Bt gene expression profiling of candidate cotton lines of Pakistan

Abstract

Bt cotton is engineered by insecticidal gene (Bt gene) taken from gram positive, soil bacterium Bacillus thuringiensis. Bt cotton contain Cry (Crystal)genes such as Cry1Ac, Cry1F and Cry2Ab that not only take part in the Bt cotton effectiveness against many cotton insect pests (Lepidoptera), but it is also human and environment friendly. Substantial information suggests that the efficiency of Bt cotton depends on controlled expression of insecticidal gene(s) that does not remain constant throughout growing season. To encounter the challenge of resistance development in targeted insect pest complex, the functionalization of Bt genes is vital and demands better understanding in context of variations in efficiency of Bt genes and their mechanism. The current study was designed to evaluate the effect of temperature on Bt gene expression in selected cotton genotypes under glasshouse conditions. A total of 124 genotypes (V-1 to V-124) were assessed, for the detection of Bt gene(s) by immunostrip assay and PCR analysis that revealed the presence of only Cry1Ac gene, except for five genotypes, among all genotypes. All the genotypes were cultivated between temperature range 25-30°C under glasshouses. Fresh leaf tissues were collected on 80 days after sowing and Cry1Ac gene expression (Bt toxin) was quantified by ELISA. Ten genotypes with the highest expression (1.00-4.17µg/g) of Bt toxin were selected and grown in three replicates under same conditions. On 70 days after sowing all the ten genotypes in three replicates were shifted into three different glasshouses under the temperature ranges of 31-35°C, 36-40°C and 41-45°C, respectively. Among the ten selected genotypes, V-76, V-112, V-106, V-102, V-93, V-96, showed the highest toxin expression (2.10, 2.65, 3.00, 3.36, 4.01 and 4.30µg/g, respectively) at the temperature range of 31-35°C on 80 day after sowing. In all the selected genotypes toxin expression level was decreased with the increase in temperature (36-40°C and 41-45°C). Compare to the Bt toxin expression at 31-35°C, high temperature range (41-45°C) reduced the Bt toxin level up to 26.82% in all the ten genotypes. It can be concluded that temperature may affect the degradation of Cry1Ac protein in leaf, with resulting decline in of Bt toxin level. To adopt the proper agronomic practices for applicable insect pest management in local genetically modified cultivar, it is indispensable to have a clear picture for the selection of best lines with Cry1Ac gene expression. This study will help in selection of candidate lines, based on insecticidal gene expression, for durable resistance against insect pests.