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作物学报 ›› 2013, Vol. 39 ›› Issue (03): 431-439.doi: 10.3724/SP.J.1006.2013.00431

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦Mlo反义基因的转化及转基因植株的白粉病抗性分析

邢莉萍,钱晨,李明浩,曹爱忠,王秀娥,陈佩度*   

  1. 南京农业大学作物遗传与种质创新国家重点实验室,江苏南京 210095
  • 收稿日期:2012-08-01 修回日期:2012-11-16 出版日期:2013-03-12 网络出版日期:2013-01-04
  • 通讯作者: 陈佩度, E-mail: pdchen@njau.edu.cn
  • 基金资助:

    本研究由国家自然科学基金项目(31101206), 全国高校博士点基金项目(20100097120036), 江苏省自然科学基金项目(BK2011636)和山东农业大学作物生物学国家重点实验室开放课题项目(2012KF09)资助。

Transformation of Antisense Wheat Mlo (Ta-Mlo) Gene and Wheat Powdery Mildew Resistance Analysis of Transgenic Plants

XING Li-Ping,QIAN Chen,LI Ming-Hao,CAO Ai-Zhong,WANG Xiu-E,CHEN Pei-Du*   

  1. National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China
  • Received:2012-08-01 Revised:2012-11-16 Published:2013-03-12 Published online:2013-01-04
  • Contact: 陈佩度, E-mail: pdchen@njau.edu.cn

摘要:

采用基因枪法将小麦反义Mlo基因导入扬麦158和济麦20的幼胚愈伤组织中,在含除草剂的分化培养基上经两轮筛选,获得抗性再生植株。PCR检测、PCR-Southern杂交、基因组DNA斑点杂交和除草剂BASTA抗性分析结果证实已获得转基因扬麦158和济麦20阳性植株,荧光定量表达分析亦证明Mlo基因发生沉默。对T0T1代转基因植株的白粉病抗性鉴定表明,有6个转基因株系高抗白粉病。对T1代转基因小麦接种白粉菌后孢子发育的显微观察结果显示,Mlo反义基因的导入明显加快了乳突的形成和维持时间,有效抑制了吸器的发育,因而使转Mlo反义基因材料表现抗病性。

关键词: 基因枪转化, 小麦反义Mlo基因, 小麦白粉病, 乳突和吸器

Abstract:

The antisense wheatMlo gene, Ta-Mlo, was transformed into wheat (Tritivum aestivum L.) varieties Yangmai 158 and Jimai 20 via biolistic transformation using immature embryo calli as explants. After two rounds of bialaphos selection and regeneration, herbicide-resistant plants were obtained, which were subsequently confirmed by PCR, PCR-Southern hybridization, genomic dot hybridization, and BASTA resistance analysis. The results showed that the Ta-Mlo antisense transgenic Yangmai 158 and Jimai 20 plants were obtained. The real time fluorescence quantitative PCR analysis proved that the transcript of Ta-Mlo was knocked down in these transgenic plants. The disease resistance test showed that the six transgenic lines appeared highly resistance to powdery mildew pathogen Blumeria graminis f. sp. tritici (Bgt). The transgenic lines showed distinct acceleration of the production and stabilization of papillae, and effective suppression to further development of haustoria of Bgt. Therefore, the transgenic lines showed high resistance to Bgt.

Key words: Biolistic transformation, Antisense wheat Mlo gene, Wheat powdery mildew, Papillae and haustoria

[1]Schulze-Lefert P, Vogel J. Closing the ranks to attack by powdery mildew. Trends Plant Sci, 2002, 5: 343–348



[2]Jeffery L D, Jonathan D G J. Plant pathogens and integrated defence responses to infection. Nuture, 2001, 411: 826–833



[3]Shirasu K, Nielsen K, Pifanelli P, Oliver R, Schulze-Lefert P. Cell-autonomous complementation of mlo resistance using a biolistic transient expression system. Plant J, 1999, 17: 293–299



[4]Kim M C, Panstruga R, Elliott C, Muller J, Devoto A, Yoon H W, Park H C, Cho M J, Schulze-Lefert P. Calinodulin interacts with MLO protein to regulate defence against mildew in barley. Nature, 2002, 416: 447–451



[5]Schweizer P, Pokorny J, Schulze-Lefert P, Dudler R. Double-stranded RNA interferes with gene function at the single-cell level in cereals. Plant J, 2000, 24: 895–903



[6]Buschges R, Hollricher K, Panstruga R, Simons G, Wolter M, Frijters A, Daelen R, Lee T, Diergaarde P, Groenendijk J, Topsch S, Vos P, Salamini F, Schulze-Lefert P. The barley Mlo gene: a novel control element of plant pathogen resistance. Cell, 1997, 88: 695–705



[7]Devoto A, Piffanelli P, Nillsson I,opsch Wallin E, Panstruga R, Gunnar H, Heijne G, Schulze-Lefert P. Topology, subcellular localization, and sequence divexsity of the Mlo family in plants. J Biol Chem, l999, 274: 34993–35009



[8]Piffanelli P, Zhou F S, Casais C, Orme J, Jarosch B, Schaffrath U, Collins N C., Panstruga R, Schulze-Lefert P. The barley MLO modulator of defense and cell death is responsive to biotic and abiotic stress stimuli. Plant Physiol, 2002, 129: 1076–1085



[9]Thordal-Christensen H, Gregersen P L, and Collinge D. The barley/Blumeria (syn. Erysiphe) graminis interaction. In: Slusarenko A, Fraser R S S, van Loon L C, eds. Mechanism of Resistance to Plant Diseases. The Netherlands, London: Kluwer Academic Publishers, 2000. pp 77–100



