干涉丹参SmORA1对植物抗病和丹参酮类次生代谢的影响

doi:10.3864/j.issn.0578-1752.2016.03.008

Abstract

Abstract:

【Objective】ERF (ethylene responsive factor) are a class of plant-specific transcription factors, which are generally involved in plant stress responses. An ERF gene (named SmORA1) has been screened from medicinal plant Salvia miltiorrhiza, which is highly homologous to CrORCA3 of Catharanthus roseus. The objective of this study is to investigate the effects of SmORA1 on the resistance to disease and on the regulation of tanshinone biosynthesis in S. miltiorrhiza through interference of SmORA1 expression in S. miltiorrhiza by RNAi method.【Method】SmORA1 was cloned and inserted into interference vector, then the constructed vector (pk-ORAi) was transformed into S. miltiorrhiza by Agrobacterium tumefaciens-mediated method and transgenic lines of SmORA1-RNAi were obtained by antibiotic resistance screening and PCR identification. Using the spectrophotometry or enzyme linked immunosorbent assay, the resistance-related enzymes (phenylalnine ammonialyase (PAL), catalase (CAT) and superoxide dismutase (SOD)) activities and physiological indexes (glutathione (GSH), proline (Pro) and malondialdehyde (MDA)) in SmORA1-RNAi transgenic lines were detected after infection by Rhizoctonia solani. The concentrations of tanshinones including dihydrotanshinone, cryptotanshinone and tanshinone II A in SmORA1-RNAi transgenic lines were detected using HPLC method. The expression levels of genes related to resistance and involved in tanshinone biosynthesis were investigated by real-time qPCR.【Result】A total of 11 positive transgenic lines were got after antibiotics and PCR screening, among of which, three lines (RNAi-11, RNAi-18, RNAi-22) with significantly down-regulated expression levels of SmORA1 were obtained after antibiotics and real-time qPCR screening, interference efficiency achieved at 80%. Afterinfection with R. solani, the disease indexes of SmORA1-RNAi lines were significantly higher than those of wild type (WT) and empty vector (VK). The contents of MDA in SmORA1-RNAi lines were also significantly higher than those of control lines, which indicated that the anti-aging ability of transgenic lines was significantly decreased. The activities of SOD, CAT and PAL in SmORA1-RNAi lines were significantly lower than those of the control. The contents of Pro and GSH were also dramatically decreased in SmORA1-RNAi lines. Moreover, the expression levels of SmPDF1.2, SmSTH-2 and SmPR-10 in SmORA1-RNAi lines were down-regulated compared with the control lines. These results implied that SmORA1 play important roles in plant resistance. Furthermore, the expression of SmHMGR1, SmHMGR2, SmGPPS, SmGGPPS1 and SmGGPPS3,encoding key enzymes involved tanshinone biosynthesis, were down-regulated in SmORA1-RNAi lines. Meanwhile, the contents of tanshinone ⅡA and cryptotanshinone were also reduced in SmORA1-RNAi lines compared with the control lines. So it was speculated that SmORA1 regulated tanshinone biosynthesis throught the regulation of the expression of SmHMGR1, SmHMGR2, SmGPPS, SmGGPPS1 and SmGGPPS3. 【Conclusion】SmORA1 is involved in the resistance response and closely related to the regulation of tanshinone biosynthesis in S. miltiorrhiza.

Key words: Salvia miltiorrhiza, ethylene responsive factor (ERF), disease resistance, tanshinone

HUA Wen-ping, LIU Wen-chao, WANG Zhe-zhi, LI Cui-qin. Effect of RNAi of SmORA1 on Disease Resistance and Tanshinones Secondary Metabolism in Salvia miltiorrhiza[J].Scientia Agricultura Sinica, 2016, 49(3): 491-502.

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References

[1] Thirugnanasambantham K, Durairaj S, Saravanan S, Karikalan K, Muralidaran S, Islam V. Role of ethylene response transcription factor (ERF) and its regulation in response to stress encountered by plants. Plant Molecular Biology Reporter, 2015, 33(3): 347-357.

[2] Klay I, Pirrello J, Riahi L, Bernadac A, Cherif A, Bouzayen M, Bouzid S. Ethylene response factor Sl-ERF.B.3 is responsive to abiotic stresses and mediates salt and cold stress response regulation in tomato. The Scientific World Journal, 2014, 2014: Article ID 167681.

