Cloning and expression analysis of a MiCO gene in mango

Abstract: 【Objective】Floral induction is an important physiological process in higher plants from vegeta⁃tive stage to reproductive growth. Photoperiod is one of the important environmental triggers of flowering,and the CONSTANS (CO) gene is a key regulator of this pathway in the model plant Arabidopsis thaliana.Mango (Mangifera indica L.) is one of the world’s important woody plants, known as“the king of tropicalfruits”. Two mango cultivars were used in present study with different flowering habits.‘SiJi’blossomsseveral times a year and‘ZiHua’blossoms once a year. Off- season production of mango has beenachieved successful by using‘SiJi’and much more income has been obtained by farmers. The molecularmechanism of different flowering habits in mango remains unclear. In present study, a MiCO gene of mango was cloned and its bioinformatics and genetic evolutionary relationships as well as the expression pat⁃tern were analyzed. The results would provide scientists with a basis to further reveal the function of COgene on flowering regulation in mango.【Methods】All materials were collected from the 8-year-old‘SiJi’ and‘ZiHua’trees grown in the park of College of Agriculture, Guangxi University. The mature leaveswere used for gene cloning. Young stems, young leaves, flowers, mature leaves and the fruits of the 7 d, 50d and 90 d post full flowering of‘SiJi’were used for studying on the expression patterns of different orga⁃nizations. Mature stems, mature leaves and young stems of‘SiJi’and‘ZiHua’were used for analyzingthe expression patterns of a year. All the materials collected were immediately stored at - 80 ℃. TotalRNA was isolated using hot-borate method, and quantified at OD260 and OD280. The RNA quality was veri⁃fied using agarose gel electrophoresis (1.2%). The single- stranded cDNA was synthesized using a Reverse Transcriptase M-M kit (TaKaRa, Dalian, China) with the primer AUP1. In the previous studies, thefull-length sequence of MiCO gene from mango was obtained from the data of transcriptome sequencing.A pair of specific primers based on the full-length sequence of CO was designed and used for gene clon⁃ing by amplified the mixed cDNA of‘SiJi’and‘ZiHua’by RT-PCR respectively. The sequence aminoacids, molecular weight and isoelectric point prediction were analyzed using software BioXM2.6. The simi⁃larity of the nucleotide and the domain structure prediction were analyzed by online NCBI BLAST pro⁃gram. The multiple alignments analysis of CO amino acids was performed using software ClustalW and aphylogenetic tree was computed using MEGA 4.0 software according to the neighbor-joining method. Inaddition, the expression characters of MiCO in different tissues and annual cycle expression were ana⁃lyzed by quantitative real-time PCR respectively. All primers used in this study were designed by Primer5.0 software.【Results】Sequence analysis showed that the full-length cDNA sequence of MiCO gene fromboth‘SiJi’and‘ZiHua’were 1 150 bp with an open reading frame (ORF) of 966 bp, encoding 322 aminoacids with a theoretical isoelectric point of 5.8. The protein molecular weight of MiCO of‘SiJi’and‘Zi⁃Hua’were 35.30 ku and 35.22 ku respectively. The conserved domain of MiCO had two B-box motifsnear by the N-terminal and a CCT domain was near by the C-terminal. Phylogenetic tree indicated thatboth MiCO proteins of mango belonged to the first group of CO proteins family, and had the closest rela⁃tionship with AtCOL4 (NM_122402) of Arabidopsis thaliana. Alignment analysis revealed that only fournucleotides and three amino acids differences were found between‘SiJi’and‘ZiHua’sequences withmore than 99% homologous. qRT-PCR showed that the MiCO gene expressed in all the tested tissues butthe expression level was different. High expression level of MiCO was observed in young leaves, flowersand the fruits in early developmental stage, while low expression level was observed in mature leaves andyoung stems. In addition, the qRT-PCR results indicated that MiCO expressed unlikely in different tissues as well as different cultivars in the annual cycle. The expression level of MiCO in‘SiJi’was highestin the young stems, while lowest in the mature stems. The expression level of MiCO in‘ZiHua’was high⁃est in the young stems in March to June, while the highest expression level was observed in mature leavesin July to March of the next year. On the whole, the expression level of MiCO gene was highest in the flowering transition period, and another small peak of the expression appeared in May to June of the next year.【Conclusion】In this study, MiCO gene was cloned from‘SiJi’and‘ZiHua’and they were conservative inboth nucleotide sequences and amino acids sequences although several differences existed in‘SiJi’and‘ZiHua’with different flowering habits. In addition, the expression level of MiCO gene was much higherin the flowering transition period than that in any other period. The results implyed that the function ofMiCO gene might be related to the flowering transition in mango.