Characterization Of MADS-box Transcription Factor Gene Family And Biological Functional Validation Of RsFLC And Micror172 In Radish (Raphanus Sativus L.)

Radish(Raphanus sativus L.,2n=2x=18)has high nutritional value,medicinal value and good storability.Therefore,it has been widely cultivated in the range of China and even the whole world.When radish was cultivated in spring,it is easy through vernalization at low temperature,which seriously affected the quality and the yield.MADS-box family played a very important role in flowering and floral organ formation,Recently,genome-wide identification of MADS-box genes have been reported in many plant species.However,the genome-wide analysis of MADS-box genes in radish remain lacking.Moreover,FLC and miR172 that played important roles in plant bolting and flowering have been studied extensively in Arabidopsis thaliana,but most of studies are in terms of gene cloning and mechanism prediction in radish.Especially their biological functions have not yet been validated effectively in radish.In the present study,MADS-box gene family was identified and characterized in radish whole genome,and the miRNA mediated gene silencing was used to validate the biological functions of RsFLC.Additionally,the biological functions of radish miR172a was validated by over-expression.Research results promote the understanding of radish MADS-box genes structure and functions,and provide a new perspective for analyzing the biological functions of radish bolting and flowering related genes and miRNA.The main results were as follows:1.In the present study,a comprehensive analysis of MADS-box genes was performed,and a total of 144 MADS-box family members were identified from entire radish genome.Through the phylogenetic analysis between radish and Arabidopsis,RsMADS genes were classified into two groups including 68 type Ⅰ(31 Mα,12 Mβ and 25Mγ)and 76 type Ⅱ(70 MIKCC,6 MIKC*).Among them,41(28.47%)RsMADS genes were located in nine linkage groups.Next,the homologous MADS-box gene pairs were identified among radish,A.thaliana,Chinese cabbage and rice.Additionally,the expression profiles of RsMADS genes were investigated in special tissues and different leaf stages based on previously reported RNA-Seq transcriptome data.Then,quantitative real-time PCR analysis indicated some MIKCC genes are differentially expressed in different parts of floral organs,while other MIKCC genes show different expression under different GA concentrations,photoperiods and vernalization.All of these results strongly demonstrate that the MADS-box genes play important roles in the development of floral organs and in the control of flowering.2.Based on the previous studies,21nt amiRFLC was designed to specifically silence radish FLC gene on the WMD website according to radish and Arabidopsis FLC gene sequence.On the basis of the Arabidopsis miR319a precursor,miR319/miR319*was replaced by amiRFLC/amiRFLC*using overlapping PCR to get the pre-amiRFLC.Subsequently,pCAMBIA2301-pre-amiRFLC vector containing amiRFLC was constructed through double enzyme digestion,then the recombinant vector was introduced into Arabidopsis by floral dip method mediated by Agrobacterium.The transgenic Arabidopsis plants were identified at the level of the genome,transcription and phenotype.The results showed that the gene pre-amiRFLC was successfully integrated into the genome of Arabidopsis,and this gene can be successful transcribed into amiRFLC.In addition,the transgenic lines showed obvious early flowering,and target gene FLC was significantly down-regulated expression.The study provided new ideas and theoretical guidance for functional validation of genes in radish.3.Radish miR172a precursor sequence(103bp)was isolated through homology cloning.The method of biological information is used to analyze the homology,phylogenetic relationship and secondary structure among radish and other plant species.At the same time,RsAP2(1305bp),RsTOEl(1314bp)and RsTOE3(1077bp)(targeted by miR172a)were isolated according to radish genome and the unigene sequences obtained from the transcriptome sequencing.Subsequently,miR172a over-expression vector(pCAMBIA2301-pre-miR172a)was constructed through double enzyme digestion,then the recombinant vector was introduced into Arabidopsis by floral dip method mediated by Agrobacterium.The transgenic Arabidopsis plants were identified at the level of the genome,transcription and phenotype.The results showed that the gene pre-miR172a was successfully integrated into the genome of Arabidopsis.This precursor can be successfully transcribed into miRl 72a,and target genes AP2,TOE1 and TOE2 were significantly down-regulated expression.In addition,the transgenic lines showed obvious early flowering.Thus radish miR172a can promote the bolting and flowering which was verified in Arabidopsis.