Cloning And Function Analysis Of ELF4 Homologous In Longan (Dimocarpus Longan Lour.)

Longan(Dimocarpus longan L.) is an important fruit tree in tropical and subtropical area. In this study, based on RNA-seq, we found 107 putative flowering-time homologues by sequence analysis and comparison, and DIELF4-1 and DIELF4-2 were found to be related to ’Sijimi’longan special flowering traits. Subsequently, full-length cDNA of DIELF4-1 and DlELF4-2 were cloned, and function was analysed them by bioinformatics softwares and real-time quantatitive PCR method. In this study, the DIELF4-1 promoter sequence was also cloned and analysed from’Sijimi’ and ’Honghezi’longan. Plant expression vectors of DIELF4-1 and DIELF4-2 were constructed. These results can help to understand the molecular regulatory mechanisms of longan flowering. The main research results were as follows:1.107 putative flowering-time homologues were found in RNA-seq database. These genes were further catalogued into circadian clock & photoperiod pathway, vernalization pathway, autonomous pathway, GA pathway, age-related pathway and floral pathway integrator genes. Compared the 107 putative flowering-time homologues by log2 (S_RPKM/L_RPKM) method in ’Sijimi’and’Lidongben’, we found the expression of ELF4 (Unigene4309 and Unigene5963) were different in two samples, and the expression of ELF4 (Unigene4309) was significantly upregulated in’Sijimi’. The change of ELF4 (Unigene4309) expression was further proved by real-time quantatitive PCR, which indicated ELF4 (Unigene4309) may have significant role in regulating longan expecial flowering traits.2. Based on our RNA-seq database, two assembly sequences of ELF4 were found, Unigene4309 and Unigene5963. Unigene4309 has complete coding region named DIELF4-1, Unigene5963 that includes partial coding region was named DIELF4-2. In this study, DIELF4-1 and DlELF4-2 were cloned from leaf buds of longan cultivar’Honghezi’. DIELF4-1 has full-length cDNA of 544 bp, encoding a acid labile subunit protein consisted of 140 amino acid residues; DIELF4-2 has full-length of cDNA 733 bp, encoding a acid labile subunit protein consisted of 144 amino acid residues. The PI of D1ELF4-1 and D1ELF4-2 proteins are 5.05 and 5.17, respectively. Sequence analysis results showed D1ELF4-1 and D1ELF4-2 proteins had a DUF1313 domain conserved in ELF4 protein. Cluster analysis results showed D1ELF4-1 got together with AtELF4, D1ELF4-2 got together with AtELF4-LIKE 1.3. The expression of two DIELF4 were analyzed by real-time quantitative PCR during flower bud differentiation of ’Honghezi’ longan. The results showed that the expression of DIELF4-1 was sharply declined in November, which implied that the function of DIELF4-1 associated with repressing flower bud differentiation; the expression of DIELF4-2 was decreased in January, which implied that the function of DIELF4-2 associated with repressing morphological differentiation of flower bud. DlELF4-1and DIELF4-2 may have different functions in regulating flowering-time of longan.4. To investigate what lead to different expression of DIELF4-1 and the function of DlELF4-1, promoters sequence of DIELF4-1 from leaves of’Sijimi’ and ’Honghezi’ longan were cloned.1330 bp in the upstream of DIELF4-1 initiation codon ATG in ’Sijimi’ and 419 bp in ’Honghezi’ were obtained. After sequence alignment, we found that -1bp--249 bp region was same between ’Sijimi’ and ’Honghezi’, but -250bp--1330 bp region of ’Sijimi’ and-250bp--419 bp region of ’Honghezi’ were different. Sequence analysis of the promoter results suggested that the expression of DIELF4-1 was regulated by light, hormone and stress conditions. Compared with ’Honghezi’, there are more light responsive elements, homone responsive elements, stress responsive elements and others responsive elements in ’Sijimi’. The longer promoter sequence of DIELF4-1 to be further cloned and analyzed in’Honghezi’.5. In this study, we constructed two recombinant plasmids pBI121-D1ELF4-1 and pCAMBIA2300-D1ELF4-2, by inserting DIELF4-1 into pBI121 vector and inserting DIELF4-2 into pCAMBIA2300-35S-OCS vector, for the further research of the functions of D1ELF4-1 and DIELF4-2.