‘湖景蜜露’桃果实酰基辅酶A氧化酶编码基因的克隆及表达分析

doi:10.3864/j.issn.0578-1752.2012.09.011

摘要/Abstract

摘要： 【目的】根据国际桃基因组序列信息，进行克隆和表达分析与桃果实内酯类合成相关的果肉组织酰基辅酶A氧化酶(Acyl-CoA oxidase，ACX)的5个候选基因，为进一步开展桃香气物质代谢分子机理的研究提供新的研究方向。【方法】根据Phytozome(www.phytozome.org/peach)发布的桃ACX全基因组序列设计特异引物对5个基因全长进行克隆测序，与参考编码序列进行比对和编码蛋白性质进行分析，并对这些成员进行组织特异性表达分析。【结果】获得了桃果肉组织中表达的5个ACX编码基因全长序列，PpACX1、PpACX3的CDS区没有突变，PpACX2核苷酸序列871位处A突变为G，导致氨基酸序列中谷氨酰胺变异为精氨酸；PpACX4的核苷酸序列在80位和368位存在两处同义突变；PpACX5在475位处A突变为C，导致蛋氨酸突变为亮氨酸。同源性分析结果显示，这些基因的氨基酸序列与番茄、南瓜、大麦、葡萄等高等植物的氨基酸序列同源性很高。系统进化树分析表明，5个基因家族成员中，除PpACX4外其余4个成员处在同一个进化分支上，且与南瓜、葡萄、番茄、大豆等的亲缘关系较近。在根、茎、叶、果实表达证明，PpACX1是果实中主要大量表达成员。【结论】‘湖景蜜露’桃果实中表达的5个桃ACX编码基因全长与基因组数据数据库有一定差异，PpACX1为5个成员中较为关键的基因成员。

关键词: 桃, 酰基辅酶A氧化酶, 基因克隆, 序列分析

Abstract: 【Objective】The aim of this study is to clone and analyse the five genes of acyl-CoA oxidase(ACX) from peach (Prunus persica L. Batsch) flesh. 【Method】Specific primers were designed based on the genome of peach of ACX published on Phytozome (www.phytozome.org), and the full-length of the five genes were cloned. 【Result】 In this study, the full-length of the five genes of ACX from peach fruit was obtained. There was no mutation in the CDS of PpACX1 and PpACX3. A mutates to G in PpACX2 on seq 871, which leads to Gln mutates to Arg. In PpACX4, there are two same sense mutations in seq 80 and 368. A mutates to c in PpACX5 on seq 475, which leads to Met mutates to Leu. The amino acid sequence of peach ACX was highly homologous with other species, especially tomato, squash, barley, and grape. Phylogenetic analysis showed that five gene family members were in the same evolutionary branch except PpACX4 which had a more closer relationship with pumpkin, grape, tomato, soybean, etc. PpACX1 was proved to be the highest expression member through the gene expression in root, stalk, leaf and fruit.【Conclusion】There are some differences between the 5 expressed genes of ACX cloned from ‘Hujingmilu’ peach fruit and the database of the genome. PpACX1 was proved to be the most important gene member in peach fruit.

Key words: Prunus persica L. Batsch, Acyl-CoA oxidase, gene cloning, sequence analysis

齐玉洁, 张鑫, 杨夏, 高中山, 贾惠娟. ‘湖景蜜露’桃果实酰基辅酶A氧化酶编码基因的克隆及表达分析[J]. 中国农业科学, 2012, 45(9): 1758-1765.

QI Yu-Jie, ZHANG Xin, YANG Xia, GAO Zhong-Shan, JIA Hui-Juan. Molecular Cloning and Sequence Analysis of Acyl-CoA Oxidase Gene from ‘Hujingmilu’ Peach Fruit[J]. Scientia Agricultura Sinica, 2012, 45(9): 1758-1765.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.09.011

https://www.chinaagrisci.com/CN/Y2012/V45/I9/1758

参考文献

[1]Jia H J, Okamoto G. Distribution of volatile compounds in peach fruit. Journal of Japanese Society for Horticultural Science, 2001, 70: 223-225.

[2]Schwab W, Davidovich-Rikanati R, Lewinsohn E. Biosynthesis of plant-derived flavor compounds. The Plant Journal, 2008, 54: 712-732.

[3]Husain Q, Hui Y H. Chemistry and biochemistry of some vegetable flavors//Handbook of Fruit and Vegetable Flavours. JohnWiley & Sons//Hoboken, New Jersey, 2010: 575-625.

[4]Arent S, Pye V E, Henriksen A. Structure and function of plant acyl-CoA oxidases. Plant Physiology and Biochemistry, 2008, 46: 292-301.

[5]Lazarow P B, Duve C A. Fatty acyl-coa oxidizing system in rat liver peroxisomes enhancement by clofibrate a hypolipidemic drug. Proceedings of the National Academy of Sciences of the USA, 1976, 73: 2043-2046.

[6]Kawamoto S, Nozaki C, Tanaka A, Fukui S. Fatty acid β-oxidation system in microbodies of n-alkane-grown Candida tropicalis. European Journal of Biochemistry, 1978, 83: 609-613.

