费尔干猪毛菜病程相关蛋白基因SfPR-1的表达规律和植物表达载体构建

摘要/Abstract

摘要： 采用RT-PCR 技术探究盐生植物费尔干猪毛菜病程相关蛋白基因SfPR-1(GenBank 登录号: JQ670917)在不同发#br#育时期、组织部位、植物激素、非生物胁迫及生物胁迫处理下的表达规律，以揭示该基因在费尔干猪毛菜生长发育和逆境胁迫下#br#的作用。结果表明，不同组织(根、茎、叶)中，SfPR-1 在根中表达量最高，预示该基因可能在根防御中发挥主要作用；SfPR-1#br#在不同发育阶段(种子、种子萌发20 d 幼苗、种子萌发30 d 幼苗、种子萌发40 d 幼苗)的表达特性显示，种子萌发30 d 幼苗时，#br#其表达差异最显著，表明其可能在植株后期生长发育中具有重要作用；SfPR-1 对不同植物激素(水杨酸SA、茉莉酸JA、脱落酸#br#ABA、乙烯合成前体ACC)均产生了响应，表明该基因在几条主要的抗病防御通路中均发挥作用。非生物胁迫(H2O2、盐、旱、冷)#br#都能够诱导SfPR-1 基因的表达，其中对盐响应程度最高。以植源性意大利青霉(Penicillium italicum)进行生物胁迫时，SfPR-1 表#br#达呈持续上升趋势。以上结果表明费尔干猪毛菜病程相关蛋白基因SfPR-1 是生物与非生物逆境胁迫下均响应的基因，推测其在植#br#物抵御逆境胁迫中发挥重要作用。

关键词: 费尔干猪毛菜, SfPR-1 基因, RT-PCR, 表达模式, 植物表达载体构建

Abstract: To investigate the potential roles of SfPR-1 at different developmental stages, tissues, phytohormones treatment, different abiotic stresses and biotic stress treatment, semi-quantitative RT-PCR method was employed to explore the expression profiles of pathogenesisrelated protein gene SfPR-1(GenBank accession number: JQ670917)from halophytes Salsola ferganica. The results displayed that SfPR-1 gene was mainly expressed in root tissue with low expression in the stem and leaves, suggesting that the gene may play a major role in the root defense. The expression characteristic of SfPR-1 at different developmental stages(seed, 20 d shoots after seed germination, 30 d shoots after seed germination, 40 d shoots after seed germination)showed the highest level was in 30 d shoots after seed germination, indicating that may play an important role in the plant late growth. The expression of SfPR-1 generated with the application of phytohormones including salicylic acid(SA), jasmonate(JA), 1-aminocyclopropane-1-carboxylate(ACC), and abscisic acid(ABA). This response can also be induced by abiotic stresses including H2O2, salt, drought, cold, and biotic stresses such as Penicillium italicum infection. The transcript levels of SfPR-1 substantially increased during biotic stress during 48 h treatment. These results suggested that SfPR-1was a key gene to the biotic and abiotic response and may play an important role in host plant defense and environmental stress.

Key words: Salsola ferganica, SfPR-1 gene, RT-PCR, Expression profiles, Construction of plant expression vectors

王艳,陈西,周莲洁,杨中敏. 费尔干猪毛菜病程相关蛋白基因SfPR-1的表达规律和植物表达载体构建[J]. 生物技术通报, 2014, 0(1): 116-124.

Wang Yan, Chen Xi, Zhou Lianjie, Yang Zhongmin. Expression Profiles of Pathogen-related Protein Gene(SfPR-1)from Salsola ferganica and Construction of Plant#br#Expression Vectors[J]. Biotechnology Bulletin, 2014, 0(1): 116-124.

