Map-based Cloning Of Mitochondrial Carrier Family Gene OsSFC1 In Rice

The chloroplast is organelle for photosynthesis in plants,its development directly affects the photosynthetic capacity and yield of plants,and then indirectly influences the quality of food crops,such as rice quality.Thus,it is very important for developing high-quality and high-yield rice materials by studying the molecular mechanism of chloroplast development and its regulation on seed quality in rice.In our previous study,the rice ossfcl mutant with spontaneous yellow green leaf phenotype was obtained through EMS-mutagenesis of cultivar indica Zhehui7954.In this study,we determined the physiological,cytological and molecular changes responsible for the ossfcl phenotype.The main results are as follows:1.Until the 5?6-leaf-seedling stage,ossfcl was practically indistinguishable from Zhehui7954 in their phenotypes.However,starting at the 1 fully-expanded leaf and heart leaf--eedling stage,yellow-green leaf was visible in ossfcl.Moreover,the plant height and root length of the mutant was also significantly shorter than its wild type.Also,the chlorophyll content of ossfcl at seedling stage was significantly lower than its wild type,while the ROS content of ossfcl was significantly higher than its wild type.TEM analysis further indicated that the ossfcl mutant displayed less and smaller chloroplasts,and less starch grains in the chloroplasts.2.Under field conditions,the chalky grain rice rate and chalkiness score of the mutant ossfcl were 93.33%and 55.83%,respectively,which were significantly higher than its wild type.Scanning electron microscopy showed that the endosperm cells of wild type were well developed,starch grains appeared angular polygonal and closely arranged,while the endosperm cells of ossfcl were poorly developed,especially in chalky parts,starch grains appeared spherical and loose arranged.3.Genetic analysis indicated that a single recessive nuclear gene determines the ossfcl phenotype.Using the F2 mapping population derived from ossfc1/02428,bulked segregant analysis mapped this gene to an 84.6-kb interval between RM14780 and RM14784 markers in rice chromosome 3.This genomic region houses 11 candidate genes according to RAP-DB Database.Sequencing of these ORFs revealed that the ossfcl mutant has a G-to-T substitution at the position 230th in the 1st exon of a mitochondrial carrier family gene LOC Os03g18160,resulting in an amino acid change from glycine to valine.Furthermore,a dCAPS marker was developed and confirm that its polymorphism co-segregated with the ossfcl phenotype.4.qRT-PCR results showed that OsSFC1 was ubiquitously expressed in all tissues examined at the young and mature stages.This gene was expressed at high level in leaves,roots and callus.The seedling leaves showed higher abundance of OsSFC1 transcripts than the mature leaves.Furthermore,temporal expression of OsSFC1 showed that its transcription increased gradually at the early and middle stage of the leaf growth and development,but decreased dramatically at its late stage.5.RNA-seq analysis indicated that a number of photosynthesis-related genes were down-regulated in ossfcl.Similar results were confirmed by qRT-PCR.6.Previous studies showed that OsSFCl gene is a homologous gene of ACR1 gene in yeast,and ACR1 gene has been proved to be closely related to the transport of succinate and fumarate.Through one-step gene replacement,ACR1 gene deletion yeast strain BY4741-?acr1 was developed.Then the complementary vectors(pYX212-OsSFC1-1 and pYX212-OsSFC1-12)containing the different ORF length of OsSFCl gene were constructed,and transformed into the yeast strains BY4741-?acrl via PEG methods.The resulting yeast could grow on the medium containing 2%alcohol as the sole carbon source,confirming that OsSFC1 could complement the function of yeast ACR1 gene.