Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (22): 4537-4549.doi: 10.3864/j.issn.0578-1752.2020.22.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Drought Resistance Analysis of GhWRKY33 in Upland Cotton

WEI Xin1,WANG HanTao2,WEI HengLing2,FU XiaoKang2,MA Liang2,LU JianHua2,WANG XingFen1(),YU ShuXun2()   

  1. 1 Hebei Agricultural University/State key Laboratory of Cotton Biology Hebei Base, Baoding 071001, Hebei
    2 Institute of Cotton Research, Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology, Anyang 455000, Henan
  • Received:2020-06-10 Accepted:2020-06-28 Online:2020-11-16 Published:2020-11-28
  • Contact: XingFen WANG,ShuXun YU E-mail:cotton@hebau.edu.cn;yu@cricaas.com.cn

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Abstract:

【Objective】In this study, GhWRKY33 gene in upland cotton was cloned, and its function was analyzed in the drought resistance, which will lay a foundation for the mechanism dissection of drought resistance and molecular breeding in cotton.【Method】The ORF of GhWRKY33 was cloned from Upland cotton CCRI10 by homologous cloning method. Bioinformatics analysis was carried out to analyze the secondary structure and hydrophobicity, and to predict the phosphorylation sites and cis-acting elements in the promoter region. In the NCBI website, the protein sequences with high homology were searched by BLASTP for sequence alignment, and the phylogenetic tree was constructed. The expression vector of 35S::GhWRKY33-GFP fusion protein was constructed and injected into tobacco leaves mediated by Agrobacterium tumefaciens to observe the fluorescence signal. qRT-PCR was conducted to detect the tissue-specific expression of the gene and the expression pattern under drought, ABA, JA and ET treatments. Plant overexpression vector was constructed and transformed into Arabidopsis thaliana. T3 transgenic lines were performed to observe the phenotype after drought treatment and to determine the physiological and biochemical indexes such as the content of proline and malondialdehyde. The expression levels of drought response genes AtP5CS, AtRD29A and AtCOR15A were identified in the wild type and transgenic lines before and after PEG treatment. 【Result】The open reading frame (ORF) of GhWRKY33 gene was 1533 bp, which encoded 510 amino acid residues. GhWRKY33 protein included two WRKY conserved domains and two C2H2 zinc finger structures, and belonged to the family of type I WRKY transcription factors. Secondary structure prediction showed that GhWRKY33 was mainly composed of random curl and contained 26 threonine phosphorylation sites, which was closely related to phosphorylation. The hydrophobicity prediction showed that the protein belonged to a hydrophilic protein. Phylogenetic tree analysis displayed that the gene had the highest homology with GrWRKY33. The GhWRKY33 protein was located in the nucleus. qRT-PCR showed that the expression of the gene possessed tissue specificity and was the highest in the apical bud tissue, and significantly increased after drought, JA and ET treatments. Compared to the wild type, the transgenic lines displayed stronger drought resistance, and the wilting degree of the plants was weaker. Under drought treatment, the content of proline was significantly higher than that of wild type, while the content of MDA was lower than that of wild type. The expression levels of GhWRKY33 and drought response genes AtP5CS, AtRD29A and AtCOR15A were significantly increased. It was speculated that PEG could induce the expression of GhWRKY33, and then regulated the up-regulation expression of the drought response genes, thus, transgenic Arabidopsis thaliana showed higher resistance to drought stress. 【Conclusion】GhWRKY33 responded to drought stress and improved the drought resistance of transgenic Arabidopsis thaliana.

Key words: upland cotton, GhWRKY33, stress, drought resistance analysis

Table 1

Primers used in this study"

引物名称 Primer name 引物序列 Primer sequence（5′-3′）
GhWRKY33-F CACGGGGGACTCTAGAATGGCTGCTTCATCATCATCT
GhWRKY33-R GACGGCCAGTGAATTCTCAAGACAGGAATCCGTCCAA
qRT-GhWRKY33-F GGGAAACCCCAATCCAAGGAGC
qRT-GhWRKY33-R AATCATGGGATGCTCGCTCCAC
qRT-GhActin-F ATCCTCCGTCTTGACCTTG
qRT-GhActin-R TGTCCGTCAGGCAACTCAT
GhWRKY33-GFP-F CACGGGGGACTCTAGAATGGCTGCTTCATCATCATCT
GhWRKY33-GFP-R CTTTACTCATACTAGTAGACAGGAATCCGTCCAAAAATG
qRT-AtRD29A-F ATCACTTGGCTCCACTGTTGTTC
qRT-AtRD29A-R AAAACACACATAAACATCCAAAGT
qRT-AtCOR15A-F ACTCAGTTCGTCGTCGTTTCT
qRT-AtCOR15A-R CTTCTTTTCCTTTCTCCTCCAC
qRT-AtP5CS-F GAGGGGGTATGACTGCAAAA
qRT-AtP5CS-R AACAGGAACGCCACCATAAG
qRT-AtUBQ1-F TGAGCCTTCCTTGATGATGCT
qRT-AtUBQ1-R GCACTTGCGGCAAATCATCT

