Generation Of Knock-out Lines For BZR1, BES1 And Their Four Paralogs In Arabidopsis Thaliana

BES1 is a critical downstream transcription factor in brassinosteroids(BR) signaling pathway. BES1 belongs to a gene family consisting of six highly homologous members, including BZR1, BES1, BEH1, BEH2, BEH3 and BEH4. Previous studies indicated that BES1 and BZR1 contribute to various important processes for plant growth and development, such as cell elongation, cell division, leaf development, root growth, xylem development, flowering time control, male fertility, stress responses, senescence, etc. At the meantime, researchers also demonstrated BZR1 and BES1 regulate the expression of hundreds of common target genes with other transcription factors such as BIM1, PIF4 by binding to the promoter regions of the target genes. However, most researchers deduced their conclusions by analyzing BES1 or BZR1 gain-of-function mutants, bes1-D or bzr1-1D, or RNAi knock-down plants. No loss-of-function genetic evidence was provided to support the importance of BZR1, BES1 and their subfamily members in regulating plant growth and development. This dissertation focues on elucidating the genetic significance of BZR1, BES1 and their paralogs by generating knock-out lines of BZR1, BES1 and their four paralogs in Arabidopsis thaliana.We purchased all available T-DNA insersion lines of BZR1, BES1 and their paralogs from ABRC. We first demonstrated that five out of six genes are completely knocked-out via RT-PCR analyses. We subsequently tried to generate double, triple, quadruple and quintuple homozygous mutants by genetic crossing. At present, we have gained double, triple, quadruple and quintuple homozygous mutants. After analyzing the phenotypes of all homozygous mutants, we found that the rosette leaf length and rosette width of three-week old plants, bolting time, branch number of 6-week old plants are all similar to those of wild type. We also treated all homozygous mutants with 100 nM BL, 500 nM BRZ to examine their effect on root or hypocotyl growth respectively. Our results showed that the root and hypocotyl length of all homozygous mutants remained unchanged compared to those of wild type seedlings. In order to further validate our conclusion, we generated BZR1-SRDX and BES1-SRDX transgenic plants and carry out similar experiments. Similar results were obtained. In addition, we also found that the change ratio of two BR downstream target genes(CPD, DWF4) in triple or quadruple mutant is also similar to wild type after treated with 100 nM BL. We also analyzed the expression patterns of all these six genes by generating their Promoter::GUS transgenic plants. In conclusion, the results obtained by analyzing the quintuple mutant, BZR1-SRDX and BES1-SRDX transgenic plants suggest that five BES1 transcription factor family may not significantly affect Arabidopsis growth and development. Further analyses are still needed to demonstrate the true biological significance of all six BZR1 subfamily members.