[3] Lu X, Zhang L, Zhang F Y, Jiang W M, Shen Q, Zhang L D, Lv Z Y, Wang G F, Tang K X. AaORA, a trichome-specific AP2/ERF transcription factor of Artemisia annua, is a positive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea. New Phytologist, 2013, 198(4): 1191-1202.

[4] Zhang Z J, Huang R F. Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor TERF2/LeERF2 is modulated by ethylene biosynthesis. Plant Molecular Biology, 2010, 73(3): 241-249.

[5] Zhang H W, Liu W, Wan L Y, Li F, Dai L Y, Li D J, Zhang Z J, Huang R F. Functional analyses of ethylene response factor JERF3 with the aim of improving tolerance to drought and osmotic stress in transgenic rice. Transgenic Research, 2010, 19(5): 809-818.

[6] Jin T C, Chang Q, Li W F, Yin D X, Li Z J, Wang D, Liu B, Liu L X. Stress-inducible expression of GmDREB1 conferred salt tolerance in transgenic alfalfa. Plant Cell, Tissue and Organ Culture, 2010, 100(2): 219-227.

[7] Berrocal-Lobo M, Molina A, Solano R. Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 Arabidopsis confers resistance to several necrotrophic fungi. The Plant Journal, 2002, 29(1): 23-32. in

[8] Onate-Sanchez L, Anderson J P, Young J, Singh K B. AtERF14, a member of the ERF family of transcription factors, plays a nonredundant role in plant defense. Plant Physiology, 2007, 143(1): 400-409.

[9] Qin J, Zuo K J, Zhao J Y, Ling H, Cao Y F, Qiu C X, Li F P, Sun X F, Tang K X. Overexpression of GbERF confers alteration of ethylene- responsive gene expression and enhanced resistance to Pseudomonas syringae in transgenic tobacco. Journal of Biosciences, 2006, 31(2): 255-263.

[10] Zuo K J, Qin J, Zhao J Y, Ling H, Zhang L D, Cao Y F, Tang K X. Over-expression GbERF2 transcription factor in tobacco enhances brown spots disease resistance by activating expression of downstream genes. Gene, 2007, 391(1): 80-90.

[11] van der Fits L, Memelink J. ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science, 2000, 289(5477): 295-297.

[12] Yu Z X, Li J X, Yang C Q, Hu W L, Wang L J, Chen X Y. The jasmonate-responsive AP2/ERF transcription factors AaERF1 and AaERF2 positively regulate artemisinin biosynthesis in Artemisia annua L.. Molecular Plant, 2012, 5(2): 353-365.

[13] Zander M, La Camera S, Lamotte O, Metraux J P, Gatz C. Arabidopsis thaliana class-II TGA transcription factors are essential activators of jasmonic acid/ethylene-induced defense responses. The Plant Journal, 2010, 61(2): 200-210.

[14] De Boer K, Tilleman S, Pauwels L, Vanden Bossche R, De Sutter V, Vanderhaeghen R, Hilson P, Hamill J D, Goossens A. APETALA2/ETHYLENE RESPONSE FACTOR and basic helix- loop-helix tobacco transcription factors cooperatively mediate jasmonate- elicited nicotine biosynthesis. The Plant Journal, 2011, 66(6): 1053-1065.

[15] 吴文燕, 蒋喜红, 刘春生, 黄璐琦, 申业. 丹参ERF转录因子SmERF1基因的表达分析和亚细胞定位. 中国中药杂志, 2013, 38(7): 957-961.

Wu W Y, Jiang X H, Liu C S, Huang L Q, Shen Y. Expression analysis and subcellular localization of an ERF transcription factor from Salvia miltiorrhiza. China Journal of Chinese Materia Medica, 2013, 38(7): 957-961. (in Chinese)

[16] 胥彬, 黄璐琦, 崔光红, 毛莹, 张辉. 丹参功能基因组学研究 Ⅳ——丹参乙烯应答因子 (ERF) 基因的分析. 中国中药杂志, 2009, 34(20): 2564-2566.

Xu B, Huang L Q, Cui G H, Mao Y, Zhang H. Functional genomics of Salvia militiorrhiza IV-analysis of ethylene responsive element binding protein gene. China Journal of Chinese Materia Medica, 2009, 34(20): 2564-2566. (in Chinese)

[17] 崔光红, 冯华, 李文渊, 汪婉宜, 黄璐琦. 丹参ERF基因组序列克隆与多态性分析. 药学学报, 2010, 45(9): 1188-1193.

