Genetic Transformation of Tobacco with Rhizobium Genes for Degradation of the Toxic Aromatic Compound 3-hydroxy-4-pyridone a Thesis Submitied to the Graduate Division of the University of Hawai'i in Partial Fulfillment
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چکیده
The tree legume leucaena (Leucaena leucocephala) is rich in protein content, but it also contains the toxin mimosine. A set of genes involved in mimosine degradation has been identified from Rhizobium TAL1145. TAL1145 degrades mimosine in two steps: the mid genes encode enzymes that break down mimosine to 3-hydroxy-4-pyridone (HP); HP is further degraded by a dioxygenase and a hydrolase, encoded by pydA and pydB, respectively. The aim of this research is to express the Rhizobium pydA and pydB genes in tobacco. pydA-pydB fusions that have properties of both bacterial genes were constructed. PydA, pydB and three types of pydA-pydB fusions were individually cloned into two binary vectors, pBI121 and pCAMBIA3201, to obtain recombinant plasmids that were used for Agrobacterium-mediated transformation. In order to first test the stable integration and expression of these genes in a higher plant, we used tobacco, which is a model plant system for genetic transformation. Stable integrations of the transgenes in tobacco were confirmed through PCR and transcription of the foreign genes analyzed by RT-PCR. Transgenic tobacco plants were grown in media containing lethal amounts of HP. The pydA and one type of pydA-pydB transformants showed greater resistance to HP compared to pydB transformants and wild-type plants. The results indicate that the bacterial enzymes are expressed in transgenic tobacco and enable them to degrade HP.
منابع مشابه
pyd genes of Rhizobium sp. strain TAL1145 are required for degradation of 3-hydroxy-4-pyridone, an aromatic intermediate in mimosine metabolism.
Rhizobium sp. strain TAL1145 degrades the Leucaena toxin mimosine and its degradation product 3-hydroxy-4-pyridone (HP). The aim of this investigation is to characterize the Rhizobium genes for HP degradation and transport. These genes were localized by subcloning and mutagenesis on a previously isolated cosmid, pUHR263, containing mid genes of TAL1145 required for mimosine degradation. Two str...
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متن کاملThe mid genes of Rhizobium sp strain TAL1145 are required for degradation of mimosine into 3-hydroxy-4-pyridone and are inducible by mimosine.
Mimosine is a toxin present in the tree-legume leucaena (Leucaena leucocephala), including its root nodules and the root exudates. The leucaena-nodulating Rhizobium sp. strain TAL1145 degrades mimosine (Mid(+)) and utilizes it as a source of carbon and nitrogen. Twelve TAL1145 mutants defective in mimosine degradation (Mid(-)) were made through Tn3Hogus, TnphoA or kanamycin-resistance-cassette ...
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