Effects Of OsGAD2 On Plant Height And Grains In Rice

Glutamate decarboxylase(GAD)is a key rate-limiting enzyme in the catabolic pathway of Glutamate,which can decarboxylate Glutamate to form gamma-aminobutyric acid(GABA).It is widely found in bacteria,plants and animals in nature.It has been reported that the plant GAD family plays an important role in the growth and development of plants and in the stress.However,there are limited reports on the role of OsGADs family in the growth and development of rice and the molecular genetic mechanism of its role.In this paper,the main characteristics of OsGADs family genes and their encoded proteins in rice were analyzed by bioinformatics method.The expression pattern of OsGAD2 in different tissues and organs of rice was analyzed by qRT-PCR.A single gene and double target OsGAD2 homozygous mutant was created using CRISPR/Cas9 technology.It was found that OsGAD2 homozygous mutant could significantly affect the plant height,grain length and grain weight of rice.This study laid an important foundation for further studying the role of OsGAD2 gene in rice growth and development and its molecular genetic mechanism.The main research results are as follows:1.The main characteristics of OsGADs gene family and its encoded proteins in rice were identifiedBioinformatics analysis showed that five OsGADs genes were distributed on chromosome 8,4 and 3,respectively.The total length of the gene varied from 2300 bp to 8500 bp,and the number of exons was 4～7.The molecular weight of the protein was 54.53-57.24 k Da.The isoelectric point(p I)is 5.54～7.12;The subcellular localization predicted that OsGAD1～OsGAD4 might be located in cytoplasm,and OsGAD5 might be located in chloroplast.However,except OsGAD2,other members of the OsGADs family have binding sites for binding Ca MBD.The promoters of OsGADs family genes in rice all contain multiple cis-acting elements related to growth and development regulation,light regulation,hormone regulation and stress response,suggesting that they may play an important role in growth and development and stress adaptation in rice.2.The expression pattern of OsGAD2 in different tissues and organs of rice was revealedThe qRT-PCR results showed that OsGAD2 was highly expressed mainly in young panicles and seeds of japonica rice ZH11 under normal conditions.The expression level was the highest in young ears at P2 stage,and decreased sharply in the subsequent development of ears.The expression of OsGAD2 was higher at 3D and5 D stages of rice grain development,but lower at other stages of rice grain development.It is suggested that OsGAD2 may play an important role in the development of tassel and seed in rice.3.A homozygous knockout mutant of OsGAD2 was createdUsing CRISPR/Cas9 technology and rice genetic transformation technology,the T0 generation single gene double target knockout mutant of OsGAD2 was successfully created,and its mutation rate was 100%.Three OsGAD2 knockout homozygous mutants have been successfully identified by generation isolation and molecular identification,and the mutant types are-3bp/+A,+T/+A and +234bp,respectively.The results of off-target analysis showed that the three mutants had no off-target effect and could be used in subsequent experiments.4.Effects of OsGAD2 knockout homozygous mutation on plant height and grain traits in ricePlant height and grain traits of rice were observed and analyzed in the mature stage of rice planted in the field.The results showed that OsGAD2 knockout mutation significantly increased the plant height,grain length and grain weight of rice.The1000-grain weight of the wild type was 24.19 g,and the 1000-grain weight of the three mutants was 28.05 g,26.14 g and 27.45 g,respectively.Compared with the wild type,the 1000-grain weight of the three mutants increased 15.96%,8.06% and13.48%,respectively.