K. Momena
Advanced Seed Research and Biotech Center, ACI Limited, Dhaka, Bangladesh
A. F. M Jamal Uddin
Associate Professor
Dept. of Horticulture, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh
P. A. Kumar
National Research Center on Plant Biotechnology, IARI, New Delhi, India
H. Mehraj
Dept. of Horticulture, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh
Helicoverpa Armigera, Cry1Ac Insecticidal Protein and Cadherin
National Research Center, Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India
Pest Management
Diseases and insects
An experiment was conducted in National Research Center on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India with a view for designing of primers for amplification of HaCad1 from Helicoverpa armigera: Based on published HaCad1 gene sequences, a set of specific primers were used to amplify the receptor binding portion of cadherin from the cDNA sample of Helicoverpa armigera midgut portion.
Total RNA isolation from H. armigera mid gut: Total RNA was isolated from the mid gut portion of the 4th instar larvae of the Helicoverpa armigera. RNA isolation was done by using TRizol reagent (Invitrogen). Quality and quantity of the total RNA was checked by gel electrophoresis and Nanodrop 1000 spectrophotometer respectively.
cDNA synthesis from the total RNA: Single stranded cDNA was prepared by using AffinityScript QPCR cDNA Synthesis Kit. (Stratagene).
Amplification of the cadherin gene fragment: Gradient PCR was performed to find out the annealing temperature for amplification of cadherin receptor region at 50-60ºC by using Taq DNA polymerase enzyme (Bangalore Genei). Pfu DNA polymerase was used to amplify the final product and avoid the mismatch of the nucleotide sequences. Amplification was performed in an eppendorf thermal cycler (Eppendorf AG, Germany).
Analysis and Purification of PCR products: After amplification of cDNA, it was checked in 1% agarose gel electrophoresis and purified the cDNA using QIAquick PCR purification kit (QIAGEN, Germany).
Cloning of cadherin gene (HaCad1) fragment into pMALc2x expression vector: For the expression of cadherin gene, 732 bp of PCR purified cadherin gene was restricted with BamHI and SacI site and cloned into correspondent site of pET29 (a+) vector. The cadherin fragment was further cloned into pMALc2X expression vector by BamHI – HindIII restriction sites for improving the protein expression. Routine lab protocols were followed for ligation, transformation and screening of transformed colonies.
Overexpression of cadherin protein: The clone was transformed into the TB1 host cells and the transformants were used for screening. Protein was isolated according to manufacture instruction (New England BioLabs) from single colony and induced by 1mM IPTG. The overexpressed protein was checked by SDS-PAGE and Western blotting.
Screening for overexpression colony by SDS-PAGE analysis and Western Blotting: Isolated protein was checked on 12% SDS-PAGE (BIO-RAD Mini-Protein-3 Electrophoresis system). Because of the small fragment of the cadherin gene the expected band size was not clearly seen under the SDS-PAGE. For further confirmation, western blotting was carried out. SDS-PAGE separated proteins were transferred to nitro cellular membrane using mini trans-blot electrophoretic transfer cell apparatus (BIO-RAD, USA). After adding primary and secondary antibody to the membrane, it was further washed with PBST for three times and air dried on what man paper and immediately photograph was taken.
Journal of Bioscience and Agriculture Research, Vol. 03 (01): 59-64, eISSN 2312-7945, 2015
Journal