| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2005: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2004: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
The genes encoding pro-form and active-form of strong fibrinolytic protease, isozme A, in the six isozymes of eartworm, Lumbricus rubellus, were subcloned into a plasmid, pEU3-NII, and expressed in the wheat germ cell-free protein synthesis system. Extra proteins with a molecular mass of about 26 and 25 kDa were produced by pro-form and active-form of plasmids, respectively, which are similar to calculated molecular mass from the amino acid sequences. These results shows that the gene from earthworm could express in plant.
The pro-form and active-form genes of isozyme A were subcloned into a plasmid pBI121, resulting the plasmids both of which the enzyme genes fused with CaMV35S promoter. These genes were transferred to Arabidopsis thaliana, cv. Columbia, and T1 seeds were obtained. T1 seeds including active-form gene were cultured on MS medium agar plate containing kanamycine and three types of plants, 1) normal shape, 2) breached leaf, 3) wrinkled leaf, were observed. All types of transgenic plants expressed extra isozyme A mRNA, but could not produce T2 seeds. The transgenic plants of T1 seeds including pro-form gene also showed the wrinkled leaves which is breached gradually, resulting the die. These results suggest that the isozyme A from earthworm is produced and works in plants, and the possibility of producing the organic waste-degrading plants if the expression of the isozyme A gene could be controlled by adequate promoters which can restrict the expression strongly or organ-specific expression affecting little on plant growth.
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