Please use this identifier to cite or link to this item:
|標題:||Characterization and function analysis of a tapetum/microspore-specific gene in Lilium longiforum
|關鍵字:||絨氈層;小胞子;小分子富含半胱胺酸蛋白;tapetum;microspore;small cysteine-rich protein||引用:||蔡淳瑩, 蔡月夏, 林學詩 (1998) 本土化百合新品系介紹. 花蓮區農業專訊 23：1415 Adie B, Chico JM, Rubio-Somoza I, Solano R (2007) Modulation of plant defenses by ethylene. J Plant Growth Regul 26:160–177 Aya K, Ueguchi-Tanaka M, Kondo M, Hamada K, Yano K, Nishimura M, Matsuoka M (2009) Gibberellin modulates anther development in rice via the transcriptional regulation of GAMYB. Plant Cell 21: 1453-1472 Bedinger PA, Pearce G, Covey PA (2010) RALFs: peptide regulators of plant growth. Plant Signal Behav 5: 1342-1346 Bleackley MR, Wiltshire JL, Perrine-Walker F, Vasa S, Burns RL, van der Weerden NL, Anderson MA (2014) Agp2p, the plasma membrane transregulator of polyamine uptake, regulates the antifungal activities of the plant defensin nad1 and other cationic peptides. Antimicrob Agents Chemother 58: 2688-2698 Bradford KJ (2008) Shang Fa Yang: Pioneer in plant ethylene biochemistry. Plant Science 175: 2-7 Chae K, Gonong BJ, Kim SC, Kieslich CA, Morikis D, Balasubramanian S, Lord EM (2010) A multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like Arabidopsis lipid transfer proteins (LTPs) suggests diversified roles for these LTPs in plant growth and reproduction. J Exp Bot 61: 4277-4290 Chae K, Kieslich CA, Morikis D, Kim SC, Lord EM (2009) A gain-of-function mutation of Arabidopsis lipid transfer protein 5 disturbs pollen tube tip growth and fertilization. Plant Cell 21: 3902-3914 Chen KC, Lin CY, Kuan CC, Sung HY, Chen CS (2002) A novel defensin encoded by a mungbean cDNA exhibits insecticidal activity against bruchid. J Agric Food Chem 50: 7258-7263 Chen Z (2001) A superfamily of proteins with novel cysteine-rich repeats. Plant Physiol 126: 473-476 Cheng H, Qin L, Lee S, Fu X, Richards DE, Cao D, Luo D, Harberd NP, Peng J (2004) Gibberellin regulates Arabidopsis floral development via suppression of DELLA protein function. Development 131: 1055-1064 Covey PA, Subbaiah CC, Parsons RL, Pearce G, Lay FT, Anderson MA, Ryan CA, Bedinger PA (2010) A pollen-specific RALF from tomato that regulates pollen tube elongation. Plant Physiol 153: 703-715 Dardelle F, Lehner A, Ramdani Y, Bardor M, Lerouge P, Driouich A, Mollet JC (2010) Biochemical and immunocytological characterizations of Arabidopsis pollen tube cell wall. Plant Physiol 153: 1563-1576 de Azevedo Souza C, Kim SS, Koch S, Kienow L, Schneider K, McKim SM, Haughn GW, Kombrink E, Douglas CJ (2009) A novel fatty Acyl-CoA Synthetase is required for pollen development and sporopollenin biosynthesis in Arabidopsis. Plant Cell 21: 507-525 De Grauwe L, Vriezen WH, Bertrand S, Phillips A, Vidal AM, Hedden P, Van Der Straeten D (2007) Reciprocal influence of ethylene and gibberellins on response-gene expression in Arabidopsis thaliana. Planta 226: 485-498 Dresselhaus T, Franklin-Tong N (2013) Male-female crosstalk during pollen germination, tube growth and guidance, and double fertilization. Mol Plant 6: 1018-1036 Endo M, Tsuchiya T, Saito H, Matsubara H, Hakozaki H, Masuko H, Kamada M, Higashitani A, Takahashi H, Fukuda H, Demura T, Watanabe M (2004) Identification and molecular characterization of novel anther-specific genes in Oryza sativa L. by using cDNA microarray. Genes Genet Syst 79: 213-226 Filichkin SA, Leonard JM, Monteros A, Liu