苹果丙二烯氧化物环化酶基因MdAOC1的克隆与表达分析

doi:10.11707/j.1001-7488.20131211

摘要/Abstract

摘要：

丙二烯氧化物环化酶（AOC）是茉莉酸生物合成途径中一个关键酶，在植物抗逆境反应中发挥重要作用。用电子克隆结合RACE技术，从苹果‘嘎拉’中克隆并鉴定1个AOC编码基因，命名为MdAOC1。MdAOC1基因开放阅读框长759 bp，编码1个由252个氨基酸组成的蛋白，该蛋白包含1个保守的Allene_ox_cyc结构域，并带有1个N-末端叶绿体前导肽。同源性分析表明，MdAOC1蛋白与水稻、拟南芥等AOC蛋白具有高度的氨基酸序列相似性，其三级结构模型是由3条链组成的疏水结合空穴，同拟南芥AOC2的蛋白三级结构相似。利用实时荧光定量RT-PCR方法测定MdAOC1基因的表达情况，发现MdAOC1具有组织特异性，在茎中表达最高；MdAOC1不仅能被机械伤所诱导，而且能被外源激素MeJA、SA处理所诱导。结果暗示MdAOC1基因可能参与苹果对生物或非生物胁迫的防御反应。

关键词: 苹果, AOC基因, 茉莉酸, 基因克隆, 基因表达

Abstract:

Allene oxide cyclase (AOC) is a key enzyme in the biosynthesis of jasmonic acid and plays an important role in plant resistance to biostress and abiostress. In the present study, the full length cDNA sequence of an apple (Malus domestica) AOC gene (named as MdAOC1 ) was amplified using in-silico cloning and RACE approaches. The ORF of MdAOC1 gene is 759 bp encoding a protein composed of 252 amino acids. MdAOC1 protein contains a conserved Allene_ox_cyc domain in C-terminus and a chloroplast transit peptide in N-terminus. Multialignment indicated that the MdAOC1 shared high identity with AOC proteins from Oryza sativa and Arabidopsis thaliana. The tertiary structure of the protein formed a hydrophobic binding cavity with two distinct polar patches, which was similar to AtAOC2. Real-time quantitative PCR analysis showed that MdAOC1 expression had tissue specificity and the highest expression was observed in the stems. Moreover, MdAOC1 expression was induced not only by the treatment of wounding, but also by salicylic acid (SA) and methyl jasmonate (MeJA). The results implied that MdAOC1 gene might be involved in the defense responses to biotic or abiotic stress.

Key words: Malus domestica, allene oxide cyclase gene, jasmonate, molecular cloning, gene expression

中图分类号:

S718.46

曹晏彬, 柏素花, 戴洪义. 苹果丙二烯氧化物环化酶基因MdAOC1的克隆与表达分析[J]. 林业科学, 2013, 49(12): 73-80.

Cao Yanbin, Bai Suhua, Dai Hongyi. Cloning and Expression Analysis of Allene Oxide Cyclase Gene MdAOC1 from Malus domestica[J]. Scientia Silvae Sinicae, 2013, 49(12): 73-80.

