苦荞麦<i>FtWRKY6</i>基因的克隆及其在低磷与激素诱导下根中的表达分析

doi:10.7668/hbnxb.20193338

华北农学报 ›› 2023, Vol. 38 ›› Issue (1): 32-39. doi: 10.7668/hbnxb.20193338

所属专题： 杂粮作物；土壤肥料；生物技术；热点文章；

• 作物遗传育种·种质资源·生物技术 • 上一篇下一篇

苦荞麦FtWRKY6基因的克隆及其在低磷与激素诱导下根中的表达分析

田建红¹, 彭喜旭¹^,², 邬清韬¹, 文碧瑶¹, 邓楚楚¹, 唐新科¹^,²^,, 王海华¹^,²^,³^,

¹ 湖南科技大学生命科学与健康学院,湖南湘潭 411201
² 经济作物遗传改良与综合利用湖南省重点实验室,湖南湘潭 411201
³ 重金属污染生态土壤修复与安全利用湖南省高校重点实验室,湖南湘潭 411201

收稿日期:2022-07-29 出版日期:2023-02-28
通讯作者:
唐新科(1971—),男,湖南湘潭人,副教授,博士,硕士生导师,主要从事荞麦种质资源与分子生物学研究。
王海华(1969—),男,湖南湘潭人,教授,博士,硕士生导师,主要从事作物抗性生理与分子生物学研究。
作者简介:
田建红(1995—),男,甘肃平凉人,在读硕士,主要从事作物抗性生理与分子生物学研究。
基金资助:
科技部国家重点研发计划(2017YFE0117600); 湖南省教育厅重点科研项目(19A176); 湖南省研究生科研创新项目(CX20211005)

The Cloning and Expression Analysis of FtWRKY6 Gene in Roots of Fagopyrum tataricum Treated by Low Phosphorus and Hormones

TIAN Jianhong¹, PENG Xixu¹^,², WU Qingtao¹, WEN Biyao¹, DENG Chuchu¹, TANG Xinke¹^,²^,, WANG Haihua¹^,²^,³^,

¹ School of Life and Health Sciences,Hunan University of Science and Technology,Xiangtan 411201,China
² Key Laboratory of Genetic Improvement and Multiple Utilization of Economic Crops in Hunan Province,Xiangtan 411201,China
³ Key Laboratory of Ecological Remediation and Safe Utilization of Heavy Metal-polluted Soils,College of Hunan Province,Xiangtan 411201,China

Received:2022-07-29 Published:2023-02-28

摘要/Abstract

摘要：

WRKY转录因子是植物非生物胁迫反应中的重要调节子。荞麦耐贫瘠,磷利用率较高。为了探究WRKY基因在荞麦磷饥饿反应中可能的调节作用,以苦荞麦为材料,采用逆转录PCR方法,从低磷处理的根RNA样品中获得了FtWRKY6基因的编码序列。FtWRKY6 cDNA长1 572 bp,编码524个氨基酸,含有2个典型的WRKY保守结构域,锌指类型为C₂H₂,归属于第Ⅰ类WRKY,与绿茶CsWRKY24在氨基酸水平上的同一性较高(55.5%)。拟南芥原生质体瞬时表达分析表明,FtWRKY6定位于细胞核中;酵母单杂交试验表明,FtWRKY6具有转录激活活性。实时荧光定量PCR分析表明,在根中FtWRKY6的表达受低磷和3种重要的低磷反应相关激素—吲哚乙酸(IAA)、赤霉素(GA)和细胞分裂素(CTK)显著诱导。上述结果提示,FtWRKY6具有转录因子的基本结构与生化特征,在根中可能通过IAA、GA、CTK信号通路的交互作用介导苦荞麦在低磷条件下的响应过程。

关键词: 苦荞麦, 低磷胁迫, WRKY基因, 基因表达, 转录激活活性, 亚细胞定位

Abstract:

WRKY transcription factors act important regulators in plant response to low phosphorus.Buckwheat performs well in under-fertilized soils with higher phosphorus use efficiency.Taking tartary buckwheat as experimental materials,this study aims to explore the possible regulatory roles of WRKY genes in phosphorus starvation response of buckwheat.The entire coding sequence(CDS)of FtWRKY6 gene was cloned from RNA samples generated from roots treated by low phosphorus using reverse transcription PCR.The obtained CDS of FtWRKY6 was 1 572 bp in length,encoded a polypeptide of 524 amino acid residues which consists of two conserved WRKY domain each with a zinc finger motif of CCHH,and belonged to the WRKY group Ⅰ.FtWRKY6 shared the highest identity(55.5%)at the amino acid level with Camellia sinensis CsWRKY24.Transient expression assay in protoplasts showed that FtWRKY6 protein was localized in nucleus.Yeast one-hybrid assay revealed that FtWRKY6 had transcription-activating activity.Real-time fluorescence quantitative PCR analysis showed that the expression of FtWRKY6 in roots was significantly induced by low phosphorus and three related hormones such as indole acetic acid(IAA),gibberellin(GA)and cytokinin(CTK).Taken together,FtWRKY6 possesses basic structural and biochemical characteristics as a putative transcription factor,and may be involved in low phosphorus response in roots possibly by crosstalk of IAA,GA and CTK signaling pathways.