PP, Nonogaki H (2004) A novel endo-beta-mannanase gene in tomato LeMAN5 is associated with anther and pollen development. Plant Physiol 134: 1080-1087 Ghag SB, Shekhawat UK, Ganapathi TR (2012) Petunia floral defensins with unique prodomains as novel candidates for development of fusarium wilt resistance in transgenic banana plants. PLoS One 7: e39557 Guan YF, Huang XY, Zhu J, Gao JF, Zhang HX, Yang ZN (2008) RUPTURED POLLEN GRAIN1, a member of the MtN3/saliva gene family, is crucial for exine pattern formation and cell integrity of microspores in Arabidopsis. Plant Physiol 147: 852-863 Haag AF, Baloban M, Sani M, Kerscher B, Pierre O, Farkas A, Longhi R, Boncompagni E, Herouart D, Dall'angelo S, Kondorosi E, Zanda M, Mergaert P, Ferguson GP (2011) Protection of sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis. PLoS Biol 9: e1001169 Hammami R, Ben Hamida J, Vergoten G, Fliss I (2009) PhytAMP: a database dedicated to antimicrobial plant peptides. Nucleic Acids Res 37: D963-968 Hara K, Kajita R, Torii KU, Bergmann DC, Kakimoto T (2007) The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rule. Genes Dev 21: 1720-1725 Hara K, Yokoo T, Kajita R, Onishi T, Yahata S, Peterson KM, Torii KU, Kakimoto T (2009) Epidermal cell density is autoregulated via a secretory peptide, EPIDERMAL PATTERNING FACTOR 2 in Arabidopsis leaves. Plant Cell Physiol 50: 1019-1031 Heslop-Harrison J (1982) Pollen-stigma interaction and cross-incompatibility in the grasses. Science 215: 1358-1364 Hirano K, Aya K, Hobo T, Sakakibara H, Kojima M, Shim RA, Hasegawa Y, Ueguchi-Tanaka M, Matsuoka M (2008) Comprehensive transcriptome analysis of phytohormone biosynthesis and signaling genes in microspore/pollen and tapetum of rice. Plant Cell Physiol 49: 1429-1450 Hofgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16: 9877 Honys D, Twell D (2004) Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biol 5: R85 Hsieh K, Huang AH (2007) Tapetosomes in Brassica tapetum accumulate endoplasmic reticulum-derived flavonoids and alkanes for delivery to the pollen surface. Plant Cell 19: 582-596 Hsu SW, Liu MC, Zen KC, Wang CS (2014) Identification of the tapetum/microspore-specific promoter of the pathogenesis-related 10 gene and its regulation in the anther of Lilium longiflorum. Plant Sci 215-216: 124-133 Hsu YF, Tzeng JD, Liu MC, Yei FL, Chung MC, Wang CS (2008) Identification of anther-specific/predominant genes regulated by gibberellin during development of lily anthers. J Plant Physiol 165: 553-563 Huang CY, Chen PY, Huang MD, Tsou CH, Jane WN, Huang AH (2013) Tandem oleosin genes in a cluster acquired in Brassicaceae created tapetosomes and conferred additive benefit of pollen vigor. Proc Natl Acad Sci USA 110: 14480-14485 Hunt L, Gray JE (2009) The signaling peptide EPF2 controls asymmetric cell divisions during stomatal development. Curr Biol 19: 864-869 Ishiguro S, Nishimori Y, Yamada M, Saito H, Suzuki T, Nakagawa T, Miyake H, Okada K, Nakamura K (2010) The Arabidopsis FLAKY POLLEN1 gene encodes a 3-hydroxy-3-methylglutaryl-coenzyme A synthase required for development of tapetum-specific organelles and fertility of pollen grains. Plant Cell Physiol 51: 896-911 Jiang J, Yu Y, Dong H, Yao L, Zhang Z, Cao J (2014) BcMF21 is important for pollen development and germination in Brassica campestris ssp. chinensis. Mol Biol Rep 41: 537-544 Johnson MA, Preuss D (2002) Plotting a course: multiple signals guide pollen tubes to their targets. Dev Cell 2: 273-281 Joshi G, Shukla A, Shukla A (2011) Synergistic response of auxin and ethylene on physiology of Jatropha curcas L. Braz. J. Plant Physiol. 