|
|
|
| Construction of the Transient Overexpression System in Eggplant (Solanum melongena) Leaves |
| WANG Peng-Fei1,2, WANG Wu-Hong2, HU Hai-Jiao2, HU Tian-Hua2, WEI Qing-Zhen2, WANG Jing-Lei2, BAO Chong-Lai2, YAN Ya-Qin2,* |
1 School of Life Sciences, Zhejiang Normal University, Jinhua 321000, China;
2 Vegetable Research Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou 310000, China |
|
|
|
|
Abstract Plant transient expression technology serves as a key tool for rapid gene function identification and is widely applied in the optimization of plant genetic transformation systems and functional gene analysis. In this study, using eggplant (Solanum melongena) germplasm 'HQ1315' as the material, overexpression vector pCNG-Smechr0901901 carrying GFP reporter gene was constructed. Transient transformation efficiency was assessed through quantitative analysis of fluorescence intensity (using ImageJ software) combined with qRT-PCR technology. Key parameters, including Agrobacterium rhizogenes strains, inoculation methods, bacterial concentration (OD600), seedling age, vacuum pressure, treatment time, and acetosyringone (AS) concentration, were systematically optimized. The results demonstrated that the gene delivery efficiency of A. tumefaciens strain C58C1 in eggplant leaves was significantly higher than that of strains GV3101 and LBA4404 (P<0.05). Employing C58C1 as the mediating strain, with an AS concentration of 200 μmol/L and a bacterial suspension OD600 of 1.0, optimal transient overexpression efficiency was achieved by vacuum infiltration treatment of true-leaf stage eggplant leaves for 3 min under a vacuum pressure of -0.08 MPa. Furthermore, the eggplant leaf transient overexpression system established in this study achieved efficient and stable expression of the target gene in 4 representative eggplant varieties, namely Lvqie, Xianqie, Sanyueqie and Yuanqie. This study successfully established a highly efficient transient overexpression system for eggplant leaves, providing crucial technical support for subsequent functional analysis of disease resistance genes in eggplant.
|
|
Received: 26 June 2025
|
|
|
|
Corresponding Authors:
*zkyyanyaqin@163.com.cn
|
|
|
|
[1] 陈思涵, 钱靖, 彭杰军, 等. 2018. 农杆菌介导的外源基因在本氏烟中瞬时表达体系优化研究[J]. 西南林业大学学报, 38(04): 6-13.
(Chen S H, Qian J, Peng J J, et al.2018. Optimization of Agrobacterium-mediated transient expression system of foreign genes in Nicotiana benthamiana[J]. Journal of Southwest Forestry University, 2018, 38(04): 6-13.)
[2] 刘洁, 张继宁, 阎世江. 2023. 茄子风味品质研究进展[J]. 中国瓜菜, 36(07): 8-11.
(Liu J, Zhang J N, Yan S J.2023. Research progress on flavor quality of eggplant[J]. China Vegetables, 36(07): 8-11.)
[3] 刘杨, 张永安, 陈火英, 等. 2021. 一种茄子原生质体的制备及瞬时转化方法[P]. 中国, CN202011458015.4.
(Liu Y, Zhang Y A, Chen H Y, et al.2021. A method for preparing eggplant protoplasts and transient transformation[P]. China, CN202011458015.4.)
[4] 廖晶晶, 牛聪聪, 解群杰, 等. 2017. 基因瞬时表达技术在园艺植物上的应用研究进展[J]. 园艺学报, 44(9): 1796-1810.
(Liao J J, Niu C C, Xie Q J, et al.2017. Research progress on application of gene transient expression technology in horticultural plants[J]. Acta Horticulturae Sinica, 44(9): 1796-1810.)
[5] 李芳, 戴素明, 李大志, 等. 2012. 一种通过农杆菌浸润柑橘瞬时表达体系的研究[C]. 中国园艺学会热带南亚热带果树分会第四届学术研讨会论文集. 中国园艺学会, 54-60.
(Li F, Dai S M, Li D Z, et al.2012. A study on transient expression system of citrus through Agrobacterium infiltration[C], Proceedings of the 4th Academic Seminar of China Horticultural Society on Tropical and Subtropical Fruits. Chinese Society for Horticultural Science, 54-60.)
