Functional Research Of A DEAD-box RNA Helicase Gene TCD33 In Rice (Oryza Sativa L.)

DIEAD-box RNA helicase can be found in many organisms,which involved in the multi-dimensional metabolism of RNA and played an important role in plant growth and development,especially in abiotic stress response.Experimental materials of this research derived from japonica rice variety Jiahua 1(WT)treated with 60Co ?-rays for the temperature sensitive albino m utant tcd33.Through the analysis of trait characteristics,map clonling,genetic complementation,subcellular localization and related functional analysis,we draw the following conclusions:1.The tcd33 seedlings displayed white leaves and died after the 4-leaf stage when grown at 20°C.However,when grown at 32?,the seedlings remained green,as did WT seedlings at both temperatures.2.The photosynthetic pigments chlorophyll(Chl)a and b and carotenoid(Car)were almost u:ndetectable in tcd33 seedlings grown at 20?,whereas there was only a slight difference in pigment levels between WT and tcd33 at 32?.Observation by transmission electron microscopy revealed the structure of the WT chloroplasts was very complete,regardless of 320C or 20?.However,at 20?,almost no intact chloroplast and a lot of bubble structure were observed in tcd33 mutants,whereas chloroplast was not significantly different from the wild type at 32?.3.Genetic analysis showed that the tcd33 mutant is a single recessive locus based on the approximately 3(green):1(white)ratio for phenotypic segregation in an F2 population generated from Pei.ai64S//tcd33.4.Using map-based cloning technology the mutant gene was localized between the InDel marker ID00216 and the SSR molecular marker MM0093 on chromosome third,with physical distant of 51 kb.We sequenced an d analyzed all candidate genes and found only a 7-bp(TTCTCAG)deletion mutation in the third exon of the gene LOC_Os03g01830(TCD33)encoding the DEAD--box RNA helicase,which led to a premature translational stop and a frame-shift mutation.we performed genetic complementation in tcd33 mutants to verify the results of map-based cloning experiment.Subcellular localization affirmed that TCD33 is localized in the chloroplast.5.Conservative analysis on amino acid sequence encoding by TCD33 revealed that it shares high sequence similarity to different higher plants.Further analysis of the homologous evolution relationsh,ip with more species,showing that TCD33 is more conserved in monocoty,ledons,rather than dicotyledons.6.The qPCR analysis of genes related to chlorophyll synthesis,photosynthesis and chloroplast development showed that TCD33 gene affects the development of chloroplast ribosomes and chloroplast development in rice.This study reported a temperature-sensitive DEAD-box RNA helicase gene TCD33 in rice,providing a new idea for the future study of chloroplast development and photosynthesis metabolism regulation when rice under the low temperature stress by taking a preliminary analysis of this gene.