转Hpa110-42小麦分子鉴定及赤霉病抗性功能评价

doi:10.3864/j.issn.0578-1752.2013.04.001

摘要/Abstract

摘要： 【目的】筛选出抗赤霉病转Hpa110-42小麦植株，为抗赤霉病小麦新品种的选育提供材料。【方法】以转Hpa110-42小麦T2植株和受体扬麦158为供试材料，通过PCR、Southern blotting、RT-PCR等分子技术进行外源基因的整合与表达检测，并采用单花滴注法鉴定转基因小麦的赤霉病抗性，同时探讨其抗性生理。【结果】经分子检测证明，外源Hpa110-42以1—3个拷贝整合到小麦基因组中并能够稳定遗传，在转录水平上也能正常表达。赤霉病抗性鉴定表明，株系T2-17、T2-15、T2-68、T2-44和T2-36的平均病小穗率极显著低于扬麦158。除T2-17外，其余株系的平均病小穗率极显著高于苏麦3号，均未达到苏麦3号的抗性水平。T2-17株系平均病小穗率显著低于T2-15、T2-68和T2-44，极显著低于T2-36、T2-11和T2-20株系。抗性生理分析显示，接种赤霉菌孢子后，所有植株的苯丙氨酸解氨酶（PAL）、几丁质酶、β-1,3-葡聚糖酶活性均上升，但转基因高抗植株比扬麦158上升更快，而感病株上升相对缓慢；尽管可溶性蛋白含量呈下降趋势，但转基因植株的高抗植株始终高于其它株。β-1,3-葡聚糖酶活性和可溶性蛋白含量与赤霉病抗性等级值呈极显著负相关。【结论】外源Hpa110-42整合到小麦基因组中，能稳定遗传并正常表达，正向参与了小麦赤霉病抗性调控，获得了抗赤霉病转Hpa110-42小麦植株。

关键词: Hpa110-42 , 转基因小麦 , 基因表达 , 赤霉病 , 抗性生理

Abstract: 【Objective】This study aims to screen Fusarium head blight (FHB) resistance plants in Hpa110-42 transgenic wheat plants and provide materials for FHB resistance breeding of wheat.【Method】T2 transgenic wheat plants were identified by PCR, Southern blot and RT-PCR, assuring whether the foreign Hpa110-42 gene was integrated and expressed in transgenic wheat plants. The resistance to FHB of transgenic wheat plants was evaluated by a dripping method to single flower, and its resistance physiology was studied.【Result】PCR, Southern blotting and RT-PCR analyses have confirmed successful integration of the foreign Hpa110-42 gene into the genome of the Yangmai 158 with 1 to 3 copies and stable inheritance and expression. Disease bioassays of transgenic plants revealed that the average percentage of diseased spikelets of transgenic lines T2-17, T2-15, T2-68, T2-44 and T2-36 were highly significantly lower than YM158. Compared with Sumai 3, the average percentage of diseased spikelets of all transgenic lines was highly significantly higher than Sumai 3 excepting line T2-17. In the transgenic lines, the average percentage of diseased spikelets of line T2-17 was significantly lower than other lines, in which T2-36, T2-11 and T2-20 have reached highly significant difference level. It is showed by physiological analysis that the activities of PAL, chitinase and β-1,3-glucanase in the high resistance transgenic plants increased faster than Yangmai 158 after inoculation. The soluble protein content in all plants had a downward trend, while its content in high resistance plants is consistently higher than others. Moreover, β-1,3-glucanase activity and soluble protein content were negatively correlated with resistance grade of FHB. 【Conclusion】 The foreign Hpa110-42 gene was stably inherited, normally expressed and positively participated in the regulation of FHB resistance, and Hpa110-42 transgenic wheat plants with resistance to FHB were obtained.

Key words: Hpa110-42 , transgenic wheat plants , gene expression , Fusarium head blight , resistance physiology

杨敏, 秦保平, 刘昌来, 蔡洪生, 王振林, 梁元存, 尹燕枰. 转Hpa110-42小麦分子鉴定及赤霉病抗性功能评价[J]. 中国农业科学, 2013, 46(4): 657-667.