Key words: Tartary buckwheat, Low phosphorus, WRKY gene, Gene expression, Transcription-activating activity, Subcellular localization

田建红, 彭喜旭, 邬清韬, 文碧瑶, 邓楚楚, 唐新科, 王海华. 苦荞麦FtWRKY6基因的克隆及其在低磷与激素诱导下根中的表达分析[J]. 华北农学报, 2023, 38(1): 32-39. doi: 10.7668/hbnxb.20193338.

TIAN Jianhong, PENG Xixu, WU Qingtao, WEN Biyao, DENG Chuchu, TANG Xinke, WANG Haihua. The Cloning and Expression Analysis of FtWRKY6 Gene in Roots of Fagopyrum tataricum Treated by Low Phosphorus and Hormones[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(1): 32-39. doi: 10.7668/hbnxb.20193338.

http://www.hbnxb.net/CN/Y2023/V38/I1/32

图/表 8

表1 本研究所用引物

Tab.1 Primers used in this study

基因(登录号) Gene (Accession number)	正向引物序列(5'—3') Forward primer sequence	反向引物序列(5'—3') Reverse primer sequence	用途 Use
FtWRKY6	ATGAATTTCACCTTCACATCCGA	GTAGAGCAATGACTCGAAGAACAAGT	cDNA克隆
(FtPinG0001577500.01)	CGCCTGCAGATGAATTTCACCTTCACATCCGA^*	GGAATTCCATATGGTAGAGCAATGACTCGAAGAACAAGT^**	构建转录激活载体
	caccATGGAGATCAATAATTATAGCC	AAAAGATCCCTTCTGCTCATAG
	TGATGGACCTCCTGATGATGG	AGACTTGGACCGACGAACC	构建亚细胞定位载体
FtHiston 3	GAAATTCGCAAGTACCAGAAGAG	CCAACAAGGTATGCCTCAGC	qPCR内参基因
(HM628903.1)

图1 FtWRKY6 编码序列PCR产物的电泳检测 Marker.DNA分子质量标准;1,2.PCR扩增产物。

Fig.1 Electrophoresis detection for PCR products of FtWRKY6 coding sequence Marker.DNA Molecular size standard;1,2.PCR amplification products.

图2 FtWRKY6基因及其编码蛋白的结构 A.FtWRKY6基因的结构:橙色实框表示外显子,黑线表示内含子,灰色实框表示5'非翻译区;预测该基因无3'非翻译区。B.FtWRKY6蛋白结构:^*.FtWRKY6 C端酸性区中密集的磷酸化位点。

Fig.2 Structures of FtWRKY6 gene and its deduced protein A.Structure of FtWRKY6 gene:Orange boxes,black lines and gray boxes indicate exons,introns,and 5' untranslated regions;this gene has no predicted 3' untranslated region.B.The structure of FtWRKY6 protein:^*.Dense phosphorylation sites in the C-terminal acidic region of FtWRKY6。

表2 FtWRKY6启动子顺式元件预测

Tab.2 Prediction of cis-elements in the promoter of FtWRKY6

顺式作用元件 cis-acting element	一致序列 Consensus sequence	功能 Function	数量 Number
GT1CONSENSUS	GRWAAW	光响应元件	4
POLLEN1LELAT52	AGAAA	花粉特异性表达元件	8
CACTFTPPCA1	YACT	叶肉特异性表达元件	19
DOFCOREZM	AAAG	茎特异性表达元件	15
RAV1AAT	CAACA	根特异性表达元件	8
GARE	TCTGTTG	GA响应元件	4
NTBBF1ARROLB	ACTTTA	IAA响应元件	2
ARR1AT	NGATT	CTK响应元件	14
Helix-loop-helix	CANNTG		4
ABRELATERD1	ACGTG	低磷响应元件	1
W-box	TGACC/T		7

图3 FtWRKY6与不同物种中同源蛋白的系统进化树分支上标注的数字为Bootstrap值,仅显示≥50%的数字。本研究的FtWRKY6用阴影凸显。蛋白登录号来源于NCBI(http://ncbi.nlm.nih.gov)。

Fig.3 Phylogenetic tree of FtWRKY6 and its homologous proteins from different species The numbers marked on the branches are Bootstrap values,of which only≥50% were shown.FtWRKY6 in this study is highlighted by shading.Protein accession numbers were retrieved from NCBI(http://ncbi.nlm.nih.gov).

图4 FtWRKY6的亚细胞定位

Fig.4 Subcellular localization of FtWRKY6

图5 FtWRKY6的转录激活分析

Fig.5 Analysis of transcription-activating activity of FtWRKY6

图6 FtWRKY6在低磷(LP)、IAA、GA₃和CTK处理下的差异表达分析数据以3次生物学重复试验的平均值±标准差表示,不同小写字母表示在0.05水平上有显著差异(n=6;邓肯多重范围检验)。

Fig.6 Analysis for differential expression of FtWRKY6 after the treatment of low phosphorus(LP),IAA,GA₃ and CTK Data are presented as the mean±standard deviation of three biological replicates,different letters indicate significant difference at P<0.05(n=6;Duncan multiple-range test).

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苦荞麦FtWRKY6基因的克隆及其在低磷与激素诱导下根中的表达分析

The Cloning and Expression Analysis of FtWRKY6 Gene in Roots of Fagopyrum tataricum Treated by Low Phosphorus and Hormones

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