欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2015, Vol. 41 ›› Issue (10): 1490-1499.doi: 10.3724/SP.J.1006.2015.01490

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

转细胞凋亡抑制基因OpIAP和p35增强小麦对纹枯病的抗性

申芳嫡1,2,洪彦涛2,杜丽璞2,徐惠君2,马翎健1,*,张增艳2,*   

  1. 1西北农林科技大学农学院, 陕西杨凌 712100；2中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程/农业部麦类生物学与遗传育种重点实验室, 北京 100081
  • 收稿日期:2015-03-31 修回日期:2015-06-01 出版日期:2015-10-12 网络出版日期:2015-06-23
  • 基金资助:

    本研究由国家转基因新品种培育科技重大专项(2013ZX08002-001-004)资助。

Expression of Apoptosis Inhibitor Genes OpIAP and p35 Enhances Resistance to Rhizoctonia cerealis in Transgenic Wheat

SHEN Fang-Di1,2,HONG Yan-Tao2,DU Li- Pu2,XU Hui-Jun2,MA Ling-Jian1,*,ZHANG Zeng-Yan2,*   

  1. 1 College of Agronomy, Northwest A&F University, Yangling 712100, China; 2  National Key Facility for Crop Gene Resources and Genetic Improvement / Key Laboratory of Crop Genetic and Breeding of Agriculture Ministry / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2015-03-31 Revised:2015-06-01 Published:2015-10-12 Published online:2015-06-23

RichHTML

PDF

940

摘要：

OpIAP (Orgyia pseudotsugata inhibitor of apoptosis protein)是黄杉毒蛾核型多角体病毒中编码细胞凋亡抑制蛋白(inhibitor of apoptosis protein, IAP)的基因，p35基因编码苜蓿斜纹夜蛾核型多角体病毒中具细胞凋亡抑制作用的35 kDa蛋白。本研究人工合成了OpIAP和p35基因，构建了同时含有OpIAP和p35表达盒的转双价基因载体pUbi:p35-RSS1P:Myc-OpIAP，两基因分别由水稻蔗糖合酶-1启动子(sucrose synthase-1 promoter, RSS1P)和玉米泛素基因(Ubiquitin, Ubi)启动子驱动。通过基因枪介导法将该载体导入小麦品种扬麦16，获得双价转基因小麦。对T0~T2代植株，利用PCR、RT-PCR、qRT-PCR及Western blot分析，确认导入的外源OpIAP和p35基因能够在4个转双价基因小麦株系中遗传并表达。用来源不同、致病力不同的禾谷丝核菌强致病株型R0301和WK207对转双价基因小麦的T1、T2代植株分别进行纹枯病抗性鉴定，结果表明，与受体扬麦16相比，双价转基因小麦的T1、T2代植株对纹枯病的抗性明显提高，说明OpIAP和p35基因的表达可以增强转基因小麦对来源不同、致病力不同的禾谷丝核菌的抗性。

关键词: 细胞凋亡抑制基因, OpIAP, p35, 转基因小麦, 小麦纹枯病

Abstract:

OpIAP (Orgyia pseudotsugata inhibitor of apoptosis protein) gene encodes an inhibitor of apoptosis protein (IAP) which comes from Orgyia pseudotsugata multicapsid polyhedrosis virus, p35 gene isolated from an Autographa californica nucleo-polyhedron virus encodes a 35 kDa apoptosis protein inhibitor. The expression of both genes displays enhanced resistance in tobacco, maize and cotton. In this study, the full-length coding sequences of OpIAP and p35 genes were synthesized, respectively. The transformation vector pUbi:p35-RSS1P:Myc-OpIAP containing two gene expression cassettes was constructed. In the expression vector pUbi:p35-RSS1P:Myc-OpIAP, the OpIAP gene was driven by the rice sucrose synthase-1 promoter and the p35 gene was driven by the maize ubiquitin promoter. Embryo calli of Yangmai 16 were bombarded by the gold particle containing pUbi:p35-RSS1P:Myc-OpIAP vector DNA. Transgenic wheat plants in T0–T2 generations were subjected to PCR, RT-PCR, qRT-PCR and Western blot analyses. The results indicated that the introduced OpIAP and p35 genes could be inherited and expressed in four transgenic wheat lines. Rhizoctonia cerealis isolate R0301 or WK207 was used to inoculate T1 and T2 plants for sharp eyespot severity assessments. The results showed that the transgenic wheat plants expressing OpIAP and p35 displayed significantly enhanced resistance to sharp eyespot compared with non-transgenic wheat Yangmai 16. Thus, the introduced OpIAP and p35 genes could be used in improving wheat resistance to wheat sharp eyespot caused by different Rhizoctonia cerealis isolates.

