OsGAD2 Influences Grain Growth And Development In Rice

Glutamate decarboxylase（GAD）is widely found in plants,animals and microorganisms.It is mainly responsible for converting glutamic acid decarboxylase into Gamma-aminobutyric acid（GABA）branch,which is also an important rate-limiting enzyme in GABA branch.Currently,the GAD family has been reported to play a regulatory role in the growth and development of Arabidopsis thaliana,tomato,melon and other plants,but whether the GAD family members regulate the growth and development of rice grains and other traits and the regulatory mechanism remains to be further explored.In this paper,OsGAD2 was used as the research object to clarify the basic characteristics of OsGAD2 expression in rice and create homozygous transgenic rice with excessive expression of OsGAD2.Combined with the existing OsGAD2knockout rice mutants,the regulation effect of OsGAD2 on grain traits such as grain shape,grain weight and germination rate was investigated.This study laid a foundation for further analysis of the role of OsGAD2 in the growth and development of rice and its mechanism,and provided a basis for molecular design breeding and cultivation of excellent rice varieties.The main results are as follows:1.The basic expression characteristics of OsGAD2 were clarified in riceGUS staining showed that OsGAD2 expression level was higher in the spikelet at the P7 and P8 stages,and in the seeds on the 1st day and 20th day after pollination,lower in the spikelet at the P6 stage,and weak in the roots,stems and leaves of rice seedlings.Subcellular localization analysis showed that OsGAD2 was mainly expressed in the nucleus.2.OsGAD2 overexpression and functional complement transgenic rice were createdUsing ZH11 and OsGAD2 knockout mutant as genetic background,three T₂transgenic homozygous rice lines with overexpression of OsGAD2 and 14 functional complement transgenic T₀ rice plants were created using RT-PCR and rice genetic transformation techniques.3.OsGAD2 positively regulates rice seed setting rateThe analysis of seed setting rate at mature stage showed that,compared with the control,the seed setting rate of the OsGAD2 knockout rice mutants was significantly reduced by 13.1-18.0%,while the seed setting rate of the OsGAD2 overexpression rice was significantly increased by more than 19.1%,indicating that OsGAD2 gene has a positive regulation effect on the seed setting rate of rice.4.OsGAD2 regulates rice grain sizeCompared with the control,the grain length,grain width,grain thickness and1000-grain weight of OsGAD2 overexpressed rice had little changes,but the yield per plant increased by more than 32.7%.The grain width of OsGAD2 knocked out mutant changed little,the grain length significantly increased by 8.4-9.0%,the grain thickness significantly decreased by 4.5-6.2%,the yield per plant decreased by13.1%-31.0%,and the 1000-grain weight did not change significantly.5.OsGAD2 regulates grain germination,seed vigor and seedling growth of riceThe test results of seed germination,seed vigor and seedling growth showed that compared with the control,OsGAD2 knockout mutants had greater effects on seed germination rate and seed vigor,while OsGAD2 overexpression rice had less effect on seed germination rate and seed vigor,and OsGAD2 had a negative regulatory effect on rice seedling growth.6.Changes of GABA content and OsGAD2 enzyme activity in rice grainsCompared with the control,the OsGAD2 enzyme activity and GABA content in the OsGAD2 knockout rice grains were significantly decreased after soaking for 24 h,while the OsGAD2 enzyme activity and GABA content in the OsGAD2overexpression rice grains were significantly increased after soaking for 24 h.These results indicated that OsGAD2 had a positive regulatory effect on the content of GABA in rice grains.7.Effects of OsGAD2 on rice qualityCompared with the control,OsGAD2 knockout mutation significantly improved the grain viscosity and chalkiness ratio.OsGAD2 overexpression significantly increased the amylose content of rice grains,but significantly decreased the chalkiness of rice grains.