The Alternative Splicing Diversity Contributes To Frameshift Mutation Adaptability In Rice

Alternative splicing is widely found in eukaryotes,which allows individual genes to produce different mRNA isoforms,thereby regulating gene expression or producing diverse proteins,which is an important factor in biodiversity.With the development of sequencing technology,more and more splice iso forms and their diverse functions have been discovered,but compared with the large number of alternative splicing events in the whole genome,our understanding of its function and biological significance is still very limited.This study intends to systematically explore the abundance and regularity of alternative splicing of each gene in rice from the perspective of genome-wide;to create a large number of gene-destroyed mutants(frameshift mutation or early termination)by CRISPR/Cas9 gene editing technology.To explore the relationship between alternative splicing and gene-destroyed mutations,and to explore the contribution of alternative splicing to the fitness of gene mutations and its potential biological significance.In this study,99 rice transcriptome data was usded to evaluate the frequency of alternative splicing events across the genome of rice,and found that the alternative splicing genes in rice accounted for 87%of all genes,much higher than previous reports and genome annotations(14%),confirmed the ubiquity of alternative splicing in rice;secondly,a total of 573923 splicing junctions were found,with an average of 15.8 junctions/gene,much larger than the average of 3.3 junctions/genes of the genome annotation;after deep sequencing of 20 gene transcripts,it was found that 53.5%of the junctions detected in 19 transcriptome data were re-detected,indicating the reproducibility and reliability of these junctions.These results indicate that in addition to the annotated junction,there are still a large number of reproducible alternative splice sites in the genome,and the function and biological significance of these alternative splicings need to be further explored.In order to study the biological function of alternative splicing,123 candidate genes were selected in this study,and they were mutated in rice using CRISPR/Cas9 gene editing technology.To explore the contribution of alternative splicing to the fitness of gene mutations.For the 146 different types of mutants obtained,PCR was used to amplify transcripts,and each site was subjected to high-throughput sequencing,statistical analysis and analysis of alternative splicing events of mutant genes and alternative splicing compared with wild type injunction ratio.The final results showed that among the 65 mutant genes,there were 42 genes in which have junctions that could partially restore the mutant protein sequence,accounting for 64.5%of the total number of genes.Among them,24 junctions that can restore the gene protein sequence account for more than 1%of the total gene expression,and belong to 17 different mutant genes.There is a significant increase in the ratio of recoverable junctions in three of the mutated genes(>10%).In order to further explore the relationship between alternative splicing and frameshift mutations,the WDA1(Os10g0471100)gene was knocked out,and five different mutation types were obtained,all showing different phenotypes(such as fertility differences,the difference in pollen viability,etc.),and the same alternative junction that can restore the protein sequence is detected in the fertile individuals,and the transcript containing the junction accounts for only 1.77%of the wild-type expression,while in the mutant The ratio increased to 37.72%-56.30%;by western blot hybridization,the presence of the junction does make the mutant protein recovery occurred,which directly proved that the WDA1 gene can completely or partially restore the gene-destroying mutation by alternative splicing,to cope with the fatal damage of the mutation to the individual.These results fully indicate that many alternative splicing in the genome may function as a "buffer pool" to alleviate the dramatic effects on gene function due to frameshift mutations,thereby increasing the suitability of the gene itself.The above results indicate that there are a large number of reproducible alternative splicing in the genome,which can improve the adaptability of functional disruptive mutations(frameshift mutation,early termination,etc.)that may occur during the evolution of genes,thereby improving the gene evolution process.The suitability,and also provides a new perspective for exploring the production of biological introns and their biological significance.At the same time,from another perspective,this study found that the use of CRISPR/Cas9 technology for the study of gene function has certain limitations,and the result of gene editing should be comprehensively analyzed and judged.