[6] 宋奕珩, 龚一富, 袁佳骜, 等. 2025. 根癌农杆菌介导的金线莲瞬时表达体系的优化[J/OL]. 分子植物育种, https://link.cnki.net/urlid/46.1068.S.20241120.1450.016.
(2025-01-10). (Song Y H, Gong Y F, Yuan J A, et al.2025. Optimization of Agrobacterium-mediated expression transient system in Anoectochilus roxburghii[J/OL]. Molecular Plant Breeding, https://link.cnki.net/urlid/46.1068.S.20241120.1450.016.)
[7] 郎遥玲, 王倩, 陈彬, 等. 2023. 农杆菌介导注射法建立番茄子叶瞬时表达系统[J]. 中国组织工程研究, 27(34): 5462.
(Lang Y L, Wang Q, Chen B, et al.2023. Establishment of tomato cotyledon transient expression system by Agrobacterium-mediated injection method[J]. Research on Organizational Engineering in China, 27(34): 5462.)
[8] 桑庆亮, 赖钟雄, 林玉玲, 等. 2014. 荔枝基因枪转化及其GUS瞬时表达研究[J]. 热带作物学报, 35(11): 2223-2229.
(Sang Q L, Lai Z X, Lin Y L, et al.2014. Litchi gene transformation with gene gun and GUS transient expression[J]. Journal of Tropical Crops, 35(11): 2223-2229.)
[9] 熊璐. 2017. 大豆原生质体瞬时表达体系的建立及大豆隐花色素的亚细胞定位[D]. 硕士学位论文, 吉林大学,导师: 左泽乘, pp. 30-35.
(Xiong L.2017. Establishment of soybean protoplast transient expression system and subcellular localization of soybean cryptochrome[D]. Thesis for M.S., Jilin University, Supervisor: Zuo Z C, pp. 30-35.)
[10] 张倩. 2009. 柑橘原生质体瞬时表达体系的建立及默科特橘橙转GFP种质的创造[D]. 硕士学位论文, 华中农业大学, 导师: 郭文武. pp. 14-19.
(Zhang Q.2009. Establishment of citrus protoplast transient expression system and creation of GFP-transformed Mentone orange germplasm[D]. Thesis for M.S., Huazhong Agricultural University, Supervisor: Guo W W, pp. 14-19.)
[11] 田壮, 房晨曦, 刘雅婷, 等. 2025. 梅花瓣瞬时转化体系构建[J]. 园艺学报, 52(04): 908-920.
(Tian Z, Fang C X, Liu Y T, et al.2025. Construction of transient transformation system for plum petals[J]. Acta Horticulturae Sinica, 52(04): 908-920.)
[12] 王悦, 吴艳菊, 孟大伟, 等. 2020. 植物瞬时表达体系真空侵染法的优化[J]. 分子植物育种, 18(20): 6743-6748.
(Wang Y, Wu Y J, Meng D W, et al.2020. Optimization of vacuum infiltration method for plant transient expression system[J]. Molecular Plant Breeding, 18(20): 6743-6748.)
[13] Alam I, Salimullah M.2021. Genetic engineering of eggplant (Solanum melongena L.): Progress, controversy and potential[J]. Horticulturae, 7(4): 78.
[14] Ahn Y K, Yoon M K, Jeon J S.2013. Development of an efficient Agrobacterium mediated transformation system and production of herbicide-resistant transgenic plants in garlic (Allium sativum L.)[J]. Molecules and Cells, 36(2): 158-162.
[15] Huang W, Zhang Y, Cao N, et al.2024. Trans-complementation of the viral movement protein mediates efficient expression of large target genes via a tobacco mosaic virus vector[J]. Plant Biotechnology Journal, 22(11): 2957-2970.
[16] Jelly N S, Valat L, Walter B, et al.2014. Transient expression assays in grapevine: A step towards genetic improvement[J]. Plant Biotechnol Journal, 12(9): 1231-1245.
[17] Khatun M, Borphukan B, Alam I, et al.2022. An improved Agrobacterium mediated transformation and regeneration protocol for successful genetic engineering and genome editing in eggplant[J]. Scientia Horticulturae, 293: 110716.
[18] Kobayashi M, Fuhrmann-Aoyagi M B, Uto A, et al.2024. Tomato mosaic virus movement protein enhances the transient expression of recombinant protein in the stem of the tomato plant Solanum lycopersicum[J]. Journal of Plant Biochemistry and Biotechnology, 33(1): 97-101.
