The Study On Clone Of Trehalose Synthesize Enzyme Gene And Construction Of High-effect Expression Vector In Cerevisiae

Trehalose ( Trehalose, a -D-glucopyranosyl- a -D-glucopyranoside ) is a kind of indeoxidized disaccharide coupled with two glucose by a , a -1,1 bond, those special creature who include trehalose can live for many years without water, for example , a kind of creature whose name is "cryptohidden life" can still resume life promptly after rehydration when 99% of their body water is dehydration. Study show that besides rehydration, high temperature, high pervasive, heavy metal and poisonous etc. can make the creature accumulate trehalose. It was thought that trehalose could respond to stress. In deeply research show that trehalose can protect biomacromolecule such as protein, enzyme, nuclear acid and so on. Therefore, the creature that include trehalose display special feature in harsh environments.Purpose of this study is clone and express Trehalose-6-phosphate phosphatase(TPS1) gene by gene engineering, which will be foundation of gene-engineering strain construction further.So main goal of this study is build a high efficient vector that can express the TPS1 in Eco li. strain.We designed a pair of primer according to TPS1 sequence that publish on Genbank and use the total RNA from Saccharomyces cerevisiae that keep in our laboratory as template, then amplified and obtained target gene(TPS1) through RT-PCR. Afterward connected TPS1 with T-vector, transformed Eschericha coli DH5 a with the recombinant plasmid. The target gene(TPS1) would amplify in the recept strain. The insertion of TPS1 gene into T-Vector was confirmed by restriction analysis and PCR, cultrued positive strains that include the recombinant plasmid whole night.Added glycerine until its concentration is 20% in order to conserve it. We knew that the cloning gene is 1507bp(include restriction endonucleases enzyme position and protect base, total are 19bp). In fact the whole gene is 1488bp, include initiation codon and termination codon, so it's a complete open reading frame, which can translate into 496 amino acids, the gene sequence is 99.6% identity to TPS1 published onGenbank, the deduced amino-acid sequence is also 99.6% identity to TPS1 amino-acid sequence. Therefore, we could express the gene next step.In the cloning experiments, we improve Trizol method of total RNA isolation when extracted RNA from Saccharomyces cerevisiae.To make the method fit in with it, moreover the RNA purification and completment was enhanced by the approach. In RT-PCR process, we designed range analysis to obtain a better PCR condition.At last the conclusion is add Taq enzyme 1.5u, template 10ng, dNTP(10mmol/L) lul, primer 60pmol each, Mg2+2.5mmol/L every reaction Reactive condition is 94C, 50s 58-52C, lmin,30s 72C, 2min,30 cycles, the last cycle,72C, 10minThe recombinant plasmid consist of T-vector and target gene was cut by Bam HI and Pst I, then insert pUC18 who cut by the same restriction endonucleases enzyme, we obtained vector that named pHY01.Meantime we cut the recombinant plasmid and pET-28a with Sac I and Bam HI, obtained pHY02 vector. The two vectors were confirmed by restriction analysis and PCR, hence we knew that the vectors were constucted successfully.Make the two vectors transform BL21, and induced with IPTG, then we gained expression protein.After quantification of extract trehalose from recombinant strain, we proved that the expression protein was active. However, if we want get more protein from it, it must be study further.In a word, this study make a steady basis for constructing gene-enginerring Eschericha coli which can produce plenty of trehalose.