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Molecular methods have been used in the study of 'Azospirillum' and other related PGPRs to carry out gene functional analysis, create gene knockouts, generate genetically engineered strains, and carry out gene expression studies. Genetic transformation has routinely been carried out using conjugation, while chromosomal modifi cations have been performed using unstable, suicide plasmids, or more stable, broad host-range vectors. Gene expression studies are often carried out using promoter-bound reporter genes; however, quantitative methods such as reverse transcribed polymerase chain reaction can now be used to directly study gene expression. In this chapter we describe the common types of vectors used in 'Azospirillum', as well as methods for transformation and mutagenesis. We also describe the use of promoter-bound reporter genes and the applications of quantitative RT-PCR for 'Azospirillum' gene expression studies. Methods for the isolation of DNA and RNA from 'Azospirillum' for use in molecular and gene expression studies are also described. |
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