Abstract Abstract Stripe rust (Puccinia striiformis f.sp tritici) is a severe fungal disease in wheat (Triticum aestivum) that results in decreased yield and quality. It is important to explore a new way of improving stripe rust resistance with introducing exogenous resistance genes in wheat breeding. In this work, the Agrobacterium-mediated transformation method was adopted to introduce eucommia ulmoides chitinase1 (EuCHIT1) gene into wheat variety 'Guizi 3', and T1 trangenic plants were obtained according to the result of β-glucuronidase (GUS) histochemical staining and PCR identification.The chitinase activities in the transgenic wheat plants and wild-type were measured, and the resistance to stripe rust, and protective enzymes activities and relative expression level of pathogenesis-related protein genes were measured. The results indicated that chitinase activity in the transgenic plants, 3 328.63 U/g FW, was 42.21% which was higher than that in the wild-type. After inoculating with race CYR32 for 7 d, the catalase (CAT), super-oxide dismutase (SOD) and peroxidase (POD) activities were 211.91, 448.37 and 81.30 U/g FW in the transgenic plants and 159.95, 294.38 and 37.87 U/g FW in the wild-type plants, respectively. This showed that the CAT, SOD and POD activities in the transgenic wheats were 32.48%, 49.76%, and 114.68%, respectively, which were higher than those in the wild-type. The results also indicated that the malondialdehyde (MDA) content in the transgenic plants was 8.69 nmol/g FW which was 29.23% lower than that in the wild-type plants (12.28 nmol/g FW). After inoculating with the fungus, the transgenic plants delayed the occurrance of stripe rust disease for 9 d compared to the wild-type. The identification of the resistance to stripe rust on wheat leaves at 14 days after inoculating showed that the transgenic plants appeared high resistance, while the wild-type was moderately susceptibel. Additionally, the lengths of flag leaves with diseases in the transgenic wheats were shorter than those in the wild-type. The relative expression levels of pathogenesis-related protein1 gene (PR-1), pathogenesis-related protein2 gene (PR-2) and pathogenesis-related protein5 gene (PR-5) in the transgenic plants were respectively 1.14, 6.61 and 3.87 folds higher than those in the wild-type before inoculation of the pathogen. After inoculation with the fungus, the expression levels of PR-1, PR-2 and PR-5 in the transgenic plants were up-regulated and were 2.14, 3.41 and 7.55 folds higher than the wild-type, respectively. The results suggested that transgenic plants increased protective enzymes activities and relative expression level of pathogenesis-related protein genes, which thereby enhanced the disease resistance of transgenic plants. This study supplies the basis for the creation of transgenic stripe rust resistant wheat materials, and it also provides a theoretical basis on further investigations about the mechanism of EuCHIT1 gene.