23(1), 67-77. Jung KH, Han MJ, Lee YS, Kim YW, Hwang I, Kim MJ, Kim YK, Nahm BH, An G (2005) Rice Undeveloped Tapetum1 is a major regulator of early tapetum development. Plant Cell 17: 2705-2722 Kaneko M, Itoh H, Inukai Y, Sakamoto T, Ueguchi-Tanaka M, Ashikari M, Matsuoka M (2003) Where do gibberellin biosynthesis and gibberellin signaling occur in rice plants? Plant J 35: 104-115 Kapoor S, Kobayashi A, Takatsuji H (2002) Silencing of the tapetum-specific zinc finger gene TAZ1 causes premature degeneration of tapetum and pollen abortion in petunia. Plant Cell 14: 2353-2367 Kapoor S, Takatsuji H (2006) Silencing of an anther-specific zinc-finger gene, MEZ1, causes aberrant meiosis and pollen abortion in petunia. Plant Mol Biol 61: 415-430 Lausser A, Kliwer I, Srilunchang KO, Dresselhaus T (2010) Sporophytic control of pollen tube growth and guidance in maize. J Exp Bot 61: 673-682 Lay FT, Schirra HJ, Scanlon MJ, Anderson MA, Craik DJ (2003) The three-dimensional solution structure of NaD1, a new floral defensin from Nicotiana alata and its application to a homology model of the crop defense protein alfAFP. J Mol Biol 325: 175-188 Liu L, Fan XD (2013) Tapetum: regulation and role in sporopollenin biosynthesis in Arabidopsis. Plant Mol Biol 83: 165-175 Liu MC, Wang BJ, Huang JK, Wang CS (2011a) Expression, localization and function of a cis-prenyltransferase in the tapetum and microspores of lily anthers. Plant Cell Physiol 52: 1487-1500 Liu MC, Yang CS, Wang CS (2011b) Expression and localization of two anther-specific genes in the tapetum and microspore of Lilium longiflorum. Bot Stud 52: 383-391 Liu MC, Yang CS, Yeh FL, Wei CH, Jane WN, Chung MC, Wang CS (2014) A novel lily anther-specific gene encodes adhesin-like proteins associated with exine formation during anther development. J Exp Bot 65: 2023-2037 Lu P, Chai M, Yang J, Ning G, Wang G, Ma H (2014) The Arabidopsis CALLOSE DEFECTIVE MICROSPORE1 gene is required for male fertility through regulating callose metabolism during microsporogenesis. Plant Physiol 164: 1893-1904 Luo H, Lee JY, Hu Q, Nelson-Vasilchik K, Eitas TK, Lickwar C, Kausch AP, Chandlee JM, Hodges TK (2006) RTS, a rice anther-specific gene is required for male fertility and its promoter sequence directs tissue-specific gene expression in different plant species. Plant Mol Biol 62: 397-408 Lunau, K (2000) The ecology and evolution of visual pollen signals. Plant Syst. Evol. 222, 89–111. Manners JM (2007) Hidden weapons of microbial destruction in plant genomes. Genome Biol 8: 225 Marshall E, Costa LM, Gutierrez-Marcos J (2011) Cysteine-rich peptides (CRPs) mediate diverse aspects of cell-cell communication in plant reproduction and development. J Exp Bot 62: 1677-1686 Muschietti J, Dircks L, Vancanneyt G, McCormick S (1994) LAT52 protein is essential for tomato pollen development: pollen expressing antisense LAT52 RNA hydrates and germinates abnormally and cannot achieve fertilization. Plant J 6: 321-338 Nallu S, Silverstein KA, Samac DA, Bucciarelli B, Vance CP, VandenBosch KA (2013) Regulatory patterns of a large family of defensin-like genes expressed in nodules of Medicago truncatula. PLoS One 8: e60355 Niu BX, He FR, He M, Ren D, Chen LT, Liu YG (2013) The ATP-binding cassette transporter OsABCG15 is required for