| Summary: | 碩士 === 元智大學 === 生物科技與工程研究所 === 99 === Abstract
Starch is the second abundant organic resources, next to cellulose, produced in plants. Leaf starch belongs to transitory starch and tends to accumulate as a direct product of photosynthesis during the day and to decrease as it is converted to sugars at night. It is known that a putative protein phosphastase encoded at the SEX4 (Starch Excess 4) locus of Arabidopsis plays an important role at starch break down in leaves, and mutation in this gene results in starch accumulation.
There are three SEX4 homologs in rice, OsSEX4 and OsLSF2 and OsLSF1. We hypothesized that similar mechanism of starch degradation exists in rice, and therefore through the strategy of RNA interference (RNAi) starch-excess transgenic rice plants can be made.
In this study, we characterized OsSEX4 and OsLSF2 during starch break down by RNAi technique. Regeneration of transformed rice callus requires months to produce leaf for the starch content assay. To reduce waiting time to analyze starch levels in leaves of transgenic plants, we developed rice suspension culture cells for starch evaluation. Rice cells were transferred into fresh sucrose-containing (+S) or sucrose-free (-S) medium for 0.5 day. In +S treatment, iOsSEX4-13 and iOsSEX4-51 (RNAi lines of OsSEX4) contained 0.8 and 0.2-fold of wild-type level. For RNAi lines of OsLSF2, iOsLSF2-7 and iOsLSF2-23, both reduced to about 0.5-fold of wild-type. In –S treatment, two OsSEX4 RNAi lines both reduced to 62% of wild type and for two OsLSF2 RNAi line both are reduced to 20% of wild-type level. In +S treatment, starch content of iOsSEX4-13 and iOsSEX4-51 lines are 1.4 and 2 folds higher than wild type.RNA lines of OsLSF2 are both about 1 fold higher than non-transformed cell lines .In -S treatment, iOsSEX4-13 and iOsSEX4-51 are 1.8 and 6.5 folds higher than the wild-type cell line and starch contents are almost the same as control cell line in the two OsLSF2 RNAi lines,.Starch contents in leaves of OsSEX4 RNAi transgenic plants, derived from iOsSEX4-13 and iOsSEX4-51 lines, both accumulated 6 times more starch than wild-type nontransgenic plant. These data supported that rice suspension cells can evaluate starch levels before regenerated RNAi of OsSEX4 plants were obtained.
Keywords: rice, starch degradation, suspension culture cells, RNA interference
|
|---|