YANG Min, QIN Bao-Ping, LIU Chang-Lai, CAI Hong-Sheng, WANG Zhen-Lin, LIANG Yuan-Cun, YIN Yan-Ping. The Molecular Identification of Transgenic Hpa110-42 Wheat and Resistance Evaluation on Fusarium Head Blight[J]. Scientia Agricultura Sinica, 2013, 46(4): 657-667.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2013.04.001

https://www.chinaagrisci.com/CN/Y2013/V46/I4/657

参考文献

[1]Buerstmayr H, Steiner B, Lemmens M, Ruckenbauer P. Resistance to fusarium head blight in winter wheat: Heritability and trait associations. Crop Science, 2000, 40: 1012-1018.

[2]Alfano J R, Collmer A. The typeⅢ (Hrp) secretion pathway of plant pathogenic bacteria: Trafficking harpins, Avr proteins, and death. Journal of Bacteriology, 1997, 179(18): 5655-5662.

[3]He S Y, Bauer D W, Collmer A, Beer S V. Hypersensitive response elicited by Ereinia amylovora harpin requires active plant metabolism. Molecular Plant-Microbe Interactions, 1994, 7(2): 289-292.

[4]He S Y, Huang H C, Collmer A. Pseudomonas syringae pv. syringae harpinPss: A protein that is secreted via the Hrp pathway and elicits the hypersensitive response in plants. Cell, 1993, 73: 1255-1266.

[5]Wei Z M, Laby R J, Zumoff C H, Bauer D W, He S Y, Collmer A, Beer S V. Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science, 1992, 257(5066): 85-88.

[6]Pandey A K, Ger M J, Huang H E, Yip M K, Zeng J Q, Feng T Y. Expression of the hypersensitive response-assisting protein in Arabidopsis results in harpin-dependent hypersensitive cell death in response to Erwinia carotovora. Plant Molecular Biology, 2005, 59: 771-780.

[7]马玲莉, 霍蓉, 高学文, 何丹, 邵敏, 王琦. 转harpinXooc蛋白编码基因hrf2对油菜抗菌核病的影响. 中国农业科学, 2008, 41(6): 1655-1660.

Ma L L, Huo R, Gao X W, He D, Shao M, Wang Q. Transgenic rape with hrf2 gene encoding harpinXooc resistant to Sclerotinia Sclerotinorium. Scientia Agricultura Sinica, 2008, 41(6): 1655-1660. (in Chinese)

[8]Peng J L, Bao Z L, Ren H Y, Wang J S, Dong H S. Expression of harpinXoo in transgenic tobacco induces pathogen defense in the absence of hypersensitive cell death. Phytopathology, 2004, 94: 1048-1055.

[9]Malnoy M, Venisse J S, Chevreau E. Expression of a bacterial effector, harpin N, causes increased resistance to fire blight in Pyrus communis. Tree Genetics and Genomes, 2005, 1(2): 41-49.

[10]Li P, Lu X Z, Shao M, Long J Y, Wang J S. Genetic diversity of Harpins from Xanthomonas oryzae and their activity to induce hypersensitive response and disease resistance in tobacco. Science in China: Life Sciences, 2004, 47(5): 461-469.

[11]Zou L F, Wang X P, Xiang Y, Zhang B, Li Y R, Xiao Y L, Wang J S, Walmsley A R, Chen G Y. Elucidation of the hrp clusters of Xanthomonas oryzae pv. oryzicola that control the hypersensitive response in nonhost tobacco and pathogenicity in susceptible host rice. Applied and Environmental Microbiology, 2006, 72(9): 6212-6224.

[12]Liu F Q, Liu H X, Jia Q, Wu X J, Guo X J, Zhang S J, Song F, Dong H S. The internal glycine-rich motif and cysteine suppress several effects of the HpaGXooc protein in plants. Phytopathology, 2006, 96: 1052-1059.

