湖北海棠病程相关蛋白MhPR8基因的克隆与表达

doi:10.3864/j.issn.0578-1752.2012.08.012

摘要/Abstract

摘要： 【目的】从湖北海棠叶片中克隆植物第Ⅲ类几丁质酶基因PR8的cDNA序列和基因组DNA序列，研究化学试剂SA、MeJA、ACC、ABA，非生物胁迫NaCl和低温（4℃），生物胁迫苹果轮纹病病原菌和苹果蚜虫诱导下MhPR8基因的表达特性。【方法】利用PCR技术分别从SA诱导的湖北海棠全长cDNA文库和基因组DNA中克隆MhPR8基因全长序列；利用生物信息学方法对其进行结构和功能的初步分析；利用实时荧光定量RT-PCR技术分析该基因的表达特性。【结果】该基因编码区为897 bp，编码298个氨基酸，蛋白质分子量为31.67 kD，等电点为4.77，命名为MhPR8。该基因在其编码区内部没有内含子。MhPR8蛋白含有保守的结构域GH18_hevamine_XipI_class_III，属于第III类几丁质酶。该蛋白含有6个保守的半胱氨基酸残基，含有植物第III类几丁质酶起催化作用的天冬氨酸和谷氨酸，分别位于第151和153位氨基酸。qRT-PCR分析结果表明，该基因在湖北海棠根中的表达量最大；SA、MeJA和ACC明显诱导该基因的表达，ABA略微诱导该基因的表达；低温诱导该基因的表达，而NaCl诱导该基因的表达不明显；生物胁迫苹果轮纹病病原菌和苹果蚜虫诱导该基因的表达。【结论】湖北海棠MhPR8可能参与SA、JA/ET介导的信号通路中，在湖北海棠抵抗生物胁迫和非生物胁迫中起着非常重要的作用。

关键词: 湖北海棠, MhPR8, 表达分析, 化学试剂, 生物胁迫, 非生物胁迫

Abstract: 【Objective】This study aimed to clone a full length cDNA and genomic DNA sequences of MhPR8 from Malus hupehensis and analyze the expression pattern of MhPR8 after treatment with the chemical reagents salicylic acid (SA), methyl jasmonate (MeJA), 1-aminocyclopropane-1-carboxylic acid (ACC), abscisic acid (ABA), abiotic stress NaCl and low temperature (4℃), and biotic factors apple ring spot pathogen (Botryosphaeria berengeriana) and apple aphids (Aphis citricota).【Method】The full length cDNA and genomic DNA sequences of MhPR8 were isolated from the M. hupehensis complete cDNA library treated by SA and genomic DNA using the method of PCR amplification, respectively. Real-time quantitative RT-PCR (qRT-PCR) was performed to determine the expression pattern of MhPR8 in M. hupehensis.【Result】The open reading frame of this gene was 897 bp, encoding 298 amino acids with a molecular weight of 31.67 kD and isoelectric point of 4.77. There was no intron in ORF of MhPR8. MhPR8 contains conserved construction domain GH18_hevamine_XipI_class_III, belongs to Class III chitinase. And this protein has six conserved cysteines, and aspartic acid and glutamic acid play a role of catalysis in plants class III chitinase, which are located in 151th and 153th amino acids, respectively. qRT-PCR results show that during the first 48 hours, MhPR8 was obviously induced by SA, MeJA, and ACC, but weakly induced by ABA. And this gene was clearly induced by low temperature, but weakly induced by NaCl. Infected with B. berengeriana resulted in obvious accumulation of MhPR8 transcripts during the first 96 h. The expression of the MhPR8 was clearly induced by the apple aphid (A. citricota) in leaves and stems of M. hupehensis. 【Conclusion】MhPR8 could be involved with the SA- signal pathway and the jasmonic acid (JA)/ ethylene (ET)-signal pathway in M. hupehensis. MhPR8 might be play a key role in resistance to biotic and abiotic stresses.

Key words: Malus hupehensis, MhPR8, expression, chemical reagents, biotic stress, abiotic stress

张计育, 渠慎春, 薛华柏, 高志红, 郭忠仁, 章镇. 湖北海棠病程相关蛋白MhPR8基因的克隆与表达[J]. 中国农业科学, 2012, 45(8): 1568-1575.

ZHANG Ji-Yu, QU Shen-Chun, XUE Hua-Bai, GAO Zhi-Hong, GUO Zhong-Ren, ZHANG Zhen. Isolation and Expression of Pathogenesis-Related Protein Gene MhPR8 from Malus hupehensis [J]. Scientia Agricultura Sinica, 2012, 45(8): 1568-1575.

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.0578-1752.2012.08.012

https://www.chinaagrisci.com/CN/Y2012/V45/I8/1568

参考文献

[1]Leon-Reyes A, Spoel S H, De Lange E S, Abe H, Kobayashi M, Tsuda S, Millenaar F F, Welschen R A M, Ritsema T, Pieterse C M J. Ethylene modulates the role of nonexpression pathogenesis-related genes 1 in cross talk between salicylate and jasmonate signaling. Plant Physiology, 2009, 149: 1797-1809.

[2]Pak J H, Chung E S, Shin S H, Jeon E H, Kim M J, Lee H Y, Jeung J U, Hyung N I, Lee J H, Chung Y S. Enhanced fungal resistance in Arabidopsis expressing wild rice PR-3 (OgChitIVa) encoding chitinase class IV. Plant Biotechnology Reporter, 2009, 3: 147-155.

[3]van Loon L C, Van Kammen A. Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var. ‘Samsun’ and ‘Samsun NN’: II. Changes in protein constitution after infection with tobacco mosaic virus. Virology, 1970, 40: 199-211.

