Molecular and functional characterization ofcry1Actransgenic pea lines

Abstract

ABSTRACT. Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses.

Highly Efficient and Reproducible Genetic Transformation in Pea for Targeted Trait Improvement

“…Primer pairs qRTDGAT, qRTOLEO, and PPT (Table ) were used to amplify the DGAT , OLEO , and PPT genes to analyze quantitative transgene expression in the transgenic plants. To normalize expression of these transgenes, the pea housekeeping gene, HMG-I/Y was used as a reference with primers HMGIII-F and HMGIII-R amplifying a 164-bp product for the quantitative expression …”

“…To normalize expression of these transgenes, the pea housekeeping gene, HMG-I/Y was used as a reference with primers HMGIII-F and HMGIII-R amplifying a 164-bp product for the quantitative expression. 28 qRT-PCR was performed in optical strip tubes using the Mx3000 qPCR system (Stratagene, US). qRT-PCR was performed in optical strip tubes using the Mx3000 qPCR system (Stratagene, US).…”

“…For RTPCR primers, HMGF and HMGR were used to amplify 350 bp and 570 bp fragments (DNA/cDNA). 28 RT-PCR amplification with cDNA was carried out for the DGAT gene using primers RTDGATF and RTDGATR, and the OLEO gene was amplified with primers RTOLEOF and RTOLEOR ( Table 2 ). 12 μL of the RT-PCR products were analyzed by electrophoresis on 1.2% agarose gels.…”

“…To normalize expression of these transgenes, the pea housekeeping gene, HMG-I/Y was used as a reference with primers HMGIII-F and HMGIII-R amplifying a 164-bp product for the quantitative expression. 28 …”