Expression of an ice plant mcSKD1Δ670 protein in rice cells and analysis of its salt tolerance ability.

• Main Authors: Wang Yung-Shun, 王永順
• Other Authors: Su Ruey-Chih
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/40926638201361707915

碩士 === 輔仁大學 === 生命科學系碩士班 === 95 === mcSKD1 gene, a salt-induced gene, has been identified from the halophyte ice plant (Mesembryanthemum crystallinum L.). The sequence of mcSKD1 is homologous to mouse SKD1 (suppressor of K+ transport growth defect) and yeast Vps4 (vacuolar protein sorting 4) that participate in the sorting of vacuolar proteins into multivesicular bodies. They all contain a highly conserved ATPase domain (the AAA domain) and are belong to the AAA (ATPase associated with a variety of cellular activities)-type ATPase family. The mcSKD1 also participates in the endoplasmic reticulum-Golgi mediated protein sorting machinery for both absorption of K+ and compartmentalization of excess Na+ under high salinity. The expression of full-length mcSKD1 in Arabidopsis was found resulted in growth inhibition of transgenic plants, so in this study we were trying to introduce a truncated mcSKD1 gene, mcSKD1Δ670 , into rice (Oryza sativa L.). The mcSKD1Δ670 gene can produce only coiled-coil motif and Walker A motif. The mcSKD1Δ670 gene was transferred into rice using an Agrobacterium-mediated method and TRF-C (Transgenic Rice F-Callus) were obtained and confirmed by PCR analysis. DNA Dot blot analysis showed that the mcSKD1670 gene has stably integrated into rice genomes and the Western blot analysis also detected the ~30 kDa mcSKD1Δ670 protein. When TRF-C was treated with different combination of K+ and NaCl solution for a week, the cell content of K+ was found to maintain above 5 mg and the Na+ content arised from 5 mg to 10 mg when increased the NaCl. The content of K+ and Na+ in Wt-C (Wild type-Callus), on the other hand, concentrations was the highest when in 100 mM NaCl. In addition, the Na/K ratios of TRF-C maintained in 0.5~2 but Wt-C was varied. The growth rate of TRF-C was slower than Wt-C. Interestingly, the Mg2+ contents of rice cell were measured to be less than 1 mg in TRF-C. We have obtained 11 transgenic rice lines. So far, Two T1 lines with were cultured hydroponically with modified Kimura B nutrient solution. In conclusion, the expression of mcSKD1Δ670 was found to enhance the absorption of Na+ and K+ in rice cell, but somehow suppress the uptake of Mg2+. Because Mg2+ play important role in many enzymes activity that may effect cell growth, a sufficient and constant supply of Mg2+ was found critical to support normal growth of our transgenic mcSKD1Δ670 rice plants.