水稻半糯基因Du8定位選殖與基因表現之分析

Abstract

稻米品質的改良為育種研究之重要指標，穀粒的澱粉占含量90 %，為影響稻米品質的重要因子。目前水稻穀粒澱粉代謝之研究雖然相當多，但尚不足以解釋水稻品種之間的直鏈性澱粉含量差異與澱粉結構多樣性間的關聯。近幾十年來研究顯示一些澱粉合成的調控基因可影響直鏈性澱粉含量與澱粉質地的差異，其中半糯 (Dull) 基因是獨立於Waxy基因但能影響直鏈性澱粉合成的調控基因。本研究利用EMS誘變出低直鏈性澱粉含量之半糯品系CNY921391-du8，進行基因定位選殖與澱粉合成相關基因表現分析，藉以探討突變基因du8在澱粉合成代謝調控上扮演之角色。經細微定位已將Du8半糯基因落在第四條染色體上，分子標幟CH0422 與 CH0426之間，含12個候選基因。定序分析結果顯示，CNY921391-du8品系在負責轉譯含有TPR（tetratricopeptide）保守區域蛋白的基因上產生了由G變成A的單點突變。此單點突變位於基因的第十一個內引子的3’端修飾位置上，利用RT-PCR進行單點突變區域的擴增，CNY921391-du8擴增出三個轉錄片段，而TK 8轉錄出單一轉錄片段。轉錄片段的定序結果發現CNY921391-du8 中在3’端修飾位置上的單點突變造成不正確的RNA修飾，降低了Du8在RNA修飾的準確率。除此之外，在其他低直鏈性澱粉突變品系中也發現另外三個Du8對偶基因。TPR區域負責蛋白質間的交互作用及多蛋白複合體的形成，在高粱、阿拉伯芥、蓖麻等高等植物中具相當高之保守性。利用real-time PCR分析du8突變品系在穀粒充實期間，半糯基因Du1與Du3的表現模式與Du8有所差異，其調控機制應與Du8有所不同。澱粉合成基因表現中，表現未有明顯差異的基因有OsAGPL3、OsBEIIa、OsISA2、OsISA3。表現有所差異但未有明顯模式的基因有OsAGPL1、OsAGPL4、OsAGPS2a、OsSSI、OsSSIIa、OsSSIIb、OsSSIIc、OsSSIIIa、OsSSIIIb、OsSSIVa、OsSSIVb、OsDPE2。而在穀粒充實時期整個表現都下降的澱粉合成基因，包含OsAGPL2、OsAGPS1、OsAGPS2b、OsGBSSI、OsGBSSII、OsBEI、OsBEIIb、OsISA1、OsPUL、OsDPE1、OsPHOL、OsPHOH。其中OsGBSSI、OsBEI與OsPUL在穀粒充實期間的表現模式與半糯基因Du8相似，但其表現量在du8突變體中顯著地降低，由此可推測Du8可調控作用上述的澱粉合成基因。

Rice quality improvement is an important issue in rice breeding. Starch is a major influence in rice quality because it composes 90% of rice grain. Though starch metabolism has been highly studied, so far non-sufficient evidence can be applied to explain the role of diverse starch textures in rice cultivars. Rice starch regulatory genes had been revealed and studied in the past few decades for better understanding in starch texture regulations. Dull genes are considered as amylose synthesis regulatory genes independent to Waxy. This study analyzed an EMS induced dull rice mutant line, CNY921391-du8, which exhibits low amylose content with opaque grain. Previous research had finely mapped Du8 between molecular markers CH0422 and CH0426 on chromosome 4, encompassing 12 candidate genes. Sequence analysis revealed a single point mutation of G to A in the gene encoding tetratricopeptide (TPR) repeat domain containing protein. The SNP mutation occurs on 3’ splice site of intron 11, leading to three truncated transcripts in the SNP of CNY921391-du8, compared to a single transcript of TK 8. In addition, three other du8 alleles were also found in other low amylose content mutants. The TPR domain mediates protein-protein interactions and multiprotein complexes assembly. Amino acid sequences of Du8 were highly conserved among higher plants such as Sorghum bicolor, Arabidopsis thaliana, and Ricinus communis. Real-time PCR analysis of Du8 mutant during rice grain developing stages showed that the Du8 expression pattern varied with the two dull genes, Du1 and Du3, indicating different regulation of Du8. Starch synthesis gene showing no significant variance expression patterns include OsAGPL3, OsBEIIa, OsISA2, and OsISA3. Genes with significant expression variance but not specified patterns include OsAGPL1, OsAGPL4, OsAGPS2a, OsSSI, OsSSIIa, OsSSIIb, OsSSIIc, OsSSIIIa, OsSSIIIb, OsSSIVa, OsSSIVb, and OsDPE2. Genes that were significantly reduced throughout the whole seed developing stages were OsAGPL2, OsAGPS1, OsAGPS2b, OsGBSSI, OsGBSSII, OsBEI, OsBEIIb, OsISA1, OsPUL, OsDPE1, OsPHOL, and OsPHOH. In addition, OsGBSSI, OsBEI, and OsPUL showed similar expression patterns as the isolated dull gene, Du8, and the expression levels were severely reduced in the overall rice grain developing stages of the du8 mutants. These result indicated the possibility role of Du8 in regulating these starch synthesis genes mentioned above.