Map-based Cloning And Functional Analysis Of Zl11,Conferring Zebra Leaf Color In Rice(oryza Sativa L.)

In the present study,a novel leaf color mutant was isolated from an indica cultivar Shuhui 527.Comparing with the wild type,the mutant has the similar phenotype at early seedling stage,but in the late growth stage,the mutant exhibits chlorotic crossbands on leaf blades in the newly grown leaves,looks like "zebra leaf".Thus,we temporarily named this mutant as zl11.Genetic analysis of the mutant was conducted based on the F1 and F2 progenies derived from crossing between zlll mutant and the indica cultivar 9311.Furthermore,the ZL11 gene was finely mapped with simple sequence repeats and newly-developed sequence tagged site(STS)markers.And the gene prediction was carried out within the located region.The main results are as follows:1.Comparing with the wild type,the zl11 mutant had the similar phenotype at early seedling stage,but after early tillering stage,the newly grown 1-2 leaves of the mutant exhibited alternating green and yellow crossbands on leaf blades(zebra leaf).However,the yellow region of zebra leaves begin to turn green at the heading stage,and disappeared completely at the early filling stage,However,the leaf color of mutant,could not be fully restored,exhibiting relatively yellower than that of wild type.The results of agronomic traits analysis showed that the plant height,the flag leaf length and width,the number of primary branches,number of secondary branches,the number of filled grains and total number of grains were significantly reduced,but the panicle length and seed-setting percentage significantly increased.Meanwhile,there was no significant change in effective tiller.2.The chlorophyll content analysis indicated that the variation of the chlorophyll content in the leaves was consistent with phenotype of leaf color.The total content of chlorophyll increased at the beginning and then decreased in followed four growth stages.Comparing with the wild type,the chlorophyll a content in the green part of the mutant decreased by 5.83%and the chlorophyll b content decreased by 72.81%at the tillering stage.Similarly,when compared to the green part,the chlorophyll a and b content in the yellow part of the mutant decreased by 45.94%and 65.19%respectively.In addition,the chloroplast ultrastructures,observed by transmission electron microscopy showed that the mesophyll cell of the green part of mutant was basically the same as that of wild type suggesting that the mesophyll cells contained normally developed chloroplasts in the mutant.However,it was observed the chloroplast increased in volume and its shape was irregular,although the number of chloroplasts did not change significantly.In contrast,the number of chloroplasts of the yellow part of mutant decreased and its shape was also irregular.There were less numbers of ganum and grana lamella,and contained starch grain and osmiophilic bodies.These results revealed that the reduction of the chlorophyll content and the abnormal development of chloroplasts may be lead to generate the zebra leaf phenotype in zl11 mutant.3.Fluorescence kinetics analysis showed that,the maximum photochemical efficiency(Fv/Fm),photochemical quenching(qP),non-photochemical quenching(NPQ),the actual photosynthetic efficiency of PSII(Y(?))and PSII electron transport rate of the green part of the mutant reduced by 8.76%,76.04%,17.91%,76.39%and 76.27%respectively and the yellow part of the mutant reduced by 29.42%,93.75%,33.50%,95.83%and 94.92%respectively,when compared to those of the wild type.These results strongly demonstrate that the photosynthetic activity,the conversion efficiency and the absolute electron transport rate of PS? in the zl11 mutant were dramatically decreased,resulting in the lower photosynthetic efficiency.4.Genetic analysis showed that the phenotype in this mutant is controlled by a single recessive nuclear gene.The fine mapping of ZL11 using recessive plants from F2 and F3 population derived from the cross between the zl11 mutant and japonica cultivar 9516 revealed that ZL11 gene was on the long arm of rice chromosome 11,and further be delimited to about 53.671 kb-long DNA fragment governed by the marker DW60 and DW46.Sequencing analysis and bioinformatics analysis showed showed that a G to A mutation occured in the second exon of LOC_Osllg37130,leading to amino acid change,replaced proline with leucine.5.The Real-time PCR results showed that LOC_Osllg37130 mainly expressed in green tissues,with the highest expression level in leaves.However,the expression level in leaves was significantly reduced in the leaves of the mutant.6.Artificial down-regulation and over-expression of LOC_Os11g37130 is involved in the regulation of leaf color.The results strongly suggested that the LOC_Osllg37130 affect leaf color.Transient expression of the ZLI1-GFP fusion protein in rice protoplasts showed that ZL11 protein is a chloroplast protein.