Key words: Apoptosis Inhibitor Gene, OpIAP, p35, Transgenic wheat, Wheat sharp eyespot

[1]Lemańczyk G, Kwa?na H. Effects of sharp eyespot (Rhizoctonia cerealis) on yield and grain quality of winter wheat. Eur J Plant Pathol, 2013, 135: 187–200



[2]Hamada M S, Yin Y, Chen H, Ma Z H. The escalating threat of Rhizoctonia cerealis, the causal agent of sharp eyespot in wheat. Pest Manag Sci, 2011, 67: 1411–1419



[3]张会云, 陈荣振, 冯国华, 刘东涛, 王静. 中国小麦纹枯病的研究现状与展望. 麦类作物学报, 2007, 27: 1150–1153



Zhang H Y, Chen R Z, Feng G H, Liu D T, Wang J. The research status and prospect of wheat sharp eyespot. J Triticeae Crops, 2007, 27: 1150–1153 (in Chinese with English abstract)



[4]吴纪中, 颜伟, 蔡士宾, 任丽娟, 汤頲. 小麦纹枯病抗性的主基因+多基因遗传分析. 江苏农业学报, 2005, 21(1): 6–11



Wu J Z, Yan W, Cai S B, Ren L J, Tang T. Genetic analysis of sharp eyespot resistance by using major gene plus polygene mixed inheritance analysis in wheat (Triticum aestivum). Jiangsu J Agric Sci, 2005, 21(1): 6–11 (in Chinese with English abstract)



[5]张小村, 李斯深, 赵新华, 李瑞军. 15个小麦重组自交系群体抗纹枯病性的遗传分析. 麦类作物学报, 2004, 24(3): 13–16



Zhang X C, Li S S, Zhao X H, Li R J. Genetic analysis on resistance to sharp eyespot by using fifteen populations of recombinant inbred lines in wheat. J Triticeae Crops, 2004, 24(3): 13–16 (in Chinese with English abstract)



[6]Shi Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell, 2002, 9: 459–470



[7]Daniel P T. Dissecting the pathways to death. Leukemia, 2000, 14: 2035–2044



[8]Behrens T W, Mueller D L. Bcl-x and the regulation of survival in the immune system. Immunol Res, 1997, 16: 149–160



[9]Zhou Q, Snipas S, Orth K, Muzio M, Dixit V M, Salvesen G S. Target protease specificity of the viral serpin CrmA. Analysis of five caspases. J Biol Chem, 1997, 272: 7797–7800



[10]Yang Y, Fang S, Jensen J P, Weissman A M, Ashwell J D. Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli. Science, 2000, 288: 874–877



[11]Wright C W, Clem R J. Sequence Requirements for Hid binding and apoptosis regulation in the baculovirus inhibitor of apoptosis Op-IAP Hid BINDS Op-IAP IN A MANNER SIMILAR TO Smac BINDING OF XIAP. J Biol Chem, 2002, 277: 2454–2462



[12]Hershberger P A, Dickson J A, Friesen P D. Site-specific mutagenesis of the 35-kilodalton protein gene encoded by Autographa californica nuclear polyhedrosis virus: cell line-specific effects on virus replication. J Virol, 1992, 66: 5525–5533



[13]Friesen P D, Miller L K. Divergent transcription of early 35-and 94-kilodalton protein genes encoded by the Hind III K genome fragment of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol, 1987, 61: 2264–2272



[14]Fisher A J, Zoog S J, Schneider C L, Friesen P D. Crystal structure of baculovirus p35: role of a novel reactive site loop in apoptotic caspase inhibition. EMBO J, 1999, 18: 2031–2039



[15]Manji G A, Hozak R R, LaCount D J, Friesen P D. Baculovirus inhibitor of apoptosis functions at or upstream of the apoptotic suppressor p35 to prevent programmed cell death. J Virol, 1997, 71: 4509–4516



