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標題: Investigation of Roles for exoPG1 and exoPG2 in Pollen Development/Elongation of Oryza sativa
作者: 洪婕瑜
Chieh-Yu Hung
關鍵字: 水稻;花粉;聚半乳糖醛酸酶;Oryza sativa;pollen;polygalacturonase
引用: Belzile, F., K.-C. Park, S.-J. Kwon, P.-H. Kim, T. Bureau and N.-S. Kim (2007). Gene structure dynamics and divergence of the polygalacturonase gene family of plants and fungus. Genome 51(1): 30-40. Brown, S. M. and M. L. Crouch (1990). Characterization of a gene family abundantly expressed in Oenothera organensis pollen that shows sequence similarity to polygalacturonase. Plant Cell 2(3): 263-274. Carvajal, F., D. Garrido, M. Jamilena and R. Rosales (2014). Cloning and characterisation of a putative pollen‐specific polygalacturonase gene (CpPG1) differentially regulated during pollen development in zucchini (Cucurbita pepo L.). Plant Biology 16(2): 457-466. Crookes, P. R. and D. Grierson (1983). Ultrastructure of tomato fruit ripening and the role of polygalacturonase isoenzymes in cell wall degradation. Plant Physiol 72(4): 1088-1093. Dal Degan, F., R. Child, I. Svendsen and P. Ulvskov (2001). The cleavable N-terminal domain of plant endopolygalacturonases from clade B may be involved in a regulated secretion mechanism. Journal of Biological Chemistry 276(38): 35297-35304. Hadfield, K. A. and A. B. Bennett (1998). Polygalacturonases: many genes in search of a function. Plant Physiol 117(2): 337-343. Han, M.-J., K.-H. Jung, G. Yi, D.-Y. Lee and G. An (2006). Rice Immature Pollen 1 (RIP1) is a regulator of late pollen development. Plant and cell physiology 47(11): 1457-1472. Harholt, J., A. Suttangkakul and H. Vibe Scheller (2010). Biosynthesis of pectin. Plant Physiol 153(2): 384-395. Huang, L., J. Cao, A. Zhang, Y. Ye, Y. Zhang and T. Liu (2009). The polygalacturonase gene BcMF2 from Brassica campestris is associated with intine development. Journal of experimental botany 60(1): 301-313. Huang, L., Y. Ye, Y. Zhang, A. Zhang, T. Liu and J. Cao (2009). BcMF9, a novel polygalacturonase gene, is required for both Brassica campestris intine and exine formation. Annals of botany: mcp244. Kalaitzis, P., T. Solomos and M. L. Tucker (1997). Three different polygalacturonases are expressed in tomato leaf and flower abscission, each with a different temporal expression pattern. Plant Physiol 113(4): 1303-1308. Lang, C. and H. Dörnenburg (2000). Perspectives in the biological function and the technological application of polygalacturonases. Applied microbiology and biotechnology 53(4): 366-375. Maceira, F. I. G., A. Di Pietro and M. I. G. Roncero (1997). Purification and characterization of a novel exopolygalacturonase from Fusarium oxysporum f. sp. lycopersici. FEMS Microbiology Letters 154(1): 37-43. Markovič, O. and Š. Janeček (2001). Pectin degrading glycoside hydrolases of family 28: sequence-structural features, specificities and evolution. Protein Engineering 14(9): 615-631. Rhee, S. Y., E. Osborne, P. D. Poindexter and C. R. Somerville (2003). Microspore separation in the quartet 3 mutants of Arabidopsis is impaired by a defect in a developmentally regulated polygalacturonase required for pollen mother cell wall degradation. Plant Physiol 133(3): 1170-1180. Rhee, S. Y. and C. R. Somerville (1998). Tetrad pollen formation in quartet mutants of Arabidopsis thaliana is associated with persistence of pectic polysaccharides of the pollen mother cell wall. The Plant Journal 15(1): 79-88. Tan, L., S. Eberhard, S. Pattathil, C. Warder, J. Glushka, C. Yuan, Z. Hao, X. Zhu, U. Avci, J. S. Miller, D. Baldwin, C. Pham, R. Orlando, A. Darvill, M. G. Hahn, M. J. Kieliszewski and D. Mohnen (2013). An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein. Plant Cell 25(1): 270-287. Tian, G.-W., M.-H. Chen, A. Zaltsman and V. Citovsky (2006). Pollen-specific pectin methylesterase involved in pollen tube growth. Developmental biology 294(1): 83-91. Torres, S., J. E. Sayago, R. M. Ordoñez and M. I. Isla (2011). A colorimetric method to quantify endo-polygalacturonase activity. Enzyme and microbial technology 48(2): 123-128. Wei, L. Q., W. Y. Xu, Z. Y. Deng, Z. Su, Y. Xue and T. Wang (2010). Genome-scale analysis and comparison of gene expression profiles in developing and germinated pollen in Oryza sativa. Bmc Genomics 11(1): 338.