参考文献

[1] Johan M. Regulation of gene expression by jasmonate hormones[J].Phytochemistry, 2009, 70: 1560-1570.
[2] Balbi V, Devoto A. Jasmonate signalling network in Arabidopsis thaliana: crucial regulatory nodes and new physiological scenarios [J]. New Phytol, 2008, 177: 301-318.
[3] Turner JG, Ellis C, Devoto A. The jasmonate signal pathway[J].Plant Cell, 2002, 14: 153-S164.
[4] Wasternack C. Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development[J]. Ann Bot, 2007, 100: 681-697.
[5] Wang KL, Li H, Ecker JR. Ethylene biosynthesis and signaling networks[J]. Plant Cell(Suppl.), 2002, 14: S131-S151.
[6] Guo H, Ecker JR. The ethylene signaling pathway: new insights[J]. Curr Opin Plant Biol, 2004, 7: 40-49.
[7] Shah J. The salicylic acid loop in plant defense[J]. Curr Opin Plant Biol, 2003, 6: 365-371.
[8] Laura MT, Flor de Dalia DF, Angela María CO, et al. Oxidative and molecular responses in Capsicum annuum L. after hydrogen peroxide,salicylic acid and chitosan foliar applications[J]. Int J Mol Sci,2013, 14: 10178-10196.
[9] Kunkel BN, Brooks DM. Cross talk between signaling pathways in pathogen defense[J]. Curr Opin Plant Biol, 2002, 5: 325-331.
[10] Pieterse CMJ, Leon-Reyes Avander ES, Van Wees SCM. Networking by small-molecule hormones in plant immunity[J]. Nat Chem Biol, 2009, 5: 308-316.
[11] Lai Y, Dang FF, Lin J, et al. Overexpression of a Chinese cabbage BrERF11 transcription factor enhances disease resistance to Ralstonia solanacearum in tobacco[J]. Plant Physiol Bioch,2013, 62: 70-78.
[12] Heath MC. Hypersensitive response-related death[J]. Plant Mol Biol, 2000, 44: 321-334.
[13] Edreva AA. Novel strategy for plant protection: induced resistance[J]. J Cell Mol Biol, 2004, 3: 61-69.
[14] Sato F, Koiwa H, Sakai Y, et al. Synthesis and secretion of tobacco neutral PR-5 protein by transgenic tobacco and yeast[J].Biochem Biophys Res Commum, 1995, 211: 909-913.
[15] Van Loon LC, Rep M and Pieterse CM. Significance of inducible defense-related proteins in infected plants[J]. Annu Rev Phytopathol, 2006, 44: 135-162.
[16] 包丽媛, 张家为. 植物病程相关蛋白1 基因家族特性的研究进展[J]. 甘肃农业科技, 2009, 1: 34-36.
[17] Hamamouch N, Li C, Seo PL, et al. Expression of Arabidopsis pathogenesis-related genes during nematode infection[J]. Mol Plant Pathol, 2011, 12(4): 355-364.
[18] Bonasera JM, Kim JF, Beer SV. PR genes of apple: identification and expression in response to elicitors and inoculation with Erwinia amylovora. BMC Plant Biol, 2006, 6: 23.
[19] 黄俊华. 中国猪毛菜属植物的地理分布特点[J]. 干旱区地理,2005, 28(3): 325-329.
[20] Guan B, Jiang GQ, Wang YX, et al. Identification of differentially expressed transcripts involved in the salt-stress response of Salsola ferganica by suppression subtractive hybridization[J]. Plant Cell Tiss Org, 2010, 103(3): 343-352.
[21] 王艳, 陈西, 杨中敏, 等. 费尔干猪毛菜病程相关蛋白基因 (SfPR-1)的克隆及在盐胁迫下的表达[J]. 植物科学学报,2013, 31(2): 164-170.
[22] Tanabe S, Hayashi N, Nishizawa Y, et al. Elicitor and catalase activity of conidia suspensions of various strains of Magnaporthe grisea in suspensions-cultured cells of rice[J]. Biosci Biotechnol Biochem, 2008, 72: 889-892.
[23] Sahebani N, Hadavi N. Induction of H2O2 and related enzymes in tomato roots infected with root knot nematode(M. javanica)by several chemical and microbial elicitors[J]. Biocontrol Sci Technol,2009, 19: 301-313.
[24] 郭金芳, 潘俊松, 王琛. 病程相关蛋白与植物抗病性关系的研究及其在草坪草抗病育种中的应用[J]. 草业学报, 2008, 17 (6): 156-163.
