Genome-wide Prediction Of Transcription Factor Families And Analysis Of ARF Family On Melon

Melon (Cucumis melo. L) belonging to Cucurbitaceae family, is one of the major horticultural crops in the world and has very important economic value. Transcription factors participate in many important regulatory processes and their functional elucidations currently are one of the important contents in functional genomics researches. Melon genome has been sequenced and the relative information has been publically released in2012, providing a good opportunity for the studies of genome-wide forecasting and characteristics analysis of transcription factor families on melon. In this study, melon transcription factors families were predicted on genome-wide scale and the reference genes were systematically validated for normalization in the expression level quantification analyses of those transcription factors by RT-qPCR technique. At last, the characteristics of ARF family genes and their expression profiling during melon fruit development were analyzed. The results obtained in the study are listed as the followings:1. By employment of46transcription factor family HMMs download from the Pfam database and10selfbuilt HMMs, a total56transcription factor families including1558genes were identified on melon whole genome with HMMER algorithm. When being ranked in size, the top ten transcription factor families are bHLH (133), ERF (126), bZIP (116), MYB_related (107), C2H2(94), NAC (82), MYB (76), G2-like (65), WRKY (60), and NF-YC(52), respectively.2. A total of15ARF transcription factors were identified on melon genome and their characteristics were further analyzed. The number of introns in ARF genes ranged from2to21and most of the ARF genes contains10to20introns. Three alternative splicing models were observed in those genes. The conserved DNA binding domain was observed in the N terminal of the proteins coded by ARF family genes, which could be further subdivided into three subgroups based on their protein sequence characteristics.3. A total of14reference genes currently used on melon or validated on other crops were selected candidates, which included ACT7, GAPC2, RAN, RPL, RPL2, ADP, TUA, EFla, RPL36aA, PP2A, UBC2, ACT,18SrRNA, and UBI-ep. Expression levels of the candidate reference genes were determined by RT-qPCR under the biological stresses (including wilt, root rot, and bacterial fruit blotch), abiotic stresses (including drought, cold, and salt stress), plant growth regulator treatments (including GA, NAA, ABA, SA, MeJA, and ethylene), and fruits at different developmental stages. Meanwhile, their expression stabilities were evaluated using GeNorm, NormFiner and BestKeeper softwares. geNorm ranked ADP and UBI-ep in all samples, ADP and RAN in root, GAPC2and RAN in leaf, TUA and ACT under biotic stresses, ADP and RPL under abiotic stresses, RAN and ACT in the leaf under plant growth regulator treatments, RAN and PP2A in fruit development, as the most stable reference genes, respectively. Except for RPL in root and RAN in fruit, ADP was recognized as the most stable reference gene under the remaining experimental conditions by Normfinder software.In BestKeeper analysis, candidate reference gene18SrRNA, RPL and RAN are stable.4. The expression profiling of ARF family genes were analyzed during melon fruit development (0day,7days,14days,21days,27days, and31days after anthesis, respectively) under artificial pollination and CPPU treatments by RT-qPCR using RAN as the reference gene. The expression levels of MELO3C011372, MELO3C014232, MELO3CO19801, and MELO3C025777, were significantly upregulated in fruit development. However, downregulations were observed on the genes of MELO3C002771, MELO3C003768, MELO3C005761, MELO3C025070, and MELO026161during fruit development. Different expression patterns of MELO3C025758, MELO3C007251, MELO3C007104, and MELO3C03938were observed under artifical pollination and CPPU treatments, respecitvly. MELO3C025758and MELO3C007251had a higher expression levels in development of artifically poUinitated fruit than that in the fruit under CPPU treatment. However, MELO3C007104and MELO3C03938exhibited a contradictory expression parttern with MELO3C025758and MELO3C007251, indicating that MELO3C007104and MELO3C03938probably participated in the response to CPPU treatment and regulation of fruit parthenocarpy.