Expressional And Functional Analysis Of OsRBR2 In Rice

RB gene is the first identified tumor suppressor gene,with its analogous gene extensively found in animals,plants and microorganism.Based on the existing research,RB gene plays an important role in the regulation of cell proliferation,cell differentiation,cell apoptosis and organogenesis.RBR,a homologous gene of RB,exists mostly in plants.The basic function of RBR is similar to RB,which regulates cell cycle.Since plants have specialized cell structures,tissue features,growth and development ways that are quite different from animals,we speculate that there may be a new specific mechanism in plants through which RBR regulates growth and development of cells.Rice contains two retinoblastoma-related genes namely OsRBR1/2.However the specific function and mechanism of action are not yet clear.Our group obtained the OsRBR1 mutant Osrbr1-1,and the expressional and functional analysis shows that OsRBR1 is a key gene that specifically controls the floral organogenesis of rice.On this basis,the project makes a primary research on the expression and function of OsRBR2.The main contents and results obtained include:(1)Expressional analysis of OsRBR2:The qRT-PCR analysis shows that OsRBR2 expresses almost in all tissues and organs.However the gene's expression level of vegetative organs(root,stem and leave)is apparently higher than that in young panicle tissue.To further analyze the overall expression characterizations of OsRBR2 in rice,we constructed fusion expression vector pCAMBIA1391Z-pOsRBR2 in which OsRBR2 promotor and GUS were expressed simultaneously.Then transformed into callus of Zhonghua 15 and obtained 9 transgenic plants.GUS histochemical assays showed that the gene was expressed in vegetative organs,such as root,stem and leaf(due to the time constraints,the gene was expressed at lower levels and the expression in panicle could not be detected).This result is roughly identical to that of qRT-PCR.It is speculated that OsRBR2 may exert a more important role in the vegetative growth of rice.(2)Functional analysis of OsRBR2 and its gene interaction with OsRBR1/2:-Construct an overexpression vector pTCK303-OsRBR2 that was driven by Ubi1 promoter.Then transformed into callus of Zhonghual5 and obtained 10 transgenic plants.At present,the plants are just growing.The analysis of morphological feathers and function could not be able to carry out.Construct twro editing vectors(that is two editing target sites)by CRISPR/Cas editing systems,transform to callus of Osrbr1-1 homozygous mutant.So far,the first editing vector has differentiated into 26 transgenic plants.Of which,6 plants are Osrbrl-1 homozygous mutants,and the rest is wild type.Sequence analysis showed that these plants realized target site editing successfully.These plants are just at vegetative growth stage.Compared with wild type,the growth and development of the transgenic plants is completely normal.(3)Subcellular localization:Construct fusion expression vector pGDG-OsRBR2 in which OsRBR2 cDNA and GFP were expressed simultaneously.Then transformed into onion epidermal cells.The results showed that fusion protein expresses specially in the nucleus.Thus we assume that OsRBR2 is a nucleoprotein that plays an important regulatory role in cell nucleus.