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A molecular approach to combat spatio-temporal variation in insecticidal gene (Cry1Ac) expression in cotton

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Abstract

Spatio-temporal expression of an insecticidal gene (Cry1Ac) in pre existing transgenic lines of transgenic cotton was studied. Seasonal decline in expression of Cry1Ac differed significantly among different cotton lines tested in the field conditions. The leaves of the Bt cotton plants were found to have the highest levels of toxin expression followed by squares, bolls, anthers and petals. Expression of the gene decreased consistently with the age of plants. Toxin expression in fruiting parts was not enough to confer full resistance against bollworms. The reduction in efficacy of transgenic cotton plants late in the season was attributed to reduction in promoter activity. For this purpose, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (rbcS) promoter was isolated from Gossypium arboreum that was further cloned upstream of an insecticidal gene (Cry1Ac) in expression vector pCAMBIA 1301. A local cotton cultivar NIAB-846 was transformed with Cry1Ac driven by rbcS promoter. The same cotton cultivar was also transformed with Cry1Ac gene driven by 35SCaMV promoter to compare the expression pattern of insecticidal gene under two different promoters. The results showed that rbcS is an efficient promoter to drive the expression of Cry1Ac gene consistent throughout the life of cotton plant as compared to 35S promoter. The use of tissue specific promoter is also useful for addressing the biosafety issues as the promoter activity is limited to green parts of plants, hence no gene expression in roots, cotton seed and other cotton products and by products.

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Abbreviations

CEMB:

Centre of Excellence in Molecular Biology

rbcS :

Rubisco small subunit

Rubisco:

Ribulose-1,5-bisphosphate carboxylase/oxygenase

ELISA:

Enzyme linked immunosorbent assay

PCR:

Polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

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Acknowledgments

The author (Dr. Allah Bakhsh) is highly thankful to Higher Education Commission (HEC), Pakistan for providing funds to CEMB, University of the Punjab for his research activities during PhD studies. Many thanks to Dr. Ghazanfar Ali Khan, Incharge Cotton Research Station Vehari for providing seed of cotton cultivar FDH-786 and Dr. Muhammad Aslam, Principal Scientist NIAB, Faisalabad for providing seed of cotton cultivar NIAB-846. The services rendered by CEMB insect rearing laboratory (Muhammad Illyas, Abdul kareem and Raza Ali Zaidi) are also acknowledged.

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  1. National Centre of Excellence in Molecular Biology, University of the Punjab, 87 West Canal Bank Road, Thokar Niaz Baig, Lahore, 53700, Pakistan

    Allah Bakhsh, Saima Siddique & Tayyab Husnain

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  1. Allah Bakhsh
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  2. Saima Siddique
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  3. Tayyab Husnain
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Correspondence to Allah Bakhsh.

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Bakhsh, A., Siddique, S. & Husnain, T. A molecular approach to combat spatio-temporal variation in insecticidal gene (Cry1Ac) expression in cotton. Euphytica 183, 65–74 (2012). https://doi.org/10.1007/s10681-011-0497-8

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