[4]van Loon L C, van striene A. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiological and Molecular Plant Pathology, 1999, 55: 85-97.

[5]van Loon L C, Rep M, Pieterse C M J. Significance of inducible defense-related proteins in infected plants. Annual Review of Phytopathology, 2006, 44: 135-162.

[6]Bartnicki-Garcia S. Cell wall chemistry, morphogenesis, and taxonomy of fungi. Annual Review Microbiology, 1968, 22:87-108.

[7]Brokaert W F, Parijs J V, Allen A K, Peumans W J. Comparison of some molecular, enzymatic and antifungal properties of chitinases from thorn-apple, tobacco and wheat. Physiological and Molecular Plant Pathology, 1988, 33: 319-331.

[8]Schlumbaum A, Mauch F, Vögeli U, Boller T. Plant chitinases are potent inhibitors of fungal growth. Nature, 1986, 324: 365-367.

[9]Barber M S, Bertram RE, Ride J P. Chitin oligosaccharides elicit lignification in wounded wheat leaves. Physiological and Molecular Plant Pathology, 1989, 34:3-12.

[10]Mauch F, Staehelin LA. Functional implications of the subcellular localization of ethylene-induced chitinase and ß-1,3-glucanase in bean leaves. The Plant Cell, 1989, 1: 447-457.

[11]Mé traux J P, Burkhart W, Moyer M, Dichner S, Middlesteadt W, Williams S, Payne G, Carnes M，Ryals J. Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase. Proceedings of the National Academy of Sciences of the USA, 1989, 86: 896-900.

[12]Samac D A, Hironaka C M, Yallay P E, Shah D M. Isolation and characterization of genes encoding basic and acidic chitinase in Arabidopsis thaliana. Plant Physiology, 1990, 93: 907-914.

[13]张计育，佟兆国，高志红，罗昌国，渠慎春，章镇. SA、MeJA、ACC 和苹果轮纹病病原菌诱导湖北海棠MhWRKY1基因的表达. 中国农业科学, 2011, 44(5): 990-999.

Zhang J Y, Tong Z G, Gao Z H, Luo C G, Qu S C, Zhang Z. Expression of MhWRKY1 gene induced by the elicitors SA, MeJA, ACC and the apple ring spot pathogen. Scientia Agricultura Sinica, 2011, 44(5): 990-999. (in Chinese)

[14]张计育，渠慎春，董畅，高志红，乔玉山，章镇. 水杨酸诱导湖北海棠全长cDNA文库的构建及应用. 西北植物学报，2010, 30(8): 1527-1533.

Zhang J Y, Qu S C, Dong C, Gao Z H, Qiao Y S, Zhang Z. Utility and construction of full-length cDNA library of Malus hupehensis post-introduced with salicylic acid. Acta Botanica Boreali –Occidentalis Sinica，2010, 30(8): 1527-1533. (in Chinese)

[15]佟兆国，王富荣, 章镇, 赵剑波, 张开春, 闫国华, 周宇, 姜立杰. 一种从果树成熟叶片提取DNA的方法. 果树学报，2008, 25:122-125.

Tong Z G, Wang F R, Zhang Z, Zhao J B, Zhang K C, Yan G H, Zhou Y, Jiang L J. A method for DNA extraction from mature leaves of fruit trees. Journal of Fruit Science, 2008, 25:122-125. (in Chinese)

[16]蔡斌华，张计育，高志红，渠慎春，佟兆国，靡林，乔玉山，章镇. 一种改良的提取草莓属叶片总RNA的方法. 江苏农业学报, 2008, 24: 875-877.

Cai B H, Zhang J Y, Gao Z H, Qu S C, Tong Z G, Mi L, Qiao Y S, Zhang Z. An improved method for isolation of total RNA from the leaves of Fragaria spp. Journal of Jiangsu Agriculture Science, 2008, 24: 875-877. (in Chinese)

[17]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 2001, 25: 402-408.

[18]Lotan T, Ori N, Fluhr R. Pathogenesis-related proteins are developmentally regulated in tobacco flowers. The Plant Cell, 1989, 1: 881-887.

[19]Memelink J , Linthorst H J M , Schilperoot R A, Hoge J H C. Tobacco genes encoding acidic and basic isoforms of pathogenesis-related proteins display different expression patterns. Plant Molecular Biology, 1990, 14: 119-126.

[20]Neale A D, Wahleithner J A, Lund M, Bonnett H T, Kelly A, Meeks-Wagner D R, Peacock W J, Dennis E S. Chitinase, β-1,3-glucanase, osmotin, and extensin are expressed in tobacco explants during flower formation. The Plant Cell, 1990, 2: 673-684.

[21]Von Dahl C C, Baldwin I T. Deciphering the role of ethylene in plant herbivore interactions. Journal of Plant Growth Regulation, 2007, 26: 201-209.

[22]Vlot A C, Klessig D F, Park SW. Systemic acquired resistance: the elusive signal(s). Current Opinion in Plant Biology, 2008, 11: 436-442.

[23]Glazebrook J. Genes controlling expression of defense responses in Arabidopsis-2001 status. Current Opinion in Plant Biology, 2001, 4: 301-308.

[24]Lamb C J, Lawton M A, Dixon R A. Signal and transduction mechanisms for activation of plant defenses against microbial attack. Cell, 1989, 56: 215-224.

[25]Bonasera J M, Kim J F, Beer S V. PR genes of apple: identification and expression in response to elicitors and inoculation with Erwinia amylovora. BMC Plant Biology, 2006, 6:23 doi:10.1186/ 1471-2229-6-23.

[26]Chinnusamy V, Schumaker K, Zhu J K. Molecular genetic perspectives on cross-talk and specificity in abiotic stress signaling in plants. Journal of Experimental Botany, 2004, 55(395): 225-236.