Abstract:microRNA171b (miR171b) is a member of miR171 family and plays an important role in plant growth and development, response to abiotic and biological stresses. To reveal the function of miR171b in eggplant (Solanum melongena) response to Verticillium dahliae, using eggplant cultivar 'Suqi No. 1' as the test material in this study, the pri-miR171b gene was cloned and its expression vector was constructed. The miR171b transgenic eggplant plants were regenerated through Agrobacterium tumefaciens-mediated genetic transformation and analyzed their resistance to Verticillium wilt. The disease resistance analysis showed that the miR171b overexpression line was more resistant to the infection of V. dahliae with disease index of 18.5, which was about 1/3 to 1/2 of the control, while miR171b antisense inhibition lines were more sensible with disease index of 68.4 and 72.6, which were 1.5~1.6 times of the control. The quantitative analysis of pathogen internal transcribed spacer (ITS)showed that the content of V. dahliae in the vascular tissues of miR171b overexpression line was significantly lower than that in the control (P<0.05), and its content in miR171b antisense lines was significantly increased (P<0.05). Antioxidant enzyme activity analysis showed that the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) enzyme in miR171b overexpression line were higher than those in the control, while their activities were lower in the antisense lines than in the control. The above results showed that miR171b was involved in the defense process of eggplant against Verticillium wilt, and played a positive regulatory role. This study provides a theoretical basis for the breeding of eggplant cultivars with the Verticillium wilt resistance.
[1] 李合生 .2000. 植物生理生化实验原理和技术[M]. 高等教育出版社, 北京 . pp. 164-165.
(Li H S.2000. Experimental Principles and Techniques of Plant Physiology and Biochemistry[M]. Higher Education Press, Beijing. pp.164-165.)
[2] 王俊红 .2020. 茄子黄萎病的防治现状及对策[J]. 中国果菜,40(3): 98-100.
(Wang J H.2020. The control status and countermeasures of eggplant Verticillium wilt[J]. China Fruits and Vegetables, 40(3): 98-100.)
[3] 王培育 .2018. 文心兰 miRNA 在生长发育及抗软腐病中的应用研究[D]. 硕士学位论文, 福建农林大学, 导师: 林玉玲, pp. 22-24.
(Wang P Y.2018. Application of oncid‐ ium hybridum miRNA in growth and development and resistance to soft rot[D]. Thesis for M. S., Fujian Agri‐ culture and Forestry University, Supervisor: Lin Y L, pp. 22-24.)
[4] 王雨 .2017. 传统农业时期茄在中国的栽培史研究[D]. 硕士学位论文 , 南京农业大学 , 导师 : 丁晓蕾 , pp. 1-2.
(Wang Y.2017. Research on the history of eggplant cul‐ tivation in China during the traditional agricultural peri‐ od[D]. Thesis for M. S., Nanjing Agricultural Universi‐ ty, Supervisor: Ding X L, pp. 1-2.)
[5] 张宪政 .1994. 植物生理学实验技术[M]. 辽宁科学技术出版社 , 沈阳 . pp. 99-100.
(Zhang X Z.1994. Experimental Technology of Plant Physiology[M]. Liaoning Science and Technology Press, Shenyang. pp. 99-100.)
[6] 仲彩莲 .2020. 黄瓜抗白粉病 miRNAs 的鉴定及功能分析[D]. 硕士学位论文, 扬州大学, 导师: 陈学好, pp. 13-17.
(Zhong C L.2020. Identification and functional anal‐ ysis of cucumber powdery mildew miRNAs[D]. Thesis for M. S., Yangzhou University, Supervisor: Chen X H, pp. 13-17.)
[7] 邹琦 .2000. 植物生理学实验指导[M]. 中国农业出版社, 北京 . pp. 161-162.
(Zou Q.2000. Experimental Guidance of Plant Physiology[M]. China Agricultural Press, Bei‐ jing. pp. 161-162.)
[8] Axtell M J, Bowman J L.2008. Evolution of plant microRNAs and their targets[J]. Trends in Plant Science, 13(7):343-349.
[9] Chen Y, Hung Y C, Chen M, et al.2019. Enhanced storability of blueberries by acidic electrolyzed oxidizing water ap‐ plication may be mediated by regulating ROS metabo‐ lism[J]. Food Chemistry, 270: 229-235.
