Cloning And Functional Analysis Of Zebra Leaf Gene ZL7 In Rice

Plant photosynthesis is the basis for plants to absorb light energy and obtain necessary substances,which has an important impact on rice yield.Improving the utilization efficiency of rice photosynthesis is an effective means to achieve high and excellent yield of rice.Leaf color mutants are ideal materials for studying plant photosynthesis because of their rich phenotypes and easy observation.The full excavation and utilization of leaf color mutants is expected to further reveal the mechanism of photosynthesis and lay a theoretical foundation for cultivating high light efficiency and high yield rice.In this study,a zebra leaf mutant zl7（zebra leaf 7）with stable inheritance was obtained from Japonica Rice Variety Chunjiang 06（CJ06）by EMS mutation.The phenotype,photosynthetic pigment content and photosynthetic parameter indexes of the mutant were observed and analyzed.The chloroplast ultrastructure was observed by transmission electron microscope,and the population separated by Indica Rice variety 93-11 and zl7 were used for genetic analysis and gene mapping;q RT-PCR method was used to analyze the expression of genes related to multiple pathways of chlorophyll metabolism and carotenoid metabolism.The main results are as follows:（1）Mutant zl7 was obtained from Japonica Rice Variety CJ06 by EMS mutation.Under natural growth conditions,the mutant zl7 has typical zebra leaf phenotype from seedling stage,the zebra leaf phenotype is the most obvious at tillering stage,and the leaves turn green at heading stage.The investigation results of agronomic characters showed that compared with the wild type,the main agronomic characters of the mutant,such as plant height,tiller,panicle length,primary branch,secondary branch and grain number per panicle,decreased significantly,while grain length,grain width and 1000-grain weight increased slightly.（2）The results of chlorophyll content showed that the content of photosynthetic pigment in the leaves of zl7 mutant was significantly lower than that of wild type at seedling stage and tillering stage;At heading stage,there was no significant difference in photosynthetic pigment content between zl7 mutant and wild type.（3）The results of photosynthetic parameters showed that the net photosynthetic rate and intercellular carbon dioxide concentration of green and yellow parts of mutant zl7 zebra leaves decreased significantly at tillering stage;At heading stage,the net photosynthetic rate of yellow part of mutant zebra leaves decreased significantly.（4）The results of transmission electron microscopy showed that the number of osmiophilic particles in the chloroplasts of the yellow part of the mutant zl7 zebra leaves increased significantly,and almost no lamellar structure was observed.It indicates that chloroplast development is abnormal.（5）Genetic analysis showed that zl7 zebra leaf mutant was controlled by a pair of recessive nuclear genes.The gene was located within 105.0kb of the molecular markers M5 and M6 on chromosome 7 by map-based cloning.The candidate gene was found by genome sequencing.LOC＿Os07g10490 has a single base substitution in the coding region,which changes from G to A,resulting in the change of amino acid from serine to asparagine.The complementary vector was constructed and transformed into mutant zl7 by Agrobacterium mediated method.The leaves of T₀positive transgenic plants recovered the phenotype,indicating that LOC＿Os07g10490is the target gene.（6）RNA-seq analysis showed that there were 2221 differentially expressed genes,of which 704 were up-regulated and 1517 were down regulated.It was found that a large number of differentially expressed genes were involved in metabolic processes,catalytic activity,stress response and so on.（7）The results of q RT-PCR showed that the expression of genes related to chlorophyll synthesis and degradation,chloroplast development and carotenoid metabolismin mutant zl7 changed in varying degrees.（8）Tissue pattern analysis showed that the expression of ZL7 gene was the highest in leaves and subcellular localization was in chloroplasts.