海岛棉几丁质酶基因GbCHI的克隆与功能分析

doi:10.3724/SP.J.1005.2012.00240

遗传 ›› 2012, Vol. 34 ›› Issue (2): 240-247.doi: 10.3724/SP.J.1005.2012.00240

海岛棉几丁质酶基因GbCHI的克隆与功能分析

马银平^1,2, 王付欣², 杨淳淋², 沈法富¹, 夏桂先²

1. 山东农业大学农学院, 作物生物学国家重点实验室, 泰安271018 2．中国科学院微生物研究所, 植物基因组学国家重点实验室, 北京100101

收稿日期:2011-03-22 修回日期:2011-05-23 出版日期:2012-02-20 发布日期:2012-02-25
通讯作者: 夏桂先 E-mail:xiagx@im.ac.cn
基金资助:
国家自然科学基金项目(编号：30671116)资助

Cloning and functional analysis of chitinase gene GbCHI from sea-island cotton (Gossypium barbadense)

MA Yin-Ping^1,2, WANG Fu-Xin², YANG Chun-Lin², SHEN Fa-Fu¹, XIA Gui-Xian²

1. State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai’an 271018, China 2. State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

Received:2011-03-22 Revised:2011-05-23 Online:2012-02-20 Published:2012-02-25

摘要/Abstract

摘要： 几丁质酶是植物主要的病程相关(PR)蛋白之一。前期工作中利用比较蛋白质组学方法, 从海岛棉7124根部蛋白中分离到一个IV型几丁质酶(GbCHI)。文章通过同源克隆获得了海岛棉GbCHI基因的cDNA序列, 并对该基因的表达特征及其蛋白的抑菌功能进行了分析鉴定。qRT-PCR实验结果表明GbCHI基因在棉花根、茎、叶、花和胚珠中均有表达, 其表达受大丽轮枝菌、水杨酸(SA)、乙烯(ACC)和茉莉酸(JA)诱导; 亚细胞定位分析显示GbCHI蛋白主要分布在细胞膜上; 体外抑菌实验证明GbCHI蛋白能显著抑制大丽轮枝菌孢子的萌发和菌丝的生长。这些研究结果为了解GbCHI的功能及其在抗黄萎病棉花分子育种中的应用提供了实验依据和思路。

关键词: 海岛棉, 大丽轮枝菌, 几丁质酶, 克隆, 抑菌

Abstract: Chitinase is one of the important pathogenesis-related (PR) proteins in plants. By comparative proteomics study, a novel pathogen-responsive chitinase (known as GbCHI) has been identified from sea-island cotton (Gossypium barbadense). The GbCHI cDNA was cloned from wilt-resistant sea-island cotton and the anti-fungal activity of the gene product was investigated. qRT-PCR analysis indicated that GbCHI was expressed constitutively in root, stem, leaf, flower, and ovule of cotton plant, and the expression could be induced by Verticillium dahliae and plant hormone SA, ACC, and JA. Subcellular localization analysis using GFP-tagged proteins showed that GbCHI-GFP fusion proteins were targeted mainly to the plasma membrane. Anti-fungal assay demonstrated that GbCHI could inhibit spore germination and hyphae growth of V. dahliae significantly. These results provide important information for under-standing the cellular function of GbCHI and for exploring the application potential of this gene in molecular breeding of wilt-tolerant cotton plants.

Key words: sea-island cotton, verticillium dahliae, chitinases, cloning, anti-fungus activity

马银平，王付欣，杨淳淋，沈法富，夏桂先. 海岛棉几丁质酶基因GbCHI的克隆与功能分析[J]. 遗传, 2012, 34(2): 240-247.

MA Yin-Ping, WANG Fu-Xin, YANG Chun-Lin, SHEN Fa-Fu, XIA Gui-Xian. Cloning and functional analysis of chitinase gene GbCHI from sea-island cotton (Gossypium barbadense)[J]. HEREDITAS, 2012, 34(2): 240-247.

