Expression Analysis And Functional Identification Of MADS-box And MYB Gene Family In Citrus

Flowering is a key stage in the whole life cycle of seed plants and has an important role in the transition from vegetative to reproductive growth.Citrus is one of the most fruit trees,but a long juvenile period and high heterozygosity greatly hinder breeding process and the study of some important economic traits.Therefore,revealling the mechanism of citrus flower development at the molecular level is important for shottening juvenile phase,improving breeding efficiency and changing agronomic traits.Previous study about transcriptome sequencing data of precocious trifoliate orange and trifoliate orange showed most of MADS-box and MYB genes were involved in flowering regulation.In this study,using the clementine mandarin and sweet orange genomes as reference genomes,a systematic analysis of the MADS-box and MYB gene family was conducted to identify candidate genes related with flower development.The verification of the function of CcPI and CsMYB120 in regulating flower development,and the study of their regulatory mechanisms provide a theoretical basis for the improvement of citrus genetic traits.The main results are as follows:1.In this study,84 and 52 MADS-box genes have been identified from clementine mandarin and sweet orange by using bioinformatic analysis,respectively.Subsequently,comparative analysis of these genes,including gene structures,phylogenetic relationships,conserved motifs,chromosome location and Gene Ontology(GO),were identified in these citrus species.According to the phylogenetic analyses,136 MADS-box genes were categorized into five groups(MIKC~C,MIKC*,Mα,Mβ,and Mγ),and MIKC~C group was further divided into 13 subfamilies.Among 128 SSRs identificated from 136 MADS-box genes,15 of 16 SSRs randomly selected were identified as true-to-type SSR loci and were highly transferable among 11 citrus species.These results provide a useful reference for comparative analysis of genetic diversity in flowering time,floral morphogenesis,fruit development.Real-time PCR showed 28 of 30 representative genes had a high variability in different tissues.Some genes displayed tissue-specific expressions,such as CcMADS1/2/3 specifically expressed in flowers and fruits,CcMADS20/21 specifically expressed in flowers,CcMADS58(AGL61)specifically expressed in fruits.CcMADS35/47/78 were constitutively expressed,indicating that the function of MADS-box genes in citrus was extensive.2.Sequence characterization,expression analysis and functional identification of CcPI belonging to MADS-box gene family.Here CcPI from Citrus Clementine isolated had a highly conserved MADS-box domain by sequence analysis.The evolutionary analysis indicated CcPI was classified as core eudicots.CcPI specifically expressed in petal and stamen functioned as B genes.CcPI protein was mainly located in the nucleus.Ectopic expression analysis of 35S::CcPI in Arabidopsis indicated transformed35S::CcPI plants had petal-like sepals lacking chlorophyll,and the length of these white/green petal-like structures in the 35S::CcPI plants was longer than the first whorl sepal in the wild-type plants.AP3 protein from citrus has high homology with At AP3 and interaction of CcPI with endogenous At AP3 might lead to abnormal phenotype of transgenic plants.3.Analysis of AP3 formed with PI.In this study,AP3 homologues CcMADS15,CcMADS17 and CcMADS18 were isolated from Citrus Clementine,of which CcMADS15had highest similarity with At AP3.The yeast two-hybrid and bimolecular fluorescence complementation experiments were performed to verify that CcPI interacted with CcMADS15 and CcMADS17,but had no interaction with CcMADS18.CcPI interaction with CcMADS15 was stronger than with CcMADS17.Only CcMADS17 could formed homodimers.4.Analysis of CcPI promoter sequence in 32 citrus species revealed five variant sites in citrus:A1a2,A1a2+Insert,A1A2,a1A2 and a1a2.Poncirus contained homozygous A1a2/A1a2,Shatian pomelo and HB pomelo contained homozygous a1a2/a1a2,Citrus Clementine and Citrus kinokuni contained homozygous A1A2/A1A2,A1a2+Insert was only existed in Gaoban pomelo and there was no homozygous species at the a1A2 locus.Most oranges were heterozygous A1A2/a1a2.Based on the characteristics of these citrus species and the function of CcPI,a1a2 mainly regulated pomolo flower and A1A2functioned in flower of some citrus with smaller flower,and the orange with middle flower size contained a1a2 and A1A2,indicating that PI gene regulated by different promoter control the size of flower in citrus.5.A total of 177 MYB gene family members(CsMYBs)were identified from sweet orange genome(Citrus sinensis).According to the number of MYB domain,these CsMYBs were categorized into four groups(4R-MYB,3R-MYB,2R-MYB and1R-MYB).The analysis of gene structure revealed that 1R-MYB genes contain relatively more introns.Investigation of their chromosomal localizations revealed that these CsMYBs were distributed in nine chromosomes and had gene duplication.In addition,the distribution of repetitive elements in different regions on the CsMYBs was also investigated.Expression analysis of CsMYBs in different tissues and response to salt stress and drought stress indicated that CsMYBs play an important role in plant development and stress response.The analysis of SSR markers developed in 177 CsMYBs were transferable in 11 citrus species.These results provide a useful reference for the selection of candidate genes and further functional analysis for citrus.6.Expression analysis and functional identification of CsMYB120.CsMYB120contained two spliced transcripts.v1 encoded full-length protein and v2 encoded truncated protein with half of MYB domain.v1 and v2 were constitutively expressed in different tissues.v1 was highly expressed in late stage of floral bud development and v2taked part in the process of flower bud differentiation and induced by hormones.Ectopic overexpression of CcMYB120 genomic DNA in Arabidopsis with high transcription level exhibited early flowering and serious defects in leaf morphology and floral organs,resulting in abortion of flower and failure to form seeds.II class phenotype similar to wild type in morphology exhibited early flowering and short flower organ,but seeds were fertile.The full-length protein v1 interacts with ARF2 and a 2R-MYB protein by yeast two-hybrid assay,indicating that CsMYB120 may respond to hormones through interaction with proteins and regulate floral organ development.