Design And Development Of PCR Genome Walking Kit And Preliminary Study On Gad Operon Of Levilactobacillus Brevis

With the advancement of technology,next-generation sequencing technology has developed rapidly,rather the strategy of whole-genome sequencing for unknown species is still expensive.Therefore,genome walking remains the method of choice for researchers to obtain the interested DNA sequence in the laboratory.Existing genome walking methods suffer from many drawbacks.For example,the method of screening genomic libraries is time-consuming and laborious,inverse PCR and digestion-ligation-mediated PCR are affected by pretreatment,resulting in low efficiency,and the high background generated by randomly primed PCR is difficult to eliminate.In this study,an efficient Semi-Site-Specific Primer PCR（3SP-PCR）was established to improve the genome walking technology;Randomly prefixed sequence-specific primer PCR（RAP-PCR）genome walking technology is proposed further improve on the basis of 3SP-PCR,and RAP-PCR genome walking kit is designed to replace commercially available kits.Finally,using the gad gene flanking sequence obtained by genome walking as the object,the formed Levilactobacillus brevis CD0817gad operon structure was analyzed and conducted relevant experiments to provide needed molecular tools for next research.The main results are as follows:（1）3SP-PCR is a based on random priming walking technology PCR method,the key of 3SP-PCR is the use of semi-outer-site-specific primer（semi-o SSP）in the secondary reaction.The 3’-end portion of semi-o SSP overlaps with the 3’-end portion of the outer site-specific primer（o SSP）used in the primary PCR,while the 5’-end portion is completely heterologous.Primary 3SP-PCR is only driven by a single o SSP,which allows partial annealing of the o SSP to the appropriate site on the formed ss DNA or genomic DNA when cycling at low stringency（25°C）.In the next medium-high stringency cycle,conversion to ds DNA is performed.Secondary 3SP-PCR using primary PCR products as templates was driven by internal inner site-specific primers（i SSP）together with semi-o SSP.Based on the principle of partial overlap,a cycle with a lower stringency（40°C）allows the semi-o SSP to pair complementary to the 3’part of the o SSP site on the target ss DNA and extend.In the next cycle of high stringency,ss DNA is converted into ds DNA contained in i SSP and semi-o SSP,while non-specific products lacking annealing sites will be diluted,thus achieving effective enrichment of the target product.After the method was validated by the gad gene of Levilactobacillus brevis and the hyg gene inserted in rice,the main bands produced were specific and clear,and could be used as an alternative to the existing genome walking method to obtain unknown sequences.（2）RAP-PCR is a genome walking technique modified on 3SP-PCR.The key lies in the design and use of the randomly prefixed sequence-specific primer（RAP-SSP）,which consists of a completely a random prefixed primer（RAP）sequence at the 5’-end and genome squence-specific primer sequence（SSP）at the 3’-end.Primary RAP-PCR,only the RAP-SSP designed for the genome sequence is used to drive.Under a low stringency cycle at 25°C,the RAP-SSP will anneal to the unknown region of the genome to form ss DNA,and then synthesize ds DNA in the subsequent high stringency（65°C）cycle target product.The internal site specific primer SSP2（SSP3）is used together with the RAP provided in the kit.After 2-3 rounds of nested high-stringency PCR cycles,the non-specific products of the primary/secondary PCR reactions are removed,and obtain the purified walking product.We tested the generality and feasibility of this method with the gad A gene of Levilactobacillus brevis and the hyg gene of rice,and the results are satisfactory,which can be used as effective data support for the genome walking kit.（3）Based on the RAP-PCR genome walking technology,a RAP-PCR genome walking kit was developed and expected to become a substitute for commercially available kits.（4）Based on the whole genome analysis of Levilactobacillus brevis CD0817 in the previous laboratory,this experiment studied the gad gene flanking related sequences obtained by genome walking technology,and analyzed the relevant functional elements of the gad operon by using bioinformatics means:promoter region,ribosome binding site,transcription initiation site,etc.And designed a shuttle vector p MG36e-Cm to explore the mechanism of gad operon.Levilactobacillus brevis competent cells was cultured to A₆₀₀ 0.4-0.6,which add p MG36e-Cm and electric shock under 1.0 kv 4 ms transformation conditions can obtain transformants.p MG36e-Cm-△gad A-Sac B containing the upstream and downstream homology arms of the gad A gene and sucrose suicide gene Sac B was constructed,and related transformation experiments were carried out to provide molecular tools for the subsequent exploration of the high-yielding GABA mechanism of Levilactobacillus brevis.