利用懸浮細胞建立快速篩選多澱粉轉殖水稻細胞株系統

Development a rapid evaluation system for starch excess RNAi transgenic rice lines by suspension cultured cells

謝依玲(Yi-Ling Hsieh)

指導教授 ： 黃麗芬

元智大學/工程學院/生物科技與工程研究所/碩士(2011年)

摘要

澱粉是自然界中植物所提供產量僅次於纖維素的有機資源，占大部分植物中儲存性的碳水化合物，葉片的澱粉是短暫性澱粉，主要是經由白天時光合作用，及碳的同化作用( Carbon assimilation ) 合成，於晚上時降解澱粉成糖類供給植物能量。前人研究發現，阿拉伯芥SEX4 ( Starch Excess 4) 蛋白參與澱粉的降解，SEX4基因突變，造成葉片澱粉累積，然而，在其他植物是否有相同的調控機制則不清楚。
目前在水稻中發現三種SEX4同源性蛋白，分別命名為OsSEX4 ( Oryza sativa Starch-Excess 4)、OsLSF2 ( Oryza sativa Starch-Excess 4- Like 2 )、OsLSF1 ( Oryza sativa Starch-Excess 4- Like 1 )。本研究主要以RNA 干擾技術研究水稻OsSEX4及OsLSF2同源基因是否參與葉片澱粉降解的調控。由於RNA干擾技術建立的水稻轉殖株需要半年以上的時間才能分析葉片澱粉含量，所以建立水稻懸浮細胞系統以加快篩選多澱粉的細胞株。首先以RT-PCR及Q-PCR的方式分析水稻野生型 ( Wild-type ) 之懸浮細胞在是否含蔗糖培養液( sucrose-containing & sucrose-free liquid medium )處理0.5天，OsSEX4三種同源性基因受糖調控的影響，結果顯示OsSEX4 與OsLSF1 mRNA含量在缺糖皆比含糖處理多2倍，但OsLSF2在含糖時卻比缺糖時低0.8倍。進一步分析OsSEX4 RNAi懸浮細胞內澱粉含量在培養液是否含糖的變化，兩個 OsSEX4 RNAi轉殖系：iOsSEX4-13與iOsSEX4-51，在有糖處理時其澱粉含量比野生型分別高1.4倍及2倍，在缺糖培養基中，其澱粉含量比野生型分別高1.8倍及6.5倍，並利用Q-PCR分析iOsSEX4-13中含糖時 OsSEX4 mRNA量相較於野生型降低0.8倍，而iOsSEX4-51下降0.2倍；於缺糖時 iOsSEX4-13及iOsSEX4-51中之OsSEX4 mRNA量相較於野生型均降低1.6倍；兩個 OsLSF2 RNAi轉殖系：iOsLSF2-7與iOsLSF2-23，在有糖處理時其澱粉含量比野生型分別高1倍及1.2倍，在缺糖培養基中，其澱粉含量和野生型差不多。利用Q-PCR分析iOsLSF2-7中含糖時 OsLSF2 mRNA量相較於野生型降低0.5倍，而在iOsLSF2-23下降0.6倍；於缺糖時iOsLSF2-7 mRNA降低0.2倍， iOsLSF2-23下降0.3倍。後續分析水稻轉殖株澱粉量，結果顯示OsSEX4 RNAi轉殖系於夜晚時澱粉降解速率變慢，因而累積澱粉。因此本研究結果證實，水稻懸浮細胞系統可在尚未長成植株前就能分析其澱粉累積量。

關鍵字

水稻懸浮細胞 ； 澱粉降解 ； RNA干擾

並列摘要

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

並列關鍵字

rice ； starch degradation ； suspension culture cells ； RNA interference

參考文獻

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