Functional Identification Of MIR156 And MIR172 Gene Family On Flowering Regulation In Citrus

microRNA(miRNA)is a class of non-coded 21-23 nt single strand RNA molecules encoded by endogenous genes,which plays an important role in regulating plant development.Citrus is the world's largest fruit tree,its long and late fruiter characteristics seriously affect its economic benefits and breeding process.Numerous studies have shown that microRNA156(miR156)and microRNA172(miR172)are involved in regulating flowering transformation in plants.In this study,3 members of the miR156 family,PtmiR156 a,PtmiR156b,PtmiR156c;and 4 members of the miR172 family,were obtained through the sequencing of the transcriptional group,which were PtmiR172-03,PtmiR172-12,PtmiR172-36,and PtmiR172-64,respectively.The overexpression vectors were constructed,then transformed into Arabidopsis and tobacco,and the phenotypes were observed,and the long distance mobility in the plants was preliminarily investigated by grafting.The main results are as follows:1.The sequence analysis of PtmiRl56 and PtmiR172 gene family members showed that the core sequence difference between PtmiR156 b and PtmiR156 c in the PtmiR156 gene family was small,only the difference between the first and the first individual bases,and the core sequence of PtmiR156 a was more different than that of them.The difference of core sequences among PtmiR172 gene family members is relatively small,with only a few base bases.The core sequences of members of miR156 gene family have little difference in Arabidopsis thaliana,tobacco,rice and common trifoliate orange.The sequence analysis of members of the miR172 gene family in these species was also found.It indicates that the miR156 and miR172 gene family members of citrus are highly conserved.2.The overexpression of PtmiR156 and PtmiR172 in Arabidopsis and tobacco showed that compared with the wild type,the PtmiR156 transgenic lines were obviously late flowers,the leaf type was round,the leaves were smaller,the branches increased,the plant type was compact,the fruit pods were shorter,and the seeds were aborted,and the PtmiR172 transgenic tobacco plants were obviously early flowers and leaves along with plant growth.The long development gradually narrowed.The flowers in the PtmiR172-12 transgenic tobacco lines showed that the pistil stigma elongated and the flower color was deep,and the PtmiR172-64 transgenic tobacco lines were all more branched in the first generation.Overexpression of PtAP2 in Arabidopsis showed obvious late flowering and abortion.It indicates that PtmiR156 delays plant flowering and prolongs vegetative growth period.PtmiR172 promotes plant flowering and affects plant floral organ development through its target genes.3.Using positive and negative grafting to explore the mobility of PtmiR156 and PtmiR172 gene family members: when overexpression of PtmiR156 b tobacco,the existence and expression of mature PtmiR156 b can be detected in the wild type tobacco scion,and the expression amount is obviously higher than that in the wild type,and the reverse grafting can still be found in the same way.When PtmiR172-12 tobacco was overexpressed as a rootstock,the presence and expression of mature PtmiR172-12 could be detected in the wild type of its wild type tobacco scion,and the expression was significantly higher than that in the wild type,and the reverse was also the same.It is preliminarily speculated that mature PtmiR156 b and PtmiR172-12 can achieve long distance movement from top to bottom and from bottom to top.