Mapping Cloning And Functional Analysis Of Low-Tillering Dwarf1(D26) Gene In Rice(Oryza Sativa L.)

As an important food crop,plant type is one of the most important factors affecting rice yield in the process of transformation from traditional experience breeding to modern precision breeding.It depends on plant height,tillering number,tillering angle,spike shape and other factors.It is a difficult scientific and technical problem to study the mechanism of rice plant type in a short period of time.Dwarfing mutant is a very ideal research object,but the regulation mechanism is poorly understood.Based on the previous work,this paper systematically studied the D26mutant of dwarfing and less tillering rice,and revealed the signal pathway mechanism of brassinolide in rice regulation.In this paper,two mutant materials of D26-1 and D26-2 with low tillering and dwarfing agronomic characters were obtained from tissue culture mutant plants in a rice mutant library.Through map based cloning,the fine location and further functional analysis were carried out.The genetic mechanism of dwarfing phenotype of D26 mutant was revealed,and the dwarfing mechanism of rice was further explored from the perspective of interaction protein screening and transcriptome sequencing analysis.The main results were as follows:D26 mutant is a recessive single gene controlling phenotype.The hybrid F₂populations were established with D26 mutant as female parent and 93-11 parent as male parent.D26 gene was precisely located in the region of 63 kb on the short arm of chromosome 3 by 425 populations and 531 populations,respectively,and the region contained 12 candidate genes.The prediction analysis,cloning and sequencing showed that D26 gene was a transcription factor which encoding GRAS family protein,and the two mutants caused the early termination with the deletion of a base C in 540 bp and 560 bp respectively.The wild-type D26 gene was transferred into D26 mutant,and it resulted in the recovery of the mutant phenotype.In addition,the second internode of D26 mutant was specifically shortened,and the plant had the erect phenotype.These suggested that D26 gene might be involved in Brassinolide（BR）signaling pathway.Furthermore,the BR hormone insensitivity experiment showed that D26 mutant was a dwarf mutant which was insensitive to exogenous BR.The results of subcellular localization of rice protoplasts showed that the expression of D26 was distributed in the whole cell.Combined with the relative quantitative analysis of the upstream and downstream related genes of BR signaling pathway,the results showed that D26 gene was not only involved in the BR response mechanism,but also a positive regulatory factor in the BR signaling pathway of rice.In order to understand the mechanism of D26 gene controlling rice grain type,we designed the over expression and gene knockout experiments of D26 gene.The results showed that in the over expression plants,the change of rice grain type was in direct proportion to the expression of D26 gene.The expression was higher,and the grain was the longer and slender.In the plants of gene knockout,the grain became the wider and shorter.The data of forward and reverse genetics showed that D26 gene could regulate rice grain development through BR positive response.The results of scanning electron microscopy showed that the expression level of D26 gene directly affected the degree of cell division of rice glume epidermis,and thus regulated the shape of rice grain.Combined with bioinformatics prediction and GST pull-down technology,the results speculated that D26 protein,as a transcription factor of GRAS family,might be mainly related to the members of rice zinc finger protein family.It was preliminarily speculated that D26 protein may be involved in brassinolide pathway regulation through the interaction with rice zinc finger protein.The analysis of GO enrichment data from transcriptome showed that the variation of differential genes mainly focused on the expression of genes in molecular function and biological process,but not in cell components.Compared with the wild type,the total number of differential genes was increased in the mutant after treated with exogenous BR.Compared with the mutant,the genes with significant changes in the differential genes were mainly down-regulated expression,and only 15 genes were up-regulated expression.The enrichment data of KEGG pathway showed that D26 mutant mainly caused significant changes in the expression of three pathways.On the one hand,it affected the gene differential expression of taurine and low calcium ammonia metabolism and glycolysis/gluconeogenesis pathway,on the other hand,it mainly affected the diterpenoid biosynthesis pathway,which corresponded to gibberellin biosynthesis,and the expression upstream genes of GA biosynthesis was regulated.Compared with the wild type,4 genes in the upstream of GA biosynthesis were up-regulated,which promoted the pathway of GA biosynthesis,and 5 genes in the upstream of GA biosynthesis were down regulated,which inhibited the pathway of GA biosynthesis.In conclusion,two D26 mutants with dwarfing and less tillering types were used in this study to elucidate the mechanism of their participation in brassinolide（BR）signaling pathway and positively regulated the rice grain type.The results of pull down protein interaction showed that D26 protein did not play an independent role in vivo,and it might work together with the rice zinc finger protein.The relationship between exogenous and endogenous brassinolide（BR）and gibberellin in rice was obtained by transcriptome sequencing of KEGG enrichment pathway,and showed that exogenous BR inhibited the biosynthesis of GA.All of these would broaden the understanding of D26 protein function,and effectively supply the regulatory mechanism of brassinolide in rice.Furthermore,it would lay a theoretical foundation for the next application research.