构建表达载体pCAMBIA1302- TaWRKY33,转入农杆菌,侵染野生型拟南芥获得转基因株系;同时利用pWMB110-TaWRKY33双元载体创建了过表达小麦株系。用转TaWRKY33的拟南芥和小麦进行耐盐性鉴定。构建诱饵载体pGBKT7-TaWRKY33,通过单转法将诱饵载体pGBKT7-TaWRKY33重组质粒和文库质粒逐步转化到酵母AH109感受态细胞。通过SD/-Trp/-Leu/-His/-Ade和X-α-gal显蓝反应筛选得到阳性克隆,进行测序和BLAST分析。制备小麦原生质体,通过瞬时表达试验共转化报告子和效应子质粒,计算相对荧光值分析转录因子的转录活性。结果实时荧光定量PCR结果显示,TaWRKY33受盐胁迫的诱导表达。双荧光素酶瞬时表达试验表明TaWRKY33在小麦细胞中可以激活相应荧光素酶的活性。从功能分析,在正常生长条件下转基因拟南芥和野生型拟南芥没有明显差异;在盐处理条件下,转基因拟南芥的根长大于野生型拟南芥的根长;过表达拟南芥的鲜重大于野生型,呈显著性差异。重要的是,在盐处理下,鲜重、相对电导率和Na ⁺含量表明,过表达TaWRKY33的小麦较其受体对照有更好的耐盐性。对TaWRKY33候选互作蛋白分析表明,这些候选互作蛋白主要参与植物体的信号转导或免疫过程,表明TaWRKY33在植物的逆境信号转导、基因转录调控过程中发挥着重要作用。结论小麦TaWRKY33受盐胁迫的诱导表达,具有潜在的转录激活活性,提高了转基因拟南芥和小麦的耐盐性;TaWRKY33可能需要其他蛋白共同作用提高小麦耐盐性。

关键词: 小麦, WRKY转录因子, 酵母双杂, 蛋白互作, 耐盐性

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

张惠媛,刘永伟,杨军峰,张双喜,于太飞,陈隽,陈明,周永斌,马有志,徐兆师,付金东. 小麦转录因子基因TaWRKY33的耐盐性分析[J]. 中国农业科学, 2018, 51(24): 4591-4602.

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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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2018.24.001

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候选基因的BLAST分析结果及其推测的功能"

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

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小麦转录因子基因TaWRKY33的耐盐性分析

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

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