Establishment Of A Noval GABA Quantitative Strategy And Screening Of Reference Genes For Quantitative Real-time PCR In Lactobacillus Brevis CD0817

Gamma-aminobutyric acid?GABA?is a natural non-protein amino acid,which possesses several important physiological functions and is a key physiologically active molecule in organisms.In recent years,many studies have focused on using lactic acid bacterial?Lactic acid bacteria,LAB?as a cell factory to produce GABA due to the fact that LAB are generally regarded as safe and have shown relatively high GABA production potential.Previously we screened a strain of Lactobacillus brevis CD0817 with high GABA production.Understanding the gene expression pattern of this organism will be useful in investigating its adaptation to different culture conditions,such as Mn²⁺stress.However,little is known concerning the appropriate reference gene which could be used for normalization in the study of gene expression of LAB.On this basis,suitable internal reference genes were screened under different stress conditions.In addition,a new and practical GABA quantitative method was established.The main results are as follows.?1?A new and efficient quantitative analysis method for GABA was established.The interaction between these two compounds severely hindered their disassociation.Herein,we present a new strategy,termed zinc acetate-assisted differential precipitation/dissolution?ZA-DPD?,for the removal of glutamate by step by step recovering pure GABA solution and discarding pure glutamate pellet,essentially attributed to the use of two core reagents?glutamate-precipitating reagent,and glutamate-rejecting reagent?.In each precipitation,the glutamate-precipitating reagent guaranteed most GABA still soluble although the rest co-precipitated with glutamate;in the coupled dissolution,the co-precipitated GABA was fully dissolved with?water used?or without?in the case of glutamate-rejecting reagent used in the final dissolution?co-dissolution of glutamate.The process was repeated twice until glutamate was thoroughly removed.Subsequently,an accurate quantitative method coupling ZA-DPD with colorimetry was thereafter established for the determination of GABA.Twenty five?L sodium acetate buffer?pH 5.8?was added into 2 mL glutamate-cleared samples,then 0.2 mL the chromogenic reagent was added and blended well followed by incubating for color generation at 50?for 50 min.After appropriate dilution?if necessary?by 20%ethanol,A₅₇₀ values were read in a UV–vis spectrophotometer.The fresh fermentation medium was treated by the same procedure as sample and used as blank control.This study may facilitate the areas associated with GABA or glutamate.?2?For accurate and reliable gene expression analysis,normalization of gene expression data using one or more appropriate reference genes is essential.In this study,we used three statistical methods?geNorm,NormFinder,and BestKeeper?to evaluate the expression levels and stability of ten candidate reference genes?FUSA[translation elongation factor G],16s rRNA,HSP60[Chaperonin GroEL],LDHD[D-lactate dehydrogenase],PYRG[CTP synthase],RPOC[pyrroline-5-carboxylate reductase],RECA[RecA/RadA recombinase],PHES[Phenylalanine--tRNA ligase alpha subunit],GAPDH[Glyceraldehyde-3-phosphate dehydrogenase],TUF[Elongation factor Tu]?under different culture conditions and different growth phases to find a suitable housekeeping gene which can be used as internal standard.The results showed that the best reference gene was RECA,and a set of two genes,TUF and PHES,is recommended for the normalization of real-time quantitative PCR experiments under all the different experimental conditions.The best gene was RECA,followed by LDHD and TUF under Mn²⁺control;the best gene was TUF,followed by PHES and RECA under glutamate control.This experiment will provide a standardized correction for the further study on the expression analysis of the target genes of CD0817 and other lactic acid bacteria?LAB?under the condition of manganese ion or glutamic acid,and also provide a basis for the selection of reference genes of other lactic acid bacteria.