| Catalase(EC1.11.1.6), which is presented in all aerobic organisms and has efficient enzyme kinetics, plays a crucial role in modulating or eliminating H2O2 from cells. Plant has multiple forms of catalase, which is encoded by a multigene family. Catalase performs multiple functions in plant tissues. Although there always have catalase isozymes that can take part in the scavenging of hydroperoxides, each of them only participates in special metabolic processes or signal pathway. So it is difficult to characterize the functions of a single gene or isozyme. In plant, H2O2 may serve as a new signal molecule in response to biotic or abiotic stress such as auxin stimulting, pathogen infection, and so on. Plant has a similar reactive manner in response to salicylic acid and pathogen infection, salicylic acid can acts by binding and inhibiting catalase activity. The H2O2 and other Active Oxygen Specieses (AOS) may serve as a second signal messengers in the activation of pathogen-related genes, which can enhance the plant's disease resistance. It has been demonstrated that special catalase in plant, which can serve as a SA-targeted protein and participate in the signal transduction pathway induced by SA, plays an important role in disease resistance. Recently, three catalase genes have been isolated from Arabidopsis, but little is known about their exact functions. Using bioinformatics we could assume what functions the unknown gene might have, but we still have to resort to mutants to characterized the unknown gene's exact function. Obtaining an useful mutant is the precondition to perform genomic research and no gene can be isolated and characterized without mutant, but the functional redundancy among these genes may limited the ability to identify and isolate Arabidopsis mutants lacking catalase activity. Researchers have found that double-stranded RNA can strongly result in the silencing of special genes, which known as RNA-interference (RNAi). The mutant's phenotypic effect resulting from RNAi could be achieved after transformed dsRNA into plants.Thus, RNAi technology has become of a powerful method for plant gene-function and signal transduction studies.Here we try to use RNAi technology to obtain a Cat3 gene-silence mutant in Arabidopsis thaliana (Columbia ecotype) by inhibiting special gene expression, and provide a basic tool for further studies on the bio-function of CAT3 and the mechanism of signal transduction induced by SA in Arabidopsis. Our experiment results are as follow:First, using bioinformatics method to analysis CAT protein coding sequences in Arabidopsis, Nicotiana plumbaginifolia, Zea mays L, Oryza sativa L, Pumpkin. It has shown that the Arabidopsis CAT3 and the N. plumbaginifolia CAT2 have the highest homology.Second, we made a blast in the whole genomic sequence of Arabidopsis by using the Cat3 gene to determine the size and location of target fragment using for constructing the gene-silence vector. After the polymerase chain reaction (PCR), an amplified fragment of 450bp was obtained, subsequently, inserted into two multi-cloning site in pFGC5941 in sense and anti-sense orientation. Thus, an intron-hp RNA gene-silence vector pFGC5941cat3ds was constructed.Third, after transforming the reconstructed plasmid into Agrobacterium LBA4404, the Arabidopsis was transformed by Floral dip. 14 lines of transforments were obtained by screening the T2 transgene seedlings with PPT.After treating the wildtype seedlings with light, darkness, ABA(100 (mol/L)and darkness, SA(1mmol/L) and darkness, total RNA was extracted from the treated plants and the transcription levels of Cat3 in response to the treatments were checked by RT-PCR. The results indicated that the Cat3 expression was increased with darkness treatment, no change occurred with ABA treatment. Interestingly, the increase of Cat3 transcriptional level induced by darkness could be partly inhibited by salicylic acid (SA) treatment.
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