[16]Dickman M, Park Y, Oltersdorf T, Li W, Clemente T, French R. Abrogation of disease development in plants expressing animal antiapoptotic genes. Proc Natl Acad Sci USA, 2001, 98: 6957–6962



[17]LaCount D J, Hanson S F, Schneider C L, Friesen P D. Caspase inhibitor p35 and inhibitor of apoptosis OpIAP block in vivo proteolytic activation of an effector caspase at different steps. J Biol Chem, 2000, 275: 15657–15664



[18]程梦兰. 转细胞凋亡抑制基因p35和iap玉米抗纹枯病的研究. 华中农业大学硕士学位论文, 湖北武汉, 2013. pp 76–79



Cheng M L. Transformation of Maize with Anti-Apoptotic Genes p35 and iap Confers Resistance to Sheath Blight. MSThesis of Huazhong Agricultural University, Wuhan, China, 2013. pp 76–79 (in Chinese with English abstract)



[19]Tian J, Zhang X, Liang B, Li S, Wu Z, Wang Q, Leng C, Dong J, Wang T. Expression of baculovirus anti-apoptotic genes p35 and OpIAP in cotton (Gossypium hirsutum L.) enhances tolerance to Verticillium wilt. PloS One, 2010, 5: e14218



[20]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, 吴宏亚. 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: 688–693



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, Wu H Y. Study on the gene transferring of Nib8 into wheat for its resistance to the Yellow mosaic virus by bombardment. Acta Agron Sin, 2001, 27: 688–694 (in Chinese with English abstract)



[21]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. Proc Natl Acad Sci USA, 1984, 81: 8014–8019



[22]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



[23]王裕中, 吴志凤, 史建荣, 陈怀谷, 邵伯坤. 小麦纹枯病流行规律研究. 江苏农业科学, 1993, (麦类纹枯病专辑): 48–53



Wang Y Z, Wu Z F, Shi J R, Chen H G, Shao B K. epidemic law research of wheat sharp eyespot. Jiangsu Agric Sci, 1993, (special issue of wheat sharp eyesport): 48–53 (in Chinese)



[24]魏学宁, 任丽娟, 张淼, 张巧凤, 刘欣, 周淼平, 马鸿翔, 吴继中, 马翎健, 张增艳. 人工合成抗纹枯病小麦新种植的鉴定. 植物遗传资源学报, 2015, 16: 373–378



Wei X N, Ren L J, Zhang M, Zhang Q F, Liu X, Zhou M P, Ma H X, Wu J Z, Ma L J, Zhang Z Y. Identification of synthetic wheat accession with resistance to wheat sharp eyespot. J Plant Genet Resour, 2015, 16: 373–378 (in Chinese with English abstract)



[25]Paul J Y, Becker D K, Dickman M B, Harding R M, Khanna H K, Dale J L. Apoptosis-related genes confer resistance to Fusarium wilt in transgenic ‘Lady Finger’ bananas. Plant Biotechnol J, 2011, 9: 1141–1148



[26]Lincoln J E, Richael C, Overduin B, Smith K, Bostock R, Gilchrist D G. Expression of the antiapoptotic baculovirus p35 gene in tomato blocks programmed cell death and provides broad spectrum resistance to disease. Proc Natl Acad Sci USA, 2002, 99: 15217–15221



[27]Wang Z, Song J, Zhang Y, Yang B, Wang Y, Chen S. Mechanism analysis of broad-spectrum disease resistance induced by expression of anti-apoptotic p35 gene in tobacco. Chin J Biotech. 2008, 24: 1707–1713



[28]Chen L, Zhang Z, Liang H, Liu H, Du L, Xu H, Xin Z. Overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat. J Exp Bot, 2008, 59: 4195–4204



[29]Zhu X, Qi L, Liu X, Cai S, Xu H, Huang R, Li J, Wei X, Zhang Z. The wheat ethylene response factor transcription factor pathogen-induced ERF1 mediates host responses to both the necrotrophic pathogen Rhizoctonia cerealis and freezing stresses. Plant Physiol, 2014, 164: 1499–1514



[30]Hansen G. Evidence for Agrobacterium-induced apoptosis in maize cells. Mol Plant-Microbe Interactions, 2000, 13: 649–657



