Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton

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 o...

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Autores principales:	Kiani,Sarfraz, Ali,Arfan, Bajwa,Kamran Shehzad, Muzaffar,Adnan, Ashraf,Muhammad Aleem, Samiullah,Tahir Rehman, Shahid,Ahmad Ali, Husnain,Tayyab
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2013
Materias:	chloroplast expression chloroplast transit peptide cotton transformation confocal microscopy ricin b-chain
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582013000600013
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spelling	oai:scielo:S0717-345820130006000132014-01-16Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cottonKiani,SarfrazAli,ArfanBajwa,Kamran ShehzadMuzaffar,AdnanAshraf,Muhammad AleemSamiullah,Tahir RehmanShahid,Ahmad AliHusnain,Tayyab chloroplast expression chloroplast transit peptide cotton transformation confocal microscopy ricin b-chain 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). 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-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.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.16 n.6 20132013-11-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582013000600013en10.2225/vol16-issue6-fulltext-2
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	chloroplast expression chloroplast transit peptide cotton transformation confocal microscopy ricin b-chain
spellingShingle	chloroplast expression chloroplast transit peptide cotton transformation confocal microscopy ricin b-chain Kiani,Sarfraz Ali,Arfan Bajwa,Kamran Shehzad Muzaffar,Adnan Ashraf,Muhammad Aleem Samiullah,Tahir Rehman Shahid,Ahmad Ali Husnain,Tayyab Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton
description	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). 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-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.
author	Kiani,Sarfraz Ali,Arfan Bajwa,Kamran Shehzad Muzaffar,Adnan Ashraf,Muhammad Aleem Samiullah,Tahir Rehman Shahid,Ahmad Ali Husnain,Tayyab
author_facet	Kiani,Sarfraz Ali,Arfan Bajwa,Kamran Shehzad Muzaffar,Adnan Ashraf,Muhammad Aleem Samiullah,Tahir Rehman Shahid,Ahmad Ali Husnain,Tayyab
author_sort	Kiani,Sarfraz
title	Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton
title_short	Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton
title_full	Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton
title_fullStr	Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton
title_full_unstemmed	Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton
title_sort	cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2013
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582013000600013
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Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton

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