engleski

Synthesis and cloning of 12 CMTI-I analogues for their expression in plants

Proteinase inhibitors play an important role in limiting and defining the range of insect pests potentially harmful to plants. The introduction of foreign proteinase inhibitor genes into agricultural crops can therefore reduce damage caused by insects. In spite of their small size, squash serine proteinase inhibitors are very strong inhibitors of bovine &#61538 ; -trypsin and their use against insect trypsins is thought to lead to some success. Among different squash inhibitor family members, CMTI-I (from Cucurbita maxima L.) has been the one most intensively studied and several biochemical and physical properties are known. In this work, 12 artificial genes that could render inhibitors sharing the CMTI-I skeleton, but having a different combination of amino acids in the reactive site and in a flanking region were synthesised using DNA polymerase I (Klenow fragment) on partially overlapping forward and reverse primers. Following treatment with Taq polymerase, genes were cloned in the phagemid vector pGEM-T ; after transformation of E. coli strain JM 101, their sequence was checked by automatic sequencer and found correct. Genes were then subcloned in the Agrobacterium plasmid vector pBI 121 in place of the uidA gene. Recombinant pBI 121 plasmids were finally transferred to A. tumefaciens strain EHA 105 by triparental mating. The last procedure made artificial genes available for plant cell transformation.

proteinase inhibitors; artificial genes; gene cloning

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