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Cloning And Fusing Of The Cholesterol Oxidase Gene And Their Expression In E. Coli. And Higher Plant

Posted on:2002-03-21Degree:DoctorType:Dissertation
Country:ChinaCandidate:R ZhangFull Text:PDF
GTID:1100360032956003Subject:Crop Genetics and Breeding
Abstract/Summary:PDF Full Text Request
Cholesterol oxidase is a type of protein that was found to have pest-resistant activityonly years ago,its toxity to such sensitive pests as cotton boll weevil is euqivalent topreviously found BT endotoxins .The protein can also inhibit the growth anddevelopment of cotton boll worm .Because of its high toxity activity andcomplementary inseticidal spectrum with Bt. endotoxin which is now being useduniversally ,the choleterol oxidase is acknowleged as one of the representitive of theinsecticidal protein of the second generation.It is reported in this study that (1) two high enzyme-yeilding strains TK54 andTi 2 have been screened from four choleterol-secrecting experimental strains,hesupernatant of which have been tested for insecticidal activity with the results showingthat cholosterol oxidase can strongly inhibit the vigor of cotton boll worm. A specificDNA fragment has been amplified from TK54 bateria strain by polymerase chainreaction(PCR) .The fragment ,after being cloned and subcloned,was sequenced to havea full length of 1.644 bp.encoding the cholesterol oxidase and showing a homology of97.6% with another choleterol oxidase gene cloned from a streptomycin bacteriastrain .They belong to a gene family .The new gene was designated as choa.The expression vectors of cholesterol oxidase full gene and mature enzyme genehave been constructed using pBV221 and expressed in BL21.The gene construct of fullgene was expressed in high level(with a ratio of 75.4% of expressed target protein /total unresolved protein).but no expression was detected in the mature enzyme geneconstruct without the leading peptide sequence. Further amplification of the leadingpeptide sequence and located at the 5' end of the mature enzyme gene, (2)the E Co/istrain transformed with the above-mentioned gene acquired the capability to expresscholesterol oxidase again. suggesting that leading peptide sequence plays a decisive rolein the expression of cholesterol oxidase gene, which has not been reported up tonow.Further analyses revealed that there was a palindmme sequence in the 3t end of theleading peptide sequence , which was thought to be an important element in theexpression of cholesterol oxidase gene with high efficiency . Up to now, no similarresults have been reported.Mono-valent plant expression vectors of cholesterol full gene and matureenzyme ttene were constructed. Along with genes Bt cr) 14 , bivalent gene expression\`ector and fusion gene expression vector were also constructed. Transgenic tobaccoplants were obtained transformed with 6 plant expression vectors by leaf disc methodmediated by A~obacteria. Results of testing transgenic plants with PCR, Southern blotmethods confirmed the successful integration of exogenous gene into tobacco plantsgenome. Although direct lethal effects on cotton boll worm couldn't be generated intobacco plants transformed with cholesterol oxidase full gene vector or mature enzymegene vector, inhibitory effects on the growth and development of cotton boll wormhave been observed to a certain degree. In addition, better inhibitory effects could beseen in transgenic tobacco plant with full enzyme gene.Among tobacco plants transformed with bivalent vectors, plants with full geneshowed relatively better worm resistance.while among tobacco plants with fusion gene,plants with mature enzyme gene had better resistance, thus further conclusion can bedrawn that full enzyme gene form is beneficial for gene expression in plant, whilemature enzyme gene form is the one that it took when cholesteroloxidase had activity.There was significant difference in the performance of pest resistance betweenearls' and later stages of plants transformed with bivalent genes. However, fusion genetransformed cotton plants showed extremely high r...
Keywords/Search Tags:cholesterol oxidase, gene fusion, insecticidal gene, expression in bateria, expression in plant
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