Phenotypic Analysis, Gene Identification And Functional Validation Of Arice Short-grain Mutant 08sg2

With the rising of the world's population and the decreasing of land resources, grain issues is more and more concerned by people as the primary issue relating to the national economy and the people's livelihood. Therefore, speeding up the development of agricultural science and technology to enhance the grain yields has become the common goal of all breeding researchers. Rice (Oryza saliva L.) is an important grain crop and a significant model plant. The mainly rice yield components are the number of effective panicle per plant, effective grains per panicle and 1000-grain weight. Otherwise, 1000-grain weight is controlled by grain width, grain length, grain thickness and length/ wide ratio. Hence, research the decisive factors of rice grain size can help us explain the genetic mechanism of rice yield from the molecular mechanisms, clarify rice growth and development process from gene level and provide a strong theoretical basis and technical support for rice high yield technology.Our study group acquired a short grain mutant sg2 (small grain2) which is selected from the EMS mutant library of the heavy panicle hybrid rice Shuhui 498, showing a phenotype of shortened grain, dwarf and shorter panicle. The result of genetic analysis and recessive plant re-sequencing proves the mutant is controled by two genes, one of which is 08sg2, the target of my research. The phenotype of 08sg2 fall in between R498 and sg2. The main conclusions of our study are as follows:1. We obtain the candidate genes from high linkage section of re-sequencing. Meanwhile, RNA-seq dates of spike at different developmental stages show that only one target gene LOC_Os08g07760 (OsBAK1) in this section obviously expressed higher in glumes. In addition, qPCR results show a lower expression of OsBAK1 in the mutant glume than wild type. Afterwards, recessive plants detected by dCAPs and the transgenic plants knocked by CRISPR/Cas9 of OsBAK1 show a consistent short grain phenotype.All the evidences above prove that the mutant 08sg2 is a weak allele mutant of OsBAK1.2. OsBAK1 is a member of the serk family which contains four homologous genes in rice. What's more, OsBAK1 is highly homologous with AtBAKl in Arabidopsis thaliana, which is a BR signaling kinase receptor interacting with receptor BRI1. Sequencing of mutational sites and bioinformatics analysis show that the mutant 08sg2 has a single base substitution G/A in the ninth exon, CDS of 929th bases of OsBAK1 causing a mutation of GGT/GAT (Gly>Asp).(The aspartate is a acidic amino acid.)3. In order to further understand the function of OsBAK1, we develop a gene evolution analysis and expression pattern research. Consequently, data for the evolution in different species shows that the gene and its mutation sites is extremely conservative. Besides, the gene still has lots of highly homologous genes in rice. In addition, expression profile collecting from the biological website and qPCR results of R498 in different parts and different developmental stages come to the same conclusion that the gene has a quite high expression in the young spike development process.4. SEM observation in the glume and epidermal of R498 and 08sg2 shows no significative change in cell size. At the same time, semi thin sections comparison of the first internode of 08sg2 and R498 has a similar result. All the datas tell us that a reduction in the number of cells leads to a dwaft and short grain in08sg2.5. Through the classical BR sensitivity tests, including the leaf angle test, the root growth experiment of coleoptile and root growth, skotomorphogenesis experiment, draw a conclusion that the mutant 08sg2 shows a BR semi insensitive phenotype relative to R498.