Mapping And Cloning Of DNL5 Gene In Rice Dwarf And Narrow Leaf Mutant And Initial Analysis Of Its Role In Disease Resistance

Plant morphology has an important influence on rice yield.Plant height and leaf shape are among the important agronomic traits of rice strains,which directly affect rice’s resistance to collapse,nutrient utilization and photosynthetic efficiency.Dwarf and disease resistant resources are very important materials in agricultural production and in the field of rice functional genomic research,and their application can substantially improve the yield and quality of rice.Therefore,the mining and utilization of new dwarf and disease resistance genes can further elucidate the molecular mechanisms of rice plant dwarfism and disease resistance,which is important for crop improvement.In this study,the dwarf and narrow leaf mutant dwarf and narrow leaf in Hejiang 5（dnl5）was obtained from T-DNA transformation of the japonica rice variety Hejiang 5,which was identified as being derived from somatic asexual lineage variation.The dnl5 mutant was mainly analyzed including phenotypic identification,histochemical section analysis,genetic analysis,gene mapping and cloning,gene expression analysis,transcriptome sequencing analysis,and disease resistance analysis.The main findings obtained through this series of studies and analyses are as follows:1.Material identification of dnl5 mutant:dnl5 is not a mutant phenotype caused by T-DNA insertion and belongs to an occasional mutation in tissue culture.The phenotype of dnl5mutant is caused by alteration of the other loci.2.Phenotypic characteristics of the dnl5 mutant:the dnl5 mutant exhibited two typical agronomic traits of plant dwarfism and narrow and curly leaf throughout development.Compared with the wild-type（WT）,the dnl5 mutant also exhibited phenotypes such as enlarged leaf angle,reduced pollen fertility to about 20%,significantly reduced spike length,uniformly shortening of the length in the first,second and third internodes,reduced seed setting rate,and altered grain shape.The observation of the paraffin sections showed that the dwarfism of the mutant plants was caused by the reduced number of cells in internode of the mutant;the narrowing and curling of the mutant leaves was due to the decreased number of small leaf veins,significantly smaller bulliform cells and changed distribution of large leaf veins.3.Genetic analysis showed that the F₁ progeny of the dnl5 mutant showed normal plant height relative to the parents.In the F₂ progeny,the segregation model of normal to dwarf stature fitted well to the expected ratio of a single inheritance,3:1,demonstrating that the dnl5phenotype was controlled by a single recessive gene.Using F₂ progeny genetic population and the parents,combined with genome sequencing,finally localized the candidate gene to the OsCSLD4 in chromosome 12.Using transgene complementation and knockout experiments,it was demonstrated that the OsCSLD4 was the causal gene of dnl5.4.RT-PCR analysis showed that the DNL5 gene was expressed at low levels in the leaf blade,leaf sheath and culm at heading stage and at high levels in the shoot and root at seedling stage.The results showed that the DNL5 gene was relatively highly expressed in young tissue.5.Transcriptome analysis of dnl5 mutant:analysis of DEGs of dnl5 mutant and WT showed that a total of 867 differentially expressed genes were detected in dnl5 mutant,of which 183 genes were down-regulated,while 684 genes were up-regulated,indicating that mutation in DNL5 affected the transcription levels of more genes in mutant plants.GO and KEGG analyses show that the vast majority of differentially expressed genes in the dnl5mutant are involved in plant growth and development,stress response and plant pathogen interaction mechanisms.6.The expression of most genes involved in plant immune response in the dnl5 mutant in CHIT and Os PR were significantly higher than that in the WT at both seedling and tillering stages using quantitative real-time polymerase chain reaction（q PCR）.The chitinase activity assays of dnl5 mutant and WT showed that the dnl5 possessd a higher chitinase activity than WT.The results of Xanthomonas oryzae pv.oryzae（Xoo）inoculation showed that the dnl5mutant showed an enhanced resistance to Xoo strain PXO99A compared with the WT.