Determination And Function Analysis Of The Candidate Gene Of The Dark Green Rice

Chlorophyll is an important pigment involved in photosynthesis in green plants, and it plays a very key and basic role in the energy capture and transfer in photosynthesis. Rice leaf color mutant is a kind of mutant whose phenotype is obvious and easy to observe. Mapping and functional analyzing of the rice leaf color mutant genes has an important guiding significance to illuminate the regulation mechanism of the leaf color genes and improve the biological yield of rice through genetic improvement of the rice leaf photosynthesis. The dark-green rice mutant we researched was mutagenized from Zhonghua 11 japonica by y-ray radiation. In previous research, we have found that the dark trait is controlled by a single gene, temporary called dg(t). Through fine-mapping and high-throughput resequencing, we found that the gene was located on chromosome 1 of rice about 88kb region.In this study, first bioinformatics analysis showed that the about 88kb region in rice genome has 10 candidate genes may related to the dark green phenotype. We planted 8 T-DNA insertion mutant strains related to the candidate genes from an existing rice T-DNA insertion mutant library. Results of both the phenotypic identifications and plant chlorophyll content determination, we found the homozygote from a T-DNA insertion mutant strain dg(t)-5, which is a T-DNA insertion mutant of dg(t)-5 gene, has the dark green phenotype similar to the mutant. Then we analyzed the gene expression of the wild type, mutant and the T-DNA insertion strain to ensure dg(t)-5 to be the candidate gene of dg(t). Function analysis of the candidate gene showed that the gene is a plant-specific domain TIGR01589 family protein which has not been reported before. Second, we use transgenic technology to transfer the candidate gene of dg(t) into a dark green mutant to clone the candidate gene of dg(t). After that, we analyzed the expression of the candidate gene of dg(t) in different tissues of rice by semi-quantitative RT-PCR, and found that the gene only highly expressed in leaves, stem and almost no expression in sheath or root. We also verified the result by GUS staining from a macro point.