[10]Chen L-G(陈利刚). Cell Biological Study on Bio-function of Barley Mlo Gene. MS Thesis of Zhejiang University, 2006 (in Chinese with English abstract)



[11]Han D-J(韩德俊), Li Z-Q(李振岐), Cao L(曹莉), Chen Y-F(陈耀锋). Advances in Mlo gene resistant to powdery mildew in barley. Acta Bot Bor-Occid Sin(西北植物学报), 2003, 23(3): 496–502 (in Chinese with English abstract)



[12]Wang M B, Waterhouse P M. Application of gene silencing in plants. Curr Opin Plant Biol, 2001, 5: 146–152



[13]Zhao T-J(赵同金), Liu H(刘恒), Zhao S-Y(赵双宜), Chen H-M(陈惠民), Xia G-M(夏光敏). Transgenic wheat progenies resistant to powdery mildew generated by Agrobacterium transformation of barley Mlo antisense gene. Plant PhysiolCommun (植物生理学通讯), 2010, 46(7): 731–736 (in Chinese with English abstract)



[14]Yu L(于玲), Niu J-S(牛吉山), Chen P-D(陈佩度), Ma Z-Q(马正强), Liu D-J(刘大钧). Cloning, physical mapping and expression analysis of a wheat Mlo-like gene. J Integr Plant Biol, 2005, 47: 214–222 (in Chinese with English abstract)



[15]Jiang Z-N(蒋正宁), Xing L-P(邢莉萍), Wang H-Z(王华忠), Yu L(于玲), Ni J-L(倪金龙), Chen P-D(陈佩度). Study on transferring wheat pathogenesis-related proteins 1 (TaPR-1) gene into wheat via micro-projectile bombardment. J Triticeae Crops (麦类作物学报), 2006, 26(3): 51–57 (in Chinese with English abstract)



[16]Wang H-Z(王华忠), Xing L-P(邢莉萍), Chen P-D(陈佩度). Transformation of powdery mildew resistance-related genes of wheat. Hereditas (遗传), 2007, 29(2): 243–249 (in Chinese with English abstract)



[17]Sheng B-Q(盛宝钦). Method for recording wheat seedling resistance to powdery mildew [Blumeria graminis (DC) Speer] using infection types. Plant Protecti (植物保护), 1988, (1): 49 (in Chinese)



[18]Wolter M, Hollricher K, Salamini F, Schulze-Lefert P. The mlo resistance alleles to powdery mildew infection in barley trigger a developmentally controlled defence mimic phenotype. Mol Gen Genet, 1993, 239: 122–128



[19]Uwe Z, Uwe S, Patrick S. Transcriptome analysis of mlo-mediated resistance in the epidermis of barley. Mol Plant Pathol, 2005, 6: 139–151



[20]Panstruga R. Serpentine plant MLO proteins as entry portals for powdery mildew fiuigi. Bioch. Soc Trans, 2005, 33: 389–392



[21]Bhat R, Miklis M, Schmelzer E, Schulze-Lefert P, Pans R. Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain. Proc Natl Acad Sci USA, 2005, 102: 3135–3140



[22]Schultheiss H, Dechert C, Kogel K, Huckelhoven R. Functional analysis of barley RAC/ROP G-protein family members in susceptibility to the powdery mildew fungus. Plant J, 2003, 36: 589–601



[23]Kim M, Lee S, Kim J, Chun H, Choi M, Chung W, Moon B, Kang C, Park C, Yoo J. Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein. J Biol Chem, 2002, 277: 19304–19314



[24]Shogo K, Tetsuo S, Tsuneo S. Identification of novel Mlo family members in wheat and their genetic characterization. Genes Genet Syst, 2010, 85: 167–175



[25]Shao B-F(邵伯飞). Cloning and Function Analysis of TaMlo3 Gene. MS Thesis of Zhejiang University, 2002 (in Chinese with English abstract)



[26]Xu H-M(徐红明), Liu H-Y (刘红彦), Wang J-M(王俊美), Tian B-M(田保明), Wang P-T(王鹏涛). Cloning and expression analysis of Mlo after Blumeria graminis f. sp. tritici infection in wheat. J Triticeae Crops (麦类作物学报), 2010, 30(3): 401–405 (in chinese with English abstract)



[27]Sun Y-F(孙燕飞), Li Y-S(李延生), Xia N(夏宁), Zhang G(张岗), Wang J-M(王俊美), Wang X-J(王晓杰), Wei G-R(魏国荣), Kang Z-S(康振生). Cloning and expression analysis of a gene TaMlo8 in wheat. J Northwest Agric For Univ (Nat Sci Edn) (西北农林科技大学学报?自然科学版), 2011, 39(10): 101–110 (in Chinese with English abstract)



[28]Wu Y-Y(吴莹莹). Construction of Wheat Gene RNAi Vector and Molecular Screening of Wheat Mlo Anti-sense Gene Transformation. MS Thesis of Shandong University, 2006 (in Chinese with English abstract)



[29]Eva V, Gabor G, Jozsef B. Virus-induced gene silencing of Mlo genes induces powdery mildew resistance in Triticum aestivum. Arch Virol, 2012, 157: 1345–1350



[30]Li X(李祥), Yi Z-L(易自力), Cai N(蔡能), Chen Z-Y(陈智勇). Antisense RNA and its application in plant genetic engineering. Lett Biotech (生物技术通讯), 2003,14(2): 162–164 (in Chinese with English abstract)

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