Molecular Cloning And Functional Analysis Of MADS-box Genes Related To Floral Development In Dianthus

The transformation from vegetative growth to reproductive growth in plants is a key developmental process.This transformation can be regulated by multiple genes.As we all know,MADS-box transcription factor is an important transcription factor,which participates in the development of flower organs and the regulation of flowering time in plants.Moreover,they play important roles in the development of fruits,stems and leaves.In this study,we analyzed the MADS gene family and confirmed candidate genes related to flower development.In addition,we studied on flower bud differentiation process and transcriptome sequencing analysis of single and double flower to reveal the regulation mechanism of single and double flower development in D.Chinensis.These results provide novel insights into the molecular mechanism of flower development in Dianthus spp..The main findings of this experiment are as follows:1.This study identified 46 MADS-box family genes in the carnation genome using bioinformatics methods,named Dca MADS1-46.The gene structure,evolutionary relationship and conserved motif of these genes were analyzed.39 genes with complete sequence were classified into four categories:30 MIKC~C,2 MIKC*,2 Mα,5 Mγ.The MIKC~C type MADS genes had been divided into 12 subfamilies:Class E(SEP),AG/STK,SOC1,AGL6,AGL17,AGL12,AGL15,AP1/FUL,SVP,Class B(AP3/PI),B sister,FLC.The results of subcellular position prediction revealed that 39genes were localized in the nucleus.Most of Dca MADS genes were more highly expressed in flowers than stems and leaves by Real-time quantitative PCR analysis.We found that these genes had different expression patterns in different developmental stages and different flowering organs.For example,Dca MADS17(FUL1)had a very high expression in bracts and ovary,Dca MADS28(PI)was specifically expressed in petals and stamens.Based on the expression patterns of these genes,we initially proposed a flower-development expression model in carnation.In this model,the Class ABCDE gene ccupied a central position.2.16 Dc MADS(ABCDE)genes were identified from D.Chinensis.The results of the Real-time PCR and in situ hybridization showed that their expression were different in different floral organs.For example,Dc AP1 is widely expressed in the early development of flower primordia,and later expressed in bracts;Dc FUL1 gene is expressed in bracts and sepals early,but concentrated in pistils and styles in the later stage;Dc AP3-1 and Dc AP3-2 are mainly expressed in petals and stamens.Later,it gradually weakened.3.Dc PI2,Dc AG2 and Dc STK genes were involved in the regulation of flower organ formation.The overexpression vectors of 10 Class A-,B-,C-and D-genes were constructed.Transgenic lines were obtained by floral dip method.Overexpression of two genes(Dc PI2,Dc AG2 and Dc STK)in Arabidopsis plants resulted in different phenotypic alteration.35S::Dc PI2 transgenic Arabidopsis showed petaliod sepals,sepals and petals enlarged.35S::Dc AG2 transgenic Arabidopsis showed early flowering,short stigma.They growed weakly,and had little rosette leaves with deformity petals and stamens;35S::Dc STK transgenic Arabidopsis showed that petal elongation restricted and its fruit set rate is reduced.These results indicated that the genes such as Dc PI2,Dc AG2 and Dc STK,were involved in the regulation of floral organ development.4.Protein interaction between ABCDEs in D.Chinensis.16 Dc MADS(ABCDE)genes were identified from Dianthus,and 11 of them were constructed into yeast two-hybrid prey and bait vector.And all of these genes were not self-activating.Yeast two-hybrid assay was used to verify the interaction between the two.Some proteins interacted with each other suggestting that they played important roles in flower development.For example,Dc FUL1 of Calss A can interact with Calss B and Calss E proteins.Dc PI2 of Class B interacts with Class A/B proteins and also with Class C/D/E genes.In Class E,Dc SEP3-1,Dc SEP3-2,Dc SEP4-2 and Dc SEP4-3 had stronger interactions with other proteins,and they formed different heterodimers,indicating that they played inportant roles in flower development.5.We performed a comparative analysis of floral morphology between single flower and double flower.The flower bud differentiation process of D.chinensis was divided into six periods:the stages 1(S1)represented stages of floral initiation and bract primordium formation,S2-S5 corresponded to sepal,petal,stamen and carpel primordium formation,and S6 represented the late flower development stage.Compared with single flower,the structure of flower organs in double flower was disorder,and its’number of stamens and petals was increased.The primary primordia of double flower produced secondary primordia resulting in more petals and stamens.6.The transcriptome sequencing analysis was carried out for the six flower bud development stages of single flower and double flower in D.chinensis.More than280.93 Gb high-quality clean data(PE150,Q30>89.95%)were generated for 36samples.The reads were mapped against the carnation genome using HISAT2 with a mapping ratio ranging from 67.72%to 71.28%.The bam files were processed via String Tie,which generated a consensus transcriptome assembly with a total of 60,409gene loci,including 56,126 known and 4,282 novel gene loci.Using WGCNA,stage-specific gene network modules were identified based on RNA-seq data.Three modules were classified,including MEblue,MEpurple and Melightyellow,which showed significant difference between single and double flowers.A number of important regulatory genes were identified as key genes operating in the double-petal formation network.The genes related to floral meristem identity,primordia position and polarity were found to be more active in double flower than single flower.Further,the results of in situ hybridization assay suggested that Dca AP2L,Dca LFY,Dca UFO genes play important roles in petal boundary and double flower formation.