anther development and pollen fertility in rice. J Integr Plant Biol 55: 710-720 Okuda S, Suzuki T, Kanaoka MM, Mori H, Sasaki N, Higashiyama T (2013) Acquisition of LURE-binding activity at the pollen tube tip of Torenia fournieri. Mol Plant 6: 1074-1090 Park JI, Hakozaki H, Endo M, Takada Y, Ito H, Uchida M, Okabe T, Watanabe M (2006) Molecular characterization of mature pollen-specific genes encoding novel small cysteine-rich proteins in rice (Oryza sativa L.). Plant Cell Rep 25: 466-474 Park SY, Jauh GY, Mollet JC, Eckard KJ, Nothnagel EA, Walling LL, Lord EM (2000) A lipid transfer-like protein is necessary for lily pollen tube adhesion to an in vitro stylar matrix. Plant Cell 12: 151-164 Pearce G, Moura DS, Stratmann J, Ryan CA (2001a) Production of multiple plant hormones from a single polyprotein precursor. Nature 411: 817-820 Pearce G, Moura DS, Stratmann J, Ryan CA, Jr. (2001b) RALF, a 5-kDa ubiquitous polypeptide in plants, arrests root growth and development. Proc Natl Acad Sci USA 98: 12843-12847 Peng ZY, Zhou X, Li L, Yu X, Li H, Jiang Z, Cao G, Bai M, Wang X, Jiang C, Lu H, Hou X, Qu L, Wang Z, Zuo J, Fu X, Su Z, Li S, Guo H (2009) Arabidopsis Hormone Database: a comprehensive genetic and phenotypic information database for plant hormone research in Arabidopsis. Nucleic Acids Res 37: D975-982 Peterson R, Slovin JP, Chen C. 2010. A simplified method for differential staining of aborted and nonaborted pollen grains. Int J Plant Biology 1:e13:66-69. Plackett AR, Thomas SG, Wilson ZA, Hedden P (2011) Gibberellin control of stamen development: a fertile field. Trends Plant Sci 16: 568-578 Qin P, Tu B, Wang Y, Deng L, Quilichini TD, Li T, Wang H, Ma B, Li S (2013) ABCG15 encodes an ABC transporter protein, and is essential for post-meiotic anther and pollen exine development in rice. Plant Cell Physiol 54: 138-154 Quilichini TD, Douglas CJ, Samuels AL (2014a) New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana. Ann Bot doi: 10.1093/aob/mcu042 Quilichini TD, Grienenberger E, Douglas CJ (2014b) The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack. Phytochemistry doi: 10.1016/j.phytochem.2014.05.002 Roberts MR, Foster GD, Blundell RP, Robinson SW, Kumar A, Draper J, Scott R (1993) Gametophytic and sporophytic expression of an anther-specific Arabidopsis thaliana gene. Plant J 3: 111-120 Rodriguez-Enriquez MJ, Mehdi S, Dickinson HG, Grant-Downton RT (2013) A novel method for efficient in vitro germination and tube growth of Arabidopsis thaliana pollen. New Phytol 197: 668-679 Rogers HJ, Bate N, Combe J, Sullivan J, Sweetman J, Swan C, Lonsdale DM, Twell D (2001a) Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10. Plant Mol Biol 45: 577-585 Rogers HJ, Maund SL, Johnson LH (2001b) A beta-galactosidase-like gene is expressed during tobacco pollen development. J Exp Bot 52: 67-75 Sambrook JE, Fritsch ET, Maniatis R (1989) Molecular cloning : a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Sanders PM, Bui AQ, Le BH, Goldberg RB (2004) Differentiation and degeneration of cells that play a major role in tobacco anther dehiscence. Sex Plant Reprod 17: 219-241 Scheres B, van Engelen F, van der Knaap E, van de Wiel C, van Kammen A, Bisseling T (1990) Sequential induction of nodulin gene expression in the developing pea nodule. Plant Cell 2: 687-700 Schmeichel KL, Beckerle MC (1997) Molecular dissection of a LIM domain. Mol Biol Cell 8: 219-230 Schopfer CR, Nasrallah ME, Nasrallah