Cloning And Expression Analysis Of Flowering Regulatory Genes SVP And AGL24in Brassica Juncea Coss

Brassica originates in the Mediterranean region, is one of the highest economic value of Cruciferous crops. Due to its high nutritional value, adaptable and multi-variant type, it has became one of the largest cultivation area and the highest production of vegetable crop groups in China. Brassica vegetables includes Brassica juncea, Chinese Cabbage, Brassica oleracea, Brassica napus and Brassia campestris etc. Since Brassica juncea is widely grown in southern China, the study on flowering time regulation of Brassica juncea will generate a significant practical value of scientific research and production.In this research, cDNA encoding flowering inhibitory factor SVP and flowering promoting factor AGL24were isolated by using homologous primers from the leaves of Brassica juncea, the homologous primers were designed with the conserved sequences of the public flowering regulatory gene. Their expression (the expression in various flowering pathways and the expression in different parts of Brassica juncea) was invesitigated by real-time RT-PCR, and the sequence-specific primers was designed according to the sequence. The main results were showed as follows:1. Cloning and analysis of flowering inhibition factor SVPAccording to the conserved sequences of public flowering regulatory genes, homologous primers were designed to amplify SVP gene from Brassica juncea. The gene fragment was739bp, its open reading frame contained726bp and estimated encoding241amino acid residues.Used methods of bioinformatics (such as Smart) to study SVP protein between Brassica juncea and other species, inclding homology and phylogenetic relationships, also predicted structural domain, phosphorylation sites, hydrophilicity, transmembrane regions, signal peptides, subcellular localization and its secondary/tertiary structure. The results were as follows:SVP encoded241amino acid residues, its molecular was about27.36kD, and the isoelectric point was about5.57. The relationship between BjSVP and BrSVP was the closest, the farthest was among BjSVP and StSVP, MnSVP. BjSVP was MIKC type protein, possible phosphorylation sites was12, including10of Ser,1of Thr and lof Tyr, hydrophobic region existed at35-50aa. BjSVP did not contain a signal peptide, did not belong to extracellular secreted proteins or membrane protein, its secondary structure mainly contained a helixsecondary structure and random coil.2. Cloning and analysis of flowering inhibition factor AGL24According to the conserved sequences of public flowering regulatory genes, homologous primers were designed to amplify AGL24gene from Brassica juncea. The gene fragment was666bp, its open reading frame contained666bp and estimated encoding221amino acid residues.Used methods of bioinformatics (such as Smart) to study AGL24protein between Brassica juncea and other species, including homology and phylogenetic relationships, also predicted structural domain, phosphorylation sites, hydrophilicity, transmembrane regions, signal peptides, subcellular localization and its secondary/tertiary structure. The results were as follows:AGL24encoded221amino acid residues, its molecular was about24.90kD, the isoelectric point was about7.67. The relationship between BjAGL24and BnAGL24was the closest, the farthest was between BjAGL24and PtAGL24. BjAGL24was MIKC type protein, possible phosphorylation sites was17, including15of Ser,1of Thr and lof Tyr, hydrophobic region existed at35-50aa. BjAGL24did not contain a signal peptide, did not belong to extracellular secreted proteins or membrane protein, its secondary structure mainly contained a helixsecondary structure and random coil.3. SVP expression analysis under different flowering pathways and in different parts of Brassica junceaSVP gene were assayed by using real-time RT-PCR, in order to analyze the expression levels under different flowering pathways (including the autonomous, vernalization and photoperiod pathways), also in leaves and shoot apex of Brassica juncea. The results showed:BjSVP mainly expressed in the vegetative growth stage, it would reach the highest point before bolting, but when bolting, the expression drastically reduced, then incressed again, overall presented "first up and then down". Also, under the three different flowering pathways,the SVP expression in the shoot apex showed a similar law,but with a different expression pattern in the leaves.4. AGL24expression analysis under different flowering pathways and in different parts of Brassica junceaAGL24gene were assayed by using real-time RT-PCR, in order to analyze the expression levels under different flowering pathways (including the autonomous, vernalization and photoperiod pathways), also in leaves and shoot apex of Brassica juncea. The results showed:Before, during and after bolting, BjAGL24all presented a high expression level, generally it reached the highest point during bolting, it also showed a "first up and then down" trend. In addition, both in vernalization and photoperiod pathways, the expression pattern of AGL24in leaves and shoot apex was similar to each other.