Study On Cloning And Function Of Genes Involved In Regulation Of Leaf Shape In Rice

Rice is one of the most important crops in the world, and has become the model crop in plant for genetics and genomic studies. Leaf is a major vegetative organ, which plays an important role in photosynthesis, transpiration and resistance to the stress and ultimately affecting development and growth.In the present study, in order to systematically dissect the molecular mechanism of leaf morphogenesis and development, two ethyl methane sulfonate (EMS)-induced rice (Oryza sativa L.) mutants with rolling leaf from the progeny of Nippobare (O. sativa ssp. japonica), namely H77 and H28, were used. We also isolated another two rolling leaf mutants K27 and K92 from Shuang Kezao (O. sativa ssp. indica) which were induced by ⁶⁰Coγ-ray. We performed anatomical analysis of all these mutants and the genetic analysis; allelic test and mapping caused genes through map-based cloning strategy were also be conducted. The main results were as follows:The identification, genetic analysis and fine mapping of the rice mutant sll11,The allelic test was performed between these four rolling leaf mutants. The identification of the phenotype of the F₁ plants that was derived from the reciprocal cross between these mutants mutually, showed that they were controlled by the same gene, which were designated as sll1-1 (shallot like leaf1-1, sll-l) and sil1-2.2,Through the anatomical and histological analysis, we have draw the conclusion that the mutation of gene SLL1, which lead to the increase of chlorophyll and reduction of the cellulose contents in rice leaf, caused the absence of the prothenchyma in the abaxial vascular bundle.3,Genetic analysis: we have constructed four segregated populations from the cross between sll1-1 and three other parents NJ6, GuangSi and nipponbare with normal leaf, and the analysis on the phenotype of the F₁ and F₂ was performed. The results showed that all the F₁ plants had normal leaf and the ratio of normal rice plants to mutant rice plants tallied with 3:1 rule. Therefore, the phenotype of the mutant sill1-1 was controlled by single recessive gene.4,The progeny from the cross between sll1-1 and a O. sativa ssp. indica parent NJ6 were used as the mapping population. The caused gene SLL1 was finely located into a region of 29.57Kb on the long arm of chromosome 9, which had 3 ORF and were presumed to be a synthetic enzyme, alike En/Spm transposon and MYB-domain containing protein, respectively, according to the annotation.The isolation of SLL1 and the gene function analysis1.The results of sequence analysis showed that there is a single substitution in the sequence of mutant Sll-1 or sll-2 (G to A). Amplification of the cDNA showed that the substitution occurred at different splice sites of the same gene, which lead to the abnormal splice.2. The SLL1 gene was located at 103061-108515 of the BAC clone, and the length of gDNA was 5455 bp. The analysis of gene structure and the amplification of the cDNA showed the length of FL-cDNA was 1134bp, with 6 exons and 5 introns, which coding a 377 amino acids protein.3. The results of RT-PCR showed that the SLL1 gene expressed to different extents in the different organs of the whole plants, which expressed highly in flower, leaf and glume, then inferiorly at root and poorly at stem and sheath.4. The transformation of SLL1::GUS was conducted. All the organ were detected to have different GUS activity in all the growth and development period, and the high level expression was detected in vascular bundle, stoma and conduit in contrast to no expression in vesicular cell. The GUS expression was detected in yang plant, leaf, stem and glume of seed, and highly at venation system, but poorly at rooting and flower which only the culm sheath, kernel and stamen could be stained.5. The result of complement test showed that SLL1 gene could recover the leaf phenotype of the mutants.The genetic analysis and fine mapping of ALM11,Allelic test showed the mutant K27 and K92 which exhibits a phenotype of rolling leaf was controlled by the same allele. Due to the gentle phenotype when compared with slll, we nominated K92,K27as alm-1(adaxialized leaf mutant-1) and alm-2, repectively.2,Anatomical and histological analysis showed that the lose of function of ALM1 caused the increases of the cell number of vesicle parenchymatous cell, though the cells in vascular bundle were normal.3,Investigation on the phenotype of the cross of alm-1/NPB,alm-1/TN1 showed that the mutant was controlled by one recessive gene.4,The population for gene mapping was established from alm-1/TN1, and about 1,200 plants with mutant phenotype were selected in the F₂ generation. The gene was finely located in 91 Kb on the chromosome 2, which was forecasted to have fourteen ORF by GENSCAN software.