杉木ClHSP70基因的克隆及表达模式分析OA北大核心

In this study,the Heat Shock Protein 70(HSP70)gene of cedar was investigated by cloning,bioinformatics and expression analysis methods to provide a scientific basis for the in-depth understanding of the function of the HSP70 gene of cedar,as well as the genetic improvement and sustainable cultivation of cedar.The annual seedlings of the excellent Chinese fir asexual line"Yang 061"were selected as test materials,and the ClHSP70 gene was cloned by reverse transcription polymerase chain reaction(RT-PCR).The physicochemical properties,transmembrane helical domain,signal peptide,secondary structure and tertiary structure of ClHSP70 protein were predicted and analysed using Expasy software and other online software;the subcellular localization of the protein was predicted using Cell-PLoc 2.0 online software;Mega 11 software was used to construct a phylogenetic tree;the ClHSP70 gene was cloned and constructed into pCAMBIA35s-EGFP vector for analysis of ClHSP70 gene.EGFP vector to analyse the subcellular localisation of the ClHSP70 protein;and its expression level was analysed using Quantitative Real-time PCR(QRP).The cloned ClHSP70 gene encoded 670 amino acids,and the molecular formula of ClHSP70 protein was C3297H5306N904O1008S26,which was unstable,did not contain signal peptide and transmembrane region,and was predicted to be localised in the cytoplasm.Phylogenetic analysis showed that Chinese fir ClHSP70 was more closely related to hazelnut(Corylus avellana).The results of subcellular localisation experiments showed that ClHSP70 protein was localised in the nucleus.qRT-PCR expression analysis showed that the relative expression of ClHSP70 gene was the highest in the leaves,and the relative expression of ClHSP70 gene reached the peak after 6 h of high temperature,and the expression of ClHSP70 gene reached the maximum after 12 h of drought treatment,and the expression of ClHSP70 gene was up-regulated by the high temperature and drought stress induced.The ClHSP70 gene was up-regulated by high temperature and drought stress.The successful cloning and sequence analysis of the ClHSP70 gene in cedar revealed its expression in different tissues of cedar and its response to high temperature and drought,and provided an important theoretical basis for the breeding of cedar resistance.