Creating New Tomato (Solanum lycopersicum) Germplasm Resistance to Gray Mold Using Eucommia ulmoides Laccase 1 (EuLAC1) Gene
YANG Jin-Yu1,3, WANG Zuo-Ri1,3, ZHAO De-Gang1,3,4, ZHAO Yi-Chen1,2,3,*
1 College of Life Sciences/Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang 550025, China; 2 College of Tea Sciences, Guizhou University, Guiyang 550025, China; 3 National-Local Joint Engineering Research Center of Karst Region Plant Resources Utilization &Breeding, Guiyang 550025, China; 4 Guizhou Key Laboratory of Agricultural Biotechnology/Plants Conservation Technology Center, Guizhou Academy of Agricultural Sciences, Guiyang 550025, China
Abstract Laccase (LAC) is a glycoprotein oxidase that influences lignin synthesis, regulates the levels of phenolic compounds in plants, and participates in plant defense mechanisms against pests and diseases. In order to obtain a new tomato (Solanum lycopersicum) germplasm with high resistance to Botrytis cinerea and in compliance with transgenic safety, in this study, based on the pGM626-Act1 vector constructed in the laboratory in the previous stage, a gene-deleter system with the fruit-specific E8 promoter driving the FLP gene and the Act1 promoter driving the Eucommia ulmoides laccase 1 (EuLAC1) gene in a plant expression vector was designed and constructed. Agrobacterium-mediated transformation technology was used to genetically transform the tomato variety 'Micro-Tom' and 5 positive tomato transgenic plants overexpressing EuLAC1 were successfully obtained. The results of resistance analysis showed that the onset time of EuLAC1 overexpressing tomato plants was significantly delayed after inoculation with B. cinerea, which was in sharp contrast with wild type and empty vector control plants. Additionally, the lesion diameter in tomato plants overexpressing the EuLAC1 gene was significantly smaller than that in the control group (P<0.05), which comprised the wild type and empty vector tomato plants. The result showed that overexpressing the EuLAC1 gene in tomato plants markedly improved their resistance to B. cinerea. Overexpression of the EuLAC1 gene also increased the activity of protective enzymes and the expression level of disease process-related protein (PR) genes in tomato plants. Deletion analysis of exogenous genes in transgenic tomato fruits revealed that no exogenous genes were detected in 22 out of 40 tomato fruits, with an exogenous gene deletion efficiency of 55%. This study provides a new technical method for the development of new disease-resistant tomato germplasm that meets transgenic safety standards.
[1] 陈炳竹. 2021. 杜仲EuDIR1过表达对番茄抗真菌病害影响研究[D]. 硕士学位论文, 贵州大学, 导师: 赵德刚, pp. 24-25. (Chen B Z.2021. Study on the resistant to fungal disease of EuDIR1 in transgenic tomato[D]. Thesis for M.S., Guizhou University, Supervisor: Zhao D G, pp. 24-25.) [2] 郭林霞, 董旋, 赵德刚. 2016. 转杜仲几丁质酶基因EuCHIT1番茄提高对灰霉病的抗性[J]. 植物生理学报, 52(5): 703-714. (Guo L X, Dong X, Zhao D G.2016. Transgenic tomato plants expressing a Eucommia ulmoides chitinase gene EuCHlT1 and their resistance to Botrytis cinerea[J]. Plant Physiology Journal, 52(5): 703-714.) [3] 韩晓雪, 韩佳轩, 姜晶. 2015. 番茄在非生物胁迫下实时定量RT-PCR中内参基因的筛选[J]. 分子植物育种, 13(4): 822-831. (Han X X, Han J X, Jiang J.2015. Screening the reference gened for the studies of quantitative real-time RT-PCR in tomato under abiotic stress[J]. Molecular Plant Breeding, 13(4): 822-831.) [4] 贾艳丽, 韩紫薇, 仇燕. 2023. 新型抗菌肽对番茄灰霉病的防治效果与机理的研究[J]. 