Generation of TaTLP1 Transgenic Wheat (Triticum aestivum) and Analysis of Its Resistance to Leaf Rust Fungus
LIANG Fang, CUI Zhong-Chi, WANG Hai-Yan*, LIU Da-Qun*
College of Plant Protection, Hebei Agricultural University /Technological Innovation Center for Biological Control of Crop Diseases and Insect Pests of Hebei/National Engineering Research Center for Agriculture in Northern mountainous Areas, Baoding 071000, China
Abstract：Thaumatin-like proteins (TLPs), which is a kind of pathogenesis-related proteins possessing antifungal activity against many plant pathogens. In recent years, the antifungal function of TLPs has received more and more attention in plant pathology research. Our previous studies have reported that one wheat (Triticum aestivum) TLP gene named TaTLP1. It is related to wheat resistance to Puccinia triticina (Pt), and the expression of TaTLP1 was significantly up-regulated at 96 h after inoculation with Pt. In this study, the full sequence of the TaTLP1 gene was inserted into the expression vector pLGY-02 driven by the strong ubiquitin (Ubi) promoter, which can be highly expressed in monocotyledons. Then the recombinant vector pLGY-TaTLP1 was transformed into susceptible material 'JW' by Agrobacterium-mediated genetic transformation. Total 140 young embryos were infested, and 31 positive lines were obtained with a transformation rate of 22.1%. The transgenic TaTLP1 wheat from T1~T4 generations were verified by qRT-PCR, and the positive plants with stable genetic expression were screened out. qRT-PCR analysis confirmed the overexpression of TaTLP1 gene in transgenic positive plants. To further confirm the function of TaTLP1 gene in wheat resistance to Pt, the impact of TaTLP1 gene on transgenic wheat including 4 consecutive generations response to leaf rust pathogen infection was analyzed, the results showed that overexpression of TaTLP1 gene increased the resistance of transgenic plants to leaf rust. In addition, the determination of physiological and biochemical content of homozygous offspring further confirmed the phenotype observations, and proline, β-1,3-glucanase, hydrogen peroxide and peroxidase were also involved in wheat resistance to leaf rust. The results indicated that overexpression of TaTLP1 gene enhanced transgenic plants resistance to wheat leaf rust and played an important role in defense against wheat leaf rust, which provides a theoretical basis for further study on the function of TaTLP1 gene and the molecular mechanism between host-pathogen interaction.
梁芳, 崔钟池, 王海燕, 刘大群. 转TaTLP1基因小麦的获得及抗叶锈性分析[J]. 农业生物技术学报, 2020, 28(6): 963-973.
LIANG Fang, CUI Zhong-Chi, WANG Hai-Yan, LIU Da-Qun. Generation of TaTLP1 Transgenic Wheat (Triticum aestivum) and Analysis of Its Resistance to Leaf Rust Fungus. 农业生物技术学报, 2020, 28(6): 963-973.
[1] 李合生. 2000. 植物生理生化试验指导[M]. 北京: 高等教育出版社. 164-167. (Li H S.2000. Plant Physiological And Biochemical Test Guide[M]. Beijing: Higher Education Press. 164-167.) [2] 李皎, 杨国武, 汪大敏, 等. 2014. DNS法测定带有保护剂的β-葡聚糖酶酶活研究[J]. 陕西农业科学, 60(07): 6-7. (Li J, Yang G W, Wang D M, et al.2014. Determination of β-glucanase enzyme activity with protective agent by DNS method[J]. Shanxi Agricultural Sciences, 60(07): 6-7.) [3] 李武. 2016. 盐胁迫下陆地棉根蛋白差异表达分析及耐盐相关基因的功能鉴定[D]. 博士学位论文, 河南大学, 导师: 房卫平. pp. 93-96. (Li W.2016. Differential expression analysis of terrestrial cotton root protein and functional identification of salt-tolerant genes under salt stress[D]. Thesis for D.C., Henan University, Supervisor: Fang W P, pp. 93-96.) [4] 刘潮, 韩利红, 王海波, 等. 2018. 植物类甜蛋白基因家族研究进展[J]. 生物技术通报, 34(03): 9-17. (Liu C, Han L H, Wang H B, et al.2018. Research progress of plant thaumatin-like protein gene family[J]. Biotechnology Bulletin, 34(03): 9-17.) [5] 王鑫. 2013. 小麦与叶锈菌互作过程中H2O2的分布及其代谢相关酶基因的表达分析[D]. 硕士学位论文, 河北农业大学, 导师: 王冬梅. pp. 14-18. (Wang X.2013. Distribution of H2O2 and expression of metabolic related enzyme genes in the interaction between wheat and leaf rust[D]. Thesis for M.S., Hebei Agricultural University, Supervisor:Wang D M, pp. 14-18.) [6] 夏金婵, 苗雨晨, 宋纯鹏, 等. 2005. 紫外分光光度技术测定植物细胞中H2O2的含量[J]. 