Adam A, Farkas T, Somlyai G, et al. Consequence of O 2·? generation during a bacterially induced hypersensitive reaction in tobacco: deterioration of membrane lipids[J]. Physiological and Molecular Plant Pathology, 1989,34(1): 13-26 Brogue K, Chet I, Holliday M, Cressman R, Biddle P, Knowlton S, Mauvais CJ, Broglie R .Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science,1991, 254:1194–1197 Bieri S, Potrykus I, Futterer J Effects of combined expression of antifungal barley seed proteins in transgenic wheat on powdery mildew infection. Mol Breed,2003 11:37–48 Bowler C, Montagu M, Inze D. Superoxide dismutase and stress tolerance[J]. Annual review of plant biology, 1992,43(1): 83-116 Collinge, D. B., Kragh, K. M., Mikkelsen, J. D., Nielsen, K. K., Rasmussen, U., and Vad, K. (1993) Plant J. 3, 31–40 Chen X (2007). Challenges and solutions for stripe rust controlin the United States. Aust J of Agric Res 58:648–655 Constabel C P, Ryan C A. A survey of wound-and methyl jasmonate-induced leaf polyphenol oxidase in crop plants[J]. Phytochemistry, 1998,47(4): 507-511 Datta K, Tu J, Oliva N, Ona I, Velazhahan R, Mew TW,Muthukrishnan S, Datta SK.Enhanced resistance to sheath blight by constitutive expression of infection related rice chitinase in transgenic elite indica rice cultivars. Plant Sci,2011,160:405–414 Gurr SJ, Rushton PJ. Engineering plants with increased disease resistance: how are we going to express it? Tibtech,2005,23:275–282 Huang X, Wang J, Du Z, et al. Enhanced resistance to stripe rust disease in transgenic wheat expressing the rice chitinase gene RC24[J]. Transgenic research, 2013,22(5): 939-947 黄芳,李文贞,陈天青,等. 贵州小麦主要品种(系)的条锈病抗性鉴定[J]. 贵州农业科学,02:81-84+88(Huang F,Li W Z,Chen T Q,et al.Identification of the Resistance to Wheat Stripe Rusr of Guizhou Major wheat Cultivars [J]Guizhou Agricultural Sciences,2014,02:81-84+88) Kovacs G, Sagi L, Jacon G, Arinaitwe G, Busogoro JP, Thiry E, Strosse H, Swennen R, Remy S. Expression of a rice chitinase gene in transgenic banana (‘Gros Michel’, AAA genome group) confers resistance to black leaf streak disease. Transgenic Res, 2013,22 (1): 117–130 Kishimoto K, Nishizawa Y, Tabei Y, Hibi T, Nakajima M,Akutsu K.Detailed analysis of rice chitinase gene expression in transgenic cucumber plants showing different levels of disease resistance to gray mold (Botrytiscinerea). Plant Sci,2002, 162:655–662 Kavroulakis N, Papadopoulou K K, Ntougias S, et al. Cytological and other aspects of pathogenesis-related gene expression in tomato plants grown on a suppressive compost[J]. Annals of botany, 2006, 98(3): 555-564. 刘太国,王保通,贾秋珍,等. 2010-2011年度我国小麦条锈菌生理专化研究[J]. 麦类作物学报,2012,03:574-578(Liu T G,Wang B T,Jia Q Z,et al.Physilogic Specialiation of Puccina striiformis f.sp.in China during 2010-2011[J]. Journal of Triticeae Crops, 2012,03:574-578) Li P, Pei Y, Sang X C, Ling Y H, Yang Z L, He G H. Transgenic indica rice expressing a bitter melon (Momordica charantia) class I chitinase gene (McCHIT1) confers enhanced resistance toMagnaporthe grisea and Rhizoctonia solani. Eur J Plant Pathol,2009, 125: 533?543 刘小烛,胡忠,李英,等.杜仲皮中抗真菌蛋白的分离和特性研究[J]. 云南植物研究,1994,04:385-391.(Liu XZ, Hu Z, Li Y, et al. Isolation and characterization of an antifungal protein from the bark of Eucommia ulmoides. Acta Bot Yunnanica,1994, 16 (4): 385–391 (in Chinese with English abstract) Liu Z, Luan Y, Li J, et al. Expression of a tomato MYB gene in transgenic tobacco increases resistance to Fusarium oxysporum and Botrytis cinerea[J]. European Journal of Plant Pathology, 2016, 144(3): 607-617 Liu SH, ZHAO DG The Effect of Eucommia Anti-fungal Peptide against Aspergillus flavus[J]. Natural ProductResearch and Development,2008,20(2): 332-334. 李振岐,商鸿生.1989.小麦锈病及其防治[M].