Map-based Cloning And Research Of TCD35,a Heat-shock Protein Gene In Rice(Oryza Sativa L.)

Chloroplast is the main site of photosynthesis in higher plants.Many genes are involved in the development and function of chloroplast,including a large number of nuclear genes and a small number of plastid genes.Moreover,cold stress is a kind of external stress that rice often encounters.Heat shock protein（HSP）is an important element for higher plants to resist and adapt to various stresses like cold.In this study,a low temperature sensitive mutant tcd35 with albino leaf color was selected by EMS mutation.The preliminary study on the mutant tcd35 obtained the following conclusions.1.At low temperature of 20℃,tcd35 showed complete albino phenotype and died at four leaf stage.At high temperature of 32℃,the whole plant of tcd35 reverted to green,which was basically the same as that of wild type.The phenotypic changes were also confirmed by the determination of photosynthetic pigment content and the observation of chloroplast sub-microstructure.2.TCD35 was a recessive nuclear gene in F₂generation of tcd35.Using the F₂generation from the cross between Pei’ai 64S and tcd35 as the mapping population,the mutant gene was located between molecular markers C3ID17742 and C3ID17857with 72 kb interval.Nine candidate genes were sequenced and the LOC＿os03g313000 was found that there was a substitution from base“G”to base“A”in exon 4 of the long transcript of gene.Complementary verification showed that the mutation of the gene may led to the appearance of abnormal albinism phenotype.3.Through the prediction of the sequence structure of the gene on bioinformatics website,we found that the gene encodes a Clp B protein located in chloroplast.Homologous sequence alignment showed that the protein was highly conserved in evolution,and the mutation site was also located in the conserved region.Phylogenetic tree analysis showed that TCD35 was more closely related to monocotyledons such as maize and millet.4.Semi-quantitative analysis showed that the gene was constitutively expressed in mature leaves;Subcellular localization results of TCD35 showed that its located on chloroplast.Fluorescence quantitative q RT-PCR analysis showed that tcd35 mutation could cause a series of changes in gene expression levels related to chloroplast development or photosynthetic pigment synthesis,among which the causing most significant effect was on the expression level of genes encoding photosystem related protein formation.5.In order to verify whether TCD35 gene has the characteristics of heat shock protein genes,was heat shock treated and the expression of two transcripts of TCD35 were detected.The results showed that high temperature may had a positive effect on the expression of long transcript.The overexpressed plants were also successfully obtained in the over-expression experiment,and the expression level was improved significantly.In conclusion,TCD35 played an important role in chloroplast development and function formation at low temperature.It was mainly involved in the formation of rice chloroplast by affecting the synthesis of photosystem related proteins.As a member of heat shock protein family,the gene TCD35 was also induced by high temperature stress.This provided a reference for studying the mechanism of chloroplast development and the action mechanism of organelle type Clp B protein at low temperature.