Please use this identifier to cite or link to this item:
標題: 百合花藥早期嶄新基因之特性分析與百合花粉與乾燥相關之LLA-23基因
Characterization of a novel early-gene during anter development in Lilium longiflorum;The lily ASR targeting to nucleus correlates with desiccation in developing pollen of lilium longiflorum.
作者: 吳志賢
Wu, Chin-Hsien
關鍵字: lily;百合;pollen;desiccation;tapetum;花粉;乾燥;絨氈層
出版社: 生物科技學研究所
引用: 余叔文、湯章城。 (1999) 植物生理與分子生物學 第二版 (科學出版社) ,493-765頁。 曾稚鐙。(2006) 鐵百合花藥發育早期基因之選殖與特性分析。 陳家全、李家維、楊瑞森。(2002) 生物電子顯微鏡學 (科儀叢書4) ,145頁。 胡適宜。 (1990) 被子植物胚胎學,曉園出版社,23-76頁。 蔡月夏、林學詩。 (1993) 台灣原生百合遺傳資源之開發利用研究。花蓮區農業改良場研究彙報9: l5-24頁 蔡月夏。 (1995) 台灣的農家要覽農作篇(二),財團法人豐年社主編,585-588頁。 Aarts, M. G. M., Hodge, R., Kalantidis, K., Florack, D., Wilson, Z. A., Mulligan, B., Stiekema, W. J., Scott, R. and Pereira, A. (1997) The Arabidopsis male sterility 2 protein shares similarity with reductases in elongation/condensation complexes. Plant J. 12: 615-623. Amitai-Zeigerson, H., Scolnik, P. A. and Bar-Zvi, D. (1994) Genomic nucleotidesequences of tomato Asr2, a second member of the stress/ripeninginduced Asr1 gene family. Plant Physiol. 106: 1699-1700. Ariizumi, T., Hatakeyama, K., Hinata, K., Inatsugi, R., Nishida, I., Sato, S., Kato, T., Tabata, S. and Torriyama, K. (2004) Disruption of the novel plant protein NEF1 affects lipid accumulation in the plastids of the tapetum and exine formation of pollen resulting in male sterility in Arobidopsis thaliana. Plant J. 39: 170-180. Banerjee, U. C. and Barghoorn, E. S. (1971) The tapetal membranes in grasses and ubish body control of mature exine pattern. Pollen Dev. Physiol. 126-127. 34 Blomstedt, C. K., Knox, R.. B. and Singh, M. B. (1996) Generative cells of Lilium logniflorum possess translatable mRNA and functional protein synthesis.Plant Mol. Boil. 31: 10831086. Bots, M., Vergeldt, F., Wolters-Arts, M., Weterings, K., Van-As, H. and Mariani, C. (2005) Aquaporins of the PIP2 class are required for efficient anther dehiscence in tobacco. Plant Physiol. 137: 1049-1056. Cakir, B., Agasse, A., Gaillard, C., Saumonneau, A., Delrot, S. and Atanassova, R. (2003) A grape ASR protein involved in sugar and abscisic acid signaling. Plant Cell. 15: 2165-2180. Canel, C., Bailey-Serres, J.N. and Roose, M.L. (1995) Pummelo fruit transcript homolgous to ripening-induce genes. Plant Physiol. 108: 1323-1324. Chang, S., Puryear, J.D., Dias, M., Funkhouser, E.A., Newton, R.J. and Cairney, J. (1996) Gene expression under water deficit in loblolly pine (Pinus taeda) : isolation and characterization of cDNA clones. Physiol. Plant. 97: 139-148. Chaubal, R. (2000) Two male-sterile mutants of Zea mays with an extra cell division in anther wall. Am. J. Bot. 87: 1193-1201. Chiang, J-Y., Shu, S-W., Ko, C-W. and Wang, C-S. (2005) Biochemical characterization of a pollen-specific cDNA encoding polygalacturonase in Lilium longiflorum. Plant Sci. 170: 433-440. Daneiil, H. (2002) Molecular strategies for gene containment in transgenic crops. Nat. biotech. 20: 581-586. Dettmer, J., Schubert, D., Calvo-Weimar, O., Stierhof, Y. D., Schmidt, R. and Schumacher, K. (2005) Essential role of the V-ATPase in male gametophyte. Plant J. 41: 117-124. Doczi, R., Csanaki, C. and Banvivi, Z. (2002) Expression and promoter activity of the desiccation-specific Solanum tubersosum gene, 35 STDS2. Plant Cell Environ. 