Phenotypic Analyses And Gene Cloning Of Rice Albino Mutant Alb35 And Dwarf And Low-Tillering Mutant S3106

Part 1:Rice is not only an important food crop,but also a monocotyledon model plant.Compared with other agronomic traits,the leaf color of rice is easy to observe,which is an ideal trait for studying chloroplast development in higher plants.Chloroplast is a special organ of plants and algae.Its development not only affects the photosynthetic rate,but also determines the yield of food crops.In rice,many key genes for chloroplast development have been identified,but little is known about the protein homeostasis system in chloroplast.In this study,we screened an albino mutant named alb35（albino mutant 35）from the chemical mutation libraries.Phenotype identification and gene cloning of alb35 mutant were studied:（1）In order to identify the key genes regulating chloroplast development,we constructed a MNU mutant libraries based on Ningjing 3 and screened a chloroplast development defective mutant from the libraries.Compared with the wild type,alb35showed complete albinism at seedling stage and death after three leaf stage.During the development of chloroplast at seedling stage,alb35 had no form chloroplast and thylakoid was seriously damaged.The content of chlorophyll and carotenoid in leaves decreased significantly.（2）In order to clone the mutant gene that causes albino phenotype of alb35 mutant,F₂was constructed by a cross between the alb35 and 9311.Genetic analysis showed that albino phenotype of alb35 is controlled by recessive single gene.Using map-based cloning,we located the mutant gene in the region of 103 kb between K5-3 and K5-4 markers in the long arm of chromosome 5.Sequencing analysis showed that a fragment of 5 bp was deleted in the exon of the 12th gene in this region which encode CRISPR/cas9 gene knockout assay proved LOC＿Os05g49970 mutation resulted in albino phenotype of alb35.（3）Through NCBI predicting LOC＿Os05g49970 encodes a chloroplast precursor like translation initiation factor cpIF2.The cpIF2 gene encodes a protein with 955 amino acids.Subcellular localization experiment showed that cpIF2 protein was located in chloroplast as dots.We found that there were two phenotypes,strong mutation albinism and weak mutation etiolated.（4）qRT-PCR of plastid development genes and Western blotting showed that cpIF2might be involved in plastid protein translation system,and cpIF2 protein was essential for chloroplast development of rice seedlings.In addition,plastid proteases Fts Hs have the function of degrading damaged proteins.The development of chloroplast requires the synergistic effect of cpIF2 and Fts Hs.The cloning of cpIF2 provides a reference for further elucidating the pathway of chloroplast protein synthesis and degradation,it also provides a new theoretical basis for the improvement of rice yield.Part 2:Plant height and tiller are important agronomic traits in rice.The high plant is prone to lodging,resulting in yield reduction The tiller also affects rice yield.In this study,we identified an endosperm defective mutant s3106 with dwarf and low-tillering in a radiation mutant libraries.Phenotype identification and gene cloning of s3106 mutant were studied:（1）In order to identify the key genes involved in the regulation of rice architecture,we constructed a radiation mutant libraries based on Ningjing 3 and screened a rice architecture mutant s3106.Under natural growth conditions,s3106 mutant showed yellow green in the whole growth period compared with the wild type.At the same time,s3106mutant also had the phenotype of dwarf,low-tillering,delayed heading and endosperm defective.Hydroponic experiment showed that s3106 mutant had more main roots and more lateral roots than the wild type.（2）In order to clone the gene causing the phenotype of s3106 mutant,F₂ was constructed by a cross between the s3106 and Dular..Genetic analysis showed that the floury endosperm phenotype of s3106 mutant was completely linked to the dwarf and low-tillering phenotypes.The mutation was located between HH 9 and HH 12,about 450kb.Sequencing analysis showed that there was about 95 KB deletion in this region.There are 12 ORFs in this region,and there is a gene FLO4 regulating endosperm development.Previous studies have shown that FLO4 mutation does not lead to dwarf and low-tillering and root morphological changes.Therefore,the other 11 ORFs were analyzed,and the fourth ORF（LOC＿Os05g33550）was found the loss of may result in the decrease of plant height,tiller number and root morphology in rice.（3）Based on NCBI forecast:LOC＿Os05g33550 encodes a 186 amino acid CW zinc finger protein.MBD4（methyl CpG binding domain protein 4）is an important domain of the gene,which indicates that the gene may have methylation function.The mutation of homologous gene At MBD4 in Arabidopsis thaliana will result in significant decrease of plant height and abnormal increase of phosphorus uptake in mutant.Excessive phosphorus uptake during the growth period of rice will lead to chlorosis.The cloning of MBD4 gene provides a reference for further elucidating the regulation of MBD4 on rice plant height and tiller,it also provides a theoretical support for the improvement of rice yield.