Cloning And Functional Analysis Of RBH1 That Affects Rice Panicle Degeneration And Mapping Of Panicle Development Gene

Rice,as monocot model plant is an important food crop,so it is of great significance to explore the genes regulating rice panicle development and clarify their functions.Panicle development regulated by multiple genes is a complex biological process,and it is one of the key factors affecting rice yield.In this study,an important gene RBH1(Rice panicle Bale Head 1)was identified by screening T-DNA insertion mutant library.The mutantion of RBH1 gene resulted in panicle degeneration.Genetic and biochemical analysis showed that RBH1 gene encodes a pectin lyase and participated in the scavenging of ROS in meristem cells.In order to find more key genes controlling panicle development,we screened EMS mutant library and identified 10panicle mutants.Mutmap method was used to clone the genes of panicle mutants.1.Cloning and functional analysis of RBH1 gene1.1 Cloning of RBH1 gene and expression pattern analysis of RBH1Two rbh1 gene mutants,named rbh1-1 and rbh1-2,were identified in this study.They showed similar mutation defect.The rbh1-1 was used as the material to carry out the detailed study.The apical spikelets of rbh1-1 were abnormal,defective and some of them degenerated into filaments.The secondary branches and grains per panicle of rbh1-1 mutant were notebly declined.The rbh1-1 showed obvious stamen and pistil defects during the formation of floral primordia.The panicle growth rate of rbh1-1mutant was dramatically decreased,which indicated that RBH1 might be essential for panicle development.TAIL-PCR confirmed that the T-DNA tag in rbh1-1 mutant was located in the LOC?Os10g31910,LOC?Os10g31910 was annotated as a pectate lyase.The complementary test showed that the transgenic plants in rbh1-1 mutant background recovered wild phenotype.The results indicated that LOC?Os10g31910 mutation resulted in the panicle degeneration phenotype of rbh1-1,LOC?Os10g31910 is the RBH1 gene in this study.The qRT-PCR result showed that RBH1 was a constitutive gene and expressed in all tissues.The in situ hybridization found that RBH1 m RNA gradually accumulated during the development of young panicle and reached the peak at the floret formation stage.1.2 Functional analysis of RBH1 geneRBH1 protein shared similar sequence and same functional domains with Arabidopsis PMR6 protein and Lotus japonicus Lj NPL protein.The in vitro enzyme activity of induced protein showed that RBH1 protein in wild type had the ability to decompose pectin.The result of immunostaining showed that pectin in rbh1-1 young panicle was notable accumulated than that in WT,which indicated that RBH1 protein had the function of pectin degradation.The results of DAB staining showed that rbh1-1 mutant had excessive accumulation of ROS.The ROS scavenging related genes in rbh1-1 mutant was remarkable promoted.These results indicated that the apical panicle defect in rbh1-1mutant resulted in excessive accumulation of ROS.Different concentrations of H₂O₂ were used as exogenous ROS to treat Zhonghua11(ZH11)callus.2 m M H₂O₂ could effectively promote the differentiation ability of callus,while 10 m M and 20 m M H₂O₂ significantly inhibited the differentiation of callus.These results indicated that the level of ROS affects cell differentiation,and appropriate ROS level can guarantee the normal differentiation of cells.RBH1 gene probably regulated cell differentiation during panicle morphogenesis by affecting ROS level.2.Mapping of panicle development genes in riceTen panicle mutants were collected from EMS mutant library.We have completed the phenotypic isolation,identification of five F₂ populations.Five panicle mutants were mapped by mutmap technique,and candidate genes of four panicle mutants were preliminarily obtained.The panicle mutant f13 was accompanied by severe growth defects and showed extreme dwarfing.F13 gene is located on chromosome 2 and encodes a functional protein containing AP2 domain.We searched the expression profile data of ricexp public database(https://ricexpro.dna.affrc.go.jp/index.html)and found that f13 was specifically expressed during the panicle development.The early termination of F13 protein translation in f13 mutant led to the destruction of F13 protein.These results indicated that F13 may be involved in the regulation of panicle development in rice.Based on the results of this study,RBH1,cloned by T-DNA tag encodes a pectin lyase,which was involved in the balance of reactive oxygen species in the process of rice young panicle differentiation and affected rice panicle morphogenesis.We mapped the candidate genes of four panicle mutants by Mutmap technology,which provided genetic resources for further study of rice panicle development.