title: Chloroplast localization of Cry1Ac and Cry2A protein--an alternative way of insect control in cotton.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, 53700, Pakistan. adnanuaf1@gmail.com.|Institute of Molecular Biology, Academia Sinica, Taipei, 115, Taiwan. adnanuaf1@gmail.com.
authors.forename: Adnan
authors.initials: A
authors.lastname: Muzaffar
authors.affiliation: National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, 53700, Pakistan. sarfrazkiani786@hotmail.com.
authors.forename: Sarfraz
authors.initials: S
authors.lastname: Kiani
authors.affiliation: National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, 53700, Pakistan. mazmatullahkhan@yahoo.com.
authors.forename: Muhammad Azmat Ullah
authors.initials: MA
authors.lastname: Khan
authors.affiliation: National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, 53700, Pakistan. qayyumabdul77@yahoo.com.
authors.forename: Abdul Qayyum
authors.initials: AQ
authors.lastname: Rao
authors.affiliation: National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, 53700, Pakistan. arfan.ali@cemb.edu.pk.
authors.forename: Arfan
authors.initials: A
authors.lastname: Ali
authors.affiliation: National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, 53700, Pakistan. phool_uaf@yahoo.com.
authors.forename: Mudassar Fareed
authors.initials: MF
authors.lastname: Awan
authors.affiliation: National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, 53700, Pakistan. aadiiq@yahoo.com.
authors.forename: Adnan
authors.initials: A
authors.lastname: Iqbal
authors.affiliation: National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, 53700, Pakistan. dr.idrees@gmail.com.
authors.forename: Idrees Ahmad
authors.initials: IA
authors.lastname: Nasir
authors.affiliation: National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, 53700, Pakistan. ahmadali.shahid@gmail.com.
authors.forename: Ahmad Ali
authors.initials: AA
authors.lastname: Shahid
authors.affiliation: National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, 53700, Pakistan. tayyabhusnain@yahoo.com.
authors.forename: Tayyab
authors.initials: T
authors.lastname: Husnain
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D004731:Endotoxins / Q000032:analysis / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D050939:Gene Fusion; D003368:Gossypium / Q000235:genetics*; D006460:Hemolysin Proteins / Q000032:analysis / Q000235:genetics*; D007150:Immunohistochemistry; D007300:Insect Control / Q000379:methods*; D007305:Insecticide Resistance / Q000235:genetics; D007306:Insecticides; D007814:Larva; D007915:Lepidoptera; D008858:Microscopy, Phase-Contrast; D018515:Plant Leaves / Q000235:genetics; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins; D019076:Transgenes / Q000502:physiology
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D017931:DNA Primers; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis