PCR-based Synthesis of Codon Optimized cry2Aa Gene for Production of Shoot and Fruit Borer (Leucinodes orbonalis) Resistant Eggplant (Solanum melongena L.) Cultivars

Simple item page
dc.contributor.authorGupta, Rahulen
dc.contributor.committeechairVeilleux, Richard E.en
dc.contributor.committeecochairWelbaum, Gregory E.en
dc.contributor.committeememberMartino-Catt, Susan Joen
dc.contributor.departmentHorticultureen
dc.date.accessioned2014-03-14T20:50:35Zen
dc.date.adate2006-01-20en
dc.date.available2014-03-14T20:50:35Zen
dc.date.issued2005-12-08en
dc.date.rdate2006-01-20en
dc.date.sdate2005-12-21en
dc.description.abstractBrinjal shoot and fruit borer (Leucinodes orbonalis Guenee) is a major limiting factor in commercial cultivation of eggplant in southeast Asia. Extensive use of pesticides as well as the conventional breeding methods have been ineffective in controlling the borer so there is a need for Integrated Pest Management (IPM) strategies for its control. Bacillus thuringiensis (Bt) is known to produce a variety of insecticidal crystal proteins toxic to lepidopteran, dipteran and coleopteran pests. The Cry2Aa protein has been found to be more toxic to brinjal shoot and fruit borer than Cry1Ab. My objective was to develop eggplant cultivars that express a codon-optimized cry2Aa gene, the sequence of which is based on that of an Indian isolate of Bt, with the eventual goal of producing fully resistant cultivars. The cry2Aa gene was modified for optimal expression in eggplant using the codon usage frequencies based on solanaceous sequences (eggplant, tomato and pepper). The GC content was increased from 34.3% in the native gene to 41.3% in the optimized gene, thus removing the AT-rich regions that are typical for Bt cry genes. Also, other mRNA destabilizing and hairpin forming structure sequences were removed. The gene was synthesized in four different parts with complementary restriction sites. A total of 152 oligonucleotides (oligos) was used to assemble the 1.9 kb gene using dual asymmetric (DA) and overlap extension (OE) PCR techniques. The individual parts were subsequently ligated using the complementary restriction sites and inserted into vector pCAMBIA 1302. Also, the transformation efficiency of 12 different eggplant cultivars was tested using plasmid pHB2892 to predict utility for transformation with the synthetic cry2Aa.en
dc.description.degreeMaster of Scienceen
dc.identifier.otheretd-12212005-234053en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-12212005-234053/en
dc.identifier.urihttp://hdl.handle.net/10919/36369en
dc.publisherVirginia Techen
dc.relation.haspartthesis.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectgenetic transformationen
dc.subjectGFPen
dc.subjectBacillus thuringiensisen
dc.subjectsynthetic geneen
dc.subjectBrinjal shoot and fruit boreren
dc.subjectgene synthesisen
dc.subjectcodon usageen
dc.subjectcodon optimizationen
dc.subjectSolanum melongenaen
dc.subjecttransgenic plantsen
dc.titlePCR-based Synthesis of Codon Optimized cry2Aa Gene for Production of Shoot and Fruit Borer (Leucinodes orbonalis) Resistant Eggplant (Solanum melongena L.) Cultivarsen
dc.typeThesisen
thesis.degree.disciplineHorticultureen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
thesis.pdf
Size:
1.81 MB
Format:
Adobe Portable Document Format
Download

Collections

Masters Theses