Transcriptome Analysis Of Sophora Alopecuroides In Response To Salt Stress And Subcellular Localization,Prokaryotic Expression Of SaLDC

Lysine Decarboxylase(LDC),whose main function is to catalyze the decarboxylase to form cadmatrine,is the first key enzyme of matrine(MA)and oxymatrine(OMA)biosynthesis pathway in Sophora alopecuroides L.,and is playing an important role in the biochemical metabolism process.The transcriptome sequencing analysis of S.alopecuroides plants under salt stress was conducted to study the defense mechanism of S.alopecuroides in response to salt stress,which provided a theoretical basis for in-depth understanding of the salt tolerance regulation of S.alopecuroidesalopecuroides.In this study,based on the full-length sequence of lysine decarboxylase gene cloned from S.alopecuroides in Ningxia,the function of the lysine decarboxylase gene was preliminarily explored through the subcellular localization of SaLDC,the relationship between gene expression and the content of its direct product,and the prokaryotic expression.The results of the study are as following:(1)The S.alopecuroides plants were stressed with 150 mmol/L NaCl solution.The leaves were collected before stress(0 d),3 d and 7 d after stress,and transcriptome sequencing was performed.A total of 58.18 Gb data was obtained,and 415804 Unigenes were spliced.There are 44177 unigenes in KEGG get comments in the database;By comparing the gene expression levels of different NaCl solution stressed days in pairs,4229(3 d vs 0 d)and 2119(7 d vs 0 d)3953(3 d vs 7 d)genes were found to be different;Wayne analysis showed that 40 differential genes were shared in three stages.GO enrichment analysis showed that these differentially expressed genes were mainly involved in catalytic stress reaction,aromatic amino acid metabolism,ion and compound transport,etc;KEGG metabolic pathways showed that the first three metabolic pathways were metabolic pathways,biosynthetic pathways of secondary metabolites and biosynthetic pathways of amino acids.Among them,the related genes in phenylpropionic biosynthesis and secondary metabolite biosynthesis pathway jointly regulate gene expression,maintain cell ion balance and osmotic balance,and improve cell salt tolerance.(2)According to the randomly selected 8 differentially expressed genes in response to salt stress metabolic pathways,the primers were designed for quantitative fluorescence verification,and the results showed that the gene expression was down-regulated,which was consistent with the change trend of the gene expression profile obtained by transcriptomic sequencing analysis,indicating that the sequencing results had high accuracy and reliability.(3)Construct the subcellular localization vector pCAM35S-GFP-SaLDC,co-inject the tobacco leaves with mt-rk CD3991 marker,observe with laser confocal microscope to confirm that SaLDC is located in the mitochondria.(4)The expression of SaLDC is tissue-specific regardless of non-salt stress or under 150mmol/L NaCl salt stress.The specific expression is that the stem has the highest expression level,followed by leaves and roots,the direct product cadaverine content change is consistent with the expression level.Correlation analysis shows that the SaLDC expression and the cadaverine content are extremely significantly positively correlated,which shows that the SaLDC expression directly affects the cadaverine content The change.(5)The results showed that the relative synonymous codon usage(RSCU)of 28 codons in SaLDC were larger than 1.00,and preference were shown with synonymous codons ending with A or T.The codon usage bias of SaLDC is the closest to that of Arabidopsis thaliana,and furthest away from that of E.coli.The codons of SaLDC were further optimized according to the codon usage bias of E.coli.The optimized gene was named optSaLDC.The recombinant expression vector pET-28a(+)-optSaLDC was constructed with the target optSaLDC and pET-28a(+)vector and then genetically transformed into E.coli BL21(DE3).After optimization,the optimum expression condition of optSaLDC in E.coli was1.0 mmol/L IPTG at 15? for 16h,and the molecular weight of the target fusion proteinwas measured to be 48990.51 Da by LC-MS/MS.The main biological function of this protein is to catalyze the decarboxylation of lysine to produce cadaverine by GO annotation.