[19] Kumar P, Banday Z Z, Riley J L, et al.2024. Agrobacterium-mediated transient gene expression optimized for the bioenergy crop camelina sativa[J]. Bio-protocol Journal, 14(7): e4964.
[20] Leite Dias S, Rizzo P, Dauria J C, et al.2025. Efficient Agrobacterium-mediated methods for transient and stable transformation in common and tartary buckwheat[J]. International Journal of Molecular Sciences, 26(9): 4425.
[21] Liu D, Zhao S, Wang J, et al.2024. Mutation in the Agrobacterium hisI gene enhances transient expression in pepper[J]. Horticultural Plant Journal, 10(3): 809-822.
[22] Mbau Y J, Iriawati I, Faizal A.2018. Transient transformation of potato plant (Solanum tuberosum L.) granola cultivar using syringe agroinfiltration[J]. AGRIVITA Journal of Agricultural Science, 40(2): 313-319.
[23] Mei F, Yang T, Chao H, et al.2025. Genomic insights into drought adaptation of the forage shrub Caragana korshinskii (Fabaceae) widely planted in drylands[J]. The Plant Journal, 121(3): e17255.
[24] Pei M S, Liu H N, Ampomah-Dwamena C, et al.2023. A simple and efficient protocol for transient transformation of sliced grape berries[J]. Protoplasma, 260(3): 757-766.
[25] Qi D, Zhang Y, Zhao Z, et al.2018. Optimization of virus-induced gene silencing system and function identification of SmIAA19 gene in eggplant[J]. Acta Horticulturae Sinica, 45(4): 691.
[26] Ramírez Preciado J A, Rodríguez-Gandarilla M G, Tapia-Campos E, et al.2025. Enhancing transient gene expression in mature leaves of Mexican lime (Citrus aurantifolia) through Agrobacterium-mediated transformation[J]. Plant Biotechnology Reports, 19: 167-180.
[27] Sun B, Huang J, Kong L, et al.2023. Alternative splicing of a potato disease resistance gene maintains homeostasis between growth and immunity[J]. Plant Cell, 36(9): 3729-3750.
[28] Tyurin A A, Suhorukova A V, Kabardaeva K V, et al.2020. Transient gene expression is an effective experimental tool for the research into the fine mechanisms of plant gene function: Advantages, limitations, and solutions[J]. Plants, 9(9): 1187.
[29] Wang Y, Dong Y, Li D, et al.2022. A highly efficient mesophyll protoplast isolation and PEG-mediated transient expression system in eggplant[J]. Scientia Horticulturae, 304: 111303.
[30] Wei Q, Wang J, Wang W, et al.2020. A high-quality chromosome-level genome assembly reveals genetics for important traits in eggplant[J]. Horticulture Research, 7(1): 153.
[31] Wen L W, Zhu H L.2015. Establishment and optimization of Agrobacterium-mediated transient gene expression system in tobacco[J]. Advances in Botanical Research, 4(2): 25-31.
[32] Wu H Y, Liu K H, Wang Y C, et al.2014. AGROBEST: An efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings[J]. Plant Methods, 10: 1-16.
[33] Wroblewski T, Tomczak A, Michelmore R.2005. Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis[J]. Plant Biotechnology Journal, 3(2): 259-273.
[34] Xian B, Xi Z, Ren C, et al.2023. The establishment of transient expression systems and their application for gene function analysis of flavonoid biosynthesis in Carthamus tinctorius L[J]. BMC Plant Biology, 23(1): 186.
[35] Yao X, Taheri A, Liu H, et al.2024. Improvement and application of vacuum-infiltration system in tomato[J]. Horticulture Research, 11(9): uhae197.
[36] Zhang Y, Qiu L, Zhang Y, et al.2024. A high-efficiency transient expression system mediated by Agrobacterium tumefaciens in Spinacia oleracea leaves[J]. Plant Methods, 20(1): 100.
[37] Zheng L, Yang J, Chen Y, et al.2021. An improved and efficient method of Agrobacterium syringe infiltration for transient transformation and its application in the elucidation of gene function in poplar[J]. BMC Plant Biology, 21: 1-19. |
|
|
|