Fig. 1

PCR amplification of GhWRKY33"

Fig. 2

Bioinformatic analysis of GhWRKY33 A: Secondary structure prediction; B: Phosphorylation site prediction; C: Hydrophobicity analysis (positive peak value represents hydrophobicity, negative peak value represents hydrophilicity)"

Table 2

Sequence analysis of GhWRKY33 gene promoter"

基序
Motif		 位置
Position (bp)		 序列
Sequence		 功能
Function
ABRE 1252 ACGTG 参与脱落酸反应的顺式作用元件Cis-acting element involved in the abscisic acid responsiveness
ARE 960 AAACCA 厌氧诱导所必需的顺式作用元件Cis-acting regulatory element essential for the anaerobic induction
1858 AAACCA
CAT-box 1446 GCCACT 与分生组织表达相关的顺式作用元件Cis-acting regulatory element related to meristem expression
G-Box 910 CACGAC 参与光响应的顺式作用元件Cis-acting regulatory element involved in light responsiveness
GCN4_motif 222 TGAGTCA 胚乳表达中的顺式作用元件
Cis-regulatory element involved in endosperm expression
1344 TGAGTCA
497 TGAGTCA
MBS 154 CAACTG 与干旱诱导相关的MYB结合位点MYB binding site involved in drought-inducibility

Fig. 3

Sequence alignment of GhWRKY33 and WRKY proteins of other species The box indicates the WRKY domain, and the star indicates the zinc finger structure. Arabidopsis thaliana AtWRKY33 (NP_181381), Gossypium raimondii GrWRKY33 (XP_012460140), Gossypium arboretum GaWRKY33 (XP_017615740), Theobroma cacao TcWRKY33 (XP_017977471), Herrania umbratica HuWRKY33 (XP_021290063), Durio zibethinus DzWRKY33 (XP_022767833), Corchorus capsularis CcWRKY33 (OMO85052), Carica papaya CpWRKY33 (XP_021908634), Hevea brasiliensis HbWRKY33 (XP_021664002), Populus alba PaWRKY17 (TKS07377), Manihot esculenta MeWRKY33 (XP_021623467), Jatropha curcas JcWRKY33 (XP_012089749), Dimocarpus longan DlWRKY17 (AEO31478), Canarium album CaWRKY33 (AXY96406), Lindera glauca LgWRKY33 (ALE71299), Vigna unguiculata VuWRKY33 (QCE01025), Morella rubra MrWRKY33 (KAB1225722), Vitis vinifera VvWRKY33 (XP_002272040), Glycine max GmWRKY49 (NP_001304523), Glycine max GmWRKY39 (NP_001348302), Gossypium hirsutum GhWRKY33 (XP_016680843). The same as below"

Fig. 4

Phylogenetic analysis of different WRKY proteins"

Fig. 5

Tissue specific expression analysis of GhWRKY33"

Fig. 6

Expression patterns of GhWRKY33 under different treatments * and **: Significant differences and significant differences at 0.05 level and 0.01 level. The same as below"

Fig. 7

Subcellular localization of 35S::GhWRKY33-GFP fusion protein"

Fig. 8

Quantitative expression level of GhWRKY33 gene in transgenic Arabidopsis"

Fig. 9

Growth of wild-type and transgenic Arabidopsis thaliana treated with PEG6000 for 12 h"

Fig. 10

Proline contents of wild-type and transgenic lines under drought treatment for 12 h A: Standard curve; B: The proline contents of wild type and of transgenic lines"

Fig. 11

MDA content of wild-type and transgenic lines under drought treatment for 12 h"

Fig. 12

Expression analyses of the genes related to drought stress in the wild-type and transgenic lines"

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