Genome-Wide Identification And Analysis Of Fruit Development-Related Four Gene Family And Function Research Of CmERFII-9IN Melon

In this study, phylogenetic analysis of fruit development-related gene family from a global point of view, exploring the origins, evolution and potential of functional differentiation of gene families; analysis of characteristization of expression and relevant analysis of regulatory protein network in family members; utilizing transient expression of target gene in function studies, providing effective and alternative direction on depth study of fruit development and ripening-related genes. Genome-wide analysis of AP2/ERF、SBP-box、EIN3/EILs、14-3-3 gene family which relate with fruit development were performed in melon. Functional research of CmERFⅡ-9 was handled in the work. The results obtained by above methods are as following:(1) Identification of 136 protein sequences of AP2/ERF family, in which ERF family proteins is 119(containing ERF subfamily proteins 64, DREB subfamily proteins 55), AP2 family proteins is 13, RAV family proteins is 4.87 genes of ERF family have no introns, the remaining 32 genes containing varying introns of 1-8. EAR motif which play a role in the negative regulation was in Group Ⅰ and Ⅹ, Cys repeats CX2CX4CX2-4C which involved in the interaction between DNA-binding and proteins was in the N-terminal of group Ⅴ, Ser/Thr motif which play a role in transcriptional activation was in group Ⅰ. Expression abundance of ERF subfamily genes in various tissues descending order is fruit, callus, flower, root, leaf, cotyledon, phloem. Expression abundance of CmERFⅢ-11 is higher in fruit than any other. cDNA full sequences of CmERFII-9 was cloned, the target gene was successfully constructed in overexpression vector pPZP221-CmERFII-9 and RNAi vector pART27-CmERFII-9. Analysis of transient expression showed that melon fruit come into being phenotype of prematurity and late-maturing via the injection of two vectors with Agrobacterium. Further analysis of fruit development and ripening-related genes indicating that the ACO gene play a positive regulatory role in process of development and ripening in melon fruit, speculating on the ACS, PDS, PFK, XTH and NCED genes play negative regulation via the activation of expression of functionally redundant genes.(2) Identification of 13 protein sequences of SBP-box family which is divided into four independent subfamily. SBP-box gene family members each contain the number of introns ranging from 1-10. SBP-box family members all have structure of double zinc-finger, and contain highly conserved bipartite nuclear localization signal. SBP-box domain in some locations is highly conserved. Resulting of sequences alignment of full length of SBP-box protein family is much variability in N-terminal and C-terminal. miRNA 156 target site is present in 6 genes. Expression abundance of SBP-box family in each tissue descending order is fruit, root, flower, callu, leaf, cotyledon, phloem. CmSBP11 is specifically expressed in the fruit.(3) Identification of 4 protein sequences of EIN3/EILs family which contain 1 intron. Melon, Arabidopsis, tomato and cucumber all have highly conserved EIN3/EILs domains, which contain most highly conserved amino acid fragments of GPVYGI, WWK, LQD, TLG. EIN3/EILs family genes is minimum in differentiation between melon and cucumber, but no homologous of CmEIL03 in the cucumber; some extent of differentiation in tomato; Arabidopsis is maximum in differentiation. Analysis of expression pattern revealed CmEIL01, CmEIL02, CmEIL04 were expressed in different developmental stages of fruit, CmEIL04 specifically expressed in the mature fruit. The results of protein interaction network showed that CmEIL01 and CmEIL02-like integrate with multiple ERF proteins, respectively.(4) Identification of 9 protein sequences of 14-3-3 which is divided into ε and non-ε subfamily, ε subfamily 5 genes, each containing 5 or 6 introns, non-ε subfamily 4 genes, containing 3 introns. ε subfamily members have high variability in the N-and C-terminal ends, non-ε subfamily members have absolutely conserved Met in N-terminal, the remaining section of protein sequences is higher variability, the homology is higher than ε subfamily members. Analysis of expression pattern in different tissues revealed that the family members were detected expression in fruit, root, flower, leaf, in which MELO3C003536, MELO3C003562, MELO3C011041, MELO3C011028, MELO3C018726 were expressed in fruit. Network of protein interaction showed 8 protein members of 14-3-3-like are in the form of dimer proteins which co-regulated activity of target protein.