转PcRS基因拟南芥的抗炭疽病研究

摘要/Abstract

摘要： 为确定虎杖（Polygonum cuspidatum Sieb. et Zucc.）白藜芦醇合酶基因PcRS（Polygonum cuspidatum resveratrol synth-ase）提高植物抗病性的有效性,以转PcRS 基因拟南芥为材料,对其进行了分子鉴定和抗炭疽病（Colletotrichum higginsianum）研究。利用Southern blot和 Northern blot 验证了外源PcRS基因在转基因拟南芥基因组中的整合和表达。对经过选育得到的T₃代纯合株系进行离体的抗病性试验。转基因拟南芥甲醇提取物对炭疽病菌具有明显抑制作用。进一步采用点滴法接种生长 4 周的转基因拟南芥离体叶片,与野生型植株相比,转基因植株叶片坏死斑直径和坏死斑上的孢子数目都显著减少,表明转基因拟南芥通过抑制孢子的繁殖来限制炭疽病菌的定植,从而提高转基因植株对炭疽病的抗性。

关键词: 虎杖, 白藜芦醇合酶, 转基因拟南芥, 炭疽菌, 抗病性

Abstract: In order to reveal the effectiveness of Polygonum cuspidatum resveratrol synthase（PcRS）gene in increasing tolerance to pathogenic microorganisms in foreign species, the PcRS transgenic Arabidopsis was used as material to identify its molecular characterization and study its resistance to Colletotrichum higginsianum. Integration and expression of transgene in the genome of Arabidopsis was confirmed by Southern blot and Northern blot analyses. Independent homozygous transgenic Arabidopsis lines with genetical stability were obtained after the selection of T₃progenies and tested in vitro for disease resistance to C. higginsianum. Agar-plate bioassays were used to test the extracts of transgenic Arabidopsis for antifungal activity against C. higginsianum. Results showed that the mycelial growth of C. higginsianum was greatly inhibited. Then the detached leaves of 4-week-old Arabidopsis plants were used to inoculate. The lesion diameter and spore production significantly reduced on transgenic Arabidopsis. Overexpression of PcRS in transgenic Arabidopsis could restrict colonization of C. higginsianum by inhibition of spore production, resulting in enhanced resistance against C. higginsianum.

Key words: Polygonum cuspidatum, Resveratrol synthase, Transgenic Arabidopsis, Colletotrichum higginsianum, Disease resistance

柳忠玉, 赵树进. 转PcRS基因拟南芥的抗炭疽病研究[J]. 生物技术通报, 2014, 0(10): 107-112.

Liu Zhongyu, Zhao Shujin. Studies on the Resistance of Transgenic Arabidopsis Overexpressing PcRS to Colletotrichum higginsianum[J]. Biotechnology Bulletin, 2014, 0(10): 107-112.

