Functional Characterization Of The MYB21-like Genes Os01g45090 And Os05g49310 In Rice(Oryza Sativa)

Transcription factors are important regulators of gene expression.In general,a transcription factor is composed of at least two discrete domains,a DNA binding domain and a activation/repression domain.On the basis of similarities in the DNA-binding domain,transcription factors have been classified into multiple families.MYB proteins form one of the largest transcription factor super-families in plants,which are characterized by a MYB domain.MYB transcription factors are involved in plant development,secondary metabolism,hormone signal transduction,disease resistance and abiotic stress tolerance.Previous studies have shown that the functions of AtMYB21,AtMYB24,AtMYB57,which group the MYB21-like lineage,are redundant,and involve in the late stamen development under the regulation of gibberellin and jasmonate.However,little is known about the function of the MYB21-like genes in rice.This study was carried out by using a closed floret mutant of rice.The mutant plant shows no obvious phenotypes during the vegetable growth.However,at anthesis the lemma and palea of the mutant flowers do not open,the anther filaments do not elongate,and the anthers are indehiscent.The expressions of Os01g45090 and Os05g49310 were not detected in mutant florets by RNA-Seq data analyses and these two genes were predicted to belong the OsMYB21-like lineage by sequence analyses.In this study,we investigated functions of Os01g45090 and Os05g49310 through the expression pattern study,the phylogenetic analyses and the protein interaction assays.Firstly,Sequence alignment proteins of AtMYB21,AtMYB24,AtMYB57,Os01g45090 and Os05g49310 showed the consensus of R2R3-MYB domain,they were known to be classical R2R3-MYB transcription factor.The phylogenetic tree of MYB21-like genes of different kinds of species showed that Os01g45090,OsO5g49310 and AtMYB21 clustered into different subclades.The genetic relationship between Os05g49310 and AtMYB21 was closer,the function of them may be more conserved.Secondly,Expression analyses showed that Os01g45090 was expressed in lemma,palea and filaments growing point;Os05g49310 was mainly expressed in stamen,weakly expressed in style and the base of ovary.Thirdly,Through the method of reverse genetics,specifically down-regulation of Os01g45090 and Os05g49310,resulted in plant dwarf,anthesis delay and immature pollen,which indicated that Os01g45090 and Os05g49310 regulated anthesis and pollen maturation.Fourthly,The mutant phenotype of closed floret mutant was not rescued by exogenous JA;Exogenous PAC made plant small,dark green leaves,anthesis delay,eventually plant completed the process of anthesis and pollination.And qPCR analyses showed that expression levels of Os01g45090 and Os05g49310 was increased.We hypothesized that jasmonate mediated the process that Os01g45090 and Os05g49310 regulated expression of anthesis genes.And there was no relationship between mutant and gibberellin.Fifthly,Yeast-two-hybrid assays show that Os01g45090 interacted with Os05g49310.These data provide evidence that the MYB21-like gene Os01g45090 and Os05g49310 played significant roles in rice anthesis and the pollen maturation,and thus their function is conserved.