25: 1197-1203. Doczi, R., Kondrak, M., Beczner, F. and Banfalvi, Z. (2005) Conservation of the drought-inducible DS2 genes and divergences from their ASR paralogues in solanaceous species. Plant Physiol. Biochem. 43: 269-276. Dong, X., Hong, Z., Sivaramakrishnan, M., Mahfouz, M. and Verna, D. P. (2005) Callose synthase (CalS5) is required for exine formation during microgametogenesis and for pollen viability in Arabidopsis. Plant Physiol. 42: 315-328. Eady, C., Lindsey, K. and Twell, D. (1994) Differential activation and conserved vegetative cell-specific activity of a late pollen promoter in species with bicelluar and tricellular pollen. Plant J. 5: 543-550. Fei, H. and Sawhney, V. K. (1999) MS32-regulated timing of callose degradation during microsporogenesis in Arabidopsis is associated with the accumulation of stacked rough ER in tapetal cells. Sex Plant Reprod. 3: 188-193. Holmes-Davis, R., Tanaka, C. K., Vensel, W. H., Hurkman, W. J., and Mccormick, S. (2005) Proteome mapping of mature pollen of Arabidopsis thaliana. Proteomics. 5: 4864-4884. Hony, D. and Twell, D. (2003) Comparative analysis of the Araidopsis Pollen Transcriptome. Plant Physiol. 132: 640-652. Hony, D. and Twell, D. (2004) Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome boil. 5: R85. Hong, S-H., Kim, I-J., Yang, D-C, and Chung, W. (2002) Characterization of an abscisic acid responsive gene homologous from Cucumis melo. J. Exp. Bot. 53: 2271–2272. Hsu, S-W. and Wang, C-S. (2006) A lily pollen-specific cDNA encoding the Cdc42/Rac-interactive-binding motif-containing protein associated with polln tube growth. Physiol. Plant. 126: 232-242. 36 Kaneko, M., Itoh, Hironori., Inukai, Y., Sakamoto, T., Ueguchi-Tanaka, M., Ashikari, M. and Matsuoka, M. (2003) Where do gibberllin biosynthesis and gibberellin signaling occur in rice plants? The Plant J. 35: 104-115. Kao, F-I., Cheng, Y-Y., Chow TY., Chen, H-W., Liu, S-M., Chen, C-H. and Chung, M-C. (2006) An integrated map of Oryza sativa. Chromosome 5. Theor. Appl. Genet. 112: 891-902. Kaul, M. L. H. (1994) Male sterility in higher plants. in monographs. theoretical and applied genetics. (Frankel, R., Grossman, M. and Maliga, P., eds.) . 10. Lin, J. J. and Dickinson, D. B. (1984) Ability of pollen to germinate prior to anthesis and effect of desiccation on germination. Plant Physiol. 74: 746-748. Mbeguie-A-Mbeguie, D., Gomez, R-M. and Fils-Lycaon, B. (1997) Molecular cloning and nucleotide sequence of an abscisic acid-, stress-, ripeninginduced (ASR) -like protein from apricot fruit (accession no. U82760) . Gene expression during fruit ripening (PGR97–161) . Plant Physiol. 115: 1288. Mariani, C., De Beuckeleer, M., Truettner, J., Leemans, J. and Golberg, R. B. (1990) Induction of male sterility in plants by a chimeric ribonuclease gene. Nature. 347: 737-741. Mariani, C., Gossele, V., De-Beuckeleer, M., De Block, M., Golberg, R. B., De-Greef, W. and Leemans, J. (1992) A chimeric ribonuclease-inhibitor gene restores fertility to male sterility plants. Nature. 357: 384-387. Mascarenhas, J. P. (1990) Gene activity during pollen development. Annu. Rev. Plant Physiol. 41: 317-338. McCormick, S. (1993) Male gametophyte development. Plant Cell. 5: 1265-1275. 