我們先前從甘藷老化葉片分離出一種天冬醯胺肽鏈內切酵素asparaginyl endopeptidase，基因表現研究顯示其在天然及誘導的老化葉片中會被活化及增加表現量(Chen et al., 2004)。本研究報告將全SPAE cDNA構築於pBI121載體內的T-DNA部分，並且受CaMV 35S敔動子的調控。利用農桿菌感染阿拉伯芥花序的方法進行植株轉殖，轉殖植株利用其抗抗生素kanamycin的特性進行篩選並且以genomic DNA擴增的方法來確認，西方默點雜交結果證實SPAE於轉殖植株中有表現。表型分析結果顯示轉殖植株比對照組表現出較早開花及葉片老化，而且平均每株轉殖植株比對照組含較少的長莢果數目及較高的發育不完全果莢百分比。根據這些結果結論甘藷天冬醯胺肽鏈內切酵素asparaginyl endopeptidase其功能與老化過程有關，且其基因表現可以增加或促進轉殖植株老化。
We have previously isolated an asparaginyl endopeptidase, SPAE, from senescent leaves of sweet potato (Ipomoea batatas cv. Tainong 57). Gene expression of SPAE was activated and enhanced in natural and induced senescent leaves (Chen et al., 2004). In this report the full-length SPAE cDNA was constructed in the T-DNA portion of recombinant pBI121 vector under the control of CaMV 35S promoter and transferred to Arabidopsis with Agrobacterium-mediated floral dip transformation. Three transgenic Arabidopsis plants were isolated and confirmed by kanamycin-resistance and genomic PCR amplification of SPAE. Protein gel blot also demonstrated sweet potato SPAE expression in these transgenic plants. Phenotypic analysis showed that transgenic plants exhibited earlier floral transition from vegetative growth and leaf senescence than control. Transgenic plants also contained fewer siliques and a higher percentage of incompletely-developed siliques per plant than control. Based on these results we conclude that sweet potato asparaginyl endopeptidase, SPAE, may function in association with the senescence process, and its expression enhances or promotes senescence in transgenic Arabidopsis plants. The altered phenotypic characteristics in transgenic plants with SPAE gene expression were also discussed.