[13]Wu X J, Wu T Q, Long J Y, Yin Q, Zhang Y, Chen L, Liu R X, Gao T C, Dong H S. Productivity and biochemical properties of green tea in response to the intact molecule and functional fragments of HpaGXooc, a harpin protein from the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola. Journal of Biosciences, 2007, 32: 1119-1131.

[14]Chen L, Zhang S J, Zhang S S, Qu S P, Ren X Y, Long J Y, Yin Q, Qian J, Sun F, Zhang C L, Wang L X, Wu X J, Wu T Q, Zhang Z K, Cheng Z Q, Hayes M, Beer S V, Dong H S. A fragment of the Xanthomonas oryzae pv. oryzicola harpin HpaGXooc reduces disease and increases yield of rice in extensive grower plantings. Phytopathology, 2008, 98: 792-802.

[15]Chen L, Qian J, Qu S P, Long J Y, Yin Q, Zhang C L, Wu X J, Sun F, Wu T Q, Hayes M, Beer S V, Dong H S. Identification of specific fragments of HpaGXooc, a harpin protein from Xanthomonas oryzae pv. oryzicola, that induce disease resistance and enhance growth in plants. Phytopathology, 2008, 98: 781-791.

[16]Saghai-Maroof M A, Soliman K M, Jorgensen R A, Allard R W. Ribosomal DNA spacer-length polymorphism in barley: Mendelian inheritance, chromosomal location and population dynamic. Proceedings of the National Academy of Sciences of the USA, 1984, 81: 8014-8018.

[17]Ma C H, Zhai C X, Wang L A, Chen X, LI Y C, Guo X L, Cui S P, Li G M. Induced resistance by the toxin filtrate of Bipolaris maydis race T cultivation. Agricultural Sciences in China, 2006, 5(9): 678-684.

[18]史树德, 孙亚卿, 魏磊. 植物生理学实验指导, 北京: 中国林业出版社, 2011, 4: 88-96.

Shi S D, Sun Y Q, Wei L. Experimental Guide of Plant Physiology. Beijing: China Forestry Press, 2011, 4: 88-96. (in Chinese)

[19]Reboutier D, Frankart C, Briand J, Biligui B, Laroche S, Rona J P, Barny M A, Bouteau F. The HrpNea harpin from Erwinia amylovora triggers differential responses on the nonhost Arabidopsis thaliana cells and on the host apple cells. Molecular Plant-Microbe Interactions, 2007, 20(1): 94-100.

[20]Peng J L, Dong H S, Dong H P, Delaney T P, Bonaserab J M, Beer S V. Harpin-elicited hypersensitive cell death and pathogen resistance require the NDR1 and EDS1 genes. Physiological and Molecular Plant Pathology, 2003, 62: 317-326.

[21]Liu R X, Chen L, Jia Z H, Lü B B, Shi H J, Shao W L, Dong H S. Transcription factor AtMYB44 regulates induced expression of the ETHYLENE INSENSITIVE2 genein Arabidopsis responding to a harpin protein. Molecular Plant-Microbe Interactions, 2011, 24(3): 377-389.

[22]Zhang C L, Shi H J, Chen L, Wang X M, Lü B B, Zhang S P, Liang Y, Liu R X, Qian J, Sun W W, You Z Z, Dong H S. Harpin-induced expression and transgenic overexpression of the phloem protein gene AtPP2-A1 in Arabidopsis repress phloem feeding of the green peach aphid Myzus persicae. BMC Plant Biology, 2011, 11: 11.

[23]Dong H S, Delaney T P, Bauer D W, Beer S V. Harpin induce disease resistance in Arabidopsis through the systemic acquired resistance pathway mediated by salicylic acid and the NIM1 gene. The Plant Journal, 1999, 20(2): 207-215.