[31]Shi Y, Wang M B, Powell K S, Van Damme E, Hilder V A, Gatehouse A M R, Boulter D, Gatehouse J A. Use of the rice sucrose synthase-1 promoter to direct phloem specific expression of β-glucuronidase and snowdrop lectin genes in transgenic tobacco plants. J Exp Bot, 1994, 45: 623−631



[32]Zhang Z, Liu X, Wang X, Zhou M, Zhou X, Ye X, Wei X. An R2R3 MYB transcription factor in wheat, TaPIMP1, mediates host resistance to Bipolaris sorokiniana and drought stresses through regulation of defense and stress-related genes. New Phytol, 2012, 196: 1155–1170



[33]Streatfield S J, Magallanes-Lundback M E, Beifuss K K, Brooks C A, Harkey R L, Love R T, Bray J, Howard J A, Jilka J M, Hood E E. Analysis of the maize polyubiquitin-1 promoter heat shock elements and generation of promoter variants with modified expression characteristics. Transg Res, 2004, 13: 299–312
[1] 苏强,荣玮,张增艳. 小麦类受体蛋白激酶基因TaPK3A的克隆与抗纹枯病功能初步分析[J]. 作物学报, 2019, 45(8): 1158-1165.
[2] 王小婷,黄锁,徐兆师,李连城,马有志,陈明,闵东红. 豌豆终止子rbc-T在转基因小麦研究中的应用[J]. 作物学报, 2017, 43(08): 1254-1258.
[3] 王永霞,杜新华,许为钢,齐学礼,李艳,王会伟,胡琳. 导入外源玉米C4型NADP-ME基因对小麦光合效能的影响[J]. 作物学报, 2016, 42(04): 600-608.
[4] 杨坤,刘欣,杜丽璞,叶兴国,张增艳. 转AcAMP-sn基因抗全蚀病小麦新种质的创制与鉴定[J]. 作物学报, 2014, 40(01): 22-28.
[5] 刘菲,杨丽华,王爱云,马小飞,杜丽璞,刘欣,李盼松,张增艳,马翎健. 转TiERF1-RC7双价基因小麦的鉴定及其全蚀病抗性[J]. 作物学报, 2013, 39(11): 2094-2098.
[6] 杨丽华,王金凤,杜丽璞,徐惠君,魏学宁,李钊,马翎健,张增艳. 抗全蚀病、根腐病的转PgPGIP1基因小麦的获得与鉴定[J]. 作物学报, 2013, 39(09): 1576-1581.
[7] 党良,宿振起,叶兴国,徐惠君,李钊,邵艳军,张增艳. BvGLP1过表达增强了转基因小麦对根腐病的抗性[J]. 作物学报, 2013, 39(02): 368-372.
[8] 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[J]. 作物学报, 2012, 38(12): 2178-2184.
[9] 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[J]. 作物学报, 2012, 38(10): 1833-1838.
[10] 王金凤，杜丽璞，李钊，黄素萍，叶兴国，冯斗，张增艳. 抗纹枯病、根腐病的转SN1基因小麦的获得与鉴定[J]. 作物学报, 2012, 38(05): 773-779.
[11] 吴琼, 许为钢, 李艳, 齐学礼, 胡琳, 张磊, 韩琳琳. 田间条件下转玉米C4型PEPC基因小麦的光合生理特性[J]. 作物学报, 2011, 37(11): 2046-2052.
[12] 孙永伟, 聂丽娜, 马有志, 徐兆师, 夏兰琴. 小麦穗发芽抗性相关Vp1基因启动子的分离及功能验证[J]. 作物学报, 2011, 37(10): 1743-1751.
[13] 李钊, 庄洪涛, 杜丽璞, 周淼平, 蔡士宾, 徐惠君, 李斯深, 张增艳. 组织特异表达启动子RSS1P在转TiERF1基因小麦中的应用[J]. 作物学报, 2011, 37(10): 1897-1903.
[14] 路妍，张增艳，任丽娟，刘宝业，廖勇，徐惠君，杜丽璞，马鸿翔，任正隆，井金学，辛志勇. 转Rs-AFP2基因小麦的分子分析及其纹枯病抗性[J]. 作物学报, 2009, 35(4): 640-646.
[15] 王瑞霞;高庆荣;崔德才;刘正斌;乔晓琳;李洪利. 转反义PLDγ基因小麦的分子检测及农艺性状分析[J]. 作物学报, 2005, 31(10): 1354-1358.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2