- 學位論文
阿拉伯芥beta澱粉水解酶及多澱粉突變株ke103之研究
Study of Arabidopsis β-amylases and a starch excess mutant ke103
摘要
暫儲性澱粉存在於葉綠體中，在每日的光照期間會合成，夜晚則會被分解並運送到植物體其他部分，有明顯的日夜週期變化。近年來的研究提供證據指出beta澱粉水解酶在暫儲性澱粉的代謝中應扮演重要的角色。阿拉伯芥中九個beta澱粉水解酶中有四個被預測會送入葉綠體中。已知其中BAM3、BAM4發生突變會造成葉片內澱粉不正常之累積，BAM1和BAM2發生突變雖不會造成澱粉累積，但葉綠體型beta澱粉水解酶四重突變的植株顯示此二基因對於澱粉降解確實有貢獻。本文利用ke103突變株進行研究，此一突變株具多澱粉性狀且在活性膠體染色時不見其BAM1活性條帶，生體外互補的實驗並發現BAM1和KE103或是受KE103活化的蛋白間有交互作用。ke103/bam1-1雙重突變株的澱粉累積量較ke103多，說明BAM1對於澱粉降解仍有作用，和先前的研究相符，亦指出ke103突變影響澱粉降解主要並非透過BAM1。為了更深入了解ke103的影響層面，本研究利用ke103 x Ler 及 Col x ke103的F2子代做為雜交植株庫進行ke103突變之定位，並開發一系列SNP分子標記檢驗植株庫內的個體基因型，計算ke103和分子標記間的互換率，結果顯示ke103位在阿拉伯芥第三號染色體上1.0 Mb和1.33 Mb之間，接近SSLP 分子標記nga172的位置。本文所設計的分子標記亦可供其他的研究利用。暫儲性澱粉降解的初期機制並不清楚，有一假說認為beta澱粉水解酶和澱粉支解酶合作直接分解完整澱粉粒，然酵母菌雙雜合實驗並未顯示BAM1、BAM3或BAM4和參與澱粉降解的異澱粉酶 ISA3間有直接或間接的交互作用。BAM1的RNA表現量會受到鹽份逆境的誘導，本研究測量bam1-1突變株受到鹽逆境處理後澱粉在黑暗中降解的情形，發現bam1-1突變株無法如Col野生型植株般因逆境刺激而分解澱粉，顯示BAM1活性在鹽分逆境時有其作用。
並列摘要
Transitory starch is synthesized in chloroplast during the light period and broken-down at night. The products of starch degradation are exported to cytosol and transported through out the plant. Recent studies suggest that beta-amylase activity is essential for transitory starch degradation. Four of the nine beta-amylases encoded in the Arabidopsis thaliana genome are predicted to be chloroplastic. Mutants of those genes were studied. Plants with mutation in BAM3 and BAM4 were reported to accumulate starch at night. Down-regulation of BAM1 or BAM2 didn’t result in the same phenotype, nevertheless, the dramatically high starch content of the quadruple mutant line bam1/bam2/bam3/bam4 suggested that the activities of BAM1 and BAM2 were not negligible. The starch excess mutant ke103 lost its BAM1 activity band in a glycogen gel-based amylase activity assay, so did the knock-out mutant of BAM1, bam1-1. Interestingly, the activity can be recovered by mixing these two samples. In vitro complementation of these mutants indicated that BAM1 can interact with a protein either encoded directly by KE103 or activated by a KE103 encoded protein. The starch content of bam1-1 is similar to that of the wild type. However, the starch content was higher in ke103/bam1-1 double mutant than in ke103, indicating that ke103 didn’t influence on starch degradation via BAM1. To further understand the function of KE103, mapping populations with F2 progeny from ke103 x Ler and Col x ke103, were established to map ke103, and a series of SNP markers were identified using CEL I nuclease. We found that ke103 co-segregated with a SSLP marker nga172. The recombination frequency between ke103 and SNP markers illustrated that ke103 located between 1.0 Mb and 1.33 Mb of chromosome 3. The early steps of transitory starch degradation remain largely unknown. It was postulated that a beta-amylase may act in concert with a debranching enzyme to attack the starch granule. The results of yeast-two-hybrid assay showed that the isoamylase ISA3 cannot interact with beta-amylases BAM1, BAM3, or BAM4. And no evidence was found to support that they would interact through the mediation of other proteins. BAM1 transcript expression was induced by salt stress. The starch of Col WT is degraded faster in salt stress than that of controls, while the starch metabolism of bam1-1 is not altered in salt stress. This result suggested that BAM1 may respond to salt stress.
參考文獻
延伸閱讀
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