Genome-wide Identification Of MADS-box Family Genes In Moso Bamboo(Phyllostachys Edulis) And Functional Analysis Of PeMADS In Flowering Regulation

China has abundant bamboo resources,but previous studies on bamboo genetic breeding were not sufficiently detailed.Bamboo has unique flowering features,such as long vegetative phase,uncertain time of flowering,and death after flowering for most species.These characteristics limit the studies on bamboo genetics and make variety breeding and genetic improvement difficult.Moso bamboo(Phyllostachys edulis)is native to China and is a large woody bamboo species with great economic value.Studying the genetic and molecular regulation of bamboo flowering has high theoretical significance and potential economic value.MADS-box genes encode a large family of transcription factors that play significant roles in plant flowering regulation and flower organ development.However,the mechanism of MADS-box gene family in P.edulis flowering regulation was not clear and thus required systematic research.The recent release of the P.edulis genome sequence enabled us to identify and comprehensively analyze its MADS-box family.We conducted systematic bioinformatics analysis and functional study on the flowering-related PeMADS genes from moso bamboo.The results of this study provide useful information in exploring the molecular mechanism of bamboo flowering.The main research results are as follows:Here,a complete genome-wide identification and characterization of the MADS-box genes in moso bamboo was conducted,and 42 MADS-box genes were found in the P.edulis genome.We found that some of the PeMADS sequences are fragmented and/or inaccurate due to the large error in the genome sequence of moso bamboo.We performed several bioinformatics techniques to obtain precise sequences by using transcriptomic assembly.By conducting phylogenetic and gene structure analyses,we classified the bamboo MADS-box gene and combined it with the homologous genes of rice,Arabidopsis,for phylogenetic analysis.The results showed that the number and grouping pattern of the type II genes are highly similar to those of the model plants,but the type I MADS-box may have experienced many loss events during the evolution of P.edulis genome.This finding suggested that bamboo may have a unique type-I subfamily evolutionary pattern but shares a common type-II subfamily evolutionary pathway with other angiosperms.Expression analysis of the bamboo MADS-box genes in floral organs and leaves revealed that several key members are involved in bamboo inflorescence development similar to their orthologous genes in Oryza.MADS-box genes with specific expression patterns may play particular functions in bamboo floral development and can be considered as candidate genes for cloning and further functional analyses.In this study,24 MADS-box genes were cloned from P.edulis.Sequence analysis showed that these 24 genes belonged to different subgroups,among which the SQUA-,AG-,and SVP-like subgroups had the most PeMADS gene members.By analyzing the PeMADS gene expression levels during bamboo flowering and the data from previous study,we selected nine PeMADS genes for overexpression in Arabidopsis.Four transgenic plants showed phenotypic changes: 35S::PeMADS3,5,19,and 29 transgenic plants had early flowering time;whereas 35S::PeMADS3 and 35S::PeMADS5 have phenotypes associated with the abnormal development of floral organs.These results suggested that these four PeMADS genes may play important roles in bamboo flowering.PeMADS3 and PeMADS5 were selected for further study.RT-qPCR was performed on 35S::PeMADS5 transgenic plants to detect the expression of Arabidopsis flowering-related genes.The results showed that the expression levels of AGL24 and SOC1 in rosette leaves and inflorescence from transgenic plants were significantly higher than those in wild type,whereas that of AP1 was down-regulated.This finding suggested that PeMADS5 influences the flowering time and floral organ development in Arabidopsis by indirectly regulating the expression levels of flowering-related genes.The overexpression of PeMADS5 in Arabidopsis svp-31 mutant(early flower)didn’t influence flowering time.However,transgenic plants developed leaf-like sepals,which indicated that PeMADS5 could also regulate floral organ development.In addition,we induced the overexpression of PeMADS5 in rice.35S::PeMADS5 transgenic rice plants were tested by PCR,and 10 transgenic lines were obtained for further study.RT-qPCR expression analysis of 35S::PeMADS3 transgenic Arabidopsis plants showed that the expression levels of AP1,FUL,and LFY genes are affected by this phenomenon and further regulate the floral organ development.Overexpression of PeMADS3 in Arabidopsis ap-1 mutant cannot rescue the petal or sepal phenotypes in the flowers,but the transgenic mutants have dense inflorescence.Twelve transgenic lines of 35S::PeMADS3 in rice were obtained for further study.STRING software was used to predict the protein interaction network of bamboo MADS-box family,and 33 interactions(including 34 PeMADS proteins)were identified.Twenty-five PeMADS proteins were selected to construct yeast two-hybrid bait and prey and to further study the interaction among the MADS-box family members in bamboo.After self-activation was detected,the 24 PeMADS proteins were combined as pairwise pairs for yeast two-hybrid analysis.Sixty-seven pairs of PeMADS proteins were detected with interaction.Cytoscape software was used to construct bamboo MADS-box gene family interaction network of the yeast two-hybrid system.This network can provide guidance for further understanding of the PeMADS gene function.