Abstract: 【Objective】 Poncirus polyandra, a relative species of citrus, is a resistant rootstock widely used in grafting. It is also a plant species with extremely small populations in Yunnan. The WRKY family, as one of the largest families of transcription factors in plants, plays a vital role in plant response to abiotic stresses such as drought, cold and salt. However, the current understandings of WRKY genes in P. polyandra are limited. The purpose of this study is to analysis the function of WRKY genes and screen the genes with potential drought resistances in P. polyandra. 【Methods】 Based on the genome-wide data of P. polyandra, WRKY genes wereidentified by HMMER, NCBI-CDD and SMART searches. Comprehensive analyses were systematically performed using bioinformatics methods, including the physicochemical properties, sequence characteristics, phylogenetic relationships, chromosome localization, collinearity, gene structure and cis-acting elements of the PpWRKY family members. In order to verify the drought resistance function of WRKY gene family members, one-year old P. polyandra seedlings were used as experimental materials. In the drought stress experiments, the seedlings were treated with 20% PEG-6000 solution. The leaves were respectively collected after 0 h, 3 h, 6 h, 12 h and 24 h, and stored in the refrigerator at -80 ℃. Real-time fluorescence quantitative analysis was carried out to study the effects of drought stress on the expression patterns of these genes. 【Results】 Amount to 46 WRKY family genes were identified in the genome of P. polyandra and named PpWRKY1-PpWRKY46. The results showed that the length of PpWRKY protein ranged from 116 to 1103 amino acids. Among them, the molecular weight of PpWRKY21 was the lowest (13213.48 kDa), whereas PpWRKY23 had the highest molecular weight (120198.96 kDa). The protein isoelectric points (pI) ranged from 4.93 (PpWRKY24) to 9.8 (PpWRKY17), and 61% of them was lower than 7.0, indicating that most of the proteins were acidic. Subcellular localization results exhibited that PpWRKY23 was located in lysosomes and vacuoles, and other PpWRKY genes were located in the nucleus. Phylogenetic analysis revealed that PpWRKY proteins could be classified into three groups, namely groups I, II and III. Group II had the most members (31), which was further divided into five subgroups (Ⅱa-Ⅱe), containing three, eight, nine, five, and six PpWRKY members separately. WRKY domain multiple sequence alignment analysis demonstrated that only the typical heptaeptides of PpWRKY15 and PpWRKY21 in subgroup Ⅱc had single amino acid variation. Chromosome localization analysis revealed that WRKY family genes were unevenly distributed on nine chromosomes in P. polyandra. The genes clustered on chromosome one were the most, with a total of nine genes. But there was only one gene distributed on chromosome four. We found 24 duplicate gene pairs in the PpWRKY family, including four tandem duplicate gene pairs and 20 fragment duplicate gene pairs in the P. polyandra genome, suggesting that fragment replication was the main driving force for the expansion of PpWRKY gene family members. The Ka/Ks values of replicated WRKY genes in P. polyandra were all less than one, suggesting that these genes might have undergone purification selection. Intergenomic collinearity analysis indicated that there were 88 pairs of collinearity genes between P. polyandra and P. trifoliata. A cis-acting element analysis of the P. polyandra WRKY gene family members identified eight types of cis-elements related to plant hormone and stress responses. Among these, 26 PpWRKY genes contained a drought inducible element (MBS), 37 PpWRKY genes contained an abscisic acid-responsive element (ABRE), and 17 PpWRKY genes contained a low temperature response element (LTR). This suggested that the PpWRKY gene plays an important role in the regulation of hormone and stress in P. polyandra. To mine the drought-resistant WRKY gene in P. polyandra, we analyzed the transcriptomic data of P. trifoliata under drought stress treaments. Reaults revealed that 37 out of the 53 WRKY genes in P. trifoliata were significantly upregulated by drought, indicating that these genes might play an important role in drought resistance. Then, we screened nine homologous drought resistance candidate genes of P. polyandra which have a high homology with P. trifoliata, such as PpWRKY8, PpWRKY12, PpWRKY13, PpWRKY19, PpWRKY23, PpWRKY27, PpWRKY28, PpWRKY30 and PpWRKY41. Their expression patterns under drought stress were detected by real-time fluorescence quantification, and the results revealed that all nine genes were significantly up-regulated under drought stress. Among these genes, the expression of PpWRKY12, PpWRKY19, PpWRKY23, PpWRKY27, PpWRKY28 and PpWRKY30 genes reached the peak after 6h of drought stress, which were 3.63-fold, 2.25-fold, 1.48-fold, 2.63-fold, 3.22-fold, and 2.89-fold of the control, respectively. PpWRKY8 and PpWRKY13 genes exhibited peak expression after 12 h of drought treatment, which were 2.6-fold and 2.9-fold of the control, separately, and the expression of PpWRKY41 was the highest after 24 h of drought treatment, which was 2.92-fold of the control. The results demonstrated that these nine WRKY genes might be involved in the defence response of P. polyandra to drought stress. The results of protein interaction prediction showed that 12 PpWRKY proteins may be involved in regulating stomatal movement through MYB transcription factor mediated ABA signal to affect drought tolerance of plants.【Conclusion】 A total of 46 WRKY genes were identified in P. polyandra. The expression levels of PpWRKY8, PpWRKY12, PpWRKY13, PpWRKY19, PpWRKY23, PpWRKY27, PpWRKY28, PpWRKY30 and PpWRKY41 significantly increased under drought stress, demonstrating that these genes may participate in the process of responses to drought. This study provides new information for analysing the function of WRKY gene and its regulatory mechanism in response to drought stress in P. polyandra, and provides excellent genetic resources for breeding drought-tolerant citrus varieties.
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