Establishment Of Two New PCR-based Genome Walking Technologies And Preliminary Study On GAD Extraction Of Levilactobacillus Brevis

Genome-walking is a molecular technique to retrieve unknown flanking sequences from known DNA,which can isolate regulatory sequences of functional genes,clon full-length open reading frames,mine new gene resources according to conserved regions and determine insertion sites of transgenes.The existing genome walking methods are limited by factors such as low specificity,high background or complex operations,which cannot meet the research needs.Therefore,it is necessary to develop more efficient genome walking technology.γ-aminobutyric acid(GABA)is a functional compound,Glutamic acid decarboxylase(GAD)can specifically convert L-glutamic acid(sodium)into GABA through α-decarboxylase reaction,and promote the growth of liver cells and regulate the metabolic function of human body.The genome walking technique can be used to isolate the complete GAD gene and its regulatory region,so as to promote the research in this field.This paper establishes Bridging PCR and Primer Extension Refractory PCR,(PER-PCR)for genome walking.At the same time,the separation and purification of GAD enzyme from Levilactobacillus brevis CD0817 were explored.On the basis of obtaining high purity GAD,it is expected to catch the complete sequence of GAD and its upstream and downstream regulatory regions by Bridging PCR or PER-PCR,in order to provide theoretical and technical support for cloning,expression regulation and other aspects of GAD gene.The main research results of this paper are as follows:Bridging PCR requires the design of three random walking primers,inner walker primer(IWP),bridging primer(BP),and outer walker primer(OWP),are involved in bridging PCR.The BP is fabricated by splicing OWP to the 5’ end of IWP’s 5’ part.A bridging PCR set is constituted by three rounds of amplification reactions,IWP was used for primary PCR,BP was used for secondary PCR,and OWP was used for tertiary PCR,which were paired with three Sequence-specific primers(SSPS).A nontarget product arising from IWP alone undergoes end-lengthening mediated by BP.This modified non-target product is preferentially formed hairpin between the lengthened ends,instead of binding with shorter OWP.Meanwhile,a non-target product,triggered by SSP alone or SSP plus IWP,is removed by nested SSP.As a result,only the target DNA is accumulated.The efficacy of bridging PCR was validated by walking the gad A/R genes of Levilactobacillus brevis CD0817 and hyg gene of rice.The PER-PCRA is characterized by the use of a walking primer set of three primers.The middle 15 nt of primary walking primer partially overlaps the 3’ parts of secondary and tertiary ones;while the 5’ parts are heterologous to each other.The overlap facilitates walking primers annealing to each other only at a low temperature,creating a lot of single-stranded DNAs.In the next high-temperature cycle,the target single-stranded DNA can be duplicated into double-stranded form by the sequencespecific primer,and thus can be exponentially amplified by the remaining hightemperature cycles.The non-target one fails to be enriched due to the lack of a perfect binding site to any primer.The heterologous 5’ part prevents creating a binding site for the walking primer on the end(s)of non-target DNA.The PER-PCR was validated digging into the unknown flanks of the hygromycin gene in rice and the L-glutamic acid decarboxylase gene in Levilactobacillus brevis CD0817.Levilactobacillus brevis CD0817 is a strain with high GABA production isolated in our laboratory,which yield can reach 321 g /L.In this paper,the purification process of GAD from Levilactobacillus brevis CD0817 was investigated.The extraction conditions were optimized and the enzyme activity was monitored by thin layer chromatography.After 2 mg/m L lysozyme treatment,the enzyme activity existed in the supernatant.Though ammonium sulfate which showed that the GAD protein with enzyme activity could not precipitate when the saturation of ammonium sulfate is less than 50%.At saturation of 70%,almost all GAD proteins are effectively precipitated.Therefore,ammonium sulfate with saturation of 70% was selected for crude enzyme precipitation.Examining GAD activity stability in phosphoric acid buffer,sodium acetate buffer,citrate-sodium citrate buffer,glutamate-sodium glutamate mixture and 10%ammonium sulfate,selecting pH 5.5 citrate-sodium citrate buffer as the ion chromatography equilibrium solution.Gradient elution experiment showed that 0.3 M NaCl could effectively elute GAD protein with enzyme activity.