Cloning and expression analysis of NF-YB transcription factors DlNF-YB1 and DlNF-YB10 in longan

Abstract: 【Objective】Longan (Dimocarpus longan L.) is a subtropical fruit species of the Sapindaceae. Longan,a typical drought-tolerant plant,is widely cultivated in tropical and subtropical region and has great economic values. Transcription factor NF-YBs are important regulators during plant growth and development, participating in regulating embryonic development, plant drought resistance, floral meristem growth and flowering time under photoperiod pathway. Two NF-YB transcription factors DlNF-YB1 and DlNF-YB10 in longan were identified and their putative functions were investigated in this study.【Methods】Two NF-YB homolog genes,UN15136 and CL2518 were identified in longan transcriptome data and named DlNF-YB1 and DlNF-YB10. Primers were designed by DNAMAN8.0. Using cDNA of‘Honghezi'longan as the template, CDS sequences of DlNF-YB1 and DlNF-YB10 were amplified by RT-PCR. The bioinformatics analysis of two DlNF-YB sequences was carried out for further understanding of their putative functions. ORF regions of two DlNF-YB genes were founded by ORF Finder program in NCBI. Phylogenetic tree analysis of two DlNF-YBs and the Arabidopsis thaliana NFYB family were obtained by MEGA6.0 software. Multiply amino acid alignment of DlNF-YB genes with other NF-YB genes of 5 species was obtained by DNAMAN8.0. Information about NF-YB protein sequence in different plants was analyzed by PredictProtein online software. The basic physicochemical properties of the proteins were analyzed by ExPASyProtParam. The secondary structure analysis about the content of α-helix, β-sheet, β-turn and random in DlNF-YB proteins were obtained by SOPMA online software. The distribution of curl and the homology modeling of the tertiary structure of DlNF-YB1 and DlNF-YB10 were performed using Swiss-Model online software. In addition, promoter sequences of DlNF-YB1 and DlNF-YB10 were analyzed by PLANTCARE online software. Further, expression patterns of DlNF-YB1 and DlNF-YB10 in different organs and different developmental period of longan were analyzed by qRT-PCR. The expression levels of DlNF-YB10 during longan flower bud differentiation and DlNF-YB1 under water stress were also analyzed by qRT-PCR. When water stress experiment was started, seeds of‘Honghezi'longan were sown in trays containing nutrient soil, vermiculite and perlite at the proportion of 2:1:1. Seedlings were cultivated under the growth chambers at a temperature of(25±2）℃, and the seedling growth was kept with 20.0%-30.0% soil moisture and 50%-55% air humidity. After 6 months, the seedlings were transplanted to 20 cm-diameter plastic pots for root water stress trial. Treatments of three water contents were set. The soil water content of 10% was set as drought treatment; the water level line was kept 5 cm above the bottom(at this time, the soil water content is more than 70%) was set as water immersion treatment; the water content between 20% and 30%was set as the control. In this study, the test was performed in a completely randomized design using Excel 2016, and the LSD test was performed using the Duncan test in SPSS 22.0(p < 0.05).【Results】The CDS sequences of DlNF-YB1(GeneBank accession number: MK372373) and DlNF-YB10(GeneBank accession number: MK359142) were cloned. Multiply protein sequence analysis of DlNF-YB1 and DlNF-YB10 with NF-YB family of Arabidopsis thaliana showed that DlNF-YB1 and DlNF-YB10 had the highest similarity with AtNF-YB1(80%) and AtNF-YB10(69%), respectively. DlNF-YB1 contained528 bp and DlNF-YB10 contained 531 bp open reading frames(ORF), encoding 175 amino acids and176 amino acids, respectively. Both DlNF-YB amino acid sequences included a typical NF-YB/HAP3 domain and five potential functional sites: N-myristoylation site, N-glycosylation site, Casein kinase Ⅱphosphorylation site, protein kinase C phosphorylation site, and cAMP and cGMP-dependent protein kinase phosphorylation sites. Multiple protein sequence alignment revealed a high similarity among DlNF-YB1, DlNF-YB10, CpNF-YB1, TcNF-YB1, AtNF-YB1, GmNF-YB10 and AdNF-YB10. Protein physicochemical properties analysis showed that DlNF-YB1 and DlNF-YB10 had similar physical and chemical properties, and both were unstable and hydrophilic proteins. The secondary structure of DlNF-YB1 and DlNF-YB10 included the form of α-helix and random coil mainly. However, the three-dimensional structure of DlNF-YB1 and DlNF-YB10 proteins differed greatly. Promoter sequence analysis showed that both of DlNF-YB1 and DlNF-YB10 promoters contained ABRE abscisic acid response elements,and specially, DlNF-YB1 promoter contained the drought response MBS cis-acting element, while the DlNF-YB10 promoter sequence contained the auxin response-related AuxRR-core cis-acting element.The expression level of both genes in‘Honghezi'seedling buds were similar with the expression level in the new shoots of‘Sijimi'longan. But the relative expression of DlNF-YB10 and DlNF-YB1 varied in different organs and months, DlNF-YB10 was highly expressed in the vegetative buds of adult longan tree and the expression level of DlNF-YB10 during physiological differentiation of flower bud gradually increased from October to December, with a peak in December, and began to decline in January of the following year. Differently, DlNF-YB1 was highly expressed in roots. Relative expression of DlNF-YB1 varied under different water stresses and it significantly increased under drought stress.【Conclusion】DlNF-YB1 and DlNF-YB10 have different functions in longan. The function of DlNF-YB10 may be related to the physiological differentiation of flower bud, while DlNF-YB1 may play an important role in the drought resistance of longan.