Cui G H, Feng H, Li W Y, Wang W Y, Huang L Q. Cloning and polymorphism analysis of SmERF in Salvia miltiorrhiza. Acta Pharmaceutica Sinica, 2010, 45(9): 1188-1193. (in Chinese)

[18] Hua W P, Zhang Y, Song J, Zhao L J, Wang Z Z. De novo transcriptome sequencing in Salvia miltiorrhiza to identify genes involved in the biosynthesis of active ingredients. Genomics, 2011, 98(4): 272-279.

[19] Yan Y P, Wang Z Z. Genetic transformation of the medicinal plant Salvia miltiorrhiza by Agrobacterium tumefaciens-mediated method. Plant Cell, Tissue and Organ Culture, 2007, 88(2): 175-184.

[20] Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 2002, 3(7): research 0034.

[21] 贺忠群, 李焕秀, 汤浩茹. 立枯丝核菌侵染下AMF对黄瓜内源激素的影响. 中国农学通报, 2010, 26(17): 187-190.

He Z Q, Li H X, Tang H R. Effect of arbuscular mycorrhizal fungi on endogenous in cucumber after Rhizoctonia solani inoculation. Chinese Agricultural Science Bulletin, 2010, 26(17): 187-190. (in Chinese)

[22] 中华人民共和国国家标准GB/T 23222-2008. 烟草病虫害分级及调查方法. 北京: 中国标准出版社, 2008.

National standards of the people’s Republic of China GB/T 23222-2008.

Grade and Investigation Method of Tobacco Diseases and Insect Pests. Beijing: China Standard Press, 2008. (in Chinese)

[23] 高俊凤. 植物生理学实验指导. 北京: 高等教育出版社, 2006.

Gao J F. Experimental Guidance for Plant Physiology. Beijing: Higher Education Press, 2006. (in Chinese)

[24] 生华, 刘梅, 化文平, 王喆之. 丹参病程相关蛋白基因 (SmPR-10) 的生物信息学及表达模式分析. 植物科学学报, 2011, 29(3): 340-346.

Sheng H, Liu M, Hua W P, Wang Z Z. Bioinformatics and expression pathogenesis-related protein 10 gene (SmPR-10) from Salvia miltiorrhiza Bunge. Plant Science Journal, 2011, 29(3): 340-346. (in Chinese)

[25] 曹晓燕, 刘丛玲, 王喆之. 丹参中病程相关蛋白基因SmSTH-2的生物信息学分析. 植物生理学通讯, 2007, 43(6): 1025-1030.

Cao X Y, Liu C L, Wang Z Z. Pathogenesis-bioinformatics analysis of pathogenesis-related protein gene SmSTH-2 in Salvia miltiorrhiza Bge.. Plant Physiology Communications, 2007, 43(6): 1025-1030. (in Chinese)

[26] 化文平, 王喆之. 丹参变应原基因 (Sam a1) 的克隆及其原核表达. 植物研究, 2008, 28(5): 574-578.

Hua W P, Wang Z Z. Cloning of an allergen gene (Sam a1) from Salvia miltiorrhiza Bunge and its prokaryotic expression. Bulletin of Botanical Research, 2008, 28(5): 574-578. (in Chinese)

[27] Liu J J, Ekramoddoullah A K M. The family 10 of plant pathogenesis-related proteins: Their structure, regulation, and function in response to biotic and abiotic stresses. Physiological and Molecular Plant Pathology, 2006, 68 (1/3): 3-13.

[28] Kavi Kishor P B, Sreenivasulu N. Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue? Plant, Cell & Environment, 2014, 37(2): 300-311.

[29] Kai G Y, Liao P, Xu H, Wang J, Zhou C C, Zhou W, Qi Y P, Xiao J B, Wang Y L, Zhang L. Molecular mechanism of elicitor-induced tanshinone accumulation in Salvia miltiorrhiza hairy root cultures. Acta Physiology Plant, 2012, 34(4): 1421-1433.

[30] Kai G Y, Xu H, Zhou C C, Liao P, Xiao J B, Luo X Y, You L J, Zhang L. Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. Metabolic Engineering, 2011, 13(3): 319-327.

[31] Ma P D, Liu J Y, Osbourn A, Dong J N, Liang Z S. Regulation and metabolic engineering of tanshinone biosynthesis. RSC Advances, 2015, 5(23): 18137-18144.

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Effect of RNAi of SmORA1 on Disease Resistance and Tanshinones Secondary Metabolism in Salvia miltiorrhiza

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