| Flowering Chinese cabbage(Brassica campestris L.ssp.chinesis var.utilis Tsen et Lee),is annual or biennial herb belonging to Brassicaceae and is one of the subspecies of Chinese cabbage.Flowering Chinese cabbage is currently one of the largest cultivated special vegetables in South China and is now cultivated throughout the country.The product organ of flowering Chinese cabbage is flowering stalk,the development of flowering stalk directly related to the yield and quality of flowering Chinese cabbage.In production and research,it is found that abnormally low temperature will lead to early bolting and flowering,which will affect the yield and quality of flowering Chinese cabbage.However,i the molecular mechanisms of abnormal temperature in bolting of flowering Chinese cabbage are not well understood,which limits the promotion of flowering Chinese cabbage and the development of high-yield technology.WRKY proteins,which plays an important regulatory role of great number of processes(e.g.stress resistance,flowering,stem development),are a kind of plant specific transcription factor and one of the largest transcription factor families.Therefore,in this experiment,the early flowering Chinese Cabbage variety “youlv 501” was selected as the material to investigate the biological characteristics of the flowering Chinese cabbage under low temperature(15℃)treatment,and the realtime fluorescence quantitative PCR was used to screen the WRKY gene of the flowering Chinese cabbage in response to low temperature.Molecular biology methods(e.g.gene cloning,bioinformatics analysis,expression analysis and functional verification)were used to analysize the regulation of WRKY transcription factors in the induction of early bolting and flowering of flowering Chinese cabbage during the process of low temperature induction.The foundation of the bolting mechanism of flowering Chinese cabbage is built and also it provides theoretical guidance for the production technology of the flowering Chinese cabbage.The main results are as follows:1.The flowering Chinese cabbage was treated with low temperature at 15°C for different time.(0 h,24 h,and 48 h).The results showed that the low temperature treatment at 15°C could early bolting and increase the plant height of the flowering Chinese cabbage,but reduce the stem diameter of flowering Chinese cabbage.With the prolonged treatment time of low temperature,the influence of low temperature on bolting and blooming of flowering Chinese cabbage was more obvious.In addition,low temperature treatment can increase expression levels of BcSOC1-1 and BcSOC1-2 genes in leaves and stems during different treatment periods.2.The WRKY transcription factors responding to low temperature stress in flowering Chinese cabbage were screened by using realtime fluorescence quantitative PCR.The results showed that there were 9 WRKY genes up-regulated in different stages,and there were 7 WRKY genes up-regulated in different stages by low temperature treatment.Both BcWRKY22 and BcWRKY70 were up-regulated in leaf and stem.3.The cDNA of BcWRKY22 and BcWRKY70 were cloned by RT-PCR methodand the genes ORFs were 897 bp and 852 bp respectively.The similarity of the BcWRKY22 and BrWRKY22 between flowering Chinese cabbage and Cabbage’s cabbage reached 98%.The similarity of the BcWRKY70 and BrWRKY70 between flowering Chinese cabbage and Cabbage’s cabbage reached 100%.Phylogenetic tree analysis showed that BcWRKY22 and BcWRKY70 had high homology with Brassicaceae.The amino acid sequence and protein structure analysis showed that both BcWRKY22 and BcWRKY70 contained the WRKY domain typical of the WRKY family.4.The heterologous expression of flowering Chinese cabbage gene BcWRKY22 in Arabidopsis thaliana revealed that transgenic plant could be bolt ahead of time compared to wild-type,and the expression levels of AtSVP and AtFLC in stems and leaves of transgenic plants were lower than those in wild-type,but the expression levels of AtSOC1 was higher than in wild-type.In addition,the expression levels of AtSVP and AtFLC were lower in stems and leaves of transgenic plant treated at low temperature,while the expression level of AtSOC1 was higher.5.In yeast one-hybrid experiments,the pGADT7-WRKY22 expression vector was transformed into the pABAi-SOC1 yeast bait yeast and yeast were grown on SD/Leu-deficient media containing the appropriate AbA-inhibitory concentration,demonstrating that WRKY transcription factors can be combined.The promoter of the flowering-related gene SOC1 indicates that the WRKY transcription factor may have a regulatory effect on the flowering-related gene SOC1.In summary,the transcription factor BcWRKY participates in the regulation of bolting and blooming of flowering Chinese cabbage at low temperature(15°C).Low temperature may up-regulate the expression of BcWRKY22 gene,and then up-regulate the expression of BcSOC1,a flowering flower related gene,and bolting and blooming of flowering Chinese cabbage in advance. |