参考文献

[1] 谢树章, 刘亚娟, 秦平伟, 张迷. 植物几丁质酶及应用研究进展. 安徽农学通报, 2009, 15(8): 58-61.
[2] 蒋红彬, 张瀛, 蒋千里, 李树品. 几丁质酶的研究概况. 山东科学, 2000, 13(4): 41-45.
[3] Meins F, Fritig B, Linthorst HJM, Mikkelsen JD, Neuhaus JM, Ryals J. Plant chitinase genes. Plant Mol Biol Rep, 1994, 12(2): S22-S28.
[4] 张志忠, 吴菁华, 吕柳新, 林义章. 植物几丁质酶及其应用研究进展. 福建农林大学学报: 自然科学版, 2005, 34(4): 494-499.
[5] Schlumbaum A, Mauch F, Vögeli U, Boller T. Plant chitinases are potent inhibitors of fungal growth. Nature, 1986, 324(6095): 365-367.
[6] Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K. Plant chitinases. Plant J, 1993, 3(1): 31-40.
[7] Brogile K, Chet I, Holliday M, Cressman R, Biddle P, Knowhon S, Mauvais CJ, Broglie R. Transgenic Plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science, 1991, 254(5035): 1194-l197.
[8] 孟亮, 李红双, 金德敏, 崔德才, 王斌. 转几丁质酶基因黑杨的获得. 生物技术通报, 2004, (3): 48-51.
[9] Grison R, Grezes-Besset B, Schneider M, Lucante N, Luellen O, Leguay JJ, Toppan A. Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimeric chitinase gene. Nat Biotechnol, 1996, 14(5): 643-646.
[10] 南相日. 菜豆几丁质酶基因转化马铃薯及后代表达. 中国农学通报, 2006, 22(2): 75-77.
[11] 王果萍, 王景雪, 孙毅, 崔贵梅, 孟玉平, 乔燕祥. 几丁质酶基因导入西瓜植株及其抗病性鉴定研究. 植物遗传资源学报, 2003, 4(2): 104-109.
[12] Nishizawa Y, Nishio Z, Nakazono K, Soma M, Nakajima E, Ugaki M, Hibi T. Enhanced resistance to blast (Mag-naporthe grisea) in transgenic japonica rice by constitutive expression of rice chitinase. Theor Appl Genet, 1999, 99(3-4): 383-390.
[13] Xiao YH, Li XB, Yang XY, Luo M, Hou L, Guo SH, Luo XY, Pei Y. Cloning and characterization of a balsam pear class I chitinase gene (Mcchit1) and its ectopic expression enhances fungal resistance in transgenic plants. Biosci Biotech Bioch, 2007, 71(5): 1211-1219.
[14] 吴家和, 张献龙, 罗晓丽, 聂以春, 田颖川, 陈正华. 转几丁质酶和葡聚糖酶基因棉花的获得及其对黄萎病的抗性. 遗传学报, 2004, 31(2): 183-188.
[15] Punja ZK, Raharjo SHT. Response of transgenic cucumber and carrot plants expressing different chitinase enzymes to inoculation with fungal pathogens. Plant Dis, 1996, 80(9): 999-1005.
[16] 殷锡圣, 刘润进. 棉花黄萎病研究进展. 中国棉花, 1996, 23(5): 2-6.
[17] Hudspeth RL, Hobbs SL, Anderson DM, Grula JW. Characterization and expression of chitinase and 1, 3-β-glucanase genes in cotton. Plant Mol Biol, 1996, 31(4): 911-916.
[18] 李骥, 刘进元. 一个新型的棉花几丁质酶基因. 植物学报, 2003, 45(12): 1489-1496.
[19] 杨郁文, 张保龙, 倪万潮, 沈新莲, 张香桂, 徐英俊. 两个棉花几丁质酶基因的克隆与表达分析. 棉花学报, 2008, 20(2): 88-93.
[20] Rasmussen U, Bojsen K, Collinge DB. Cloning and characterization of a pathogen-induced chitinase in Bras-sica napus. Plant Mol Biol, 1992, 20(2): 277-287.
[21] Li YZ, Zheng XH, Tang HL, Zhu JW, Yang JM. Increase of β-1, 3-glucanase and chitinase activities in cotton callus cells treated by salicylic acid and toxin of verticillium dahliae. Acta Bot Sin, 2003, 45(7): 802-808.
[22] Koga D. Application of chitinase in agriculture. J Met Mater Miner, 2005, 15(1): 33-36.
[23] Neuhaus JM, Sticher L, Meins F Jr, Boller T. A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole. Proc Natl Acad Sci USA, 1991, 88(22): 10362-10366.
[24] Mikkelsen JD, Berglund L, Nielsen KK, Christiansen H, Bojsen K. Structure of endochitinase genes from sugar beets. In: Brine CJ, Sandford PA, Zikakis JP, eds. Advances in Chitin and Chitosan. Amsterdam: Elsevier, 1992: 344-353.
[25] Nishimura MT, Stein M, Hou BH, Vogel JP, Edwards H, Somerville SC. Loss of a callose synthase results in sali-cylic acid-dependent disease resistance. Science, 2003, 301(5635): 969-972.
[26] Penninckx IAMA, Thomma BPHJ, Buchala A, Métraux JP, Broekaert WF. Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell, 1998, 10(12): 2103-2113.
[27] Maleck K, Levine A, Eulgem T, Morgan A, Schmid J, Lawton KA, Dangl JL, Dietrich RA. The transcriptome of Arabidopsis thaliana during systemic acquired resistance. Nat Genet, 2000, 26(4): 403-410.
[28] Reymond P, Weber H, Damond M, Farmer EE. Differen-tial gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell, 2000, 12(5): 707-720.
[29] Vannini A, Caruso C, Leonardi L, Rugini E, Chiarot E, Buoncore V. Antifungal properties of chitinases from Castanea sativa against hypovirulent and virulent strains of the chestnut blight fungus Cryphonectria parasitica. Physiol Mol Plant P, 1999, 55(1): 29-35.
[30] Gomes VM, Oliveira AEA, Xavier FJ. A chitinase and a beta-1, 3-glucanase isolated from the seeds of cowpea (Vigna unguiculata L. Walp) inhibit the growth of fungi and insect pests of the seed. J Sci Food Agr, 1996, 72(1): 86-90.
[31] Lawrence CB, Joosten MHAJ, Tuzun S. Differential in-duction of pathogenesis-related proteins in tomato by Alternaria solani and the association of a basic chitinase isozyme with resistance. Physiol Mol Plant P, 1996, 48(6): 361-377.

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海岛棉几丁质酶基因GbCHI的克隆与功能分析

Cloning and functional analysis of chitinase gene GbCHI from sea-island cotton (Gossypium barbadense)

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