Map-based Cloning And Functional Analysis Of DCFL1 Genes In Rice(Oryza Sativa L.)

As the most important food and economic crop,rice has been widely planted in China.Thus,it is critical to thoroughly undersanding the mechianism contribute to rice yield.Among those factors,Plant height and the quality of flag leaf play decisive role.Therefore,the understanding of the genetic model of plant height and the molecular mechanism of flag leaf development has vital significance in improving rice type and fertility.A dwarf and curled flag leaf(dcfl1)mutant was discovered from the progeny of indica restorer line Jinhui10 with seeds treated by ethyl methane sulfonate(EMS).This paper performed systematic studies in morphological characteristics detection,identification of photosynthetic pigments and characteristics,cytological observation,genetic analysis,gene fine mapping,candidate gene identification,map-based cloning and gene function research etc.The main results were as follows:1.Morphological analysis and agronomic traitThe dcfl1 was dwarf in all phases of plant development,and the cell length of the 2nd leaf sheath surface of the dcfl1 was significantly shorter than the wild type.The dcfl1 displayed a severe curl at the base of flag leaf blade after the heading stage,while the upper part was normal.Besides,other leaf blades appeared as normal as the wild type.The results of agronomic analysis indicated that there are no significant differences in grain number per panicle,filled grain number per panicle,seed setting rate and 1000-seed weight between the dcfl1 and the wild type.However,the tiller number and the efficient panicle per plant were both increased in the dcfl1 mutant when compared with the wild type.2.Photosynthetic pigment and characteristicsDuring the heading stage,the photosynthetic pigment content was increased,and the results showed that the contents of chlorophyll a and total chlorophyll in the dcfl1 increased significantly compared with those of the wild type for the flag leaves,second and the third leaves.In addition,the photosynthetic parameter of the photosynthetic rate(Pn),stomatal conductance(Gs)and transpiration rate(Tr)in the dcfl1 flag leaves were declined significantly.3.Genetic analysis and and molecule mapping of DCFL1Genetic analysis indicated that the dwarf and curled flag leaf traits of dcfl1 were controlled by a recessive nuclear gene.Based on the F2 population derived from a cross between the dcfl1 and an indica sterile line,Xinong 1A,the gene was fine mapped on chromosome 3 between InDel marker Ind03-11 and Ind03-6 with the physical distance 78 kb.4.Cloning and protein analysis of the DCFL1After sequencing of the fifteen annotated genes in the molecule mapping,a single nucleotide substitution from G to A within LOC_Os03g04680(causing a Cys to Tyr substitution)has been found in dcfl1.So,LOC_Os03g04680 was predicted as the candidate gene.DCFL1 encoding cytochrome P450 and belonging to CYP96 subfamily,was designated as OsCYP96B4 according to the proposed nomenclature for P450.5.Function analysis of DCFL1The DCFL1 complementation vector has been constructed and transformed into the dcfl1 mutant.Twelve positive transgenic plants were acquired and detected.The mutant phenotype has been completely rescued in transgenic plants,suggesting that LOC_Os03g04680 is DCFL1 gene.6.Expression patterns analysis of DCFL1Semi-quantitative RT-PCR and quantitative RT-PCR(qPCR)analysis exhibited that DCFL1 was universally expressed in various tissues including roots,stems,leaves and panicles at varying degrees with the highest level in flag sheath.To further characterize the expression patterns of DCFL1,GUS-test was carried out.The GUS activity was ubiquitously detected in the all organ,which was consistent with the RT-PCR and qPCR results.7.The relationship between DCFL1 and phytohormoneThe dcfl1 and the dcfl1-1 did not show pronounced effect or sensitivity to BR and GA3 when compared with WT grown under similar conditions.Exogenous treatment of BR and GA3 also failed to fully complement the dcfl1 dwarf phenotype.However,we still cannot exclude the possibility that DCFL1 may be involved in phytohormone metabolism without investigating the response of dcfl1 to other exogenous phytohormone and testing the endogenous hormone levels.8.DCFL1 may be involved in the cuticular wax biosynthesisThe scanning electron microscopy(SEM)analysis showed that there was a ravine between two small veins which decrease the area of cuticular wax in the base of flag leaf of the dcfl1 when compared with wild-type.The chlorophyll leaching assay showed that chlorophyll was more readily extracted from dcfl1 flag leaf than from the wild-type ones.Thus,the dcfl1 cuticle was more permeable than the wild-type,suggesting reduced cuticle wax content in dcfl1.These results suggest that DCFL1 may be involved in the cuticular wax biosynthesis.