JB (1999) The male determinant of self-incompatibility in Brassica. Science 286: 1697-1700 Shiba H, Takayama S, Iwano M, Shimosato H, Funato M, Nakagawa T, Che FS, Suzuki G, Watanabe M, Hinata K, Isogai A (2001) A pollen coat protein, SP11/SCR, determines the pollen S-specificity in the self-incompatibility of Brassica species. Plant Physiol 125: 2095-2103 Silverstein KA, Moskal WA, Jr., Wu HC, Underwood BA, Graham MA, Town CD, VandenBosch KA (2007) Small cysteine-rich peptides resembling antimicrobial peptides have been under-predicted in plants. Plant J 51: 262-280 Singh DP, Jermakow AM, Swain SM (2002) Gibberellins are required for seed development and pollen tube growth in Arabidopsis. Plant Cell 14: 3133-3147 Song JH, Cao JS, Wang CG (2013a) BcMF11, a novel non-coding RNA gene from Brassica campestris, is required for pollen development and male fertility. Plant Cell Rep 32: 21-30 Song S, Qi T, Huang H, Xie D (2013b) Regulation of stamen development by coordinated actions of jasmonate, auxin, and gibberellin in Arabidopsis. Mol Plant 6: 1065-1073 Steffens, B, Wang, J, Sauter, M (2006) Interactions between ethylene, gibberellin and abscisic acid regulate emergence and growth rate of adventitious roots in deepwater rice. Planta:223, 604–612. Stieglitz H (1977) Role of beta-1,3-glucanase in postmeiotic microspore release. Dev Biol 57: 87-97 Suen DF, Wu SS, Chang HC, Dhugga KS, Huang AH (2003) Cell wall reactive proteins in the coat and wall of maize pollen: potential role in pollen tube growth on the stigma and through the style. J Biol Chem 278: 43672-43681 Suwabe K, Suzuki G, Takahashi H, Shiono K, Endo M, Yano K, Fujita M, Masuko H, Saito H, Fujioka T, Kaneko F, Kazama T, Mizuta Y, Kawagishi-Kobayashi M, Tsutsumi N, Kurata N, Nakazono M, Watanabe M (2008) Separated transcriptomes of male gametophyte and tapetum in rice: validity of a laser microdissection (LM) microarray. Plant Cell Physiol 49: 1407-1416 Swain SM, Muller AJ, Singh DP (2004) The gar2 and rga alleles increase the growth of gibberellin-deficient pollen tubes in Arabidopsis. Plant Physiol 134: 694-705 Takayama S, Shimosato H, Shiba H, Funato M, Che FS, Watanabe M, Iwano M, Isogai A (2001) Direct ligand-receptor complex interaction controls Brassica self-incompatibility. Nature 413: 534-538 Takeuchi H, Higashiyama T (2012) A species-specific cluster of defensin-like genes encodes diffusible pollen tube attractants in Arabidopsis. PLoS Biol 10: e1001449 Tan H, Liang W, Hu J, Zhang D (2012) MTR1 encodes a secretory fasciclin glycoprotein required for male reproductive development in rice. Dev Cell 22: 1127-1137 Tang W, Ezcurra I, Muschietti J, McCormick S (2002) A cysteine-rich extracellular protein, LAT52, interacts with the extracellular domain of the pollen receptor kinase LePRK2. Plant Cell 14: 2277-2287 Terauchi R, Kahl G (2000) Rapid isolation of promoter sequences by TAIL-PCR: the 5'-flanking regions of Pal and Pgi genes from yams (Dioscorea). Mol Gen Genet 263: 554-560 Twell D, Yamaguchi J, Wing RA, Ushiba J, McCormick S (1991) Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements. Genes Dev 5: 496-507 Tzeng JD, Hsu SW, Chung MC, Yeh FL, Yang CY, Liu MC, Hsu YF, Wang CS (2009) Expression and regulation of two novel anther-specific genes in Lilium longiflorum. J Plant Physiol 166: 417-427 Verwoerd TC, Dekker BM, Hoekema A (1989) A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17: 2362 Vizcay-Barrena G, Wilson ZA (2006) Altered tapetal PCD and pollen wall development in the Arabidopsis ms1 mutant. J Exp Bot 57: 2709-2717 Wang A, Xia Q, Xie W, Datla R, Selvaraj G (2003) The classical Ubisch bodies carry a sporophytically produced structural protein (RAFTIN) that is essential for pollen development. Proc Natl Acad Sci USA 100: 14487-14492 White CN, Proebsting WM, Hedden P, Rivin CJ (2000) Gibberellins and seed development in maize. I. Evidence that gibberellin/abscisic acid balance governs germination versus maturation pathways. Plant Physiol 122: 1081-1088 Yi B, Zeng F, Lei S, Chen Y, Yao X, Zhu Y, Wen J, Shen J, Ma C, Tu J, Fu T (2010) Two duplicate CYP704B1-homologous genes BnMs1 and BnMs2 are required for pollen exine formation and tapetal development in Brassica napus. Plant J 63: 925-938 Zhang D, Yang L (2014) Specification of tapetum and microsporocyte cells within the anther. Curr Opin Plant Biol 17: 49-55 Zhang DS, Liang WQ, Yuan Z, Li N, Shi J, Wang J, Liu YM, Yu WJ, Zhang DB (2008) Tapetum degeneration retardation is critical for aliphatic metabolism and gene regulation during rice pollen development. Mol Plant 1: 599-610 Zhang W, Sun Y, Timofejeva L, Chen C, Grossniklaus U, Ma H (2006) Regulation of Arabidopsis tapetum development and function by DYSFUNCTIONAL TAPETUM1 (DYT1) encoding a putative bHLH transcription factor. Development 133: 3085-3095 Zhang X, Feng B, Zhang Q, Zhang D, Altman N, Ma H (2005) Genome-wide expression profiling and identification of gene activities during early flower development in Arabidopsis. Plant Mol Biol 58: 401-419||摘要:||
鐵炮百合(Lilium longiflorum) LLA139是利用扣除抑制雜合(suppression subtractive hybridization)的方法自花藥小胞子發育時期cDNA集合庫中選殖出的基因。LLA139 cDNA可轉譯出102個胺基酸，扣除N端30個胺基酸的訊息胜?，估計成熟蛋白的分子量為7.3 kDa。推測其可形成兩個finger motifs的蛋白質。序列比對得知與LLA67及HvLEM1兩蛋白質分別有42%及47%的相同度。特別的是三者蛋白質序列之半胱胺酸呈現高度保守，屬於小分子富含半胱胺酸的蛋白質，但三者功能皆尚未知。原位雜合及北方墨漬分析顯示LLA139為絨氈層和小孢子特有的基因，且不受外加激勃素的誘導。利用激勃素生合成抑制劑uniconazole和乙烯競爭抑制劑 2,5-norbornadiene (NBD)處理後LLA139 mRNA會明顯增加，顯示LLA139分別受乙烯和激勃素的逆調控。然而，同時添加uniconazole和NBD時，LLA139 mRNA的量和未添加的對照組相比反而減少，顯示乙烯與激勃素之間的相互對話(cross-talk)反而使發育花藥中的LLA139基因表現下降。為了進一步探討LLA139的功能，利用絨氈層專一的TAP啟動子建構了三種不同的重組基因構築，分別為full-length (TAP::FL)、刪除N-finger motif (TAP::△N)、刪除C-finger motif (TAP::△C)的載體，以農桿菌方式感染轉型至阿拉伯芥，篩選出T2 homologous子代植株，觀察LLA139蛋白對阿拉伯芥轉殖株花藥發育的影響。三個轉殖株莢果皆很明顯的較野生型短小，然而只有LLA139::△C轉殖株的花粉萌發率較野生型少。藉由掃描式電子顯微鏡觀察，三個轉殖株花粉外壁皆有異物堆積的現象，特別是LLA139::△C花粉外壁部分區域的網格狀結構喪失，呈現平滑的表面。為了找到LLA139的啟動子，進行兩次TAIL-PCR (thermal asymmetric interlaced PCR)向5'UTR總共延伸了1953 bp。LLA139啟動子具有花藥專一性的調控區，包括9個AGAAA、15個GTGA motif、2個LAT56/59 box以及多個植物荷爾蒙調控區。將LLA139基因的啟動子依不同長度之刪除片段分別構築含有β-glucuronidase (GUS)基因轉殖至阿拉伯芥中，發現所有轉殖株GUS訊號皆專一表現在花藥當中。以切片觀察，進一步得知GUS訊號專一表現在絨氈層中。最小刪除片段373 bp中具有一個預測的LAT56/59 box及兩個GTGA motif可能為調控小孢子/絨氈層專一表現的調控區。另外，LLA139f-1612::barnase植株花藥在塑膠切片下觀察，可以觀察到阿拉伯芥轉殖株之花粉發生空泡的異常現象，而且後續裂孔及藥隔的發育皆受到影響，導致花藥無法正常開裂。轉殖株果莢長度短亦無種子產生。將LLA139f-1612::barnase植株雌蕊與野生型花粉雜交後，果莢長度復原，種子產量也恢復正常。LLA139為百合花藥中絨氈層及小孢子專一性的小分子富含半胱胺酸蛋白基因，其啟動子能夠造成雄不孕，具有實際的農業應用價值。