37 McCormick, S. (2004) Control of male gametophyte development. Plant Cell. 16: 142-153. Millar, A. A. and Gubler, F. (2005) The Arabidopsis GAMYE-LIKE genes, MYB33 and MYB65 , are microRNA-regulated genes, that redundantly facilitate anther development. Plant Cell. 17: 705-721. Nacken, W., Huijser. P., Beltran. J. P. Saedler. H. and Sommer. Hans. (1991) Molecular characterization of two stamen-specific genes, tap1 and fil1, that are expressed in the wild type, but not in the deificens mutant of Antirrhinum majus. Mol. Gen. Genet. 229: 129-136. Padmanabhan, V., Dias, D. and Newton, R. J. (1997) Expression analysis of a gene family in a loblolly pine (Pinus taeda L.) induced by water deficit stress. Plant Mol. Biol. 35: 801–807. Pan, Y-Y., Wang, X., Ma, L-G. and Sun, D-Y. (2005) Characterization of Phosphatidylinositol-Specific Phospholipase C (PI-PLC) form Lilium daviddi pollen. Plant Cell Physiol. 46: 1657-1665. Rhee, S. Y., Osborne, E., Poindexter, P. D. and Somerville, C. R. (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: 70-1180. Riccardi, F., Gazeau, P., De-Vienne, D. and Zivy, M. (1998) Protein changes in response to progressive water deficit in maize. Plant Physiol. 117: 1253-1263. Rogers, H. J., Maund, S. L. and Johnson, L. H. (2001) Aβ-galactosidase-like gene is expressed during tobacco pollen development. J. Exp. Bot. 52: 67-75. Sandra, C., Emidio, A., Serena, V., Erika, F., Marica, S., Gianpiero, M., Sergio, S., Margherita, L. and Gianni, B. (2005) Alfalfa Mob1-like 38 genes are expressed in reproductive organs during meiosis and gametogenesis. Plant mol. boil. 58: 789-807. Scott, R. (1994) Anther development: a molecular perspective. In the molecular biology of flowering. Brian R. J. Eds. CAB International. UK. 141-193. Shen, G., Pang, Y-Z., Wu, W-S., Deng, Z-X., Liu, X-F., Lin, J., Zhao, L-X., Sun, X-F. and Tang, K-X. (2005) Molecular cloning, characterization and expression of a novel Asr gene from Ginkgo biloba. Plant Physiol. Biochem. 43: 836-843. Silhavy, D., Hutva´gner, G., Barta, E. and Ba´nfalvi, Z. (1995) Isolation and characterization of a water-stress-inducible cDNA clone from Solanum chacoense. Plant Mol. Biol. 27: 587–595. Skaden. R., Sathish. P., Federico. M., Abebe. T., Fu. J. and Kaeppler. H. (2002) Cloning of the promoter a novel barely gene, Lem1, and its organ-specific promotion of Gfp expression of lemma and palea. Plant mol. Boil. 49: 545-555. Stacy, R. A. and Aelen, R. B. (1998) Identification of sequcence homology between internal hydrophilic repeated motifis of group 1 late-embryogenesis-abundant proteins in plants and hydrophilic repeats of the general stress protein GsiB of Bacillus subtilis. Planta. 206: 476-478. Staiger, D., Kappeler, S., Muller, M. and Apel, K. (1994) The proteins encoded by two tapetum-specific transcripts, Satap35 and Satap44, from Sinapis alba L. are localized in the exine cell wall layer of developing microspores. Planta. 192: 221-31. Steedman, H. F. (1957). A new ribboning embedding medium for histology, Nature. 179: 1345. Suzuki, K., Takeda, H., Tsukaguchi, T. and Egawa, Y. (2001) Ultrastructural study on degeneration of tapetum in anther of snap 39 bean(Phaseolus vulgaris L.) under heat stress. Sex. Plant Reprod. 13: 293-299. Suzuki, K., Tsukaguchi, T., Takeda, H. and Egawa, Y. (2001) Decrease of pollen stainability of green bean at high temperatures and relationship to heat tolerance. J. Am. Soc. for Horticultural Sci. 126: 571-574. Taylor, P. E., Glover, J. A., Lavithis, M., Craig, S., Singh, M. B., Knox, R. B., Dennis, E. S. and Chaudhury, A. M. (1998) Genetic control of male fertility in Arabidopsis thaliana: structural analyses of post-meiotic developmental mutants. Planta. 