Functional Analysis Of Related Rice Grain Size Genes And Strategic Research On In-Frame Mutants To Create Elite Varieties

Rice yield is mainly composed of several basic components,including the number of effective panicles per unit area,grain number per panicle and grain weight.The grain size is the main factor affecting the grain weight,but also an important indicator to measure the rice appearance quality.Explaining the genetic regulation mechanism of grain size is conducive to the improvement of rice yield and rice quality.Although several genetic systems for controlling rice grain types have been identified,their regulatory networks are still not perfect.Using bioinformatics methods and genome editing technologies to mine more rice grain type genes helps to understand the molecular regulation mechanism of rice yield formation,and provides more genetic resources for high-yield and high-quality rice breeding.1.Functional analysis of OsCKX8Rice cytokinin oxidase OsCKX8 was a functional gene that negatively regulates rice grain size.The knockout mutants of OsCKX8 obtained by the CRISPR/Cas9 system increased the grain length and the 1,000-grain weight,while the over-expressed lines showed the opposite traits.The outer epidermal cells of mature seeds and the outer parenchyma cell layer of pre-flowering glumes in OsCKX8 mutants were observed by scanning electron microscopy and paraffin section respectively.The results showed that the OsCKX8 mutants mainly increased the grain length mainly by promoting cell division and increasing the cell number.OsCKX8 transcripts were detected in stem,leaf and sheath,but relatively stronger in panicles.When the young panicles developed to 12-14 cm,the expression levels reached a peak.Subcellular localization analysis indicated that OsCKX8 was mainly expressed in cell membrane,nucleus and cytosol.The level of cytokinin in the OsCKX8 knockout mutants was increased.Thus,the sensitivity to exogenous 6-BA was enhanced,and the elongation of the shoot and the development of the root were more restrained.In contrast,the OsCKX8 mutants exhibited a slower response to gibberellin GA3.It was speculated that OsCKX8 may be involved in the regulation of grain size by cytokinin and gibberellin pathways.2.Cloning and functional analysis of OsGRL1 geneThe possible target genes in QTL-qGRL1.1 were analyzed by bioinformatics,which had been finely mapped to rice chromosome 1.The candidate gene LOC_Os01g66970(OsGRL1)for controlling grain length in qGRL1.1 was identified by the CRISPR/Cas9 system.The mutant plants with knockout OsGRL1 gene showed the traits of increased grain length and 1,000-grain weight.However,the grains of transgenic lines overexpressing OsGRL1 became smaller and lighter.The results indicated that OsGRL1 was a functional gene that negatively regulated grain size in rice.OsGRL1 encoded a C4HC3 type RING v domain and an unknown functional DUF3675 domain,which was distributed on both nucleus and cell membrane.The integrity of RING v domain was necessary for transcriptional activation.Analysis of RNA-Sequencing data and the verification of differentially expressed genes revealed that besides the changes of the expression level in the grain size genes,there were a large number of differentially expressed genes related to abiotic stress in the knockout mutants and overexpression lines of OsGRL1.The results of stress treatment suggested that OsGRL1 may be involved in the response of rice to salt stress.The stress treatment experiment preliminarily speculated that OsGRL1 may be involved in the response to salt stress.These findings suggested that OsGRL1 may affect rice grain size and salt tolerance in a variety of pathways.3.Strategic research on in-frame mutants to create elite varietiesThe CRISPR/Cas9 technology was used to efficiently edit the miRNA recognition sequence of the target gene,eascaping from the miRNA suppression.Then,the in-frame mutants that enhanced the expression levels of the target gene in the miRNA-resistant mutant strains were screend for improveing the elit traits.The OsGRF4 gene,which was inhibited by miR396d and regulated the grain size,was selected to obtain a series of miR396d-resistant mutants through the CRISPR/Cas9 system.There were two types of OsGRF4 mutants in the miR396d-resistant mutants:the in-frame mutations and frame-shift mutations.Among them,the expression level of in-frame OsGRF4 mutants was increased,enlarging the grain size.While the frame-shift mutants exhibited the opposite phenotypes.The results suggested that the in-frame OsGRF4 mutants successfully escaped from the miR396d supression to enlarge grain size.Simultaneously,the rc gene with frame-shift mutation and functional silence was successfully restored by the CRISPR/Cas9 system.Hence,the white rice was converted to the anthocyanin-rich red rice in the in-frame mutants.Taken together,the stragegy for employing CRISPR/Cas9 system to obtain the in-frame mutation of both the miRNA recognition sequence and the frame-shift mutants to create elite varieties was feasible.