| Summary: | 碩士 === 國立虎尾科技大學 === 生物科技研究所 === 101 === The soil borne micro-organisms may secrete phytase to decompose insoluble phytate and release phosphate, subsequently promote plant growth. There is limited study with phytase from Trichoderma harzianum, a well know biological control against plant pathogens. In this study, the full-length of DNA fragment (phytase-full) encoding T. harzianum ETS323 phytase was obtained by RT-PCR and 5ˊ-RACE, as well as ETS323 phytase gene by PCR (Genomic-phytase-full ). The 1918 bp DNA fragment of Genomic-phytase-full is composed of five exons, separated by four introns. phytase-full comprises 1680 bp and its predicted protein has N-terminal signal peptide . The molecular mass of predicted mature ETS323 phytase is 58.4 kDa with 5.3 of pI. A high conserved regin (RHGARYPT) is found in ETS323 phytase, which is conserved among histidine acid phosphatases. ETS323 phytase is probably a glycoprotein as there are ten putative N-glycosylation sites. ETS323 phytase was expressed as soluble proteins in Escherichia coli; however, no phytase activity was detected. ETS323 phytase was not successfully expressed in yeast Pichia. The crude proteins were prepared from T. harzianum ETS323 liquid-cultured in phyate containing medium. Western blot analyses showed that the antibodies raised against EST323 phytase could recognize a protein bands from this culture medium. Moreover, Quantitive RT-PCR analyses indicated that the transcript level of ETS323 phytase could be induced by phytate.
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