Studies On Construction Of Chloramphenicol Based Saccharomyces Cerevisiae Mitochondria Shuttle Vector

Mitochondria are the main energy supply unit of eukaryotic organisms which including human being,that provide energy through oxidative phosphorylation for cells.Mitochondria are semi-autonomous organelles,containing covalently closed circular DNA.The molecules can synthesize a variety of gene products under the regulation of the nuclear genome.Mitochondrial DNA(mtDNA)mutations and deletions can lead to series of human genetic diseases.Mitochondrial genetic transformation is considered to be a possible treatment strategy.Therefore,the establishment of an effective system of mitochondria transformation may have a good prospect for the treatment of mitochondrial diseases and may provide some ideas.Further more,since mitochondria have a complete transcription,translation system,it has the potential of being a protein bioreactor.Currently studies of mitochondrial exogenous transformation and screening maintain still in the experimental validation.The work includes 4 parts:(1)Chloramphenicol inhibit the growth of yeast;(2)Construction of a chloramphenicol based Pichia pastoris screening system;(3)Construction of a gene trap vector for yeast mitochondrial replicon;(4)Construction of an E.coli-S.cerevisiae mitochondria shuttle vector.(1)Chloramphenicol inhibit the growth of yeastIn this paper,we mainly use the yeast Saccharomyces cerevisiae(S.cerevisiae)INVSc 1 and Pichia pastoris(P.pastoris)GS115 as the research objects.Firstly,we verified theirs sensitivity of chloramphenicol(Chl).Using Yeast Extract Peptone Glycerine Medium(YPG or YPEG)which containing different concentrations of chloramphenicol,and cultured at 30℃,determination the minimum inhibitory dose of chloramphenicol which inhibit these two strains.The results showed that on the YPG medium which containing chloramphenicol,S.cerevisiae INVSc 1 and P.pastoris GS115 had a significant growth inhibition,and this inhibition correlated with the concentration of chloramphenicol.These result description chloramphenicol can inhibit S.cerevisiae INVSc 1 and P.pastoris GS115.More ever,we measured the minimum inhibitory dose was 0.1 mg/mL and 2 mg/mL respectively.Then the chloramphenicol resistance gene,the chloramphenicol acetyltransferase gene(CAT),was amplified from the plasmid pACYC 184 by means of PCR,cloned into a P.pastoris expression vector pPIC 3.5K,that constructed vector named CAT-3.5K.It was transformed by the method of electricity conversion into P.pastoris GS115,and screened.CAT gene expression was induced by methanol,and then measured for growth in the case of YPG medium containing chloramphenicol.CAT gene expression after induction,can significantly increase the growth of P.pastoris GS115,there are significant differences with the control group.RT-PCR was also detected in the expression of the CAT gene,those demonstrate that chloramphenicol can inhibit the growth of yeasts,vector that contain chloramphenicol resistance gene can obtain chloramphenicol-resistant yeast host.(2)Construction of a chloramphenicol based Pichia pastoris screening systemCAT gene was amplified by PCR from the plasmid pACYC 184,and then amplified kanamycin resistance gene(Kanr)upstream regulatory sequences and downstream regulatory sequences from pPIC 3.5K,obtained the functional CAT gene sequences by the method of fusion PCR,and cloned into pET 43.1a,which constructed CAT-pET 43.1a.Finally,the digested fragments were ligated to pPIC 3.5K,which replace Kanr gene,constructed a chloramphenicol screening carrier named pPIC 3.5C.The sequencing results show that the vector was constructed.Linearized vector pPIC 3.5C was transformed into GS115 by electro-transformation,using chloramphenicol-resistant screening,and colony PCR to identify transformants.After confirming of the chloramphenicol transformants,we determination the growth of P.pastoris in the chloramphenicol medium,which had a significant difference compared with the control group.To further validate the ability of the vector using for stransforming and creening exogenous proteins,we constructed vecror which contain an enhanced green fluorescent protein(EGFP)as reporter gene.EGFP was obtained amplification from pEGFP-N2 vector,ligated to pPIC 3.5C,construct to be EGFP-3.5C.Sequencing results showed the vector was successfully constructed.Electric conversion it into GS115,induced by methanol.Using RT-PCR,fluorescence microscopy and Western blotting(WB)observe the expression.The results showed that EGFP protein in this paper can be constructed into vector pPIC 3.5 C and transformed,screened using the corresponding screening system successfully.(3)Construction of a gene trap vector for yeast mitochondrial repliconBased on the above experimental results,we optimized the CAT gene codon optimized by yeast mitochondrial preference codon,and then obtained mitochondrial specific expression CAT’by PCR,while adding yeast mitochondrial cytochrome oxidase subunit ii(cox 2)gene promoter(Pcox2)terminator(Tcox2)by PCR,which named P-CAT’-T.It was connected to pMD18-T by using of TA cloning,structured tobe P-CAT’-T-18T.Using pUC 18 as template,PCR amplification DNA fragment containing the ampicillin resistance gene(Ampr)and E.coli replicon origin(Ori)pBR322 origin as the vector frame,digestion by restriction enzyme and then linked with to CAT’.Which formed a vector comprising a multi-cloning site(MCS)and mitochondrial specific expression genes CAT’,named pMT-D.It lack mitochondrial replicon and may replication in E.coli.Sequence analysis showed that the vector sequences are consistent with expectations.In the study,the yeast mitochondrial vector can be used for replicon trap and studing the function of mitochondrial replicon.(4)Construction of an E.coli-S.cerevisiae mitochondria shuttle vectorFor studing the function of S.cerevisiae mitochondria replicon,this thesis constructed a S.cerevisiae mitochondria replicon trapping vector,pMT-D.Nest PCR amplified NDA fragment containing the mitochondria replicon ori5 from S.cerevisiae mitochondrial genome.Since the mitochondrial genome has the characteristics of high AT,it is difficult to obtain the fragment.In this study,we used an improved PCR cycles and an improved buffer system,obtained a DNA fragment of 90.4%AT content.Which resolved the effect of high AT content in amplification.Sequencing and blast in GenBank showed that we achieve the desired DNA sequence.And then we ligated the amplified replicon into pMT-D,named pMT 1.INVSc 1 was transformed by the method of electrotransformation will pMT 1,screened though chloramphenicol resistance plate.At last we obtain the transformants.Bifunctional CAT analysis method which presented in this study was used for detection of CAT’ gene expression.After then,we extracted the mitochondrial genome of transformants,transformed into E.coli.On Ampr screened plate,we observed bacterial colony containing pMT 1.That described pMT 1 has the ablity of replication in INVSc 1,and the vector that contains CAT’ gene correctly expressed can give the strains chloramphenicol resistance.This study found that chloramphenicol can suppress the growth of S.cerevisiae and using the chloramphenicol resistance gene CAT,we constructed a P.pastoris screening system of chloramphenicol.Also we used codon optimization and molecular cloning methods that constructed a yeast replicon trapping vector,pMT-D,and using this trapping vector obtain a mitochondrial expressing vector based on ori5,named pMT 1.Using bifunctional CAT analysis method which presented in this study,we studied the function of ori5,successfully constructed a vector and transformation,screening system of S.cerevisiae mitochondria.This study provides a tool for mitochondrial replicon function research,and theoretical and experimental supports for mitochondrial theory and practical application of exogenous protein expression in S.cerevisiae mitochondria,laid the foundation for related research.