Cis-Regulatory Elements Analysis And Interacting Protein Screening Of CsGABP Promoter In Citrus

Organic acid is one of the most important components which are contributed to the fruit quality. Moreover, dynamic changes of organic acid is closely related to the senescence process and storage performance. A high concentration of organic acid is quite important for keeping fresh of citrus fruit. Therefore, developing a way to maintain high level of organic acid and/or to slow down degradation process of organic acid is necessary to retard senescence process and keep quality of stored citrus fruit, and is valuable for postharvest citrus research. Citric acid is the main organic acid in citrus fruit. The content of citric acid showed a decline trend with fluctuations during postharvest storage. GABA shows closely link with TCA cycle, in which the main citric acids were synthesized, and is participated in citric acid catabolism during postharvest in citrus fruit. GABP(mitochondrial GABA permease) transfer the GABA from cytosol into mitochondrion,and then closely link the GABA shunt and TCA cycle, it can be therefore as a key point for studying the transcriptional regulation of citric acid metabolism during storage in citrus fruit. Exploring the upstream regulatory factors and regulatory mechanism of GABP on citric acid metabolism would be the theoretical basis for developing techniques to control the concentration of organic acid and redard senescence process of postharvest citrus fruits. In present study, we cloned the mitochondrial GABP gene(CsGABP) from citrus and analyzed the cis-regulatory elements in the promoter. The candidate TFs which might be upstream regulatory factors of CsGABP, were obtained by screening the transcriptome data and vrified by Y1 H experiements. The regulatory mechanisms of these candidate TFs were discussed. Main results were following:1. The full length gDNA and corresponding promoter sequences of mitochondria GABP(CsGABP) were cloned from Hirado Butun Pumelo and acidless pumelo, and the ORF region of CsGABP was respectively cloned from five citrus varieties including Hirado Butun Pumelo, Acidless pumelo, Bingtang orange, Jin orange and Shatian Pumelo.The results showed that Hirado Butun and Acidless pumelo contain two different CsGABP gene sequences, the former one is CsGABP-a, CsGABP-b and the later CsGABP-a, CsGABP-c. The similarity of gDNA and promoter sequences were up to 99%and 98%, respectively. CsGABP-a has a consensus sequence in both varieties. On contrary, the sequences of ORF region in five varieties is high conservative, suggesting only CsGABP-a was perhaps transcripted.2. CsGABP-a promoter sequences were predicted to be with several cis-regulatory elements: light response elements(G-box, GT1-motif, TCT-motif, etc.), stress responsive elements(G-box, W-box, GCC-box, TGACG-motif), hormone responsive elements Gbox and ABRE(ABA responsiveness), GCC-box(ethylene responsiveness), TGACGmotif and CGTCA-motif(MeJA-responsiveness), GARE-motif(gibberellin responsiveness)and TCA-element(salicylic acid responsiveness). The presence of abovementioned regulatory elements indicated that CsGABP-a gene expression may be induced by light, stress and hormone.3. Total of 23 transcriptional factors were confirmed to co-expression with CsGABP gene based on transcriptome data analysis. Five types, a total of nine candidate transcription factors(CsWRKY40, CsWRKY33, CsbHLH, CsERF72, CsZF-B, CsBZIP11,CsABR1, CsBZIP21, CsZF-A2) were significantly correlated with CsGABP gene expression through the qRT-PCR verification and correlation analysis, and were selected for further research.4. The bHLHs, WRKYs, AP2/EREBPs and bZIPs were probably bind to G-box, Wbox, GCC-box, TGACG-motif, respectively, by transcriptome data prediction and qRTPCR confirmation. Eight element-bait and segments-bait vectors containing these elements within CsGABP-a promoter and nine prey vectors of candidate transcriptional factors were designed and constructed. Yeast one hybrid screening using cDNA library and ‘Point to point’ validation showed that CsWRKY33 and CsABR1 interacted with WBox and GCC-Box elements, respectively, i.e. CsbHLH interacted with QbHLH fragment;CsZF-B and CsbHLH13 both interacted with QAP2-bHLH fragment. QbHLH and QAP2-bHLH fragments contained the core sequence of G-Box. W-Box, GCC-Box and G-Box elements were predicted to be involving in stress, ethylene, and ABA responses. Summery,these results illustrate that CsGABP gene is interacted with CsWRKY33, CsABR1,CsbHLH, CsbHLH13 and CsZF-B, and is probably induced by the stress, ethylene, ABA and light.