Gene Mapping And Clone Of Dwarf Small Seed1 Mutant Of Rice Variety Lipingzabianhe

As one of the world major food crops,more than 50% people on earth live were on rice.The plant height of the rice is an important agronomic trait and closely related to its yield and quality of rice.Identifying,researching and using more plant height and grain trait mutants resources to separate more relevant functional genes in rice are important for further elucidating the growth and development mechanisms and production practices of the rice.Previous study of our research group had obtained a stably inherited dwarf small seed mutants of the rice(tentatively named as Dwarf small seed-1,dss1)by chemical mutagen Ethylmethane Sulfonate(EMS)to treat the Guizhou local japonica rice varieties Liping heteroptera.In order to identify the location and function of the mutated genes in dss1 mutation plant,Mut Map technology were sued to finely locate candidate genes.We constructed gene over-expression vectors for genetic transformation of dss1 based on the preliminary mapping.The results were obtained as follows:1 Fine Mapping of dss1 gene and screening of candidate genesCompared with the wild-type,the dss1 mutant displayed pleiotropic abnormal phenotype similar to those of brassinosteroid-deficient mutants,such as dwarfing,dark green,rugose erect leaves,small seed and longer neck internode panicles.By using Mut Map technique,14 SNP polymorphic markers were obtained on rice chromosome 3.After sequencing,it was found that the gene of Os DWARF was mainly due to the mutation of the coding base at position 1004 in exon 5 from C to T,which resulted in the change of encoded amino acid from threonine(ACT)to isoleucine(ATT).A new allele which coding the gene Os DWARF for the key enzyme BR-6 oxidase in rice rapesinolide bio-synthesis pathway.2 Candidate gene validation of dss1In order to further determine whether the dwarf small seed traits of dss1 were caused by the mutation of Os DWARF gene,a plant over-expression vector was constructed,in which the p Cambia1301 plasmid was used as the Basic Vector,the10 kda gliadin termination sequence as Terminator and expression of Os DWARF Gene driven by 35 s Promoter as Plant Over-expression Vector.The method of Agrobacterium mediated transformation of rice stem tip was used to transform Os DWARF gene over-expression vector into dss1.Chlorophyll content was measured at tillering stage,and result shown that the chlorophyll content in the transgenic plants was restored to the wild type.There were no significant differences in net photosynthetic rate,inter-cellular CO2 concentration,stomatal conductance and transpiration rate between transgenic plants and wild type at heading stage.Similarly,there were no obvious difference in plant height,seed size,spike length and so on between the transgenic plants and wild type at natural maturation stage.The mature seeds of transgenic plant,wild type and dss1 mutant were obtained to study the dark morphology.The result shown that under normal light condition,the internodes and mesocotyls all three plant were not elongated,but after 10 days culturing under dark conditions,the mesocotyls of transgenic plant and wild type were obviously elongated.However,there was no change in internodes and mesocotyls of dss1.Therefore,the mutation character of the dss1 is determined by the mutation of the Os DWARF gene.3 Studying of Chlorophyll precursorsIn addition,the dss1 showed a deeper color of leaf from tillering stage and maintained this characteristic throughout the growth period.The chlorophyll a and chlorophyll b content was 1.4 and 1.3 times of the wild type respectively,and the photosynthetic pigment content was 25.6% higher than the wild type.After determine the precursors in the chlorophyll synthesis pathway of dss1,the proto IX and Mg-Proto content was 0.475 and 1.71 times of the wild type respectively.At the same time,the relative expression levels of key genes in the chlorophyll synthesis pathway of dss1 were analyzed,and it showed that the CHLH gene which encoding the key enzyme(Magnesium ion chelate synthase)in the chlorophyll bio-synthetic pathway had a higher expression level,which was 4.1 times that of the wild type,but there was no significant difference in the expression of other key genes compared with the wild type.The results indicated that the leaf color deepening of dss1 was mainly due to the difference in the step of Mg-Proto synthesis by Proto IX in chlorophyll synthesis pathway,and the over-expression of CHLH gene encoding magnesium ion chelate synthase.