LLA139 transcript was identified from a suppression subtractive cDNA library at the microspore satge of lily (Lilium longiflorum) anthers. The LLA139 cDNA encodes 102 amino acids. Taking out the signal peptide of 30 amino acids at the N-terminus, the estimated molecular mass of mature LLA139 protein is 7.3 kDa. The protein may form a structure of two finger motifs. Sequence analysis indicates that the protein shares 42% and 47% identity with LLA67 and HvLEM1 protein, respectively. Particularly the cysteine residues in the sequence of these proteins are highly conserved. They are classified as small cysteine-rich proteins, but the function is still unknown. In situ hybridization and Northern blot analysis showed that LLA139 mRNA was expressed at the tapetum and microspore. The LLA139 gene was not induced by exogenous gibberellins (GA). With the treatment of uniconazole, an inhibitor of GA biosynthesis and 2,5-norbornadiene (NBD), an inhibitor of ethylene action, we revealed that the level of LLA139 mRNA was significantly elevated, suggesting LLA139 is negatively regulated by either GA or ethylene. However, with the treatment of both uniconazole and NBD, the level of LLA139 mRNA was reduced when compared with the control without treatment. It suggested that a cross-talk between GA and ethylene occurring in the developing anther reduced the expression of LLA139. To further explore the function of LLA139, three constructs: TAP::LLA139 full length (TAP::FL), TAP::deleted N-finger motif (TAP::△N), and TAP::deleted C-finger motif (TAP::△C) were ligated with a rice tapetum-specific promoter (TAP) and transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation system. The T2 homologous lines were obtained and their phenotypes were exemined. The three transgenic lines had shorter siliques than wild-type. The germination percentage of TAP::△C pollen is significantly less than that of the wild type. Scanning electron microscopy revealed the observed amorphous extrabacular protrusions on the exine surface of these transgenic lines. In particular, the TAP::△C pollen showed patches of smooth exine without a visible exine network. A region of LLA139 promoter sequence with 1953 bp upstream from start codon was identified using two runs of TAIL-PCR approach. The LLA139 promoter contains putative anther-specific cis-elements, including nine AGAAA, fifteen GTGA and two LAT56/59 boxes. The LLA139 promoter was subjected to a series of 5'-deletion by which the full-length and three 5'-deleted fragments were fused with the GUS (β-glucuronidase) reporter gene, and introduced into Arabidopsis for functional assays. The LLA139 transgenic lines with various 5'-deleted fragments exhibited GUS signals in the anther, indicating of anther-specificity. The cross-sections of LLA139f-1612::GUS and LLA139f-373::GUS detected GUS signal only in the tapetum. The GUS signal in the anther of LLA139f-373::GUS line is reflected by the sequence contains two GTGA motifs and a LAT56/59 box. In addition, the anthers of the LLA139f-1612::barnase transgenic lines contained vacuolated pollen which differred from the normal pollen of wild-type. The development of stomium and septum in LLA139f-1612::barnase transgenic lines were accordingly affected, resulting in an inability of dehiscence. The LLA139f-1612::barnase transgenic siliques were short and seedless. When the female organs of LLA139f-1612::barnase transgenic lines were cross-pollinated with wild-type pollen, the siliques were reverted to the normal size with regular seed production, similar to wild type. LLA139 gene is a tapetum and microspore-specific gene that encodes a small cysteine-rich protein in Lilium longiflorum, and the identified promoter that causes male sterility has a practical value of agricultural application.
|Appears in Collections:||生物科技學研究所|
Show full item record
Files in This Item:
TAIR Related Article
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.