一个甘蔗Ⅱd类WRKY转录因子基因的克隆和表达分析

doi:10.3864/j.issn.0578-1752.2018.23.002

Abstract

Abstract:

【Objective】 WRKY, a group of unique transcription factors in plants, plays an important role in plant physiological regulation and stress response. Through analysis of the role of transcription factor WRKY in sugarcane growth and development and stress resistance, this study will provide excellent gene resources for sugarcane resistance molecular breeding. 【Method】 A unigene sequence of WRKY gene was extracted from the sugarcane transcriptome database, and its full-length cDNA sequence was obtained by RT-PCR amplification. Bioinformatics analysis of this gene sequence and its encoded protein sequence was performed using ORF finder, Smart, ExPaSy, Prabi, NetPhos, Cell-PLOC 2.0 and DNAMAN6.0 softwares, and the phylogenetic tree analysis was constructed using MEGA6.0 software. 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 in tobacco leaves. Yeast hybridization assay was used to verify whether WRKY possess transcriptional self-activation activity. The tissue specific expression (root, bud, leaf, stem pith and epidermis) of WRKY and its dynamic expression under MeJA (100 μmol·L ^-1), SA (5 mmol·L ^-1), PEG (25%), NaCl (250 mmol·L ^-1), CuCl₂(500 mmol·L ^-1) and CdCl₂(500 mmol·L ^-1) stresses in sugarcane variety ROC22 were analyzed by quantitative real-time PCR (qRT-PCR) technique.【Result】 A WRKY transcription factor gene, named ScWRKY6 (GenBank Accession Number: MH393927), was cloned from the sugarcane variety ROC22. This gene sequence was 1 289 bp in full length with a 1 059 bp ORF, encoding 352 amino acids, and contained 45 phosphorylation sites. The theoretical isoelectric point, the instability index and the hydrophilicity of ScWRKY6 protein was 9.73, 50.23 and -0.579, respectively, which is supposed to be an alkaline unstable hydrophilic protein. The ScWRKY6 protein has one WRKY domain and one zinc finger motif (CX5CX23HXH), and its amino acid sequence has the highest homology with Sorghum bicolor WRKY (XP_002464211.1). It is speculated that this gene belongs to the Ⅱd sub-group of WRKY family according to phylogenetic tree analysis. Subcellular localization results showed that the ScWRKY6::GFP fusion protein was located in the nucleus. Yeast transcriptional activation verification experiments indicated that ScWRKY6 protein did not have transcriptional auto-activation activity. qRT-PCR analysis revealed that ScWRKY6 was constitutively expressed in sugarcane, and the expression level in order from high to low were in bud, leaf, root, stem epidermis and stem pith. Its expression in bud, leaf and root were 2.05, 1.55 and 1.37 times higher than that in stem pith, respectively. The expression level of ScWRKY6 was up-regulated under the stresses of NaCl, PEG, MeJA, Cu ²⁺ and Cd ²⁺. Its highest expression was 4.18, 6.88, 3.63, 4.86 times higher than of the control when treated with NaCl for 12 h, PEG for 3 h, CuCl₂ for 24 h and CdCl₂ for 24 h, respectively.【Conclusion】 ScWRKY6 protein was located in the nucleus and did not have transcriptional auto-activation activity. The gene was expressed in different sugarcane tissues and was induced by NaCl, PEG, CuCl₂ and CdCl₂treatments. It is presumed that the ScWRKY6 may play a role in response to drought stress, salt tolerance and metal ion stress in sugarcane.

Key words: sugarcane, WRKY transcription factor, exogenous stress, qRT-PCR

ZHANG Xu,LING Hui,LIU Feng,HUANG Ning,WANG Ling,MAO HuaYing,LI CongNa,TANG HanChen,SU WeiHua,SU YaChun,QUE YouXiong. Cloning and Expression Analysis of a Ⅱd Sub-Group WRKY Transcription Factor Gene from Sugarcane[J].Scientia Agricultura Sinica, 2018, 51(23): 4409-4423.

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引物名称Primer name	引物序列Primer sequence (5°-3°)	用途Usage
ScWRKY6	F: GCTGCTGAGGGAGTTGCATA	扩增全长 Full length amplification
ScWRKY6	R: AAGCAGGGCCAAACATCTCA	扩增全长 Full length amplification
GFP-ScWRKY6	F: GGGGTACCATGGAGGAAGTGGAGGAGGC	亚细胞定位 Subcellular localization
GFP-ScWRKY6	R: GCTCTAGACACCTGTGCTGACTGAGTTGGC	亚细胞定位 Subcellular localization
BD-ScWRKY6	F: CATGCCATGGAGATGGAGGAAGTGGAGGAGGC	转录激活 Transactivation
BD-ScWRKY6	R: CGCGGATCCCACCTGTGCTGACTGAGTTGGC	转录激活 Transactivation
qScWRKY6	F: GCGAGGTCCTTCTTGTCGTC	实时荧光定量PCR qRT-PCR
qScWRKY6	R: CGTTCACCGGATCGCTCATT	实时荧光定量PCR qRT-PCR
CAC	F: ACAACGTCAGGCAAAGCAAA	内参基因 Actin gene
CAC	R: AGATCAACTCCACCTCTGCG	内参基因 Actin gene
CUL	F: TGCTGAATGTGTTGAGCAGC	内参基因 Actin gene
CUL	R: TTGTCGCGCTCCAAGTAGTC	内参基因 Actin gene

Cloning and Expression Analysis of a Ⅱd Sub-Group WRKY Transcription Factor Gene from Sugarcane

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