Molecular Mechanism Of Rice Grain Shape Control By OsRhoGAP2 Over-expression

The high yield and quality breeding of rice has always been an important target of rice research.Grain shape is one of the key factors determining rice yield,and is closely related to the quality of rice.Although a variety of signaling pathways have been found to be associated with grain shape control in rice,and many QTLs controlling grain shape have been identified,the molecular mechanism of regulating grain size is still largely unknown.Previously,taking rice Rop gene OsRacD as bait,the putative interacting protein encoding genes were screening by yeast two hybrid from rice young panicle cDNA library,and a unknown Rho GTPase activating protein encoding gene was isolated,named OsRhoGAP2.To identify its function,the pCAMBIA1302-OsRhoGAP2-eGFP vector was constructed,and obtained the over-expression transgenic rice(OE-OsRhoGAP2).It was found that the grain shape of T2 transgenic rice changed significantly,which showed that the grain length was longer,the grain width was narrower and the grain thickness became thinner.In this study,we focus on the relationship between OsRhoGAP2 and rice grain shape which taking the T3 and T4 generations of OE-OsRhoGAP2 rice as materials.T3 generations of OE-Os RhoGAP2 rice were screened,and verified by qRT-PCR,the results showed that 7 transgenic lines were obtained,which the expression level of OsRhoGAP2 were up 2.48~17.55 times compared with control.The phenotypic analysis showed that the morphology of the mature seeds of the transgenic rice was the same as that of the T2 generation transgenic rice,also characterized by the long grain length,the narrow grain width and the thinner grain thickness.The identification of T4 generations of OE-Os RhoGAP2 rice were the same as that of the T3 mentioned above,the results showed that 95% of the T4 transgenic rice were positive.qRT-PCR analysis showed that in the 19 individual rice,the expression of OsRhoGAP2 gene was up to 1.35~26.29 times.The expression of OsRhoGAP2 during the 14 days after the florescence were detected,the abundance of its transcripts were accumulated in a gradual decline in 8 days after flowering,but then increased,and up to the peak in 14 th days,which up to 3.28 times of that in 1st day after flowering,suggest that OsRhoGAP2 gene may involved in rice seed development processe.The expression of 5 amyloid synthase encoding genes OsSSIIa,OsSSIVb,OsSSSI,OsGBSSI andOsSBEI in the immature seeds of T4 transgenic rice was detected by qRT-PCR.The results showed that the expression of OsSSIIa and OsGBSSI increased in immature seeds,while the expression of OsSSIVb decreased slightly,but the expression of OsSSSI and OsSBEI genes had no significant changes.The rice seed quality analysis showed that,compared with the wild type seeds,the water content of the transgenic rice seeds increased by 3.5%,and the content of the total starch and amylose increased by 12.3% and3.12%,respectively,but the protein decreased by 18.52%.There was no significant difference in fat content and gel consistency.It is suggested that the over-expression of OsRhoGAP2 gene may improve the accumulation of starch in seed by promoting the expression of OsSSIIa and Os GBSSI in the development of seed.The expression of cell proliferation related genes were analyzed by qRT-PCR.The results showed that the expression of cyclin-dependent kinase gene R2 and histone encoding gene H3 increased significantly in T4 transgenic plants,which were 2.32 times and 3.15 times compared with that of control,respectively.The glumes surface of the transgenic rice seeds was observed by scanning electron microscope.The results showed that compared with the wild type rice,the epidermal cells of the transgenic rice hull were shortened longitudinally and the number of cells increased significantly.It can be deduced that seed shape change in OE-OsRhoGAP2 transgenic rice were due to the increase the expression of cell proliferation gene,and the increase the number of glume shell cells.The expression of known grain marker genes GS3,GS5,GW2 and GW8 were also analyzed by qRT-PCR.The results showed that the expression of grain width gene GW2 was significantly enhanced to3.07 times,while the expression of grain length gene GS3 was significantly drop to 0.79 times,suggested that the length of grain length,narrow grain width and thinning of OE-OsRhoGAP2 transgenic rice may be related to the expression changes of GS3 and GW2 genes.In vitro rice pollen staining and pollen germination results showed that the fertile pollen and the pollen germination rate of the transgenic rice were all higher than that of control,suggested that over-expression of OsRhoGAP2 may enhance pollen activity by promoting starch accumulation.Statistical analysis of rice agronomic traits showed that the number of tillers and seed setting rates of the transgenic plants of the T4 generation increased significantly compared with the wild type,and the spike length,the number of primary branches,the number of spikes and the thousand grain weight were significantly different,but the number of effective tillers and the total number of total grains of the T4 plants were not significantlychanged,suggested that Os RhoGAP2 gene is related to the control of rice yield traits.In this paper,the seed morphological changes in T3 and T4 generation of OE-OsRhoGAP2 transgenic rice were detected and analyzed by molecular and cytological methods.It was found that the over-expression of OsRhoGAP2 gene up-regulated the expression of OsSSIIa,OsGBSSI,R2 and H3 genes,and promoted the accumulation of starch in the seeds and the proliferation of glume cells.As a result,the grain shape and seed nutritive constituents both changed in OE-OsRhoGAP2 rice.Meanwhile,the statistical analysis of agronomic characters of transgenic rice suggested that OsRhoGAP2 gene may be of potential value in the breeding high yield and high quality rice.