Application Potential Assessment Of OsRhoGAP2 Gene And Its Promoter In Rice Molecular Breeding

Rice is the main food crop and is also an important model for plants.To authenticate the functional genes of important agronomic traits in rice has an important value.At the early stage of our laboratory,we mainly study the signaling pathway related to rice sterility.We isolated a number of genes from a cDNA library of young spike of rice which interact with OsRacD,including a Rho GTPase,named OsRhoGAP2?GenBank accession number: 364311?.In this study,we want to evaluate the application potential of this gene in molecular design breeding through authenticating the gene's fuction and analysing its promoter.At the early stage of our laboratory,we cloned the promoter of OsRhoGAP2 gene and constructed plant expression vector,fusing OsRhoGAP2 promoter and five 5'-terminal deletion fragmentto the GUS reporter gene.And then we transfered them to Arabidopsis and obtained transgenic Arabidopsis.We want to understand the function of different segments of the promoter through detecting the activity of GUS gene in T3 generation of transgenic Arabidopsis.GUS histochemical analysis,enzyme assay and deletion analysis showed that there might be an important anther-specific element between-703 bp to-289 bp at the upstream of the promoter's 5' end,which drived GUS gene to express in the anther of transgenic Arabidopsis,especially in the early stage of microspore development,but not in root,stem,leave and fruit pod.We speculate that OsRhoGAP2 genes may express in the early stage of microspore development and have an important role in maintaining the normal development of anther.Through spraying five kinds of hormones?IAA,6-BA,GA,SA,MeJA?and five stress treatments?high temperature,low temperature,drought,high salt and dark?to 10 days transgenitic Arabidopsis combined with GUS staining results,we found that the promoter of this gene may be a promoter associated with stress induction.It can drive downstream gene to express under IAA induction and high temperature,drought,high salt,but not respond to other hormones.Then we further speculated that the promoter may be an adversity-inducible promoter.When combined with deletion analysis,it showed that there might be an important MeJA response element at-1292 bp to-948 bp at the upstream of the promoter's 5' end,which may be involved in the MeJA signaling pathway.Based on the earlier work of our laboratory,this study aimed at understanding the application potential in production through the molecular detection and phenotypic analysis of T₂ generation of transgenic rice.We found that more than 90% of them were positive plants by extracting rice genomic DNA combined with PCR;the gene was overexpressed in transgenic rice by means of qRT-PCR;the effective tiller number increased significantly,which was 32.35%-38.75% higher than that of the wild type by agronomy traits statistical analysis.So,in the molecular design breeding,we can improve the effective tiller number and then improve rice yield by improving the OsRhoGAP2 expression level.In this study,we found OsRhoGAP2 and its promoter have important application potential in molecular design breeding.We found that this gene may express specifically in anther and respond to IAA and high temperature,drought,high salt abiotic stress.So,this gene can be used in the plant genetic engineering,plant strain improvement and improve the resistance of plants.At the same time,we found a method to improve the effective tiller number,and then to increase the yield of rice.