[10] Gao J, Luo M, Peng H, et al.2019. Characterization of cadmium-responsive microRNAs and their target genes in maize (Zea mays) roots[J]. BMC Molecular Biology, 20(14): 1-9
[11] Gobbato E, Marsh J, Vernié T, et al.2012. A GRAS-type tran‐ scription factor with a specific function in mycorrhizal signaling[J]. Current Biology, 22(23): 2236-2241.
[12] Häffner E, Karlovsky P, Splivallo R, et al.2014. ERECTA, salicylic acid, abscisic acid, and jasmonic acid modulate quan‐ titative disease resistance of Arabidopsis thaliana to Verticillium longisporum[J]. BMC Plant Biology, 14(85):1-16.
[13] Hendelman A, Kravchik M, Stav R, et al.2016. Tomato HAIRY MERISTEM genes are involved in meristem maintenance and compound leaf morphogenesis[J]. Journal of Experimental Botany, 67(21): 6187-6200.
[14] Lan X J, Zhang J, Zong Z F, et al.2017. Evaluation of the bio‐ control potential of Purpureocillium lilacinum QLP12 against Verticillium dahliae in gggplant[J]. BioMed Research International, 2017(2): 1-8.
[15] Lin Y F, Hu Y H, Lin H T, et al.2013. Inhibitory effects of propyl gallate on tyrosinase and its application in con‐ trolling pericarp browning of harvested longan fruits[J]. Journal of Agricultural and Food Chemistry, 61(11): 2889-2895.
[16] Liu H H, Tian X, Li Y J, et al.2008. Microarray-based analy‐ sis of stress-regulated microRNAs in Arabidopsis thali- ana[J]. A RNA Publication of the RNA Society, 14(5): 836-843.
[17] Liu S P, Zhu Y P, Xie C, et al.2012. Transgenic potato plants expressing StoVe1 exhibit enhanced resistance to Verticil- lium dahliae[J]. Plant Molecular Biology Reporter, 30(4), 1032-1039.
[18] Llave C.2002. Endogenous and silencing-associated small RNAs in plants[J]. Plant Cell, 14(7): 1605-1619.
[19] Mishra A K, Duraisamy G S, Matoušek J, et al.2016. Identifi‐ cation and characterization of microRNAs in Humulus lupulus using high-throughput sequencing and their response to Citrus bark cracking viroid (CBCVd) infection[J]. BMC Genomics, 17(919): 1-19.
[20] Mu X Y, Liu X R, Cai J H, et al.2018. MiR395 overexpression increases eggplant sensibility to Verticillium dahliae infection[J]. Russian Journal of Plant Physiology, 65(2): 203-210.
[21] Pantelides I S, Tjamos S E, Paplomatas E J.2010. Ethylene perception via ETR1 is required in Arabidopsis infection by Verticillium dahliae[J]. Molecular Plant Pathology, 11(2): 191-202.
[22] Reinhart B J, Slack F J, Basson M, et al.2000. The 21-nucleo‐ tide let-7 RNA regulates developmental timing in Cae- norhabditis elegans[J]. Nature, 403(6772): 901-906.
[23] Sun J, Zhang E, Xu L X, et al.2010. Comparison on charac‐ terization of longan (Dimocarpus longan Lour.) polyphe‐ noloxidase using endogenous and exogenous substrates[J]. Journal of Agricultural and Food Chemistry, 58(18): 10195-10201.
[24] Tong A, Quan Y, Shu W, et al.2017. Altered accumulation of osa-miR171b contributes to Rice stripe virus infection by regulating disease symptoms[J]. Journal of Experimen‐ tal Botany, 68(15): 4357-4367.
[25] Wang H, Chen Y, Lin H, et al.2018. Phomopsis longanae chi- Induced change in ROS metabolism and its relation to pericarp browning and disease development of harvest‐ ed longan fruit[J]. Front Microbiol, 9(2466): 1-8.
[26] Yang L, Jue D W, Li W, et al.2013. Identification of miRNA from eggplant (Solanum melongena L.) by small RNA deep sequencing and their response to Verticillium dahli- ae infection[J]. PLOS ONE, 8(8): e72840.
[27] Zhou L, Quan S, Xu H, et al.2018. Identification and expression of miRNAs related to female flower induction in walnut (Juglans regia L.)[J]. Molecules, 23(5): 1-15.