Map-based Cloning Of A Rice Mutant Gene WP4 Controlling White Stripe Leaf And Panicle

Rice(Oryza sativa L.)is the main food crop for over half of the global population.As a model plant for monocots,functional genomics of rice has been a research focus in plant biology.Because its relatively high mutation frequency and easily accessible phenotype,rice leaf color mutant has become an ideal material to study the genetic and regulation mechanism of plant photosynthesis,chlorophyll biosynthesis,degradation and chloroplast development.In this thesis,a white stripe leaf and white panicle mutant wp4 deriving from Japonica rice cultivar ‘Zaoxaingjing' was physiological,cytological and genetically characterized.The leaves of wp4 exhibited a reversible white striped phenotype,which could be gradually turned into normal green like a wild-type.However,the flag leaves and its sheaths appeared to be white striped.After flowering,the young panicles of wp4 became completely white.Besides the difference on the color of leaves and panicles,the plant height of wp4 was shorter,the grains per spike and the yield per plant were also lower than that of the wild-type.Nevertheless,agronomic traits such as flowering time,number of tillers and 1000-grain-weigh showed no obvious differences from that of the wild type.The contents of chlorophyll a,chlorophyll b and total chlorophyll in wp4 seeding,young panicle,flag leaf and flag leaf sheaths at heading stage were drastically lower than those in the wild-type,and consequently made the net photosynthetic rate of flag leaves of wp4 significant lower than that of wild-type.A transmission electron microscopy analysis revealed that wp4 cells had much fewer and smaller undifferentiated chloroplasts.,the granal stacks in the mesophyll cells were also smaller and lacked well structured thylakoid membranes.All F1 plants from the three crosses between wp4 and cv.‘Nanjing11' or ‘Peiai64' or ‘Nipponbare' displayed wild-type phenotype,and their F2 progenies all showed a segregation ratio of 3:1(green: white panicle plants).Therefore,the mutant phenotype in the wp4 was controlled by a single recessive nuclear gene.Genetic mapping of the wp4 was performed using the F2 population of a cross of wp4 mutant and cv.‘Nanjing11'.The wp4 locus was final localized at an 8.65 kb region interval between two markers dcaps8-4 and z29 on Chr.8,based on 2937 mutant phenotype individuals.Within this region,only one open reading frame(ORF)(Os08g01099300)encoding an adenylate kinase OsAK1 was predicted.Sequencing of the 8.65 kb region around OsAK1 failed to find any differences between the wp4 mutant and its wild-type,but the mRNA level of OsAK1 in wp4 was significantly lower than that of its wild-type.We introduced a fragment carrying the OsAK1 genomic DNA and cDNA under the driven of a Ubi promoter into the wp4 background,and found that the OsAK1 fragment rescued the plant phenotype.Hence,we concluded that OsAK1,which encodes an adenylate kinase,was responsible for the phenotype of wp4.RT-PCR analysis revealed that OsAK1 is constitutively expressed in all the examined tissues with relatively higher expression levels in green tissues,such as leaves,leaf sheaths and young panicles.The GUS gene driven by the OsAK1 promoter was transformed into rice.The staining of transgenic plants verified the OsAK1 expression pattern as indicated by the RT-PCR results.Subcellular localization of OsAK1 protein was carried out and the result suggested that the GFP signal was localized in the chloroplast.The expression levels of some genes associated with Chl biosynthesis and photosynthesis in wp1 were up-or down-regulated when compared with wild-type.Taken together,our results indicated that OsAK1 plays an important role in regulating gene expression associated with chlorophyll biosynthesis and photosynthesis in leaves of rice.