Cloning Of SOC1 Promoter And The Interactions Between SOC1 Promoter And FLC Or SVP In Brassica Juncea Coss

The appropriate timing of flowering is very pivotal for reproductive success in plants, so the plants have evolved complex and sophisticated regulatory mechanisms to control bloom at the most optimal time. Brassica juncea Coss as an important cruciferae brassica vegetable originated in Asia and has a wide range of cultivated area in South China. Therefore, it is necessary to study the flowering genetic regulatory networks of Brassica juncea Coss for production and scientific research. Nearly 30 years studies using Arabidopsis thaliana as a model plant have revealed that flowering genetic regulatory network contains 6 major pathways:photoperiod pathway, autonomous pathway, vernalization pathway, gibberellin (GA) pathway, ambient temperature, and age pathway. These six pathways converage to regulate a small number of "floral integrator genes", which encode different classes of proteins that govern the flowering time by merging signals from multiple pathways. For example, flowering-time integrator gene SOC1 can merge the key regulatory factors of FLOWERING LOCUS C (FLC) from vernalization pathway and SHORT VEGETATIVE PHASE (SVP) from photoperiod pathway. However, it remains unknown whether the expression of SOC1 gene is controlled by FLC and SVP via DNA-protein interactions in mustard.Using Genome Walking method in this research, the SOC1 promoter has been successfully amplified from the mustard genomic DNA via specific primers, which are designed in accordance with the known SOCl gene sequence, from the mustard genomic DNA. The key regulatory elements of the promoter are analyzed via bioinformatics. Subsequently, we have constructed recombinant plasmid of pAbAi-SOC1, pGADT7-FLC and pGADT7-SVP. Then we have also studied the interactions of SOC1 promoter and SVP or FLC proteins, using yeast one-hybrid system, which can provide foundation for the further research of SOCl transcriptional regulation. The results are as follows:1. Cloning and bioinformatics analysis of the SO1l promoterWe extrated genomic DNA from the leaves of Brassica juncea and designed the specific primers in accordance with the SOC1 known sequence. Using Genome Walking method, we successfully obtained 782bp SOC1 promoter sequence. Phylogenetic analysis of the SOCl promoter showed it had the closed relationship with Arabidopsis thaliana SOCl (AtSOCl), but had a distant relationship with Arabis alpine SOC1 (AaSOC1). Bioinformatics analysis of cis-acting elements indicated that the SOCl promoter sequence in mustand contains a core promoter element of TATA-box, an upstream promoter element of CAAT-box and a variety of responsive elements.2. Detection of the interactions between SOC1 promoter and FLC or SVP proteinsWe designed primers with double restriction sits of HindⅢXhoI and subcloned the SOC1 promoter from Brassica juncea gDNA. Then we constructed the yeast recombinant plasmids: pAbAi-SOC1, pGADT7-FLC and pGADT7-SVP. Using yeast transformation system user manual, we inegrated the linearized pAbAi-SOC1 plasmid into YIH yeast genome and got yeast transformant of YIH (pAbAi-SOCl) which could grow normally on SD/-Ura plate with agar after 3-5 days at 30℃. It suggested that the linearized pAbAi-5OC1 plasmid had been successfully integrated into yeast genome. Additionally, we tested the bait strain for AbAr expression on SD/-Ura/AbA* plates and chosed a minimal inhibitory concentration of AbA. The results showed that false-positives could be fully suppressed under AbA100ng/ml. Then we prepared yeast competent cells with Y1H (pAbAi-SOC1) for the co-transformation of another yeast plasmid pGADT7-FLC or pGADT7-SVP. We found that the yeast hybrid combinations of Y1H (pAbAi-SOC1×pGADT7-FLC) and Y1H (pAbAi-SOC1 ×PGADT7-SVP) could grow normally on SD/-Leu/AbA100. It suggested that the SOC1 promoter in Brassica juncea could interact with FLC or SVP proteins. Our results have provided valuable information and solid theoretical basis for further investigation of their interaction sites.3. Prediction of the key interaction domain between SOC1 promoter and FLC or SVP proteinsThe previous research in Arabidopsis thaliana showed that MADS-box proteins can specifically bind to the CArG-boxes of target genes, then activate or inhibit the expression of downstream genes. SVP and FLC are MIKC type MADS-box proteins, so we analyzed the BjSOCl promoter sequence. However, SOC1 promoter sequence in mustard contains three CArG-boxes, which are similar but not equal with that of Arabidopsis thaliana SOC1 promoter. Therefore, we infer the three sites have the same function as Arabidopsis thaliana CArG-boxes and they probably are the interaction sites between SOC1 promoter and FLC or SVP proteins.