Construction of a recombinant pBI121-CHGLU containing chitinase and gluganase genes.
2007
Tuhid Far, Mas`ud | Mapar, Mona
Production of transgenic cotton (Gossypium hirsutum L.) was studied utilizing three Agrobucterium–mediated strains: EHA101, LBA4404 and C58. All Agrobacterium strains harboured a binary vector, pBI121. In order to construct a plasmid containing chitinase gene (pBI121-BCH) a 1.0 Kbp bean chitinase gene (chti) was subcloned into EcoRI site of LacZ´ operon in pGEM-7Zf(+), an intermediate cloning vector, followed by transformation of E. coli strain XL1-Blue and screening for a LacZ- transformant cells. The insert was then digested with two different restriction enzymes, XbaI and SacI, and subsequently ligated into the homologous sites in pBI121. In this case, the chti gene was placed under the control of a constitutive CaMV35S promoter. Gene orientation was verified by double restriction digestion pattern. Hypocotyl pieces were inocolated with Bacteria suspension. Thus hypocotyl pieces were transferred to calli induction medium. Kanamycin resistant calli were transferred into regeneration medium. After germination, these embroyoes were transferred to the regeneration medium. Majority of Kanamycin – resistant calli were obtained using strain A.tumefaciens LBA4404. Expression of gus in leaf of transgenic plants derived from variety Coker, was the result of successful transformation by gus gene. PCR analysis showed that transgenic plants contained the chitinase. Integration of chiti gene into the genome of putative transgenics was further confirmed by southern blot analysis. &lsquoWestern&rsquoimmunoblot analysis showed a strong expression of chitinase gene in transgenic lines as campared to non-transformed cotton.
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