Cloning,Expression And Functional Analysis Of RPS13 And HSP17.8 Genes In Sophora Flavescens

Sophora flavescens is used as a Chinese traditional medicine for clearing away heat and eliminating dampness.Now it widely used in clinical treatment for chronic viral hepatitis,female vaginal inflammation and various human cancers.The chemical active components of Sophora flavescens have shown good application prospects.Ribosomal proteins and heat shock proteins are known to play a very important role in maintaining the normal life activities of plants and alleviating the damage caused by adversity stress,respectively.The ribosomal protein S13 and heat shock protein 17.8 of Sophora flavescens were cloned,expressed in vitro and their biological functions were predicted in this study.Objective:The RPS13 gene and HSP17.8 gene were cloned and induced to express the target protein in vitro.The physical and chemical properties,secondary structure,tertiary structure and other biological information of the target protein were analyzed systematically.The structure and function of the two proteins were preliminarily discussed.Methods:Based on the unigene datas of Sophora flavescens transcriptome,specific amplification primers were designed by primer premier 5.0 software.The total RNA of Sophora flavescens was extracted and the integrity of RNA was detected by microplate reader and 1%agarose gel electrophoresis.The ORFs of RPS13 genes and HSP17.8gene were amplified by RT-PCR.The amplified fragments were ligated into the pMD19-T vectors.The recombinant plasmid was identified by double enzyme digestion?Nde I/Xho I?,bacterial liquid PCR and sequencing.The amplified fragments and the pET22b?+?vector were ligated,and the recombinant plasmid was also identified by double enzyme digestion?Nde I/Xho I?,bacterial liquid PCR and sequencing.The bioinformatics analysis of RPS13 and HSP17.8 was systematically performed.The proteins were induced by different concentration of IPTG at different temperature andtime.The expression levels of RPS13 and HSP17.8 of Sophora flavescens were analyzed by western blot.Results:?1?The total RNA extraction:the ratio of OD_260nm/OD_280nm80nm was 1.92,and the bands of 28S,18S and 5S of RNA were clearly visible by 1%agarose gel electrophoresis.?2?RT-PCR:the three RPS13 showed bright bands at a molecular weight of about500bp?theoretical molecular weight 456 bp?in 1%agarose gel electrophoresis.Similarly,HSP17.8 showed a bright band at a molecular weight of about 500bp?theoretical molecular weight 501 bp?.?3?Double enzyme digestion?Nde I/Xho I?,bacterial liquid PCR and sequencing:the results of double enzyme digestion and bacterial liquid PCR of three RPS13 showed clear target bands at the molecular weight of about 500bp,and the sequencing results were consistent with the original transcriptome data by mRNA sequencing.The double enzyme digestion and bacterial liquid PCR of HSP17.8 showed clear target bands at the molecular weight of about 500bp,and the sequencing results were also consistent with the original transcriptome data.?4?The results of double enzyme digestion?Nde I/Xho I?,bacterial liquid PCR and sequencing were correct.?5?Bioinformatics analysis showed that the three RPS13 were unstable protein,their theoretical molecular weight were about 17.2 KD,the genetic relationship of RPS13s in Sophora flavescens were closed to that of Elaeis guineensis.The tertiary structure showed that the three RPS13s had four?-helix at the N-terminus and three?-helix at the C-terminus,which were linked by a long random coil.HSP17.8 was a stable protein,the theoretical molecular weight was about 17.8 KD,the genetic relationship of HSP17.8 in Sophora flavescens was closed to that of Lupinus angustifolius.The tertiary structure showed that the HSP17.8 had four?-helix and five?-sheets,which formed the folding pattern of?1-?1-?2-?2-?3-?3-?4-?4-?5.The five?-sheets were folded in the samedirection and surrounded by?-helix,forming the internal framework of the three-dimensional structure of the protein.?6?The expression levels of RPS13 and HSP17.8 of Sophora flavescens were detected by western blot.The best expression condition was 0.5mmol/L IPTG,16?,24 hours for RPS13-1,1mmol/L IPTG,25?,8 hours for RPS13-2,0.1mmol/L IPTG,25?,8hours for RPS13-3,respectively.0.5mmol/L IPTG,25?,8 hours for HSP17.8.Conclusion:The full-length ORFs of RPS13s and HSP17.8 genes from Sophora flavescens were successfully cloned,and expressed in vitro.The study of physicochemical properties,evolution,secondary and three-dimensional structures were provided a theoretical foundation for the further structural and functional researches of RPS13s and HSP17.8.