四川大学学报(自然科学版), 42: 1263-1265. (Xia J Q, Miao Y C, Song C P, et al.2005. Determination of H2O2 in plant cells by ultraviolet spectrophotometry[J]. Journal of Sichuan University(Natural Science Edition), 42: 1263-1265.) [7] Cheng M, Fry J E, Pang S, et al.1997. Genetic transformation of wheat mediated by Agrobacterium tumefaciens[J]. Plant Physiology, 115(3): 971-981. [8] Datta K, Velazhahan R, Oliva N, et al.1999. Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease[J]. Theoretical & Applied Genetics, 98(6-7): 1138-1145. [9] Fierens E, Rombouts S, Gebruers K, et al.2007. TLXI, a novel type of xylanase inhibitor from wheat (Triticum aestivum) belonging to the thaumatin family[J]. Biochemical Journal, 403(3): 583-591. [10] Cao J, Lv Y Q, Hou Z R, et al.2016. Expansion and evolution of thaumatin-like protein (TLP) gene family in six plants[J]. Plant Growth Regulation, 79: 299-307. [11] Koiwa H, Kato H, Nakatsu T, et al.1999. Crystal structure of tobacco PR-5d protein at 1. 8 Å resolution reveals a conserved acidic cleft structure in antifungal thaumatin-like proteins[J]. Journal of Molecular Biology, 286: 1137-1145. [12] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[M]. Methods, 25: 402-408. [13] Li X Y, Gao L, Zhang W H.et al.2015. Characteristic expression of wheat PR5 gene in response to infection by the leaf rust pathogen, Puccinia triticina[J]. Journal of Plant Interactions, 10(1): 132-141. [14] Mccormac A C, Elliott M C, Chen D F.et al.1997. A flexible series of binary vectors for Agrobacterium-mediated plant transformation[J]. Molecular Biotechnology, 8(3): 199. [15] Menu-Bouaouiche L, Vriet C, Peumans W J, et al.2003. A molecular basisor the endo-β 1, 3-glucanase activity of the thaumatin-like proteinsfrom edible fruits[J]. Biochimie, 85(1): 123-131. [16] Monteiro S, Barakat M, Picarra-Pereira MA, et,al.2003. Osmotin and thaumatin from grape: A putative general defense mechanism against pathogenic fungi[J]. Phytopathology, 93: 1505-1512. [17] Munis M F, Tu L, Deng F, et al.2010. A thaumatin-like protein gene involved in cotton fiber secondary cell wall development enhances resistance against Verticillium dahliae and other stresses in transgenic tobacco[J]. Biochemical and Biophysical Research Communication, 393(1): 38-44. [18] Ordoñez M E, Germán S E, Kolmer J A.2010. Genetic differentiation within the Puccinia triticina population in South America and comparison with the North American population suggests common ancestry and intercontinental migration[J]. Phytopathology, 100(4): 376-383. [19] Osmond R I W, Hrmova M, Fontaine F, et al.2001. Binding interactionsbetween barley thaumatin-like proteins and (1, 3)-β-D-glucans[J]. European Journal of Biochemistry, 268(15): 4190-4199. [20] Parkhi V, Kumar, Sunilkumar G, et al.2009. Expression of apoplastically secreted tobacco osmotin in cotton confers drought tolerance[J]. Molecular Breeding, 23: 625-639. [21] Rajam M V, Chandola N, Saiprasad Goud P, et al.2007. Thaumatin gene confers resistance to fungal pathogen as well as tolerance to abiotic stresses in transgenic tobacco plants[J]. Biologia Plantarum, 51(1): 135-141. [22] Ramos M V, de Oliveira R S B, Pereira H M, et al.2015. Crystal structure of an antifungal osmotin-like protein from Calotropis procera and its effects on Fusarium solani spores, as revealed by atomic force microscopy: Insights into the mechanism of action[J]. Phytochemistry, 119: 5-18. [23] Roelfs A P, Martell L B.1984. Uredospore dispersal from a point source within a wheat canopy[J]. Phytopathology, 74(10): 1262-1267. [24] Shi G Q, Fu J Y, Rong L J, et al.2018. TaMIR1119, a miRNA family member of wheat (Triticum aestivum), is essential in the regulation of plant drought tolerance[J]. Journal of Integrative Agriculture, 17(11): 2369-2378. [25] Singh N K, Kumar K, Kumar D.2013. Characterization of a pathogen induced thaumatin-like protein gene AdTLP from Arachis diogoi, a wild peanut[J]. PLOS ONE, 8(12): e83963. [26] van Damme E J, Charels D, Menu-Bouaouiche L, et al.2002. Biochemical, molecular and structural analysis of multiple thaumatin-like proteins from the elderberry tree (Sambucus nigra L.)[J]. Planta, 214(6): 853-862. [27] Zhang J R, Wang F, Liang F, et al.2018. Functional analysis of a pathogenesis-related thaumatin-like protein gene TaLr35PR5 from wheat induced by leaf rust fungus[J]. BMC Plant Biology, 18(1): 76.
