| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1994: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1993: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
In order to clarify the mechanism of plant self-defense and to create the pathogene-resistant plants, gene analysis of the pahogenesis-related chitinase which has srong lytic acitivity against plant pathogens was performed.
(1) Nucleotide sequence of pathogenesis-related chitinase.
Yam callus was inoculated with Fusarium oxysporum to express mRNA of pathogenesis-related chitinase, and cDNA library was constructed. On the other hand, the partial amino acid sequence was analyzed, and the primers for PCR were synthesized. By using these primers and cDNA library as the template, PCR was done to amplify the pathogenesis-related chitinase.This amplified cDNA was used for screening of pathogenesis-related chitinase from both cDNA and genome DNA libraies, and followed by nucleotide sequence analysis. As the result, pathogenesis-related chitinase obtained fromyam callus was classified into a new class, Class IV,which is similar to Class I having N-terminal chitin binding region, but secreted.
(2) Enzymatic properties.
This chitinase showed double optimum pHs, srong lytic activity and substrate inhibition in kinetic behavior. Furthermore, this chitinase hydrolyzed chitin to form alpha-anomeric product, whereas other chitinolytic enzymes formed beta-anomeric product.
(3) Transgenic plant.
In order to clarify the role N-terminal chitin-binding region of this pathogenesis-related chitinase and to create pathogen-resistant plant, the gene of this chitinase was transformed into tobacco by using Tiplasmid system of Agrobacterium tumefaciens. As the result, the transgenic tobacco transformed by insertion of full length of this pathogenesis-related chitinase was created.
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