[24]刘香利, 金伟波, 刘缙, 赵惠贤, 郭蔼光. 高分子量麦谷蛋白亚基基因1Bx14转化小麦. 中国农业科学, 2011, 44(21): 4350-4357.

Liu X L, Jin W B, Liu J, Zhao H X, Guo A G. Transformation of wheat with high molecular weight glutenin subunit gene 1Bx14. Scientia Agricultura Sinica, 2011, 44(21): 4350-4357. (in Chinese)

[25]王利国, 李玲, 范冬雨. 墨兰炭疽菌侵染过程与几丁质酶、β-1,3-葡聚糖酶活性的变化. 园艺学报, 2005, 32(6): 1133-1135.

Wang L G, Li L, Fan D Y. Studies on infection processes of colletotrichum gloeosporioides and activities of chitinase and β-1,3-glucanase in leaves of cymbidium sinense. Acta Horticulturae Sinica, 2005, 32(6): 1133-1135. (in Chinese)

[26]程红梅, 简桂良, 倪万潮, 杨红华, 王志兴, 孙文姬, 张保龙, 王晓峰, 马存, 贾士荣. 转几丁质酶和β-1,3-葡聚糖酶基因提高棉花对枯萎病和黄萎病的抗性. 中国农业科学, 2005, 38(6): 1160-1166.

Cheng H M, Jian G L, Ni W C, Yang H H, Wang Z X, Sun W J, Zhang B L, Wang X F, Ma C, Jia S R. Increase of fusarium- and verticillium- resistance by transferring chitinase and glucanase gene into cotton. Scientia Agricultura Sinica, 2005, 38(6): 1160-1166. (in Chinese)

[27]左豫虎, 康振生, 杨传平, 芮海英, 娄树宝, 刘惕若. β-1,3-葡聚糖酶和几丁质酶活性与大豆对疫霉根腐病抗性的关系. 植物病理学报, 2009, 39(6): 600-607.

Zuo Y H, Kang Z S, Yang C P, Rui H Y, Lou S B, Liu X R. Relationship between activities of β-1,3-glacanase and chitinase and resistance to phytophthora root rot in soybean. Acta Phytopathologica Sinica, 2009, 39(6): 600-607. (in Chinese)

[28]张长伟, 凌英华, 桑贤春, 李平, 赵芳明, 杨正林, 李云峰, 方立魁, 何光华. 转苦瓜几丁质酶基因McCHIT1水稻及其稻瘟病抗性. 作物学报, 2011, 37(11): 1991-2000.

Zhang C W, Ling Y H, Sang X C, Li P, Zhao F M, Yang Z L, Li Y F, Fang L K, He G H. Transgenic rice lines harboring McCHIT1 gene from balsam pear (Momordica charantia L.) and their blast resistance. Acta Agronomica Sinica, 2011, 37(11): 1991-2000. (in Chinese)

[29]冯洁, 陈其煐. 棉株体内几种生化物质与抗枯萎病之间关系的初步研究. 植物病理学报, 1991, 21(4): 291-297.

Feng J, Chen Q H. On the biochemical substances in cotton plants related to the resistance to fusarium wilt. Acta Phytopathologica Sinica, 1991, 21(4): 291-297. (in Chinese)

[30]李淼, 檀根甲, 李瑶, 丁克坚, 产祝龙, 凌云. 猕猴桃品种酚类物质及可溶性蛋白含量与抗溃疡病的关系. 植物保护, 2009, 35(1): 37-41.

Li M, Tan G J, Li Y, Ding K J, Chan Z L, Ling Y. Relationships between the contents of phenolics, soluble proteins in plants of kiwifruit cultivars and their resistance to kiwifruit bacterial canker by Pseudomonas syringae pv. actinidiae. Plant Protection, 2009, 35(1): 37-41. (in Chinese)

[31]章元寿. 植物病理生理学. 南京: 江苏科学技术出版社, 1996: 34-38.

Zhang Y S. Physiological Plant Pathology. Nanjing: Jiangsu Science and Technology Press, 1996: 34-38. (in Chinese)