Metabolic Engineering of Berberine in Plants Can Confer Resistance to Insects
Anila Latif, Zaheer Abbas, Farhatullah and Ghulam Muhammad Ali*
National Institute for Genomics and Advanced Biotechnology, Pakistan
drgmali5@gmail.com
Figure 1
Isolation and cloning of BBE gene. a) Agarose gel electrophoresis of amplified product from DNA of B. lyceum; b) Agarose gel electrophoresis of pTZ57 R/T harboring BBE gene.
Figure 2
Developments of plant transformation construct. a) Agarose gel electrophoresis of entry clone isolated from transformed colonies; b) Agarose gel electrophoresis of plant transformation vector isolated from transformed colonies.
Figure 3
Physical map of complete cassette. LB, Left boarder; RB, Right boarder; CaMV 35S, Cauliflower mosaic virus 35 S promoter; YFP, Yeast fluorescent protein; BBE, Cloned barbarian bridge enzyme gene; P nose, P nose promoter; Pat, Phosphinothricin acetyle transferase gene; NOS, NOS terminator.
Figure 4
Transformation of Agrobacterium strain GV 3101 (pMP90RK). Lane M, 1kb DNA ladder (Fermentas); lane 1 and 2, 3 and 4 are amplified products from transformed colonies of Agrobacterium Lane 6 is non transformed colony.
Figure 5
Different steps of floral dip transformation. a, Muffin preparation and seeds sowing; b, Maximum bolting stage prior to infection with transformed Agrobacterium; c, Infection with transformed Agrobacterium culture; d, Screening of transformants with basta spray.
Figure 6
Agarose gel electrophoresis of amplified products from transformed plants.
Figure 7
Expression analysis of BBE gene. Lane E1 to E9, amplifications of BBE gene from the reverse transcription reactions product from different transformed events; lane C, no amplification of BBE gene from the reverse transcription reaction product of wild type isogenic line. Lower panel shows the expression of 18S rRNA gene.
Figure 8
Insect bioassay of transformed and wild type isogenic line. a, wild type isogenic line exposed to 2nd instar larvae (photograph was taken just after introduction of larvae); b, wild type isogenic line (photograph was taken after 72 hours); c, e and g are transformed events 5, 8 and 9 respectively (photographs were taken just after introduction of insects); d, f and h are transformed events 5, 8 and 9 respectively (photographs were taken after 72 hours).