Polygalacturonases (PG) 廣泛存在於細菌、真菌及植物的果實和花粉中,屬於果膠水解酶,在水解果膠的主鏈-聚半乳糖醛酸 (polygalacturonic acids,PGA)上扮演關鍵角色。富於果實中的PG已被了解到其在果實成熟上的重要性,然而,花粉中的PG卻尚未被研究透徹。水稻基因體中有45個基因被預測為PG,其中七個豐富存在於花藥,我們著重研究FPKM值最高的LOC_Os02g10300這個基因,並稱它為exoPG1,在花粉前期有高表現的LOC_Os08g23790在此也另做探討,稱之為exoPG2。即時聚合酶鏈鎖反應的結果指出這七個PG主要表現在花粉,為了調查PG的角色,構築PG靜默基因轉殖水稻,獲得十棵轉殖水稻後,以顯微鏡觀察轉殖水稻之花粉,結果顯現除了SiPG#8,其餘轉殖株之成熟花粉的形狀和活性均和vector轉殖株無明顯差異。經偵測exoPG1蛋白之後,發現SiPG#1及SiPG#3之訊號低落,且透過計算他們子代的hygromycin抗性比例來檢查其T-DNA遺傳性,結果亦大大低於預期,SiPG#1及SiPG#3分別只有6%及37%,這表示帶有T-DNA的花粉在傳送過程中發生缺失。為檢查其萌發過程,分別進行in vitro及in vivo germination實驗,由in vitro germination之結果,發現大部分花粉皆無異狀,但由in vivo germination之結果卻發現SiPG#8的柱頭中有多數屬於empty stigma,SiPG#1及SipG#3中則是有為數不少的aborted pollen。在百合花粉及水稻花粉之PG活性測試中,發現培養液均無活性,推測PG可能為一膜蛋白。即時聚合酶鏈鎖反應亦指出SiPG#1,SiPG#3及SiPG#8之表現量確實皆下降了。由這些分析結果,我們總結出exoPG2可能會影響花粉之生長發育,而exoPG1則可能於花粉管的延長過程扮演重要角色。

Polygalacturonases (PGs), also known as pectinase, play critical roles in hydrolyzing polygalacturonic acids (PGA) which is the main chain component of pectin in plant cell wall. PGs are found ubiquitously in phytopathgens as well as fruit and pollen of plants. PGs enriched in fruit were known to be critical for ripening, however, remain much less investigated for pollen. Rice genome has 45 PG genes, however, in which only 7 members are actively transcribed and are all expressed exclusively in anther. To investigate roles of these PGs, we focus on LOC_Os02g10300 (exoPG1) and LOC_Os08g23790 (exoPG2) that expressed predominantly in the mature/germinating and early bicellular pollen, respectively. ExoPG-silenced rice lines, aimed on silencing all seven exoPGs together, were constructed and 10 independent lines were evaluated. Western blotting revealed that line SiPG#1 and SiPG#3 have much lowered exoPG proteins. However, all lines, except for SiPG#8, exhibited normal plant growth and pollen development in terms of shape and viability. Interestingly, the T-DNA inheritability was severely lower than expected in line SiPG#1 and #3, indicate a defect in their pollen transmission processes. In vivo germination revealed a significant retardation of pollen elongation on stigma in line SiPG#1 and #3, but not in the vector-transformed rice. Moreover, SiPG#8 was defected in pollen development, likely disturbed the followed anther dehiscence indirectly and caused an 'empty stigma' phenotype, eventually decreased the fertility rate to be only 20%. Real-time PCR examinations confirmed a severe reduction of transcript of exoPG1, exoPG2 as well as other exoPGs. Together with the expression profiles of exoPG1 and exoPG2, we conclude that exoPG2 may affect pollen development while exoPG1 involve in pollen tube elongation.
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