Cloning of WRKY transcription factors and their expression analysis under low temperature stress in Canarium album (Lour.) Raeusch.

Abstract: 【Objective】Canarium album (Lour.) Raeusch. is one of the characteristic and precious fruit in tropical and subtropical regions of China. Its fruit is highly nutritional and healthy for the people.Generally, C. album is poor in cold resistance, and often suffers from low-temperature damage, which greatly limites the development of the species. Recently, accumulated evidences have revealed the important regulatory roles of WRKY transcription factors (WRKY TFs) in cold stress response of many plants. In the present study, four members of CaWRKY TF family were cloned, and were then subjected to series of bioinformatics analysis in order to investigate the regulatory functions of WRKY TFs in the growth and cold stress response of C. album. Their expressions in different organs were investigated using quantitative real time PCR (qRT-PCR) . Fourthermore, their expression patterns together with the total antioxidant capacity (T-AOC) changes in leaves under different low temperature treatments were also studied.【Methods】For low temperature treatment, four-month-old young seedlings of C. album 'Fulan-1'were treated at-3 ℃, 4 ℃ and 25 ℃, for 24 h, respectively representing treatments of cold dam-age, chilling injury and control. T-AOC in leaves under different low temperature treatments was measured using a T-AOC detection kit. For the expression analysis of the four CaWRKY TFs in roots, stems, leave, flowers, fruits and sees, samples were collected from a mature C. album'Fulan-1'tree.EZNATM Plant RNA Kit was used for the extraction of total RNA from leaves and different organs of young seedlings treated with low temperature.cDNA for PCR amplification and qRT-PCR were respectively synthesized using Thermo Scientific RevertAid First Strand cDNA Synthesis Kit and TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR. Based on our transcriptome data, four possible CaWRKY TFs sequences were selected, and primers for PCR amplification and c DNA sequencing were designed according to their sequences. Tthe bioinformatic characteristics, codon bias of the four cloned CaWRKY TFs were analyzed using online softwares. Subsequently, TransStart®Top Green qPCR SuperMix Kit was used to profile expression patterns of these CaWRKY TFs in different organs and low temperature treated leaf samples by using ACTB7 and TUB5 as reference genes.【Results】The open reading frames (ORF) of CaWRKY21, 33, 50 and 55 were 561, 1 644, 357 and 1 044 bp, encoding186, 547, 118 and 347 amino acids respectively (The GenBank accession numbers were MG356652, MG356653, MG356654 and MG356655) . Bioinformatics analysis results showed that CaWRKY21, 33, 50, 55 were all unstable hydrophobic proteins, but their molecular weight, atomic number, isoelectric point, instability and fat solubility differed a lot. Besides, CaWRKY21 belonged to basic protein, which was distinct from others. All the four CaWRKY members contained conserved WRKY domain, but the domain types and members varied. CaWRKY21 and 55 contained one WRKYGQK domain and one C2 H2 zinc finger structure (CX5 CX23 HXH and CX7 CX23 HX2 H) , belonging to plant class Ⅱ WRKY TFs.CaWRKY33 contained two typical WRKYGQK domains and one C2 H2 zinc finger structure (CX4 CX23 HXH) , belonging to plant class I WRKY TFs. CaWRKY50, however, contained one WRKYGKK variable domain and variant zinc finger structure, and belonged to plant class III WRKY TFs. Subcellular localization prediction result revealed that CaWRKY21 and CaWRKY55 might locate in the nucleus, while CaWRKY33 and CaWRKY50 were located in endoplasmic reticulum membrane and cytoplasm respectively. Cluster analysis result showed that the two class II WRKY TFs, CaWRKY21 and CaWRKY55 shared high similarity, and CaWRKY33 and CaWRKY50 had closer relationship. Besides, very high WRKY TFs homologies were found among C. album and Carica papaya, Citrus sinensis and Eucalyptus grandis. The codon bias analysis revealed that the effective number of codons (ENc) in CaWRKY21, 33, 50 and 55 were 45.78, 50.04, 52.19 and 54.41 respectively, which suggested that the codon bias levels of CaWRKY TFs would be low. Additionally, both GC and GC3 s value were less than0.5, indicating that they were biased toward the synonymous codons with A or T on the 3 rdsite. qRTPCR analysis showed that CaWRKY21, 33, 50 and 55 presented organ-specific expression patterns in C.album. CaWRKY21 showed high expression in the fruits and seeds, while CaWRKY33 and 55 were mainly expressed in the leaves, and the expression of CaWRKY50 in the stems was the highest. Under low temperature treatment, T-AOC increased gradually with the decrease of temperature and peaked at -3 ℃ (Significantly differences of T-AOC were found among the treatments at p-value < 0.01) . Consistently, the expression levels of CaWRKY21, 33, 50 and 55 were also up-regulated greatly during this process, indicating that CaWRKY21, 33, 50 and 55 might function in low temperature response of C. album.【Conclusion】The treatment of chilling injury at 4 ℃ might not reach the critical temperature of C.album, but when treated in freezing injury temperature at-3 ℃, the antioxidant capacity of C. album increased rapidly and its cold resistance system responded quickly. During this process, CaWRKY21, 33, 50, 55, which belonged to plant WRKY TF gene family, responded to low temperature stress observably.In addition, CaWRKY21, 33, 50, 55 were probably involved in the organ development of C.album.Our study could provide a basis for the future research and application of WRKY TF family genes of C.album.