Down-Regulated Expression Of OsDCL3b Affects Grain Yield And Quality In Rice

Rice is one of the three most important cereal crops in the world,directly feeding half of the world population.With the rapid growth of global population and improved living condition,studies focusing on improvement in rice yield and quality have become research hotspots.High-yield and better nutritional quality targeted rice breeding based on genetic engineering will be a chief part of them.Plant DCL(Dicer-like)protein is a type of ribonuclease type III endonucleases,which is involved in the biogenesis of non-coding RNA(ncRNA)via double-stranded RNA processing,and thus influence plant growth and development as well as stress adaptation through regulation of gene expression.In this study,we detected significantly enhanced rice quality and reduced grain yield per plant in OsDCL3 b down-regulated plants.Using comparative small ncRNA and mRNA transcriptome analysis on young panicles in OsDCL3 b down-regulated mutants and wild-type(ZH11)rice plants,followed by qRT-PCR and functional analysis in transgenic plants,we also identified differentially expressed ncRNA and mRNA genes associated with rice grain yield and quality.Our study will provide a basis for revealing the genetic and molecular mechanism of rice grain yield and quality formation,and also a theoretical support for genetic engineering based breeding of high-yield and grain-quality rice cultivars.The results are as follows:(1)Investigation on rice grain yield-related traits.Under regular field crop management,grain yield-associated traits of wild-type and OsDCL3 b down-regulated plants were investigated during maturation.Wild-type(ZH11)produced more pollen from each spikelet and the pollen fertility reached nearly 99%,while two representative down-regulated mutant lines 5-3 and 6-1 showed reduced number of pollen grains from each spikelet and significantly decreased pollen fertility,which lead to significantly reduced rice seed setting rate of 20% and 64% in contrast to 92% in wild-type(ZH11).Rice grain yield per mutant 5-3 and 6-1 plant decreased significantly to 7.9g and 19.4g,respectively,compared to 41.3 g per wild-type plant.Significantly reduced rice seed setting rate in CRISPR/cas9-mediated OsDCL3 b knockout plants corroborate that OsDCL3 b might be involved in the regulation of rice yield-related traits.(2)Investigation on rice grain quality-related traits.We tested several rice grain quality indexes in wild-type(ZH11)and OsDCL3 b down-regulated mutant(6-1)according to National Standards of the People’s Republic of China(GB).Compared with wild-type(ZH11),mutant(6-1)rice showed an increase in protein content by approximate 13.9%,in which contents of many of amino acid,especially lysine(~11.9%)increased substantially.No significant change of grain fat and Cr content was detected in transgenic lines.Contents of Pb and Cu were shown to decrease significantly by 32% and 10%.(3)Small ncRNA transcriptome analysis.The biogenesis of 21 nt and 24 nt small ncRNA was shown to be influenced by down-regulation of OsDCL3 b.Using GO enrichment analysis,we found that genes regulated by the down-regulated 21 nt small ncRNAs were categorized into functional groups primarily as cellular process modification,cellular response to stimulus,signal transduction,and biological process modification,and that genes regulated by the down-regulated 24 nt small ncRNAs were categorized as metabolic process,hydrolase activity,and nucleic acid binding.Small ncRNA candidates,including differentially expressed important candidates involved in rice grain yield and quality formation,were identified using qRT-PCR test,which validated the small ncRNA transcriptome data and laid a foundation for identifying the biological function of differentially expressed important small ncRNA candidates based on transgenic techniques.(4)mRNA transcriptome analysis.In comparison with wild-type(ZH11),394 and 644 genes were significantly up-regulated in line 5-3 and 6-1,respectively,in which,178 genes were up-regulated in both lines.102 and 530 genes were down-regulated significantly in line 5-3 and 6-1,respectively,in which,54 were shared in both lines.GO enrichment analysis showed that the up-regulated genes were categorized as metabolic process,catalytic activity,and response to stress,while the down-regulated genes as response to stimulus,developmental process,external and internal cellular component,and translation factor activity.Protein encoding gene candidates,including important differentially expressed ones involved in rice grain yield and quality formation,were identified using qRT-PCR test,verifying the reliability of the mRNA transcriptome sequencing data.(5)Differentially expressed candidate gene analysis.We have purchased rice TDNA insertion mutant seeds for the identified differentially expressed gene candidates,and detected three mutants altered in rice yield traits,one of them for candidate gene(LOC_Os02g01030)encoding HSP40 protein showed a merely 50% seed setting rate in comparison with about 90% in wild-type(Dongjin),and the mechanism of which remains to be studied.Overall,alteration in the expression of OsDCL3 b affected rice grain yield and quality formation through biogenesis of the 21 nt and 24 nt small ncRNA in rice young panicles via regulation on the expression levels of related genes.