Construction Of Transciption Factor Interaction Network In Brassica Napus Developing Seed

Brassica napus is one of the most important oil crops in the world,it's also one of the main oil crops in China,the seeds oil of Brassica napus accounts for 47% of the total yield of vegetable oils.Although in recent years,the oil yield per plant is constantly rising,but Chinese Brassica napus' oil shortage is still huge.Thus,improving oil content is one of the main research hotspots in the area of crop planting.Transcription factors can regulate gene expression in the form of complexs and some studies demonstrated that some transcription factors are involved in the regulation of oil synthesis in rapeseed.Therefore,constructing interaction networks among transcription factors in rapeseeds can reveal the regulation mechanisms of oil synthesis,and it can also make great influence in raising the oil yield of Brassica napus.Through the combination of yeast two-hybrid technology and nextgeneration sequencing method,this study intends to screen interactions among transcreiption factors in Brassica napus in a large-scale and multiple-repetition basis.The main results are as follows:(1)Successful cloning of 279 specifically and highly expressed transcription factors in Brassica napus seeds.Using the transcriptome database and the gene chip database,we screened transcription factors with a relatively high level of specific expression in Arabidopsis seeds,and sorted them on the basis their expression level.Combining oil synthesis related candidate transcription factors that we tracked via quantitative trait locus(QTL)technology in our laboratory,we finally got 500 transcription factors.We identified homologous genes corresponding to Arabidopsis transcription factors from rapeseed database,carried out GO enrichment analysis of these transcription factors,and successfully cloned 279 transcription factors.(2)Expression library and interaction screening construction.Using TOPO directional cloning technology,we transferred transcription factors into entry vectors,and obtained 168 entry vectors.We transferred transcription factors from the entry vector into p ADLOX and p DBLOX expression vectors respectively by using Getway technology and got 127 AD expression vectors and 129 DB expression vectors.AD and DB expression vectors were transferred into Y8800 and CRY8930 yeast strains respectively based on positive identification of bacterial liquid PCR.Finally we obtained AD yeast library containing 105 transcription factors and DB yeast library containing 116 transcription factors.Strains containing interaction pairs were selected through the conjugation of the expression library,and their interaction efficiency was calculated as well.In addition,we analyzed the interaction protein pairs using second-generation high-throughput large-scale sequencing.(3)Interaction network construction and key transcription factors analysis.We screened out 34 interaction pairs of 33 transcription factors according to the sequencing result,constructed corresponding networks based on interaction results.According to their corresponding metabolic pathways analysis,we found some proven interactions,such as the interaction between ABI5 and DPBF4,we also found some new interactions like the interaction between GIF1 and FLC which is related with seed size.In summary,this study successfully constructed transcription factors libraries of Brassica napus seeds and their interaction network.By mapping the transcription factors interaction network,we found out some important transcription factors,which make a difference in regulating Brassica napus characteristics such as seed size,oil synthesis,plants stress resistance ability,etc.It has laid a foundation for related research oil content increasement and stress resistance of Brassica napus.