北方园艺, 18: 18-27. (Jia Y L, Han Z W, Qiu Y.2023. Novel antimicrobial peptide on control of tomato gray mold and its mechanism[J]. Northern Horticulture, 18: 18-27.) [5] 梁思博. 2023. SlatpA基因在番茄灰霉病及细菌性叶斑病抗性中的功能研究[D]. 硕士学位论文, 东北农业大学, 导师: 王傲雪, pp. 41-51. (Liang S B.2023. Functional study of SlatpA gene in resisitance against Botrytis cinerea and Pseudomonas syringae in tomato[D]. Thesis for M.S., Northeast Agricultural University, Supervisor: Wang A X, pp. 41-51.) [6] 马晨, 周欣玥, 王全. 2018. 番茄灰霉病生物防治的研究进展[J]. 园艺与种苗, 38(2): 61-62. (Ma C, Zhou X Y, Wang Q.2018. Research progress on biological control of Botrytis cinerea[J]. Horticulture & Seed, 38(2): 61-62.) [7] 齐学礼, 陈艳艳, 王永霞. 2024. 中国作物育种先进技术的研发现状与发展建议[J/OL]. 分子植物育种, 1-11. (Qi X L, Wang Y Y, Wang Y X.2024. Current status and development recommendations of advanced crop breeding technology in china[J/OL]. Molecular Plant Breeding, 1-11. [8] 孙翠. 2020. 酵母细胞壁对梨和番茄果实采后病原真菌抗性的诱导作用及相关机理研究[D]. 博士学位论文, 浙江大学, 导师: 余挺, pp. 29-48. (Sun C.2020. Effect of wall of yeast on inhibiting postharvest pathogenic fungi in pear and tomato fruits and the possible defense mechaniams involved[D]. Thesis for Ph.D., Zhejiang University, Supervisor: Yu T, pp. 29-48.) [9] 孙妍, 陈思宇, 肖健, 等. 2021. 不同果实形状番茄品种茎部内生细菌群落结构及代谢功能特征[J]. 西南农业学报, 34(12): 2586-2595. (Sun Y, Chen S Y, Xiao J, et al.2021. Characteristics of endophytic bacterial community structure and metabolic function in stems of tomato varieties with different fruit shapes[J]. Southwest China Journal of Agricultural Sciences, 34(12): 2586-2595.) [10] 王丽, 周增强, 侯珲. 2016. 4种杀菌剂对葡萄灰霉病菌的毒力测定及复配试验[J]. 中国农学通报, 32(20): 40-43. (Wang L, Zhou Z Q, Hou H.2016. Toxicity determination and synergistic effect of four fungicides against grape Botrytis cinerea[J]. Chinese Agricultural Science Bulletin, 32(20): 40-43.) [11] 王素. 2023. 生物安全法规与监管措施应对转基因技术挑战: 全球经验与启示[J]. 分子植物育种, 21(20): 6746-6751. (Wang S.2023. Biosafety regulations and regulatory measures to cope with challenges of GM technology: Global experience and enlightenment[J]. Molecular Plant Breeding, 21(20): 6746-6751.) [12] 王欣凯, 王硕. 2020. 探究番茄灰霉病生物防治的研究进展[J]. 科技资讯, 18(13): 74-75. (Wang X K, Wang S.2020. Research progress on biological control of Botrytis cinerea[J]. Science & Technology Information, 18(13): 74-75.) [13] 王艳情. 2021. 棉花HDAC和ERF基因抗黄萎病功能的初步研究[D]. 硕士学位论文, 河北农业大学, 导师: 李志坤, pp. 12. (Wang Y Q. 2021. Preliminary study of cotton HDAC and ERF genes anti-verticillium wilt[D]. Thesis for M.S., Hebei Agricultural University, Supervisor: Li Z K, pp. 12.) [14] 徐艳, 杨金玉, 陈炳竹. 2024. 过表达杜仲EuDIR1基因对番茄灰霉病的抗性影响研究[J]. 种子, 43(02): 10-17; 29.(Xu Y,Yang J Y,Chen B Z. 2024. Research on the effect of overexpression Eucommia ulmoides EuDIR1 gene on tomato resistance to Botrytis cinerea[J]. Seed, 43(02): 10-17; 29.) [15] 杨心彪, 李兴需, 刘睿, 等. 2022. 鲜食番茄成熟过程中果实营养成分的动态变化[J]. 华中农业大学学报(自然科学版), 41(3): 191-199. (Yang X B, Li X X, Liu R, et al.2022. Dynamic changes of fruit nutrient components during ripening of fresh tomatoes[J]. Journal of Huazhong Agricultural University (Natural Science Edition), 41(3): 191-199.) [16] Barros J, Serk H, Granlund I, et al.2015. The cell biology of lignification in higher plants[J]. Annals of Botany, 115(7): 1053-1074. [17] Capriotti L, Baraldi E, Mezzetti B, et al.2020. Biotechnological approaches: Gene overexpression, gene silencing, and genome editing to control fungal and oomycete diseases in grapevine[J]. International Journal of Molecular Sciences, 21(16): 5701. [18] Cheng X, Li G, Ma