Isolation and expression analysis of Citrus transcription factor CitMYB40

Abstract: 【Objective】The MYB transcription factors are widespread in plants. According to the numberof repeats in the MYB binding domain, they can be divided into four categories: R1-MYB, R2R3MYB,R1R2R3-MYB and 4R-MYB. Of these, R2R3MYB is the largest subfamily. Previous studies found thatthe R2R3MYB transcription factors were widely involved in the regulation of plant physiological and biochemical processes, including secondary metabolic reaction, pollen development and tube growth, cellmorphogenesis and stress responses. CitMYB40 protein sequence contains two repeats in the MYB bind⁃ing domain (R₂ and R₃), which belongs to the R2R3-MYB subfamily. In this study, we predict the biological function of CitMYB40 in order to lay a foundation for the genetic improvement of citrus resistance tostresses.【Methods】Based on the genomic sequences of citrus, one R2R3-MYB transcription factor Cit⁃MYB40 was analyzed in Citrus junos‘Ziyang’. We obtained seeds from mature fruits that were sown inMS Solid medium to germinate at 28 ℃ under a 16/8 h light/dark photoperiod. When the seedlings were atthe two-true-leaf stage, we cleaned the seedlings and put them in sealed bottles. Seven treatments wereimplemented, respectively: 100 μmol·L^-1 abscisic acid (ABA), 20 mg·L^-1 ethylene-responsive (ACC), 2mg·L^-1 salicylic acid (SA), 100 mg·L^-1 jasmonic acid (MeJA), 250 μmol·L^-1 NaCl, 10% PEG6000 and4 ℃ low temperature. Roots and leaves were harvested at 0, 2, 4, 8, 12 and 24 h after treatments in orderto extract the RNA. In addition, we collected the roots, leaves, flowers and fruitlets of the‘Ziyang’for tissue specific expression analysis. All the samples were stored at low temperature in a refrigerator (-80 ℃ ).We used the Plant Genomic DNA Kit to extract DNA from‘Ziyang’and used the RNA prep plant Kit (TIANGEN) to isolate the total RNA from the processed materials. The first strand cdna was synthesizedby using the Prime ScriptTM RT reagent Kit. All the above operation processes were carried out accordingto the kit instructions. The CDS and the corresponding genomic sequence of CitMYB40 were obtainedfrom the phytozome database. We used the GSDS (http://gsds.cbi.pku.edu.cn/) tool to analyze the genestructure; ExPASy-ProtParam (http://au.expasy.org/tools/protparam.html) to analyze the coding protein relative molecular mass, isoelectric point and other physical and chemical properties; Wolfpsort (http://wolf⁃psort.seq.cbrc.jp) to predict protein subcellular localization; plantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to analyze promoter elements; Conserved Domains (http://www.ncbi.nlm.nih.gov/cdd) to conserve protein motifs; DNAMAN to analyze CitMYB40 amino acid sequence homology;CLUSTALX software to perform the multiple sequence alignments; MEGA 6.0 to construct the Phylogenetic tree by the neighbor joining method (neighbor-joining, NJ); and qRT-PCR to analyze the expression ofCitMYB40 under various plant hormone and abiotic stresses. The relative expression levels of CitMYB40were calculated by the 2^-△△Ct (ΔCt=CtCitMYB40 - CtCitActin) method. The data obtained was statistically analyzedby using Microsoft Office Excel 2007 and the one-way ANOVA was calculated by using spass17.0.【Results】The sequence analysis showed that CitMYB40 contained four exons and three introns. It’s openreading frame (ORF) was 984 bp through encoding a protein with 297 amino acid residues, and it’s upstream 2 000 bp contained a plurality of hormones and osmotic stress elements, such as abscisic acid responsive elements (abres), jasmonic acid responsive element (CGTCA-motif), ethylene responsive element (ERE), salicylic acid responsive element (TCA) and was rich in TC repeats of the stress responsiveelement (TC rich repeats). The sequence alignment showed that CitMYB40 shared 45.57%-53.51% amino acid identities with the homology of amino acids sequences from Vitis vinifera, Populus euphratica, Jatropha curcas, Prunus mume and Solanum tuberosum. The phylogenetic tree with CitMYB40 and Arabidopsis thaliana R2R3-MYB proteins was constructed with the results showing that CitMYB40 was groupedwith the Arabidopsis AtMYB41and AtMYB49 proteins. Real time quantitative analysis revealed that theexpression of CitMYB40 in different tissues exhibited different patterns; the expression level of CitMYB40 in flower was higher than that in the root, leaf and stem. Under plant hormone stress, CitMYB40was up-regulated by ABA，ACC，SA and MeJA. Moreover, we found that the expression in the leaf washigher than in the root. Under the various abiotic stresses, similar expression patterns were shown in theroot by PEG6000 and NaCl treatment, which were first increased and then decreased. The expression patterns observed in the leaves exhibited two peaks. Under the 4 ℃ low temperature, the expression patternwas first increased and then decreased in the root and leaf.【Conclusion】The expression of CitMYB40 existed in the tissue specificity, and CitMYB40 could be induced by plant hormones and abiotic stresses.All the results suggest that CitMYB40 could play an important role in the growth processes and stress responses.