Screening And Functional Analysis Of Flowering Regulatory Genes From The MADS Box Family In Wild Radish

Wild radish(Raphanus raphanistrum)is a cruciferous radish plant,mainly distributed in North America,Australia and Europe,and has also been found in Sichuan Province,Qinghai Province,Taiwan and other places in China,at present,this type of weed has become one of the important malignant weeds in wheat fields and rape fields around the world.Under the pressure of long-term natural evolution and artificial selection,some wild radish populations have phenotypic differentiation of early and late flowers in terms of flowering time.In order to explore the genes that play a key regulatory role under this differentiation,the transcriptome sequencing results were first analyzed,and a number of MADS-box family candidate genes that may be related to the regulation of early and late flowers were screened;the expression of the candidate genes was verified by fluorescence quantitative PCR;finally,the expression of two candidate genes with significant differences was obtained by Agrobacterium-mediated method and the function was verified.The following results were obtained:1.Taking the population(NF)with no change in flowering time as the control,taking the early flower(EF)population and the late flower(LF)population as the study object,201,045 differential genes were initially obtained by transcriptome sequencing analysis.Among them,under the same growth period,1657 early flower upregulation differential genes and 1625 late flower downregulation differential genes were obtained.2.The transcriptional regulator(TF)family classification of the above differentially expressed genes was found to be 2838 unigenes divided into 59 TF families.The MYB family had the highest(382,13.5%),followed by the AP2-EREBP family(275,9.7%)and the b HLH family(231,8.1%).In addition,MADS-box family genes(149,5.3%)associated with plant flowering regulation were also found.3.Fluorescence quantitative PCR verification results show that there is differential expression of flowering regulator genes in 6 MADS-box families.Among them,compared with normal populations and late-flowering populations,the expression of Rrc25＿p9 genes and Rrc12115＿p1 genes in early-flowering populations increased significantly,indicating that Rrc25＿p9 genes and Rrc12115＿p1 genes may regulate the flowering process by increasing their expression in wild radish.4.Through gene cloning,the full length of the candidate gene was obtained,and it was found that the Rrc25＿p9 gene consisted of 594 nucleotides,encoding a total of 197 amino acids,and the Rrc25＿p9 protein had 1 SRF-TF functional domain and a k-box domain,and the subcellular results showed that the gene was more likely to be localized in the plasma membrane and nucleus,without secretion of signal peptides,and without transmembrane domain.Rrc12115＿p1 gene consists of 648 nucleotides,encoding a total of 215 amino acids,Rrc12115＿p1 protein has an SRF-TF functional domain,has a 6-amino acid nuclear localization signal,no secretion signal peptide,no transmembrane domain.5.Through the construction of phylogenetic trees between different species,it is found that the Rrc12115＿p1 gene and the AGL61 gene are clustered into one class,and the Bootstrap value is high,indicating that the Rrc12115＿p1 gene is closely related to AGL61,which may be related to the guidance of the pollen tube and the initiation of endosperm development of plants involved in AGL61,as well as the regulation of partial flowering.It was found that the Rrc25＿p9 gene and the MAF5 subfamily were clustered into the same category and the self-expanding value was high,indicating that the Rrc25＿p9 gene was closely related to the MAF family and may be similar to the late flower function performed by MAF5.6.Using Agrobacterium-mediated method,the Rrc25＿p9 and Rrc12115＿p1 were overexpressed in Arabidopsis thaliana,and it was found that Rrc25＿p9 overexpressed Arabidopsis plants were delayed in flowering by 3-6 days compared with WT and no-loaded Mock,and the number of rosette leaves was reduced by 3-5 pieces,indicating that the Rrc25＿p9 gene may have the function of participating in the regulation of late flowers of plants.Rrc12115＿p1 overexpressed Arabidopsis plants had no significant differences in flowering time,number of rosette leaves,and plant height compared with WT and no-loaded Mock,indicating that Rrc12115＿p1 gene is not truly overexpressed in Arabidopsis thaliana or its single overexpression cannot have an important impact on the phenotype of Arabidopsis thaliana.