205: 492-505. Tsuchiya, T., Toriyama, K., Ejiri, S. and Hinata, K. (1994) Molecular characterization of rice genes specifically expressed in the anther tapetum. Plant Mol. Biol. 26: 1737-1746. Tsuchiya, T., Toriyama, D., Yoshikawa, M., Ejiri, S. I. and Hinata, K. (1995) Tapetum-specific expression of the gene for an Endo-β-1,3- glucanase causes male sterility in transgenic tobacco. Plant Cell Physiol. 36: 487-494. Twell, D., Soon, K. P. and Eric, L. (1998) Asymmertric division and cell-fate determination in developing pollen. Trends Plant Sci. 3: 305-310. Vaidyanathan, R., Kuruvilla, S. and Thomas, G. (1999) Characterization andexpression pattern of an abscisic acid and osmotic stress responsive gene from rice. Plant Sci. 140: 25-36. Von den Heuvel. K. J., Van-Lipzig. R. H., Barendse. G. W. and Wullems. G. J. (2002) Regulation of expression of two novel flower-specific genes from tomato (Solanum lycopersicum) by gibberellin. 53: 51-59. Von Heijne. G. (1986) A new method for predicting signal sequence cleavage sits. Nucleic Acids Res. 14: 4683-4690. 40 Wang, A., Xia, Q., Xie, W., Datla, R. and Selvargj, G. (2003) The classical ubisch bodies carry a sporophytically produced structural protein (RAFTIN) that is essential for pollen development. Proc. Natl. Acad. Sci. 24: 14487-14492. Wang, C-S. (1992b) Immuno-logical characterization of a tapetal protein in developing andthers of Lilium longiflorum. Plant Physiol. 99: 822-829. Wang, C-S., Liau, Y-E., Wu, T-D., Su, C-C., Huang, J-C. and Lin, C-H. (1998) Characterization of a desiccation-related in lily pollen during development and stress. Plant Cell Physiol. 39: 1307-1314. Wang, C-S., Walling, L-L., Eckard, K-J. and Lord, E. M. (1992a) Patterns of protein accumulation in developing anters of Lilium longiflorum correlate with histological events. Am. J. Bot. 79: 118-127. Wang, C-S., Wu, T-D., Chung, C-K. and Lord, E. M. (1996) Two classes of pollen-specific, heat-stable proteins in Lilium longiflorum, Physiol. Plant. 97: 643-650. Wang, H-J., Jauh, G-Y., Hsu, Y-H. and Wang, C-S. (2003) The nuclear localization signal of a pollen-specific, and desiccation-associated protein of lily is necessary and sufficient for nuclear targeting. Bot. Bull. Acad. Sin. 44: 123-128. Wang, X., Cui, S-J., Ma, L-G. and Sun, D-Y. (2000) The involvement of PI-PLC-IP3 signaling pathway in pollen tube growth. Acta. Bot. Sin. 42: 697-702. Wolkers, W. F., McCready, S., Brandt, W. F., Lindsey, G. G. and Hoekstra, F. A. (2001) Isolation and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro. Biochem. Et. Biophysica. Acta. 1544: 194-206. Worrall, D., Hird, D. L., Hodge, R., Paul, W., Draper, J. and Scott, R. (1992) Premature dissolution of the microsporocyte callose wall 41 causes male sterility in transgenic tobacco. Plant Cell. 4: 759-771. Yang, C-Y., Chen, Y-C., Jauh, G-Y. and Wang, C-S. (2005) A Lily ASR Protein Involves Abscisic Acid Signaling and Salt Resistance in Arabidopsis. Plant Physiol. 139: 836-846. Zhang, C-H., Guinel, F. C. and Moffatt, B. A. (2002) A comparative ultrastructural study of pollen development in Arabidopsis thaliana ecotype Columbia and male-sterile mutant apt1-3. Protoplasma. 219: 59-71.
