search
for
 About Bioline  All Journals  Testimonials  Membership  News  Donations


Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 16, No. 6, 2013, pp. 1-14
Bioline Code: ej13074
Full paper language: English
Document type: Research Article
Document available free of charge

Electronic Journal of Biotechnology, Vol. 16, No. 6, 2013, pp. 1-14

 en Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton
Kiani, Sarfraz; Ali, Arfan; Bajwa, Kamran Shehzad; Muzaffar, Adnan; Ashraf, Muhammad Aleem; Samiullah, Tahir Rehman; Shahid, Ahmad Ali & Husnain, Tayyab

Abstract

Background: Transgenic plants inhabiting single Bt gene are prone to develop insect resistance and this resistance has been reported in case of some important yield-devastating insect larvae of commercial crops, such as cotton and rice. Therefore, it has become essential to adapt new strategies to overcome the problem of insect resistance and these new strategies should be sophisticated enough to target such resistant larvae in broad spectrum. Among these, plants may be transformed with Bt gene tagged with some fusion-protein gene that possesses lectin-binding capability to boost the binding sites for crystal protein gene within insect mid-gut in order to overcome any chances of insect tolerance against Bt toxin. Enhanced chloroplast-targeted Bt gene expression can also help in the reduction of insect resistance.
Results: In the present investigation, a combined effect of both these strategies was successfully used in cotton ( G. hirsutum check for this species in other resources ). For this purpose, plant expression vector pKian-1 was created, after a series of cloning steps, carrying Cry1Ac gene ligated with chloroplast transit peptide towards N-terminal and Ricin B-Chain towards C-terminal, generating TP-Cry1Ac-RB construct.
Conclusions: Efficacy of pKian-1 plasmid vector was confirmed by in-planta Agrobacterium check for this species in other resources -mediated leaf GUS assay in tobacco. Cotton (G. hirsutum) local variety MNH-786 was transformed with pKian-1 and the stable integration of TP-Cry1Ac-RB construct in putative transgenic plants was confirmed by PCR; while fusion-protein expression in cytosol as well as chloroplast was substantiated by Western blot analysis. Whereas, confocal microscopy of leaf-sections of transgenic plants exposed that hybrid-Bt protein was expressing inside chloroplasts.

Keywords
chloroplast expression; chloroplast transit peptide; cotton transformation; confocal microscopy; ricin b-chain

 
© Electronic Journal of Biotechnology
Alternative site location: http://www.ejbiotechnology.info

Home Faq Resources Email Bioline
© Bioline International, 1989 - 2017, Site last up-dated on 05-Dec-2017.
Site created and maintained by the Reference Center on Environmental Information, CRIA, Brazil
System hosted by the Internet Data Center of Rede Nacional de Ensino e Pesquisa, RNP, Brazil