C, et al.2019. Comprehensive genome-wide analysis of the pear (Pyrus bretschneideri) laccase gene (PbLAC) family and functional identification of PbLAC1 involved in lignin biosynthesis[J]. The Public Library of Science, 14(2): e0210892. [19] Chou E Y, Schuetz M, Hoffmann N, et al.2018. Distribution, mobility, and anchoring of lignin-related oxidative enzymes in Arabidopsis secondary cell walls[J]. Journal of Experimental Botany, 69: 1849-1859. [20] Hu Q, Min L, Yang X, et al.2018. Laccase GhLac1 modulates broad-spectrum biotic stress tolerance via manipulating phenylpropanoid pathway and jasmonic acid synthesis[J]. Plant Physiology, 176(2): 1808-1823. [21] Konig A.2013. A framework for designing transgenic crops: Science, safety and citizen's concerns[J]. Nature Biotechnology, 21: 1274-1279. [22] Kovacs G, Sagi L, Jacon G, et al.2013. Expression of a rice chitinase gene in transgenic banana ('Gros Michel', AAA genome group) confers resistance to black leaf streak disease[J]. Transgenic Research, 22(1): 117-130. [23] Li Y.2012. Gene deletor: A new tool to address gene flow and food safety concerns over transgenic crop plants[J]. Frontiers in Biology, 7(6): 557-565. [24] Li Y, Duan H, Smith W.2007. Gene-deletor: A new tool to address concerns over GE crops[J]. USDA Information Systems for Biotechnology News Report, http://www.isb.vt.edu/news/2007/news07.jun.htm. [25] Liu M, Zhang Z, Xu Z, et al.2021. Overexpression of SlMYB75 enhances resistance to Botrytis cinerea and prolongs fruit storage life in tomato[J]. Plant Cell Reports, 40(1): 43-58. [26] Liu Q, Luo L, Zheng L.2018. Lignins: Biosynthesis and biological functions in plants[J]. International Journal of Molecular Sciences, 19(2): 335. [27] Luo D, Sun W, Cai J, et al.2023. SlBBX20 attenuates JA signalling and regulates resistance to Botrytis cinerea by inhibiting SlMED25 in tomato[J]. Plant Biotechnology Journal, 21(4): 792-805. [28] Luo K, Duan H, Zhao D, et al.2007. 'GM-gene-deletor': Fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants[J]. Plant Biotechnology Journal, 5(2): 263-274. [29] Morrison I M.1972. A semi-micro method for the determination of lignin and its use in predicting the digestibility of forage crops[J]. Journal of the Science of Food and Agriculture, 23(6): 791. [30] Naim F, Dugdale B, Kleidon J, et al.2018. Gene editing the phytoene desaturase alleles of Cavendish banana using CRISPR/Cas9[J]. Transgenic Research, 27(5): 451-460. [31] Nishibayashi S, Hayakawa T, Nakajima T, et al.1996. CMV protecton in transgenic cucumber plants with an introduced CMV-O cp gene[J]. Theoretical and Applied Genetics, 93: 672-678. [32] Stewart C N, Halfhill J M D, Warwick S I.2003. Trans- gene introgression from genetically modified crops to their wild relatives[J]. Nature Reviews Genetics, 4(10): 806-817. [33] Tobimatsu Y, Schuetz M.2019. Lignin polymerization: How do plants manage the chemistry so well?[J]. Current Opinion in Biotechnology, 56: 75-81. [34] Vanholme R, Demedts B, Morreel K, et al.2010. Lignin biosynthesis and structure[J]. Plant Physiology, 153(3): 895-905. [35] Vats S, Bansal R, Rana N, et al.2022. Unexplored nutritive potential of tomato to combat global malnutrition[J]. Critical Reviews in Food Science and Nutrition, 62(4): 1003-1034. [36] Zhao Y, Liu Y, Dong X, et al.2022. Identification of a novel laccase gene EuLAC1 and its potential resistance against Botrytis cinerea[J]. Transgenic Research, 31(2): 215-225.
