Map-based Cloning And Functional Analysis Of A Low Temperature Sensitive Mutant Tcd7 In Rice(Oryza Sativa L.)

The chloroplast development is controlled by both the nuclear genes and the plastid genes.PEP polymerase is one of the two RNA polymerases responsible for plastid gene transcription in higher plants,and the other enzyme is referred to as the nucleus-encoded plastid RNA polymerase（NEP）.NEP is a single-subunit enzyme,which is largely responsible for the transcription of housekeeping genes.PEP predominantly mediates the transcription of the photosynthesis genes.A lack of genetic function of the complex regulatory process will result in leaf color mutation.A rice thermo-sensertive leaf color mutant tcd7 derived from japonica variety Jiahua No.1 treated by ⁶⁰Coγ-rays was used in this study.Characterizations of the tcd7 mutant could be conclued as follows:1.Compared with the wild type,the mutant tcd7 exhibited a therno-sensitivy phenotype:In the condition of 18℃,the 3rd leaf of the mutant presents white green stripes or partial albino phenotype,the 4th leaf of the mutant appears green leaf and has no difference with wild type;however,when under temperrature of 32℃,all the leaves turn green,completely,and consistent with that of wild type.2.By observating the photosynthetic pigment content determination and electron microscope of 3-leaf-stage seedlings grown on at 18℃and 32℃,which shows mutation tcd7 phenotype be related to the development of chloroplast and the accumulation of photosynthetic pigment abnormalities.At 18℃,the photosynthetic pigment contents of tcd7 mutants at the 3-leaf stage was drastically lower than those in WT plants.However,which was consistent with WT plants at 32℃.3.Genetic analysis on individuals from cross of Pei’ai 64S and tcd7 be correlated that the phenotype was controlled by a single recessive nuclear gene.4.A map-based cloning of the mutant gene was conducted.The mutant gene was localized between the markers ID14867 and RM15419 on chromosome 3 about 123KB range,with eight of the scope of candidate genes for subcellular prediction analysis.After whole genome sequencing analysis found that the coding of pfkB-type carbohydrate kinase family protein gene LOC＿Os03g40550 third exon nine bases on continuous absence,caused the genetic encoding of two alpha helix of hydrophobic amino acid,alanine,valine,eventually lead to the change of the structure of the senior protein.Then on the gene function complementary,to verify whether the phenotypic abnormalities caused by genetic mutations.5.Through yeast two hybrid experiment and mutant tcd7 on MS medium without sugar,and MS medium with 2%、3%sucrose to observe whether the leaves turn green.To make sure that have been reported in arabidopsis thaliana fructokinase-like protein FLN1/FLN2 family proteins encoded protein interaction and function is consistent.6.By the expression of tissue specificity,subcellular localization,the encoded amino acid sequence of conservative and evolutionary tree analysis,which showed that quantity of the mutant TCD7 gene expression in the leaf is higher than the root,the encoding protein localization in chloroplasts,and monocotyledon related nearly with dicotyledonous plants such as arabidopsis thaliana.7.By analyzing the transcript levels of genes associated with chlorophyll synthesis,photosynthesis and chloroplast development in the tcd7 mutant under different temperature,found that depend on the PEP polymerase gene regulation pathway appear cut,and TCD7 gene also showed significantly lower under low temperature condition,therefore deducing TCD7 gene was involved in interaction of the chlorophyll photosynthesis genes.This research discovered a new rice temperature sensitive genes TCD7,the results conducts a preliminary analysis to the function of the gene,then will be directed to the further research of gene function,as well as to improve the chloroplast development and regulation mechanism provides a novel insights.