Evolutionary characteristic and functional research of miR159 gene fami- ly in hickory

Abstract:【Objective】MiR159 is a conserved gene family, which plays an important role in vegetative and reproductive growth in plants, especially in the development of plant floral organs. In the miR159 gene family, there are usually multiple members, which regulate plant growth and development by act-ing on their target genes. There has been no any report on the research of miR159 gene family in hicko-ry so far. Therefore, we conducted a research on miR159 family in hickory. The aim of the study was to reveal the secondary structure of its precursor sequence and analyze its stem-loop structure, in order to explore the phylogenetic relationship of miR159 and the conservation of miR159 mature bases and to verify the cleavage site of miR159 on the target gene and master its cleavage base position. It is neces- sary to explore the function of miR159 in hickory, and the role of miR159 in plant development through genetic modification to provide a theoretical basis for the study of the flowering mechanism of hickory. 【Methods】The precursor sequence of miR159 was obtained from the previously identified miR159 fami-ly members of hickory. The RNA Folding from online website was used to analyze the secondary struc-ture of the precursor sequence. The precursor sequences of Arabidopsis, rice, soybean, poplar, and grape in miRBase were downloaded to perform multiple alignments of miRNA genes. Then the bootstrap is set to 1000 times using the neighbor joining method, and the evolutionary tree of miR159 precursor se- quence was constructed using MEGA 5.0. The conservation of hickory miR159 sequence was analyzed through the online platform WebLOGO, and the target gene of the cca- miR159 was predicted by PsRNATarget software. While the cleavage effect of the cca-miR159 on target genes was verified using 5’RACE technology with the male and female flower buds of hickory as test materials. The cca-miR159 precursor sequence was amplified with PCR, and positive transgenic plants were obtained by transgenic technology to verify their gene function.【Results】In this study, two precursor sequences of the miR159, cca-miR159.1 and cca-miR159.2, were cloned in hickory. The cca-miR159.1 precursor se-quence contained only one cca-miR159a mature body, the cca-miR159.2 precursor sequence contained two mature bodies, cca-miR159a and cca-miR159b. The bases conservation of cca-miR159a were obvi- ously higher than later, the base changes of the cca-miR159b at positions 4, 8, 9, 11, 12, 13, and 20 were quite different. Evolutionary analysis found that hickory, poplars and grapes were clustered in the same subfamily, indicating that the cca-miR159 was more closely related to the Ptc-miR159. At the same time, it was found that the two precursor sequences of the cca- miR159 were clustered on the same branch, and there might have been a duplication event during the evolution process, and the phenome- non of gene doubling occurred. It was predicted that the target genes of the cca-miR159a were mainly MYB family genes. The MYB proteins plays an important role in regulating the cell cycle. MYB proteins mainly control the cell cycle by controlling the different periods of cell division, while the target genes of the cca-miR159b are AP1 and UBX, and contig780 corresponds to the AP1, which plays an important role in the formation of plant flower meristems and floral organs. The expression of the AP1 promotes plant flowering. Contig00675 corresponds to the UBX protein, which regulates the formation of growth hormone. The cleavage site of the target gene of hickory miR159 was verified and the cleavage site of the target gene of the cca-miR159 was basically between the 11th and 12th base, and its cleavage site was different in different species. Through genetic modification, it was found that the number of leaves of Arabidopsis thaliana positive plants overexpressing cca-miR159.1 was less than that of the wild type, while the number of leaves of positive plants overexpressing cca-miR159.2 was reduced by about 10 days earlier than that of the wild type.【Conclusion】miR159 contained two mature forms of the cca-miR159a/b in hickory, and the two members were mainly cleaved between the 11th and 12th base of the target gene. The target genes of the cca-miR159a were mainly MYB family genes, among them MYB33 and MYB65 would promote the development of anthers and pollen. The cca-miR159a might inhibit the growth of plant leaves, but the cca-miR159b would advance the flowering period of the plant. Based on the above experiments, the next step could be to explore mechanism of miRNA and target genes regu- late plant growth and development through the relationship between miR159 and target genes.