Gene Cloning Of Yellow-green Leaf Mutant 577ys In Rice

Leaf is the main organ of plant photosynthesis.Chloroplast is an important organelle of photosynthesis.Chlorophyll is the major component of plant photosynthetic pigrments,and it also the main carrier of plant photosynthesis to absorb the light energy.Many Ieaf color mutations are related to chloroplast development and chlorophyll anabolism abnormality.In higher plants,chlorophyll synthesis is a very complicated process.The reaction of the biosynthesis of chlorophyll a and chlorophyll b from the glutamine-tRNA needs a series of enzymes.Magnesium protoporphyrin IX monomethyl ester cyclase(MgPME)is the key enzyme of chlorophyll biosynthesis that converts magnesium protoporphyrin monoester Ⅸ to divinyl protochlorophyllide a,so it plays an very important role in the formation of carbon ring.In this study,we had obtained a yellow-green leaf mutant 577ys based on EMS mutagenesis of the japonica rice variety Nippobare.We finished the candidate gene selection we will clone the gene and analysis its function based on the mapping of the mutant.The major results were as follows:(1)Phenotypic characteristics and agronomic traits:the 577ys mutant exhibited yellow-green leaf and slow-growth throughout the whole growth period.The leaves appeared the rust spots at the seedling stage,and the rust spots were obviously reduced at the heading stage,while the rust spots were unconspicuous at the mature stage.Compared with the wild type,the plant height,grains per panicle and seeds setting rate were significantly decesased in 577ys mutant,and decreased by 18.3%,31.7%and 11.8%,respectively.Number of productive panicles per plant,panicle length and seed setting rate were reduced by 10.8%,7.1%and 7.4%in 577ys mutant.(2)Determination of photosynthetic pigment content:Compared with the wild type,the total chlorophyll,chlorophyll a,chlorophyll b and carotenoid were decreased by 81.9%,79.1%,91.9%and 64.7%in 577ys mutant,respectively,at seedling stage.The total chlorophyll,chlorophyll a,chlorophyll b and carotenoid decreased by 59.5%,53.2%,82.1%and 46.4%in 577ys mutant,respectively,at booting stage.Indicating that 577ys gene mutation seriously affected the synthesis of photosynthetic pigments.(3)The ultrastructure of chloroplast was observed by transmission electron microscopy(TEM).It showed that,577ys chloroplast was swelling,thylakoids develpment disorder,the number of chloroplast grana decreased obviously,choloroplast Irregular and had no obvious grana lamella stacks.The number of osmiophilic granule in chloroplast increased significantly.The resulted indicated that chloroplast development of 577ys mutant was inhibited.(4)Map-based cloning of the 577ys mutant gene:the preliminary work of our laboratory has localized 577ys at about 105.4kb on the short arm of chrl,which contains 13 predicted genes.On the basis of this,PCR amplification and sequencing of LOC_Os01g17170,showed that a single nucletide C to T substitution at position 419 of its DNA,correspond with the 419 of its CDS,which lead to mutation of the amino acid at position 140 by threonine to isoleucine.LOC_Os01g17170 encodes the magnesium protoporphyrin IX monomethyl cyclase.(5)Complementation analysis:construction of pC2300-LOC_Os01g17170 transgenic expression vector was transformed into the mutant 577ys by Agrobacterium-mediated method,the transgenic positive plants leaves were green,indicating that the 577ys yellow mutant was caused by the mutation of LOC_Os01g17170 gene.(6)Expression analysis:The expression of 577YS gene in different organs at different stages was detected by RT-PCR.The results showed that the 577YS gene expressed in leaves,stems,leaf sheaths and young panicles,and which in leaves was at highest.(7)Subcellular localization:transient expression by PEG mediated vector which is construction of pC2300-LOC_Os01g17170-eGFP in protoplast,the results showed that the pC2300-LOC_Os01g17170-eGFP fusion protein was clearly co-localized with chlorophyll autofluorescence,indicating that 577YS protein was located on chloroplast.(8)Trypan blue staining:using trypan blue stained the 577ys and wild type leaf of tillering stage at the same position.The results showed that there were dark blue spots in the leaves of the mutant,indicating that the mutation cell dead on the rusty spot.