抗全蚀病的转PvPGIP2-TaLTP5双价基因小麦的创制及鉴定

doi:10.3864/j.issn.0578-1752.2013.23.002

摘要/Abstract

摘要： 【目的】全蚀病是小麦的毁灭性病害。创制转PvPGIP2和TaLTP5双价基因小麦，分析外源PvPGIP2和TaLTP5在转基因小麦中的遗传与表达情况，选育抗全蚀病的转基因小麦新种质。【方法】采用基因重组技术构建了PvPGIP2和TaLTP5双价表达转基因载体pA25-PvPGIP2-TaLTP5，利用基因枪介导法将该载体转入小麦品种扬麦18，采用PCR、RT-PCR与qRT-PCR方法分析转基因小麦T0—T4植株中目的基因及其表达量，并对T3和T4逐株进行全蚀病接种与抗性鉴定。【结果】创制并选育出稳定的抗全蚀病转PvPGIP2和TaLTP5小麦株系6个。PvPGIP2和TaLTP5能够在这6个转基因小麦株系中稳定遗传，并高水平表达。对转PvPGIP2和TaLTP5小麦的全蚀病抗性鉴定结果表明，与受体扬麦18相比，转PvPGIP2和TaLTP5小麦对全蚀病抗性明显提高。【结论】创制了转PvPGIP2和TaLTP5抗全蚀病的小麦新种质，其对全蚀病具有一定的抗性。

关键词: 小麦 , PvPGIP2 , TaLTP5 , 双价基因 , 全蚀病 , 转基因

Abstract: 【Objective】 Take-all is a destructive disease of wheat production worldwide. The aim of this study is to develop and select stable PvPGIP2 and TaLTP5 transgenic wheat lines with resistance to take-all pathogen Gaeumannomyces graminis. 【Method】Gene recombination technology was used to prepare the transformation vector pA25-PvPGIP2-TaLTP5 expressing both PvPGIP2 and TaLTP5. Particle bombardment method was used to introduce both PvPGIP2 and TaLTP5 into wheat cultivar Yangmai 18. PCR, RT-PCR and qRT-PCR methods were used to detect the presence and transcript levels of PvPGIP2 and TaLTP5 in the transgenic wheat plants of T0-T4 generations. G. graminis mycelia plug inoculation and take-all severity and the disease index were used to score the resistance degrees of these transgenic wheat lines and non-transgenic wheat Yangmai 18 at young seedling-stage. 【Result】The results indicated that the introduced PvPGIP2 and TaLTP5 genes were stably inherited, and could be highly expressed in six transgenic wheat lines. The transgenic wheat plants expressing PvPGIP2 and TaLTP5 showed significantly enhanced resistance to G. graminis compared with non-transgenic wheat Yangmai 18. 【Conclusion】 These results suggested that the introduced PvPGIP2 and TaLTP5 genes can be used for improving wheat resistance to take-all.

Key words: wheat , PvPGIP2 , TaLTP5 , binary tansgene , take-all , transgenic

荣玮12, 王金凤2, 李钊2, 王爱云1, 杜丽璞2, 叶兴国2, 魏学宁2, 张增艳2. 抗全蚀病的转PvPGIP2-TaLTP5双价基因小麦的创制及鉴定[J]. 中国农业科学, 2013, 46(23): 4858-4867.

RONG Wei-12, WANG Jin-Feng-2, LI Zhao-2, WANG Ai-Yun-1, DU Li-Pu-2, YE Xing-Guo-2, WEI Xue-Ning-2, ZHANG Zeng-Yan-2. Development and Characterization of Both PvPGIP2 and TaLTP5 Transgenic Wheat Lines with Resistance to Take-all[J]. Scientia Agricultura Sinica, 2013, 46(23): 4858-4867.

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参考文献

[1]Gutteridge R J, Bateman G L, Todd A D. Variation in the effects of take-all disease on grain yield and quality of winter cereals in field experiments. Pest Management Science, 2003, 59: 215-224.

[2]祝秀亮, 李钊, 杜丽璞, 徐惠君, 杨丽华, 庄洪涛, 马翎健, 张增艳. 兼抗全蚀病和白粉病小麦新种质的创制与鉴定. 作物学报, 2012, 38(12): 2178-2184.

Zhu X L, Li Z, Du L P, Xu H J, Yang L H, Zhuang H T, Ma L J, Zhang Z Y. Development and characterization of wheat lines with resistance to take-all and powdery mildew diseases. Acta Agronomica Sinica, 2012, 38(12): 2178-2184. (in Chinese)

[3]Desiderio A, Aracri B, Leckie F, Mattei B, Salvi G, Tigelaar H, Van Roekel J S C, Baulcombe D C, Melchers L S, De Lorenzo G, Cervone F. Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris. Molecular Plant-Microbe Interactions, 1997, 10(7): 852-860.