参考文献

[1] M, Back JH, Tang X, et al. Resveratrol：a review of preclinical studies for human cancer protection[J]. Toxicol Appl Pharmacol, 2007, 224（3）：274-283.
[2] Anekonda TS. Resveratrol-a boon for treating Alzheimer’s disease?[J]. Brain Res Rev, 2006, 52（2）：316-326.
[3] P, Szakmary A, Jaeger W, et al. Resveratrol and its analogs：defense against cancer, coronary disease and neurodegenerative maladies or just a fad?[J]. Mutat Res, 2008, 658（1-2）：68-94.
[4] J, Yun S, Chung I, et al. Resveratrol synthase transgene expression and accumulation of resveratrol glycoside in Rehmannia glutinosa[J]. Molecular Breeding, 2005, 16（3）：219-233.
[5] S, Page JE, Schmidt J, et al. Aromatic and pyrone polyketides synthesized by a stilbene synthase from Rheum tataricum[J]. Phytochemistry, 2003, 62（3）：313-323.
[6] CK, Lam CN, Springob K, et al. Constitutive accumulation of cis-piceid in transgenic Arabidopsis overexpressing a sorghum stilbene synthase gene[J]. Plant Cell Physiology, 2006, 47（7）：1017-1021.
[7] S, Hu Y, Wang X, et al. High content of resveratrol in lettuce transformed with a stilbene synthase gene of Parthenocissus henryana[J]. Journal of Agriculture and Food Chemistry, 2006, 54（21）：8082-8085.
[8] O, Aury JM, Noel B, et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla[J]. Nature, 2007, 449（7161）：463-467.
[9] Hipskind JD, Paiva NL. Constitutive accumulation of a resveratrol glucoside in transgenic alfalfa increases resistance to Phoma medicaginis[J]. Mol Plant Microbe Interact, 2000, 13（5）：551-562.
[10] Serazetdinova L, Oldach K, Lörz H. Expression of transgenic stilbene synthases in wheat causes the accumulation of unknown stilbene derivatives with antifungal activity[J]. J Plant Physiol, 2005, 162（9）：985-1002.
[11] Zhu YJ, Agbayani R, Jackson MC, et al. Expression of the grapevine stilbene synthase gene VST1 in papaya provides increased resistance against diseases caused by Phytophthora palmivora[J]. Planta, 2004, 220（2）：241-250.
[12] Kobayashi S, Ding CK, Nakamura Y, et al. Kiwifruits（Actinidia deliciosa）transformed with a Vitis stilbene synthase gene produce piceid（resveratrol-glucoside）[J]. Plant Cell Rep, 2000, 19（9）：904-910.
[13] A, Sparvoli F, Mattivi F, et al. Expression of the stilbene synthase（StSy）gene from grapevine in transgenic white poplar results in high accumulation of the antioxidant resveratrol glucosides[J]. Transgenic Res, 2004, 13（3）：203-214.
[14] 庄楚雄, 生书晶, 等. 虎杖白藜芦醇合酶基因转化拟南芥的研究[J].华南理工大学学报, 2011, 39（5）：138-142.
[15] 张悦丽, 张博, 等. 11 种植物精油对6 种植物病原真菌的抑菌活性研究[J]. 植物科学学报, 2013, 31（2）：198-202.
[16] KD, Oh BJ, Yang JM, et al. Differential interactions of a Colletotrichum gloeosporioides isolate with green and red pepper fruits[J]. Phytoparasitica, 1999, 27（2）：1-10.
[17] SL, Warkentin TD, DeLong CM, et al. Transgene copy number can be positively or negatively associated with transgene expression[J]. Plant Mol Biol, 1993, 21（1）：17-26.
[18] W, Newton RJ, Weidner DA, et al. Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine[J]. J Exp Bot, 2007, 58（3）：545-554.
[19] L, Oldach K, Lörz H. Expression of transgenic stilbene synthases in wheat causes the accumulation of unknown stilbene derivatives with antifungal activity[J]. J Plant Physiol, 2005, 162（9）：985-1002.
[20] F, Kindl H. Coordinate- and elicitor-dependent expression of stilbene synthase and phenylalanine ammonialyase genes in Vitis cv. Optima[J]. Arch Biochem Biophys, 1991, 288（2）：552-557.
[21] W, Vornam B, Krause E, et al. Structural organization and differential expression of three stilbene synthase genes located on a 13 kb grapevine DNA fragment[J]. Plant Mol Biol, 1994, 26（2）：667-677.
[22] IM, Park MR, Chun JC, et al. Resveratrol accumulation and resveratrol synthase gene expression in response to abiotic stresses and hormones in peanut plants[J]. Plant Sci, 2003, 164（1）：103-109.
[23] Thomma BPHJ, Eggermont K, Penninckx IAMA, et al. Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens[J]. Proc Natl Acad Sci USA, 1998, 95（25）：15107-15111.
[24] Glazebrook J. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens[J]. Annu Rev Phytopathol, 2005, 43（7）：205-227.