Analysis Of Transcription Characteristics For Floral Development And Functional Study Of Relevant MADS Box Genes In Rong’An Kumquat

Kumquat (Fortunella crassifolia Swingle) belongs to Fortunella plants of Rutaceae, having a closely relationship with sweet orange (Citrus sinensis Osbeck) and satsuma orange (Citrus unshiu Marc). The flowering and fruiting Characteristics of kumquat differ from other citrus,which can shoot, bloom on the new branches and bear fruit for several times in only one year. It is a distinctive material for the study floral induction and development in citrus. Rong’an kumquat which is the main product in Guangxi province was used as the plant material in this study. By high throughput sequencing analysis, basic transcription characteristics for floral development were figured out. At the same time, we found some MADS box family genes with crucial regulatory function involved in floral development. By homology-based cloning, sequence information of 37 MADS box genes was obtained and series of bioinformatics analyzes were performed as the same as cluster analysis. The expression levels of all MADS box genes were then tested using Semi-quantitative RT PCR and RT-qPCR. The results showed that 12 of the 37 genes had directely relationship with floral development. Construction of plant expression vectors of 6 key genes were all completed and 2 of the genes were transformated into Arabidopsis thaliana to perform more function studies. Besides, in order to obtain more accurate quantitative results of gene expression, we selected several reference genes for different tissues and abiotic stress conditions. The main results were summerised as follows:1. Transcriptome sequencing analysis of Rong’an kumquatBy microscopic observation, processes of morphogenesis and differentiation during floral development were analyzed and the samples for anslysis of transcriptome sequencing and expression profile were final determined.88,712 transcripts and 46,361unigenes were obtained by high-throughput sequencing with Rong’an kumquat flower samples. Distribution characteristics of SNP and SSR in the transcriptome were then preliminarily analyzed. Based on the results of sequence alignment to transcriptome data in the database of whole-genome of sweet orange, it was verified that this transcriptome data of Rong’an kumquat had the better representative and reliability and could be used as reference sequences for further expression profiling studies.2. Digital Gene Expression Profiling Analysis during floral development of Rong’an kumquat.Digital Gene Expression Profiling was performed in five samples from differently floral development. By clustering analysis in GO and KEGG, the gene expression characteristics was campared in different developmental stage and some genes and pathways were found to play key roles in the regulation of floral organ formation, such as genes involed in plant hormone signaling, sugar metabolismand, and transcription factors. During this analysis, MADS box family genes encoding series of transcription factors were found that they might play some crucial roles in floral developmetal process.3. Selection of reference genes for gene expression analysis of Rong’an kumquat in various tissues and under abiotic stressIn order to obtain more accurate results of the data in testing gene expression levels, this study evaluated the stability of 16 candidate reference genes of Rong’an kumquat using the methods of geNorm, Normfinder, and Bestkeeper in different tissues of Rong’an kumquat under some type of abiotic stress. The results showed that that ACT7 alone or the combination of ACT7+ACT1+EF1-α or ACT1+ACT8 could meet the requirements for the normalization of RT-qPCR studies under the experimental conditions tested here.4. Cloning, bioinformation analysis, and expression analysis of MADS box genes from Rong’an kumquat37 MADS box family genes were cloned by homology cloning strategy. Bioinformation analysis of nucleic acid and amino acid sequences were performed. Clustering analysis of their amino acid sequence showed that the 37 genes distributed in 13 subfamilies of MADS box genes. Testing of Semi-quantitative RT PCR in different tissues showed that 15 of the 37 MADS box genes had directly relationship with regulation of floral development in Rong’an kumquat. Expressiong level testing of RT-qPCR showed that each of 12 key MADS box genes had a certain expression characteristic and played various and important rolein floral regulation process.5. Functional verification of some key MADS box genes for floral development by genetic transformation into ArabidopsisThe six key MADS box genes such as FcSEPl, FcSEP1-2, FcGLO, FcAGL11, FcDEFH21, and FcAGL61-2 were selected for further functional verification studies. Eukaryotic expression vectors of all the 6 genes were constructed, and FcSEP1-2 and FcGLO were transformed into Arabidopsis by Agrobacterium-mediated transformation. Functional analysis showed that vegetative growth of transgenetic seedlings was inhibited and expression levels of some genes invloed in flowral development in Arabidopsis were extremely changed. Functional verification of FcSEP1-2 and FcGLO was preliminarily studied.