| The WRKY gene family is one of the most important transcription factor family in plants,which is widely involved in plant growth and development.As important regulatory factors at the transcriptional level,WRKY gene can be used as the activator or suppressor upstream of many genes involved in growth and development to participate in complex regulatory response networks.On the one hand,with the advent of the whole genome sequencing era,more and more species of genetic databases have been established,and there are more and more reports on the the classification,structural characteristics and evolutional characteristics of WRKY gene family;On the other hand,more and more studies showed that many WRKY transcription factors were involved in the regulatory pathways of plant response to environmental stress,and played important roles in these pathways.The function of WRKY transcription factors is not only confined to the process of stress response,but also involved in the reproductive growth of plants.In this research,We found a few WRKY genes related to pollen development in e FP of Arabidopsis thaliana and we seeked out the homologous genes in Brassica campestris through BRAD.We found that BcWRKY31 is the only transcript of At WRKY31 in Brassica campestris.We explored the function of BcWRKY31 through evolution analysis of WRKY gene family,promoter expression analysis and WRKY31 overexpression transgenic plant analysis.The main results are summarized as following:(1)The evolution characteristics of WRKY family was identified by the evolutionary analysis.There were 138 members of the WRKY genes in Brassica campestris.These WRKY genes all went through WGT and WGD,The WRKY transcription factors were divided into three families Ⅰ,Ⅱ,Ⅲ and the family Ⅱ was divided into five sub – families(Ⅱa,Ⅱb,Ⅱc,Ⅱd,Ⅱe).(2)During the evolution of the WRKY gene family of Brassica campestris,most of new WRKY genes after WGT and WGD were preserved.Phylogenetic analysis and co-linear analysis of WRKY gene family between Arabidopsis and Brassica campestris,the number of genes in Brassica campestris has changed greatly after the whole genome replication event and the tripling of the genome,and it is found that the loss rate of the gene is far less than the average loss rate of the Brassica campestris gene 60%,this suggests that most of the new WRKY genes are preserved after the replication event.(3)There was a functional complementation of WRKY genes associated with pollen development in Arabidopsis thaliana.Pollen viability of wrky31 was observed by pollen staining,and there was no significant difference between wild type pollen and mutant pollen.The q RT-PCR results showed that the expression of the other WRKY genes was up-regulated while At WRKY31 was down-regulated at the same time.Therefore,the up-regulated genes can make up for the defect of At WRKY31.(4)We found the promoter of BcWRKY31 had a high transcriptional activity in maturity pollen and the abnormal development of pollen morphology in BcWRKY31 overexpression plants suggested that BcWRKY31 might be involved in the development of pollen grains.First,we cloned the promoter of BcWRKY31 and construced the promoter expression vector successfully,and the analysis result showed the promoter of BcWRK31 showed transcriptional activity specificly in maturity pollen grains.Then we constructed BcWRKY31 overexpression transgenic plants by genetic transformation in Brassica campestris.After that,the morphology and cytology of transgenic plants were observed,the results showed that there There was no significant difference between transgenic plants and control plants in vegetative growth stage,neither of the morphological development of floral organs.But we found that about 70 % of the pollen grains of over-expression plants showed abnormal appearance when scanned by SEM.It is suggested that BcWRKY31 plays an important role in the pollen development of Brassica campestris. |
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