Expression analysis of CmMYB330 gene from Citrus maxima ‘Hongroumiyou’

Abstract: 【Objective】Citrus maxima‘Hongroumiyou’is an important fruit with high economic value inFujian province, however, juice sacs granulation occurred during the later stage of fruit ripening and postharvest storage and seriously influeced the quality. Some studies showed that juice sacs granulation wasclosely correlated with lignin metabolism. Regulation of transcription factors (TFs) on lignin metabolismwas studied in recent years. Some MYB TFs involved in lignin metabolism were reported, and the R2R3MYB subgroup 4 was deeply studied. This study aimed at isolating R2R3 MYB subgroup 4 MYB relatingto juice sacs granulation from C. maxima‘Hongroumiyou’. The genetic characteristics and expressionpattern of one MYB TF were analyzed for investigating the role of MYB TFs in regulating lignin metabo⁃lism in juice sacs and to providing a reference for the study of juice sacs granulation mechanism,【Meth⁃ods】A coding region sequence and regulatory sequence of the 5’terminal of MYB TFs CmMYB330 werecloned by bioinformatics analysis and PCR technology from C. maxima‘Hongroumiyou’with reference tothe genome-wide analysis of MYB TFs in Citrus sinensis. The bioinformatic analysis of their sequenceswas carried out by ProtParam, PSORT, DNAMAN, Pfam and PlantCARE. Expression patterns ofCmMYB330 during different developmental periods and in different parts of C. maxima‘Hongroumiyou’juice sacs were analyzed by quantitative real-time PCR technology. CmMYB330-GFP fusion expressionvector was transformed into tobacco leaves for the analysis of subcellular localization by agrobacteriummediatedmethod. The transcriptional activation activities of CmMYB330- CDS, CmMYB330- N andCmMYB330- C were detected by yeast two hybrid system.【Results】Results of sequences analysis ofCmMYB330 characteristics showed that a CmMYB330 gene with length 942 bp, encoding 313 amino ac⁃ids was cloned from C. maxima‘Hongroumiyou’juice sacs. The encoding protein of CmMYB330 with mo⁃lecular weight of 34.873 8 ku, isoelectric point 8.97, projected as nuclear localization protein, and the ami⁃no acid sequence of CmMYB330 had two Myb_DNA-binding domains, suggesting that CmMYB330 was atypical R2R3 MYB TFs. Results of the amino acid sequence of CmMYB330 blast in NCBI showed thatCmMYB330 was similar to CsMYB308-like (Citrus sinensis, XP_006466200.1), CICLE_v10026112mg hy⁃pothetical protein (Citrus clementina, XP_006426418.1), SlMYB330 (Solanum lycopersicum, XP_004241841.1), SiMYB330 (Sesamum indicum, XP_011096483.1), MdMYB3 (Malus domestica,AEX08668.1) with the homology rate 99%, 98%, 55%, 54%, 51% respectively. Construction of amino ac⁃id sequences alignment and phylogenetic tree showed that CmMYB330, AmMYB330 (Antirrhinum majus,P81395.1), AmMYB308 (Antirrhinum majus, P81393.1), AtMYB4 (Arabidopsis thaliana, AT4G38620.1),Cs8g02740.1 (Citrus sinensis, Cs8g02740.1), Cs1g19400.1 (Citrus sinensis, Cs1g19400.1) were classed asR2R3 MYB subgroup 4. The C2 motif, specific to R2R3 MYB subgroup 4, containing a transcriptional re⁃pression domain EAR motif located at the C terminal was found in CmMYB330, AmMYB330, AmMYB308,AtMYB4, Cs8g02740.1 and Cs1g19400.1. In addition, the bHLH protein interaction motif, C1 motif, zincfinger motif were also found in their sequences. CmMYB330 was also closer to Cs1g19400.1 and Am⁃MYB330 in phylogenetic tree analysis. It was speculated that CmMYB330 might be functionally similar toother R2R3 MYB subgroup 4 TFs which was involved in the regulation of lignin metabolism genes. Predic⁃ition results of cis-acting elements of the regulatory sequence of the 5’terminal of CmMYB330 by Plant⁃CARE showed that the initiation codon upstream sequence -1 748 bp was the regulatory sequence of the5’terminal of CmMYB330, containing promoter core elements TATA-box in the upstream of initiation co⁃don(-103)-(-112) bp and CAAT-box in (-191) bp - (-195) bp, 3 MBS, 1 AC-I, 1 5UTR Py-rich stretch,and a large number of hormone-sponsive elements, such as gibberellin, ethylene and salicylic acid. Quan⁃titative real-time PCR analysis showed that with the advancement of juice sacs development in C. maxi⁃ma‘Hongroumiyou’, the expression level of CmMYB330 had gone up at first and then down afterward,but it was increased in general and reached the maximum expression level on 208 days after flowering(DAF), and then declined. Analysis of subcellular localization and transcriptional activation activityshowed that the green fluorescence signal was detected in the nucleus of tobacco leaves of transformedp- super1300- CmMYB330- GFP, which was consistent with the predicted result, indicating thatCmMYB330 was localized in the nucleus. CmMYB330 was a nuclear localization protein with one of thecharacteristics of TFs. And Y2H Gold yeast strains of transformed pGBKT7-CmMYB330-CDS, pGBKT7-CmMYB330- N and pGBKT7-CmMYB330- C could not grow on the SD-Trp-His-Ade (TDO) mediumand the SD-Trp-His-Ade+X-α-Gal (TDO/X) medium. CmMYB330 fusion protein did not activate down⁃stream reporter genes His3 and Ade2. They had no activity of transcription activation in Y2H Gold yeast.【Conclusion】MYB TF CmMYB330 coding region sequence and regulatory sequence of the 5’terminalwas isolated from C. maxima‘Hongroumiyou’. CmMYB330 belonged to R2R3 MYB subgroup 4, whichwas related to the juice sacs granulation and was localized in the nucleus, and with the advancement ofjuice sacs development in C. maxima‘Hongroumiyou’，the expression level of CmMYB330 was increasedin general. This study would be valuable for further study on the relationship between CmMYB330 andjuice sac granulation.