Map-based Cloning Of A Rolling Leaf Mutant And Functional Analysis Of Gene Ocu5 In Rice

Rice is one of main crop in China, even in the world. In reality, human is faced with several problems, including reduced ploughs, worsen environment and increased population size, that could be resolved depending on enhancing per unit area yield of rice. There are some drawbacks in traditional breeding, such as long period and high risk. With the development of molecular technologies, we hope to breed super rice to resolve those problem. Therefore, building rice ideotype become an aim scientists pursued around the word. It is proved that curl leaf could solve a contradiction between leaf length and stem, which is good for increasing sunlight areas, improving ventilation conditions and then accumulate rice photosynthate.We screened one plant mutant ocu5 of rolled leaf becoming tubular from our T-DNA insert mutant library in our laboratory. Else, we did a series of corresponding experiments for the mutant: phenotype investigation, genetical analysis, gene mapping, genetics identification, specificity expression, sub-cellular localization, Mutations functional verification. Relevant research is as follows:1.Character of mutant ocu5 from seedling stage to maturation stage: stable rolled leaf phenotype, plant height as wild type(WT), reduced tiller number, tubular leaf curl, deepen leaf color, normal fructification, lower wizened rate, thousand seed weight as same as WT. On the cell morphology, the alveolar cell number and area of mutant ocu5 accordingly dropped compared with WT so that the mutant presented leaf inner-rolling.2.Phenotype of hybrid F1 from WT and mutant ocu5 presented all rolled leaf, segregation ratio of F2 of rolled leaf and WT is 3:1, which indicated that the character is controlled by single dominant genes.3.We located mutant gene in 80 kb area on No.6 chromosome by map-based cloning method, combined with two BAC, and selected five candidate genes by analyzing the gene. In sequencing, we found that the 3’end of LOC_Os06g10600 gene loses 50 bases apart 633 bp from terminator.4.It is proved that phenotype of the mutant is indeed controlled the gene after doing overexpression and carrier interference experiments.5.Subcellular localization, building fusion protein vecter both the gene and GFP and transforming rice protoplast, showed that the gene is located in nucleus.6.In-situ hybridization experiment showed that the gene expresses in the apical meristem outside or once the outer and doesn’t express in mature leaf and stem.7.Taking apical meristem both Nipponbare and mutant sample, it showed that expression quantity of the mutant gene is little more high than Nipponbare, but it is not evident, and quantity of protein is obviously higher than Nipponbare after RT-PCR and Western blot.8.Contrasting the result of instantaneous transformation of tobacco, it showed that mutation site really affect gene quantity.This experiment not only found a dominant gene controlling rolled leaf character and study its function, but also proved 3’end mutation site’s influence on gene expression.