[4]Lafitte C, Barthe J P, Montillet J L, Touze A. Glycoprotein inhibitors of Colletotrichum lindemuthianum endopolygalacturonase in near isogenic lines of Phaseolus vulgaris resistant and susceptible to anthracnose. Physiologial Plant Pathology, 1984, 25: 39-53.

[5]Bergmann C W, Ito Y, Singer D. Ploygalacturcuase-inhibing protein accumulates in Phaseouls vulgaris L., in response to wounding, elicitors and fungal infection. The Plant Journal, 1994, 5(5): 625-634.

[6]Cervone F, De Lorenzo, Darvll A. Can Phaseolus PGIP inhibit pectic enzymes from microbes and plants? Phytochemistry, 1990, 29(2): 447-449.

[7]Frediani M, Cremonini R, Salvi G, Caprari C, Desiderio A, D’Ovidio R, Cervone F, De Lorenzo G. Cytological localization of the PGIP genes in the embryo suspensor cells of Phaseolus vulgavis L.. Theoretical and Applied Genetics, 1993, 87: 369-373.

[8]D’Ovidio R, Raiola A, Capodicasa C, Devoto A, Pontiggia D, Roberti S, Galletti R, Conti E, O’Sullivan D, De Lorenzo G. Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects. Plant Physiology, 2004, 135: 2424-2435.

[9]Farina A, Rocchi V, Janni M, Benedettelli S, De Lorenzo G, Renato D’Ovidio. The bean polygalacturonase-inhibiting protein 2 (PvPGIP2) is highly conserved in common bean (Phaseolus vulgaris L.) germplasm and related species. Theoretical and Applied Genetics, 2009, 18: 1371-1379.

[10]D’Ovidio R, Roberti S, Di Giovanni M, Capodicasa C, Melaragni L M, Sella L, Tosi P, Favaron F. The characterization of the soybean polygalacturonase-inhibiting proteins (Pgip) gene family reveals that a single member is responsible for the activity detected in soybean tissues. Planta, 2006, 224: 633-645.

[11]党良, 王爱云, 徐惠君, 祝秀亮, 杜丽璞, 邵艳军, 张增艳. 抗根腐病的转GmPGIP3基因小麦扬麦18的获得与鉴定. 作物学报, 2012, 38(10): 1833-1838.

Dang L, Wang A Y, Xu H J, Zhu X L, Du L P, Shao Y J, Zhang Z Y. Development and characterization of GmPGIP3 transgenic Yangmai 18 with enhanced resistance to wheat common root rot. Acta Agronomica Sinica, 2012, 38(10):1833-1838. (in Chinese)

[12]王金凤, 李钊, 杜丽璞, 黄素萍, 徐惠君, 冯斗, 张增艳. 转PvPGIP2基因小麦的获得与纹枯病抗性鉴定. 植物遗传资源学报, 2013, 14(1): 179-183.

Wang J F, Li Z, Du L P, Huang S P, Xu H J, Feng D, Zhang Z Y. Development and characterization of PvPGIP2 transgenic wheat plants with enhanced resistance to Rhizoctonia cerelis. Journal of Plant Genetic Resources, 2013, 14(1): 179-183. (in Chinese)

[13]Van Loon L C, Van Strien E A. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiological and Molecular Plant Pathology, 1999, 55: 85-97.

[14]Kader J C. Lipid-transfer proteins in plants. Annual Review Plant Physiological Plant Molecular Biology, 1996, 47: 627-654.

[15]Maldonado A M, Doerner P, Dixon R A, Lamb C J, Cameron R K. A putative lipid transfer protein involved in systemic resistance signaling in Arabidopsis. Nature, 2002, 419: 399-403.

[16]Sujon S, Young J K, Ki D K, Byung K H, Sung H O, Jeong S S. Overexpression of lipid transfer protein (LTP) genes enhances resistance to plant pathogens and LTP functions in long-distance systemic signaling in tobacco. Plant Cell and Reports, 2009, 28: 419-427.

[17]King G J, Turner V A, Hussey C E, Wurtele E S, Lee S M. Isolation and characterization of a tomato cDNA clone which codes for a salt-induced protein. Plant Molecular Biology, 1988, 10: 401-412.

