Map-Based Gene Cloning And Functional Analysis Of Two Types Of Rice Narrow-leaf Mutants

Rice is one of the most important grain crops. Leaf is the main organ for photosynthesis and plays an important role in plant growth and development. Leaf morphology directly influences photosynthesis, transpiration, and crop yield. At present, only a few narrow-leaf genes have been cloned. Therefore, cloning more leaf width-related genes will lay a solid foundation for further research on the molecular mechanism of leaf development. In this study, we carried out phenotypic characterization, microscopic observation, map-based gene cloning and functional analysis of two rice leaf shape mutants. This thesis consists of two following parts:Part 1: Recessive narrow leaf mutant sd110 was isolated from an EMS mutated japonica cultivar SD808. Results about the sd110 mutant were listed below:(1)The phenotype of sd110 mainly showed a 50% reduction in leaf width(more severe on the top three leaf), reduced leaf length and plant height, increased tiller number and delayed root development.(2)Through the observation of the leaf-cross sections, we found the number of veins was reduced in mutant leaves. Microscopic observation of leaf epidermal cells showed that the cell size has no obvious change, while decreased cell number is the main cause of narrow-leaf shape.(3)Genetic analysis showed that the narrow leaf phenotype is caused by a recessive gene. The candidate gene was mapped within a 59-kb region on chromosome 3. Sequencing results revealed that one single-base mutation occurred at the junction of the second exon and intron of a ribosomal protein(RP) gene, changing GT to AT. Sequence analysis of the mutant cDNA revealed that the second intron was not spliced or excised at a new junction eight bases in front of that in the wild type, leading to a frame-shift mutation. The mutant phenotype can be rescued by transformation of the wild-type gene into the sd110 mutant. RNA interference of this gene in wild type plants mimicked the sd110 mutant phenotype, which confirmed that the RP gene controls the final leaf size.(4)The results of RT-PCR and histological staining of the promoter:GUS transgenic rice plants showed that the RP gene was widely expressed in various organs, including leaf, seed, column and panicle.(5)The transient expression vector containing the RP gene coding sequence fused in-frame with the GFP gene was constructed and transformed into rice protoplast. Laser confocal scanning microscope observation showed that the RP protein was located in the endoplasmic reticulum where protein synthesis occurs.Part 2: The recessive narrow leaf mutant F2-195 was obtained by 60Co-γ radiation of the japonica cultivar Chun Jiang 06. This study focused on the phenotype observation, microscopic observation, gene mapping and functional analysis. Results about F2-195 are as follows:(1)The mutant phenotype mainly includes narrow leaf, open-spikelet, smaller seed and increased tiller number. However, little change in the plant height and leaf length was observed. Stereoscopic microscope observation of leaf-cross section showed that number of veins was significant reduced by 50%, compared to the wild-type. Through scrapping the upper epidermis, we found that the narrow leaf was due to decreased cell number. Paraffin sectioning revealed that the mutant leaf margin development was abnormal.(2)With map-based cloning method, we realized that F2-195 is a double mutant. The mutation loci were located within 12.7 kb on the short arms of chromosome 11 and 12. Through analysis of target region on the website(http://rice.plantbiology.msu.edu/index.shtml), we found out that the candidate gene was actually the WOX3 gene, which was located in the 3.0 Mb segment duplications between chromosome 11 and 12. Sequencing results showed that there was a large insertion in the WOX3 gene copy on chromosome 11(named as NAL2) both in the wild-type and F2-195 mutant. A large deletion was detected in the WOX3 gene copy on chromosome 12(named as NAL3) in the F2-195 mutant, leading to the loss function of NAL2/3.(3)The promoter of WOX3 was fused with GUS reporter gene and transformed to a japonica rice variety Shiokari. GUS staining results showed that there was a high expression in young tissues, such as young leaf, young leaf sheath, young panicle, culm node and pulvinus.(4)WOX3 belongs to Wuschel related homeobox family transcription factor. Previous studies showed that WOX3 is a transcriptional activator. To investigate its real role, the transcriptional activity of WOX3 was examined using the luciferase transient expression assays in rice protoplasts. Through detecting the activity of luciferase, we found that WOX3 may functions as a transcriptional repressor and point mutation in the WUS box(WOX3M) compromised its transcriptional repression activity. To further confirm this result, WOX3 M was fused in-frame with the transcription activation motif VP64 and repression motif EAR respectively and transformed into the F2-195 mutant. Phenotypic analysis of the transgenic lines showed that WOX3 function as a transcriptional repressor.(5)Kinematic analysis was taken on the first leave of the wild type and F2-195 mutant from day 4 to day 13 after sowing. The data revealed that there was small difference in cell size of WT and mutant, on the contrary, remarkable lower cell number and cell division rate. The mutation influences cell proliferative activity, and the final narrow leaf phenotype was mainly caused by the cell division rather than cell expansion. Flow cytometry analysis revealed that cells in the G1 phase was increased while those in the S and G2/M phase was reduced in the F2-195 mutant. Real-time PCR analysis revealed that expression of several genes that inhibit the G1/S phase transition was upregulated in the F2-195 mutant.(6)The whole plant phenotype and leaf size of the triple mutant of F2-195(nal2/3) and nal1 was more similar to the nal1 mutant, indicating that NAL1 likely locates downstream of NAL2/3.(7)The whole plant phenotype and leaf size of the triple mutant of F2-195(nal2/3) and sd110 was more similar to the nal2/3 mutant, indicated that sd110 likely locates upstream of NAL2/3.