[7]Kawaguchi A, Tsubotani S, Seyama Y, Yamakawa T, Osumi T, Hashimoto T, Kikuchi T, Ando M, Okuda S. Stereochemistry of dehydrogenation catalyzed by Acyl-CoA oxidase. The Journal of Biochemistry, 1980, 88: 1481-1486.

[8]Coopers T G, Harry B. β-Oxidation in glyoxysomes from castor bean endosperm. The Journal of Biological Chemistry, 1969, 244(13): 3514-3520.

[9]Wang H J, Dall M T, Waché Y. Evaluation of acyl coenzyme A oxidase (ACOX) isozyme function in the n-alkane-assimilating yeast Yarrowia lipolytica. Journal of Bacteriology, 1999, 181: 5140-5148.

[10]Waché Y, Pagot Y, Nicaud J M, Belin J M. Acyl-CoA oxidase, a key step for lactone production by Yarrowia lipolytica. Journal of Molecular Catalysis B: Enzymatic, 1998, 165-169.

[11]Waché Y, Aguedo M, Choquet A, Gatfield I L, Nicaud J M, Belin J M. Role of beta-oxidation enzymes in gamma-decalactone production by the yeast Yarrowia lipolytica. Applied and Environmental Microbiology, 2001, 67: 5700-5704.

[12]Waché Y, Husson F, Feron G. Yeast as an efficient biocatalyst for the production of lipid-derived flavours. Antonie van Leeuwenhoek, 2006, 89: 405-416.

[13]Hooks M A, Bode K, Couee I. Regulation of Acyl-CoA oxidases in maize seedlings. Phychemistry, 1995, 40(3): 657-660.

[14]Hayashi H, Bellis D L, Yamaguchi K, Kato A. Molecular characterization of a glyoxysomal long chain acyl-CoA oxidase that is synthesized as a precursor of higher molecular mass in pumpkin. Journal of Biochemistry, 1998, 273: 8301-8307.

[15]Rachel A, Christopher L, Anthony L. Structure determination and analysis of acyl-CoA oxidase (ACOX1) from tomato. Acta Crystallographica, 2006, D62: 683-686.

[16]Ameeta K A, Youlin Q, Deepti G B, Woerner B M, Brown S M. Gene isolation and characterization of two acyl-CoA oxidases from soybean with broad substrate specificities and enhanced expression in the growing seedling axis. Plant Molecular Biology, 2001, 47: 519-531.

[17]Xi W P, Zhang B, Liang L, Shen J Y, Wei W W, Xu C J, Allan A C, Ferguson I B, Chen K S. Postharvest temperature influences volatile lactone production via regulation of acyl-CoA oxidases in peach fruit. Plant, Cell and Environment, 2012, 35(3): 534-545.

[18]陈长宝, 朱树华, 周杰. 改良CTAB法提取成熟肥城桃果实的总RNA. 山东农业科学, 2009(5): 102-104.

Chen C B, Zhu S H, Zhou J. Improved CTAB method for total RNA extraction of mature Feicheng peach fruit. Shandong Agricultural Science, 2009(5): 102-104. (in Chinese)

[19]Sudhir K, Koichiro T, Matoshi N. MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Briefings in Bioinformatics, 2004, 5(2): 150-163.

[20]Tong Z, Gao Z, Wang F, Zhou J, Zhang Z. Selection of reliable reference genes for gene expression studies in peach using real-time PCR. Molecular Biology, 2009, 10: 71.

[21]Jose M O, Ana G P, Jose J R, Luis C S. Aroma of virgin olive oil: Biogenesis of the “Green” odor notes. Journal of Agricultural and Food Chemistry, 1993, 41(12): 2368-2373.

[22]Helena P, Mario R S. Plant lipoxygenases. physiological and molecular features. Plant Physiology, 2002, 130(9): 15-21.

[23]Akikazu H. The biogeneration of green odour by green leaves. Phytochemistry, 1993, 34(5): 1201-1218.

[24]Baker A, Graham I A, Holdsworth M, Smith S M, Theodoulou F L. Chewing the fat: oxidation in signaling and development. Trends in Plant Science, 2006, 11: 124-132.

[25]Kirsch T, Loffler H G, Kindl H. Plant acyl-CoA oxidase: Purification, characterization and monomeric apoprotein. Journal of Biochemistry, 1986, 18: 8570-8575.

[26]Hooks M A, Bode K, Couee I. Higher-plant medium- and short-chain acyl-CoA oxidases: Identification, purification and characterization of two novel enzymes of eukaryotic peroxisomal beta-oxidation. Biochemical Journal, 1996, 320(2): 607-614.

[27]刘巍立. 桃果实成熟软化过程中特征香气物质形成相关酶活性的变化研究[D]. 杭州: 浙江大学, 2010.

Liu W L. Study on the characteristics aroma and related enzyme activity during ripening of peach (Prunus persica L.) fruit[D]. Hangzhou: Zhejiang University, 2010.(in Chinese)