Genome-wide Characterization,Expression Patterns And Functional Analysis Of The NF-Y Gene Family In Watermelon

During their life,plants are constantly affected by a variety of biotic and abiotic stresses,which seriously affect the growth and development of plants.Nuclear factor Y(NF-Y)is a heterotrimeric transcription factor that binds to the CCAAT box in the promoter region of its target genes.The NF-Y complex consists of three distinct subunits,NF-YA,NF-YB and NF-YC.Numerous studies have shown that NF-Y complexes in Arabidopsis and rice are involved in multiple biological processes such as embryo development,seed development,flowering time regulation,abscisic acid signaling.Suggesting that NF-Y gene family is sophisticate and mysterious.However,the reports of related genes in watermelon are very limited.In this study,we focused on the NF-Y gene family in watermelon.We identified the NF-Y family genes in watermelon,analyzed their expression patterns and explored their possible functions in response to stresses.(1)Bioinformatics analysis identified a total of 25 members(7 NF-YA,10 NF-YB and 8 NF-YC)from watermelon genome.22 of the watermelon NF-Y family genes were cloned and identified.Conserved domain analysis of the proteins indicated that 7 members of the NF-YA subunit contain the CBFB_NFYA domain,10 members of the NF-YB subunit and 8 members of the NF-YC subunit contain the CBFD_NFYB_HMF domain.(2)qRT-PCR analysis showed that most of the NF-Y genes in watermelon were up-regulated during the induction of SA,ABA and ACC.In the process of MeJA induction,most of the watermelon NF-Y genes were down-regulated.After Fusarium oxysporum f sp.niveum inoculation,defense genes were abundantly expressed,a large number of NF-Y genes were significantly up-regulated in the root and leaf tissues of watermelon,and their expression even reached a higher level in root tissues.(3)The subcellular localization of ClNF-Y family proteins indicated that the ClNF-YA proteins are mainly localized in the nucleus of cells,the ClNF-YB and the CINF-YC proteins are localized in the membrane and nucleus of cells.(4)Bimolecular fluorescence complementation results showed that ClNF-YB4,ClNF-YB5,ClNF-YB6,C1NF-YB7,ClNF-YB8 and ClNF-YB10 interact with ClNF-YC1,ClNF-YC2,ClNF-YC3,ClNF-YC4,ClNF-YC5,ClNF-YC7 and ClNF-YC8 respectively.The interactions are localized in the membrane and nucleus of cells.ClNF-YB1 and ClNF-YB9 interact with ClNF-YC6 respectively and the interactions are localized in the nucleus of cells.The yeast three-hybrid method was used to study possible combinations of heterotrimeric complexes in ClNF-Y family.(5)The effects of the 10 CINF-Y genes on plant growth and their roles in defense reponses were studied by transgenic over-expressing(OE)Arabidopsis.Compared with wild-type,CINF-YB7-OE plants were less susceptible to Botrytis cinerea,and the relative expression level of PR1 and PR5 genes in ClNF-YB7-OE plants were up-regulated.CINF-YA2-OE and CINF-YC5-OE plants showed significant increased susceptibility to B.cinerea than wild type and the relative expression level of PR1 and PR5 genes in CINF-YA2-OE and ClNF-YC5-OE plants were down-regulated.These results demonstrate that ClNF-YB7 may plays a positive role in the regulation of resistance to B.cinerea through activation of defense genes expression,while ClNF-YA2 and ClNF-YC5 are negative regulators.As compared with the Co1-0,the growth of bacteria in ClNF-YA3-OE,ClNF-YB1-OE and ClNF-YC2-OE plants were significantly reduced after Pseudomonas syrinage pv.tomato(Pst)DC3000 inoculation,expression of PR1 and PR5 genes in CINF-YA3-OE,ClNF-YB1-OE and ClNF-YC2-OE plants were up-regulated.The growth of bacteria in leaves of the CINF-YA2-OE and ClNF-YB7-OE plants were significantly increased after inoculation with Pst DC3000,expression of PR1 and PR5 genes in CINF-YA2-OE and ClNF-YB7-OE plants were down-regulated.These results indicate that ClNF-YA3,ClNF-YB1 and ClNF-YC2 are positive regulators of defense responses against Pst DC3000,ClNF-YA2 and ClNF-YB7 are negative regulators.During the seed germination stage,compared with the wild type,the sensitivity of CINF-YA2-OE Arabidopsis to ABA,mannitol and NaCl were reduced,the susceptibility of CINF-YA3-OE Arabidopsis to mannitol and NaCl were stronger,the susceptibility of ClNF-YB5-OE Arabidopsis to ABA was stronger,and the sensitivity of ClNF-YC4-OE Arabidopsis to NaCl was reduced.