Effects Of OsGAS2 On Pollen And Seed-setting Rate In Rice

GAS2 family protein widely exists in eukaryotes,which is the glucosidase IIβ-subunit and plays an important role in glycoprotein processing,cell apoptosis and some signal transduction.At present,there are few reports on the phenotypic function and molecular mechanism of GAS2 genes in plant growth and development,while the phenotypic function of OsGAS2 gene in rice growth and development and its molecular mechanism have not been reported.In this study,the basic characteristics of OsGAS2 gene and its encoded protein were analyzed by bioinformatics.The expression differences of OsGAS2 gene in different tissues and organs of rice were identified by qRT-PCR.Based on CRISPR technology,RNA interference technology and rice genetic transformation technology,the T₀ and T₁ generation mutants of OsGAS2 and the T₀ generation down-regulation transgenic rice of OsGAS2 gene were created.It was found that the T₀ heterozygous knockout mutation of OsGAS2 gene seriously affected the pollen development and seed-setting rate in rice,and the T₁ homozygous mutation also seriously affected the vegetative growth of rice.It laid a foundation for further exploring the phenotypic function of OsGAS2 in growth and development of rice and its molecular mechanism,and provided theoretical guidance for molecular breeding and breeding of excellent rice varieties.The main results are as follows:1.The main characteristics of rice OsGAS2 gene and its encoded proteinOsGAS2 is located on chromosome 1 of rice,with a total genome length of 6963bp,16 exons,a transcript length of 2408 bp and a CDS length of 1845 bp.The molecular weight of OsGAS2 is about 69.3 k Da,and the p I of OsGAS2 is 4.76.OsGAS2 has many conserved domains of GAS2 family protein in plant,including LDLa domain,signal peptide and PRKCSH domain.2.The expression pattern of OsGAS2 in riceThe results of qRT-PCR showed that OsGAS2 gene may be constitutively expressed in rice,with the highest expression in leaves and higher expression in panicles and grains at different developmental stages.3.Establisment of T0,T1 knockout mutants of OsGAS2 and RNA interference T0 transgenic rice plantsBy CRISPR/Cas9 gene editing technology and rice genetic transformation technology,three types of non off-target T₀ generation heterozygous mutants（11plants）were established,and the three types of mutants were-45 bp,-34 bp and-41bp,respectively.The seeds of T₁ from T₀ generation were germinated and planted.A total of 6 T₁ knockout plants with two homozygous mutation types were obtained.The mutation types were-45 bp and+T,respectively.Five T₀ transgenic rice plants of OsGAS2 down-regulated expression were established by RNA interference and rice genetic transformation.It laid a material foundation for further study on the role of OsGAS2 in rice and its molecular mechanism.4.The serious effect of OsGAS2 T0 heterozygous mutation on the pollen development and seed-setting rate in riceThe results of I₂-KI staining showed that the proportion of fertile pollen in T₀mutant of OsGAS2 was significantly decreased,and the fertile pollen rate was less than 30%.The seed setting rate of wild-type rice and OsGAS2 T₀ knockout heterozygous mutant was observed at maturity.The seed setting rate of wild type was about 69.7%,while the seed setting rate of 8 heterozygous mutants was 0%,and the number of grains per plant of the other 3 mutants was only 7,3 and 4,respectively.These results suggest that OsGAS2 gene may affect rice seed setting by regulating pollen development.5.Obtaining a number of candidate genes co-regulating rice pollen development with OsGAS2The results of qRT-PCR showed that the expression levels of OsGAS2 and DPW in the wild type were significantly higher than those in the OsGAS2 mutant,and the expression levels of the other seven reported genes related to pollen development regulation in the wild type were significantly lower than those in the OsGAS2 mutant,indicating that OsGAS2 and some differentially expressed genes may co-regulate pollen development in rice.6.The serious effect of OsGAS2 T1 homozygous mutation on the vegetative growth of riceThe seeds harvested from T₀ knockout plants of OsGAS2 were germinated and planted in the field at the same time as the wild type.The observation at tillering stage showed that T₁ homozygous mutation of OsGAS2 seriously affected the vegetative growth of rice.We speculate that the T₁ homozygous mutation of OsGAS2 will also seriously affect the reproductive growth of rice,but it needs to be confirmed by later experimental results.