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Chloroplast localization of Cry1Ac and Cry2A protein--an alternative way of insect control in cotton.

Muzaffar A, Kiani S, Khan MA, Rao AQ, Ali A, Awan MF, Iqbal A, Nasir IA, Shahid AA, Husnain T - Biol. Res. (2015)

Bottom Line: The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation.The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system.The results of this study will also be of great value from a biosafety point of view.

View Article: PubMed Central - PubMed

Affiliation: National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, 53700, Pakistan. adnanuaf1@gmail.com.

ABSTRACT

Background: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants.

Results: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 μg/g tissue of Cry1Ac and 0.568 μg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein.

Conclusion: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

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Gus Expression in experimental plants. A: transgenic plant leaves having blue-green color B: Non transgenic plant leaves as negative control with no color change.
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Fig5: Gus Expression in experimental plants. A: transgenic plant leaves having blue-green color B: Non transgenic plant leaves as negative control with no color change.

Mentions: A total of 1000 embryos were transformed with MUZ_01 (TP-Cry1Ac + CryIIA) and subjected to transient expression of the GUS gene. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the MUZ_01 construct vector in the plant expression system because Cry1Ac, Cry2A, and GUS gene expression were under the same promoter (Figures 5 and 6). A bluish green color was apparent in transgenic embryos but not in nontransgenic ones (Figure 6). Thirty plants that survived and passed screening were moved to selection free medium. In the end, seven plants survived soil acclimatization and were moved to the field. The overall transformation efficiency was calculated to be 0.7% (Table 1).Figure 5


Chloroplast localization of Cry1Ac and Cry2A protein--an alternative way of insect control in cotton.

Muzaffar A, Kiani S, Khan MA, Rao AQ, Ali A, Awan MF, Iqbal A, Nasir IA, Shahid AA, Husnain T - Biol. Res. (2015)

Gus Expression in experimental plants. A: transgenic plant leaves having blue-green color B: Non transgenic plant leaves as negative control with no color change.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389580&req=5

Fig5: Gus Expression in experimental plants. A: transgenic plant leaves having blue-green color B: Non transgenic plant leaves as negative control with no color change.
Mentions: A total of 1000 embryos were transformed with MUZ_01 (TP-Cry1Ac + CryIIA) and subjected to transient expression of the GUS gene. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the MUZ_01 construct vector in the plant expression system because Cry1Ac, Cry2A, and GUS gene expression were under the same promoter (Figures 5 and 6). A bluish green color was apparent in transgenic embryos but not in nontransgenic ones (Figure 6). Thirty plants that survived and passed screening were moved to selection free medium. In the end, seven plants survived soil acclimatization and were moved to the field. The overall transformation efficiency was calculated to be 0.7% (Table 1).Figure 5

Bottom Line: The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation.The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system.The results of this study will also be of great value from a biosafety point of view.

View Article: PubMed Central - PubMed

Affiliation: National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, 53700, Pakistan. adnanuaf1@gmail.com.

ABSTRACT

Background: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants.

Results: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 μg/g tissue of Cry1Ac and 0.568 μg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein.

Conclusion: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

Show MeSH
Related in: MedlinePlus