[18]White A J, Dunn M A, Brown K, Hughes M A. Comparative analysis of genomic sequence and expression of a lipid transfer protein gene family in winter barley. Journal of Experimental Botany, 1994, 45: 1885-1892.

[19]Sterk P, Booij H, Schellkens G A, Van Kammen A, De Vries S C. Cell-specific expression of the carrot EP2 lipid transfer protein gene. The Plant Cell, 1991, 3: 907-921.

[20]Kader J C. Lipid-transfer proteins: A puzzling family of plant proteins. Trends in Plant Science, 1997, 2(2): 66-70.

[21]Cammue B P, Thevissen K, Hendriks M, Hendriks M, Eggermont K, Goderis I J, Osbron R W, Gerdette F, Kader J C. A potent antimicrobial protein from onion seeds showing sequence homology to plant lipid transfer proteins. Plant Physiology, 1995, 109: 445-455.

[22]Sun J Y, Gaudet D A, Lu Z X, Frick M, Puchalski B, Laroche A. Characterization and antifungal properties of wheat nonspecific lipid transfer proteins. Molecular Plant-Microbe Interactions, 2008, 21(3): 346-360.

[23]Zhu X L, Li Z, Xu H J, Zhou M P, Du L P, Zhang Z Y. Overexpression of wheat lipid transfer protein gene TaLTP5 increases resistances to Cochliobolus sativus and Fusarium graminearum in transgenic wheat. Function Integrative Genomics, 2012, 12: 481-488.

[24]崔洪志, 郭三堆. 双价杀虫基因植物表达载体的构建及其在烟草中的表达. 农业生物技术学报, 1998, 6(1): 8-12.

Cui H Z ,Guo S D. Construction of plant expression vectors harboring insecticidal genes and expression in tobacco. Journal of Agricultural Biotechnology, 1998, 6(1): 8-12. (in Chinese)

[25]冯道荣, 许新萍, 邱国华, 李宝健. 多个抗病抗虫基因在水稻中的遗传和表达. 科学通报, 2000, 45(15): 1593-1599.

Feng D R, Xu X P, Qiu G H, Li B J. Expression and inheritance of multiple transgenes in rice plants. Chinese Science Bulletin, 2000, 45(15): 1593-1599. (in Chinese)

[26]Ye X D, Al-Balibi S, Klflti A, Zhang J, Lucca P, Beyer P, Potrykus I. Engineering the Provitamin A (β-Carotene) biosynthetic pathway into (Carotenoid-free) rice endosperm. Science, 2000, 287: 303-305.

[27]徐惠君, 庞俊兰, 叶兴国, 杜丽璞, 李连城, 辛志勇, 马有志, 陈剑平, 陈炯, 程顺和. 基因枪介导法向小麦导入黄花叶病毒复制酶基因的研究. 作物学报, 2001, 27(6): 684-689.

Xu H J, Pang J L, Ye X G, Du L P, Li L C, Xin Z Y, Ma Y Z, Chen J P, Chen J, Cheng S H. Study on the gene transferring of Nib8 into wheat for its resistance to the yellow mosaic virus by bombardment. Acta Agronomica Sinica, 2001, 27(6): 684-689. (in Chinese)

[28]Saghai-Maroof M A, Soliman K M, Jorgensen R A, Allard R W. Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proceedings of the National Academy of Sciences of the USA, 1984, 81: 8014-8019.

[29]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods, 2001, 25: 402-408.

[30]Daval S, Lebreton L, Gazengel K, Boutin M, Guillerm-Erckelboudt A, Sarniguet A. The biocontrol bacterium Pseudomonas fluorescens Pf29Arp strain affects the pathogenesis-related gene expression of the take-all Gaeumannomyces graminis var. tritici on wheat roots. Molecular Plant Pathology, 2011, 12(9): 839-854.

[31]Bithell S L, Butler R C, Harrow S, McKay A, Cromey M G. Susceptibility to take-all of cereal and gross species, and their effects on pathogen inoculums. Annals of Applied Biology, 2011, 159: 252-266.

[32]Liu X, Yang L H, Zhou X Y, Zhou M P, Lu Y, Ma H X, Zhang H Y, Ma J L, Zhang Z Y. Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease. Journal of Experimental Botany, 2013, 64(8): 2243-2253.

[33]Cao A Z, Wang X E, Chen Y P, Zou X W, Chen P D. A sequence-specfic PCR marker linked with PM21 distinguisher chromosomes 6AS, 6BS, 6DS of Triticum aestivum and 6VS of Haynaldia villosa. Plant Breed, 2006, 125: 201-205.