上海:上海科技出版社,1989:1-150(Li Z Q,Shang H S.1989. Wheat rust control[M]. Shanghai:Shanghai Scientific Technical Publishers,1989:1-150) Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCT Method. Methods 25:402–408 Lamb C, Dixon R AThe oxidative burst in plant disease resistance[J].Annual review of plant biology, 1997,48(1): 251-275 Mellersh D G, Foulds I V, Higgins V J, et al. H2O2 plays different roles in determining penetration failure in three diverse plant–fungal interactions[J].2002, The Plant Journal, 29(3): 257-268 Mauch F, Hadwiger LA, Boller T (1984). Ethylene: symptom, not signal for the induction of chitinase and β-1,3-glucanase in pea pods by pathogens and elicitors. Plant Physiol, 76 (3): 607–61 Naz R, Bano A, Wilson N L, et al. Pathogenesis-Related Protein Expression in the Apoplast of Wheat Leaves Protected Against Leaf Rust Following Application of Plant Extracts[J]. Phytopathology, 2014, 104(9): 933-944 Sancho MA, de Forchetti SM, Pliego F, Valpuesta V, Quesada MA. Peroxidase activity and isoenzymes in the culture medium of NaCl adapted tomato suspension cells. Plant Cell Tiss Org,1996, 44 (2): 161–167 Schlumbaum A, Mauch F, V og eli U, et al. Plant chitinases are potent inhibitors of fungal g row th[ J]. Nature,1986,324( 27): 365–367 Shin S, Mackintosh CA, Lewis J, Heinen SJ, Radmer L, DillMacky R, Baldridge GD, Zeyen RJ, Muehlbauer GJ.Transgenic wheat expressing a barley ClassII chitinase gene has enhanced resistance against Fusarium graminearum. J Exp Bot,2008 59:2371–2378 Tayeh C, Randoux B, Vincent D, et al. Exogenous trehalose induces defenses in wheat before and during a biotic stress caused by powdery mildew[J]. Phytopathology, 2014, 104(3): 293-305 Wan AM, Chen XM, He ZH (2007). Wheat stripe rust in China.Aus J Agric Res 58:605–619 XiaoY H, Li X B, Yang X Y, Luo M, Hou L, Guo S H, Luo X Y,Pei Y. Cloning and characterization of a balsam pear class I chitinase gene (Mcchit1) and its ectopic expression enhances fungal resistance in transgenic plants. Biosci Biotechnol Biochem, 2007,71: 1211?1219 魏国荣,韩德俊,赵杰,等.小麦成株期抗条锈病种质筛选与评价[J]. 麦类作物学报,201102:376-3819（Wei G,Han D J,Zhao J,et al.Identification andEvaluation of Adult Plant Resistance to Stripe Rusr in Wheat Germplasms[J].Journal of Triticeae Crops,2011, 02:376-3819）. 王军娟,陶飞,安菲,等.温度与条锈病菌双胁迫下小麦内参基因的选择[J]. 植物病理学报,2014,05:497-503(Wang J J,Tao F,AN F,et al. Selection of reference genes in wheat stressed by temperature and Puccinia striiformis f．sp．tritici[J] Acta Phytopathologica Sinica,2014,05:497-503) 张长伟,凌英华,桑贤春,等. 转苦瓜几丁质酶基因McCHIT1水稻及其稻瘟病抗性[J].2011,作物学报, 11:1991-2000(Zhang C W, Ling Y H, Sang X C,et al.Transgenic Rice Lines Harboring McCHIT1 Gene from Balsam Pear (Momordica charantia L.) and Their Blast Resistance[J]. Acta Agronomica Sinica, 2011,11:1991-2000.) Zeng XF, Li L, Li JR, Zhao DG. Constitutive expression of McCHIT1-PAT enhances resistance to rice blast and herbicide, but does not affect grain yield in transgenic glutinous rice. Biotechnol Appl Bioc,2015, 1–9 Zhao TJ, Zhao SY, Chen HM, Zhao QZ, Hu ZM, Hou BK,Xia GM. Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling. Plant Cell Rep, 2006,25(11):1199–1204 Zhou X L, Han D J, Chen X M, et al. Characterization and molecular mapping of stripe rust resistance gene Yr61 in winter wheat cultivar Pindong 34[J]. Theoretical and applied genetics, 2014, 127(11): 2349-2358.