[25] Hwang HJ, Kim H. Gene encoding PR-10 protein of Lithospermum erythrorhizom is responsive to exogenous stimuli related to the plant defense system[J]. Plant Sci, 2003, 165: 1297-1302.
[26] Zhao SQ, Guo JB. Systemic acquired disease resistance and signal transduction in plant[J]. Aca Bot sin, 2003, 2(5): 539-548.
[27] 王勇刚. 植物诱导抗病性与病程相关蛋白[J]. 湖南农业大学学报, 2002, 28(2): 77-82
[28] Cheong JJ, Yang DD. Methyl jasmonate as vital substance in plants[J]. Trends Genet, 2003, 19(7): 409-413.
[29] 蔡昆争, 董桃杏, 徐涛. 茉莉酸类物质(JAs)的生理特性及其在逆境胁迫中的抗性作用[J]. 生态环境, 2006, 15(2): 397-404.
[30] Fones H, Preston GM. Reactive oxygen and oxidative stress tolerance in plant pathogenic Pseudomonas[J]. FEMS Microbiol Lett, 2011,327: 1-8
[31] Inzé A, Vanderauwera S, Hoeberichts FA, et al. Subcellular localization compendium of hydrogen peroxide-induced proteins[J]. Plant Cell Environ, 2012, 35: 308-320.
[32] Chen Z, Silva H. Active oxygen species and induction of plant systemic acquired resistance by SA[J]. Science, 2003, 262: 1883-1886.
[33] Glazebrook J. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens[J]. Annu Rev Phytopathol, 2005,43: 205-227.
[34] Walling LL. Adaptive defense responses to pathogens and pests Adv[J]. Bot Res, 2009, 51: 551-612.
[35] Mur LAJ, Kentom P, Atzorn R. The outcomes of concentrationspecific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death[J]. Plant Physiol, 2006, 140: 249-262.
[36] Koornneef A, Pieterse CMJ. Cross talk in defense signaling[J].Plant Physiol, 2008, 146: 839-844.
[37] Lopez MA, Bannenberg G, Casterana C. Controlling hormone signaling is a plant and pathogen challenge for growth and survival[J]. Curr Opin Plant Biol, 2008, 11: 420-427.
[38] Li ZT, Dhekney SA, Gray DJ. PR-1 gene family of grapevine: a uniquely duplicated PR-1 gene from a Vitis interspecific hybrid confers high level resistance to bacterial disease in transgenic tobacco[J]. Plant Cell Rep, 2011, 30: 1-11.
[39] Hon WC, Griffith M, Mlynarz A, et al. Antifreeze proteins in winter rye are similar to pathogenesis-related proteins[J]. Plant Physiol,1995, 109: 879-889.
[40] Broekaert WF, Terras FRG, Cammue BPA. Induced and performed antimicrobial protein[C] //Slusarenko AJ, Fraser RSS, van Loon LC. Mechanisms of resisitance to plant diseases. Dordrecht: Kluwer Academic Publishers, 2000, 371-477.
[41] Zeier J, Delledonne M, Mishina T, et al. Genetic elucidation of nitric oxide signaling in incompatible plant-pathogen interactions[J].Plant Physiol, 2004, 136, 2875-2886.
[42] Griffith M, Yaish WF. Antifreeze proteins in overwintering plants: a tale of two activities[J]. Trends Plant Sci, 2004, 9: 399-405.
[43] Liu B, Zhang SH, Zhu XY, et al. Candidate defense genes as predictors of quantitative blast resistance in rice[J]. Mol Plant- Microbe Interact, 2004, 17: 1146-1152.
[44] Seo PJ, Lee AK, Xiang FN. Molecular and functional profiling of Arabidopsis pathogenesis-related genes: insights into their roles in salt response of seed germination[J]. Plant Cell Physiol, 2008,39(3): 334-344.
[45] Ghanta S, Bhattacharyya D, Sinha R, et al. Nicotiana tabacum overexpressing γ-ECS exhibits biotic stress tolerance likely through NPR1-dependent salicylic acid-mediated pathway[J]. Planta,2011, 233: 895-910.
[46] Sharma R, Vleesschauwer DD, Manoj K, et al. Recent advances in dissecting stress regulatory crosstalk in rice[J]. Mol Plant, 2013,6(2): 250-260.