在花藥發育前期絨氈層細胞,對於花粉的發育十分的重要。一旦絨氈層細胞不正常,會導致花粉發育不正常。因此我們利用小孢子發育時期經抑制扣除雜合 (suppression-subtractive hybridization) 所建立的百合花藥cDNA集合庫。得到一個LLA-67 cDNA選殖體。LLA-67 cDNA經5''-RACE得到全長cDNA,若去除掉poly A共有565 鹼基對,可轉譯出101個胺基酸的蛋白質。LLA-67蛋白質的等電點為3.6,分子量為10.1 kDa。經過序列比對得知為一嶄新未知的蛋白質。此蛋白質N端具有一個訊息胜,推測可能為一個分泌性蛋白質。利用北方點墨法,發現LLA-67為花藥專一基因。而且花藥發育時期中,LLA-67 mRNA在前減數分裂時期和花粉成熟時期均偵測不到,只有在小孢子發育時期中偵測到,此時期正是絨氈層細胞活動十分旺盛。經原位雜合定位法,LLA-67 mRNA在絨氈層偵測到,其它藥壁細胞則無,因此確定LLA-67為絨氈層專一的基因。另外,LLA-67不似其他的絨氈層細胞基因並不會受到吉貝素誘導。
另一個主題是花粉專一性ASR基因 (LLA-23) 。此基因編譯的蛋白質C端含有一個進核序列。實驗中取不同時期的花粉做超薄切片,當百合花苞長度6公分時的小孢子為單核細胞,此時澱粉體開始產生。在花苞長到8-10公分時,小孢子隨而進行有絲分裂而產生花粉。此時花粉開始有生殖細胞,並且其營養細胞質的澱粉體呈現聚集的現象。當花苞繼續長到12公分時,聚集的澱粉體轉而分散。以免疫金粒定位法,觀察到ASR蛋白質在早期的花苞6公分時還未產生。當花苞長到8公分長度時,ASR蛋白質開始出現。花苞長度在8-10公分時,營養細胞質所偵測到的ASR蛋白比在花粉的細胞核都多。當花苞長度達到12-14公分時,ASR蛋白質明顯的出現在細胞核裡,而且以生殖細胞核裡偵測到的ASR蛋白質最多,營養細胞核次之,而營養細胞質所含的ASR蛋白質最少。由上觀察的結果,ASR蛋白質進核的時間點應在開花前一天 (13公分花苞),而此時正是花粉開始自行脫水乾燥的時候。

The LLA-67 clone was identified from the suppression-subtractive cDNA library at the stage of microspore development in lily (Lilium longiflorum) anthers. Using 5''-RACE, a full-length of LLA-67 with a size of 565 bp was obtained. The LLA-67 encodes a protein of 101 amino acids, having a calculated molecular mass of 10.1 kDa and a calculated isoelectric point of 3.6. Sequence alignment indicates that LLA-67 shares no sequence similarity with known proteins. The protein contains a signal peptide at N-terminus, suggesting a secretory protein. Northern analysis indicates that LLA-67 transcripts are anther-specific and temporally expressed only at the phase of microspore development. In situ hybridization suggested that LLA-67 transcripts were only detected in tapetum, a confirmation of tapetum origin. Unlike other tapetum-specific genes induced by gibberellin, LLA-67 was not induced by GA3.
LLA-23, an ASR (abscisic acid-, stress-, ripening-induced) protein was isolated from lily pollen. The ASR protein contains a nuclear localization sequence at C-terminus. Histological differences of various developmental stages of pollen grains were examined. At 6-cm buds, microspore is in a uni-nucleate form in the anther and amyloplasts of the vegetative cell begin to form. Mitosis occurs in microspores at 8-10-cm buds, giving rise to generative cells, and amyloplasts in the vegetative cells tend to aggregate. As the lily buds grow to 12 cm, aggregated form of amyloplasts becomes disperse. Immunogold labeling analysis indicated that ASR protein was not detected at microspores of 6-cm buds. At 8-10-cm buds, higher amount of ASR proteins were detected at the vegetative cytoplasm than at the vegetative and generative nuclei. As buds grow to 12-14 cm, ASR protein in the generative nuclei reaches to the highest amount, vegetative nuclei the next, and vegetative cytoplasm displays the least amount of ASR protein. Taken together, our results provide a clear-cut evidence that ASR protein enters into the nucleus at 12-cm buds a stage immediately before desiccation occurs in the anther (13-cm buds).
其他識別: U0005-3108200608061200
Appears in Collections:生物科技學研究所

Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.