参考文献

柏素花, 祝军, 戴洪义. 2012. 苹果酰基辅酶 A 结合蛋白 2 编码基因MdACBP2的克隆和表达分析. 园艺学报, 39(10): 1893-1902.
蒋科技. 2007. 药用植物茉莉酸合成途径关键酶基因的克隆与研究.上海: 复旦大学博士学位论文.
蒋科技, 皮妍, 侯嵘, 等.2010. 植物内源茉莉酸类物质的生物合成途径及其生物学意义.植物学报, 45(2): 137-148.
刘艳, 潘秋红, 战吉宬, 等.2008. 豌豆叶片内源水杨酸和茉莉酸类物质对机械伤害的响应.中国农业科学, 41(3): 808-815.
Agrawal G K, Jwa N S, Agrawal S K, et al. 2003. Cloning of novel rice allene oxide cyclase (OsAOC): mRNA expression and comparative analysis with allene oxide synthase (OsAOS) gene provides insight into the transcriptional regulation of octadecanoid pathway biosynthetic genes in rice. Plant Science, 164(6): 979-992.
Anderson J M. 1988. Jasmonic acid-dependent increases in the level of specific polypeptides in soybean suspension cultures and seedlings. Journal of Plant Growth Regulation, 7(4): 203-211.
Creelman R A, Mullet J E. 1997. Biosynthesis and action of jasmonates in plants. Annual Review of Plant Biology, 48(1): 355-381.
Creelman R A, Tierney M L, Mullet J E. 1992. Jasmonic acid/methyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression. Proceedings of the National Academy of Sciences, 89(11): 4938-4941.
Emanuelsson O, Nielsen H, Heijne G V. 1999. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Science, 8(5): 978-984.
Gfeller A, Baerenfaller K, Loscos J, et al. 2011. Jasmonate controls polypeptide patterning in undamaged tissue in wounded Arabidopsis leaves. Plant Physiology, 156(4): 1797-1807.
Hamberg M, Fahlstadius P. 1990. Allene oxide cyclase: a new enzyme in plant lipid metabolism. Archives of Biochemistry and Biophysics, 276(2): 518-526.
Hofmann E, Zerbe P, Schaller F. 2006. The crystal structure of Arabidopsis thaliana allene oxide cyclase: insights into the oxylipin cyclization reaction. The Plant Cell Online, 18(11): 3201-3217.
Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔC_T method. Methods, 25(4): 402-408.
Laudert D, Weiler E W. 1998. Allene oxide synthase: a major control point in Arabidopsis thaliana octadecanoid signalling. The Plant Journal, 15(5): 675-684.
Maucher H, Hause B, Feussner I, et al. 2000. Allene oxide synthases of barley (Hordeum vulgare cv. Salome): tissue specific regulation in seedling development. The Plant Journal, 21(2): 199-213.
Metodiev M V, Tsonev T D, Popova L P. 1996. Effect of jasmonic acid on the stomatal and nonstomatal limitation of leaf photosynthesis in barley leaves. Journal of Plant Growth Regulation, 15(2): 75-80.
Meyer A, Miersch O, BÜttner C, et al. 1984. Occurrence of the plant growth regulator jasmonic acid in plants. Journal of Plant Growth Regulation, 3(1-4): 1-8.
Ortel B, Atzorn R, Hause B, et al. 1999. Jasmonate-induced gene expression of barley (Hordeum vulgare) leaves—the link between jasmonate and abscisic acid. Plant Growth Regulation, 29(1/2): 113-122.
Parthier B. 1990. Jasmonates: Hormonal regulators or stress factors in leaf senescence?. Journal of Plant Growth Regulation, 9(1-4): 57-63.
Pena-Cortés H, Albrecht T, Prat S, et al. 1993. Aspirin prevents wound-induced gene expression in tomato leaves by blocking jasmonic acid biosynthesis. Planta, 191(1): 123-128.
Pi Y, Liao Z, Jiang K, et al. 2008. Molecular cloning, characterization and expression of a jasmonate biosynthetic pathway gene encoding allene oxide cyclase from Camptotheca acuminata. Bioscience Reports, 28: 349-355.
Reinbothe C, Springer A, Samol I, et al. 2009. Plant oxylipins: role of jasmonic acid during programmed cell death, defence and leaf senescence. Febs Journal, 276(17): 4666-4681.
Salzman R A, Brady J A, Finlayson S A, et al. 2005. Transcriptional profiling of sorghum induced by methyl jasmonate, salicylic acid, and aminocyclopropane carboxylic acid reveals cooperative regulation and novel gene responses. Plant Physiology, 138(1): 352-368.
Sasaki Y, Asamizu E, Shibata D, et al. 2001. Monitoring of methyl jasmonate-responsive genes in Arabidopsis by cDNA macroarray: self-activation of jasmonic acid biosynthesis and crosstalk with other phytohormone signaling pathways. DNA Research, 8(4): 153-161.
Schaller F. 2001. Enzymes of the biosynthesis of octadecanoid-derived signalling molecules. Journal of Experimental Botany, 52(354): 11-23.
Shan X Y, Wang Z L, Xie D. 2007. Jasmonate signal pathway in Arabidopsis. Journal of Integrative Plant Biology, 49(1): 81-86.
Stenzel I, Hause B, Maucher H, et al. 2003a. Allene oxide cyclase dependence of the wound response and vascular bundle-specific generation of jasmonates in tomato-amplification in wound signalling. The Plant Journal, 33(3): 577-589.
Stenzel I, Hause B, Miersch O, et al. 2003b. Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana. Plant Molecular Biology, 51(6): 895-911.
Stenzel I, Otto M, Delker C, et al. 2012. ALLENE OXIDE CYCLASE (AOC) gene family members of Arabidopsis thaliana: tissue-and organ-specific promoter activities and in vivo heteromerization. Journal of Experimental Botany, 63(17): 6125-6138.
Tsonev T D, Lazova G N, Stoinova Z G, et al. 1998. A possible role for jasmonic acid in adaptation of barley seedlings to salinity stress. Journal of Plant Growth Regulation, 17(3): 153-159.
Turner J G, Ellis C, Devoto A. 2002. The jasmonate signal pathway. The Plant Cell Online, 14(suppl 1): S153-S164.
Wasternack C, Hause B. 2002. Jasmonates and octadecanoids: signals in plant stress responses and development. Progress in Nucleic Acid Research and Molecular Biology, 72: 165-221.
Wu Q, Wu J, Sun H, et al. 2011. Sequence and expression divergence of the AOC gene family in soybean: insights into functional diversity for stress responses. Biotechnology Letters, 33(7): 1351-1359.
Yamada A, Saitoh T, Mimura T, et al. 2002. Expression of mangrove allene oxide cyclase enhances salt tolerance in Escherichia coli, yeast, and tobacco cells. Plant and Cell Physiology, 43(8): 903-910.
Yao J L, Cohen D, Atkinson R, et al. 1995. Regeneration of transgenic plants from the commercial apple cultivar Royal Gala. Plant Cell Reports, 14(7): 407-412.
Ziegler J, Hamberg M, Miersch O, et al. 1997. Purification and characterization of allene oxide cyclase from dry corn seeds. Plant Physiology, 114(2): 565-573.
Ziegler J, Stenzel I, Hause B, et al. 2000. Molecular cloning of allene oxide cyclase. The enzyme establishing the stereochemistry of octadecanoids and jasmonates. Journal of Biological Chemistry, 275(25): 19132-19138.

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