Screening Of Brus - 301 Inhibitors Of Rhesus Slicitrosteroid Mutant, Gene Cloning And Function

Brassinosteroids (BRs) are a type of vital phytohormone and play important roles in a wide range of developmental and physiological processes such as stem, elongation, vascular differentiation, male fertility, seed size and germination, flowering time, senescence, and resistance to biotic and abiotic stresses. Researches have figured out the main biosynthesis and the signal transduction pathway through several-decade effort. They have begun paying attention to the crosstalk between BR signaling and other signal pathways in recent years. In order to find novel components which participate in BR or abscisic acid (ABA) signaling we carried out suppressor screening in bril-301,a BR receptor weak allele.We used ethyl methanesulfonate (EMS) to mutate about ten thousands of bril-301 seeds and M1 were divided into 22 groups. Then we sowed M2 seeds on 1/2 MS with 0.75μM ABA to screen ABA insensitive or larger candidates according to the germination rate and growth phenotypes. Finally, we obtained four types as follows:First, four candidates are hyper insensitive to ABA and have obvious growth phenotypes; Second, two candidates are hyper insensitive to ABA and have no obvious growth phenotypes; Third, six candidates are slightly insensitive to ABA and have no obvious growth phenotypes; Fourth, twelve candidates have obvious growth phenotypes.We select one mutant numbered 16-19 to study further from above candidates. 16-19 can suppress the phenotype of bril-301 and shows higher hypocotyl, longer petiole, taller height and earlier flowering. By the methods of map-based cloning and next generation sequencing we identified the defects of red light receptor, PHYB resulted in the phenotype of this mutant. The transition of guanine to adenine caused 599th tryptophan became a stop codon and resulted in early termination of PHYB protein. We named it phyB-69.We studied the relationship between phyB-69 and BR utilizing the methods of physiology, biochemistry and molecular biology and came to following conclusions: The output of BR signal is enhanced in PHYB deficient mutant phyB-69; The defects of PhyB affects the transcriptional level of BR biosynthesis genes, up-regulates CPD and DET2 significantly and therefore promotes BR biosynthesis; light signaling and BR have antagonistic roles in regulating Arabidopsis hypocotyls and petioles during photomorphogenesis.