Cloning, Expression and Functional Analysis of LoNAC48 Gene in Lilium Oriental Hybrid 'Siberia'
HU Xiao-Cong1, WANG Ke1,2, SUN Liang1,*
1 School of Agriculture, Jinhua Polytechnic, Jinhua 321017, China; 2 College of Landscape Architecture, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China
Abstract：The long cultivation cycle and poor quality of lily bulbs by produced in China is a important factors that lead to long-term dependence on imports. The plant-specific transcription factor NAC plays an important role in regulating plant growth and development, resistance establishment, etc., and mining, identifying and studying related genes in lily is a strategy to solve the above problems. In this study, one gene transcript carrying NAC domain was screened in the lily transcriptome database, and the gene was cloned and identified as LoNAC48 gene (GenBank No. PP078617) based on PCR. Bioinformatics analysis showed that LoNAC48 gene contained a complete open reading frame with a length of 777 bp encoding 258 amino acids, which was an unstable hydrophobic protein, and phylogenetic tree analysis showed that it was highly homologous to Lilium regale LrNAC48 protein. The results of subcellular localization showed that LoNAC48 was a nuclear transcription factor, and tissue expression analysis showed that LoNAC48 gene was present in bulbs, roots, flowers and other tissues and organs expression, among which the expression level was the highest in leaves; The results of different stress responses showed that the expression of LoNAC48 gene was susceptible to low temperature, high temperature, drought, salt and hydrogen peroxide (H2O2) stresses, Under low temperature, salt, and hydrogen peroxide stress, the expression level was significantly upregulated at 12 hours compared to the control, and significantly decreased at 24 hours of drought stress, and the expression pattern of LoNAC48 was almost the same under salt stress and hydrogen peroxide stress. Phenotypic analysis of transgenic Arabidopsis thaliana lines showed that overexpression of LoNAC48 gene could significantly promote the growth of Arabidopsis thaliana and increase the leaf area. These results indicated that LoNAC48 gene had the dual functions of stress resistance and regulation of plant growth and development. This study provides genetic resources for molecular-assisted breeding of lily and improvement of lily traits.
[1] 郭方其, 吕萍, 吴超, 等. 2020. 浙江主栽盆栽百合种质资源表型性状遗传多样性分析[J]. 分子植物育种, 18(14): 4802-4811. (Guo F Q, Lv P, Wu C, et al.2020. Genetic diversity of pot flower Lilies (Lilium) germplasm resources by morphological traits[J]. Molecular Plant Breeding, 18(14): 4802-4811.) [2] 蒋瑶, 陈文波, 陈其兵. 2022. 野生湖北百合HSFA2耐热基因的克隆及表达分析[J]. 北方园艺, (22): 67-73. (Jiang Y, Chen W B, Chen Q B, 2022. Cloning and expression characterization of heat-resistant gene HSFA2 in wild lily[J]. Northern Horticulture, (22): 67-73.) [3] 凌吴, 李泽, 陈志文, 等. 2023. LA百合与亚洲百合远缘杂交及杂种后代的鉴定[J]. 分子植物育种, 1-13. (Ling W, Li Z, Chen Z, et al.2023. Distant hybridization between Lilium longiflorum thunb.×Lilium asiatic hybrida and Lilum asiatic hybrida and identification of their hybrid progeny[J]. Molecular Plant Breeding, 1-13.) [4] 赵秦. 2021. LrWRKY3基因在岷江百合对尖孢镰刀菌防卫反应中的功能分析[D]. 硕士学位论文, 昆明理工大学, 导师: 刘迪秋. pp. 47-p52. (Zhao Q.2021. Functional analysis of LrWRKY3 gene in defense response of Lilium regale wilson to Fusarium oxysporum[D]. Thesis for M.S., Kunming University of Science and Technology, Supervisor: Liu D Q, pp. 47-52.) [5] Abbas F, Ke Y, Zhou Y, et al.2020. Cloning, functional characterization and expression analysis of LoTPS5 from Lilium 'Siberia'[J]. Gene, 756: 921-927. [6] Breuer C, Ishida T, Sugimoto K.2010. Developmental control of endocycles and cell growth in plants[J]. Current Opinion in Plant Bology, 13(6): 654-660. [7] Du F, Fan J, Wang T, et al.2017. Identification of differentially expressed genes in flower, leaf and bulb scale of Lilium oriental hybrid 'Sorbonne' and putative control network for scent genes[J]. BMC Genomics, 18: 899-904. [8] Gao Y, Fan Z Q, Zhang Q, et al.2021. A tomato NAC transcription factor, SlNAM1, positively regulates ethylene biosynthesis and the onset of tomato fruit ripening[J]. Plant Journal, 108(5): 1317-1331. [9] Gong B, Yi J, Wu J, et al., 2014. LlHSFA1, a novel heat stress transcription factor in lily (Lilium longiflorum), can interact with LlHSFA2 and enhance the thermotolerance of transgenic Arabidopsis thaliana[J]. Plant Cell Reports, 33(9): 1519-1533. [10] Han K, Zhao Y, Sun Y, et al.2023. NACs, generalist in plant life[J]. Plant Biotechnology Journal, 21(12): 2433-2457. [11] Hu P, Zhang K, Yang C.2019. BpNAC012 positively regulates abiotic stress responses and secondary wall biosynthesis[J]. Plant Physiology, 179(2): 700-717. [12] Lazare S, Bechar D, Fernie A R, et al.2019. The proof is in the bulb: Glycerol influences key stages of lily development[J]. Plant Journal, 97(2): 321-340. [13] Li J, Xie L, Tian X, et al.2021. TaNAC100 acts as an integrator of seed protein and starch synthesis exerting pleiotropic effects on agronomic traits in wheat[J]. Plant Journal, 108(3): 829-840. [14] Li X, Wang Q, Guo C, et al.2022. NtNAC053, a novel NAC transcription factor, confers drought and salt tolerances in tobacco[J]. Frontiers in Plant Science, 13: 817106. [15] Li W, Zeng Y, Yin F, et al.Genome-wide identification and comprehensive analysis of the NAC transcription factor family in sunflower during salt and drought stress[J]. Scientific Reports, 11(1): 19865. [16] Liu B, Santo D M, Mayobre C, et al.2022. Knock-out of CmNAC-NOR affects melon climacteric fruit ripening[J]. Frontiers in Plant Science, 13: 878037. [17] Lu X, Zhang X, Duan H, et al.2018. Three stress-responsive NAC transcription factors from Populus euphratica differentially regulate salt and drought tolerance in transgenic plants[J]. Plant Physiology, 162(1): 73-97. [18] Liu J, Qiao Y, Li C, et al.2023. The NAC transcription factors play core roles in flowering and ripening fundamental to fruit yield and quality[J]. Frontiers in Plant Science, 14(1): 1095967. [19] Maki H, Sakaoka S, Itaya T, et al.2019. ANAC032 regulates root growth through the MYB30 gene regulatory network[J]. Scientific Reports, 9(1): 11358. [20] Singh, S, Koyama H, Bhati K K, et al.2021. The biotechnological importance of the plant-specific NAC transcription factor family in crop improvement[J]. Journal of Plant Research, 134: 475-495. [21] Sun L, Yan R, Yang R, et al.2022. Melatonin promotes scale primordium initiation and basal root regeneration and slows starch degradation in Lilium 'Siberia'[J]. Scientia Horticulturae, 294: 220798. [22] Sun Y, Zang Y, Ma Y, et al.2023. Identification and functional analysis of LpNAC37 associated with somatic embryogenesis in Lilium pumilum DC. Fisch. based on transcriptome analysis[J]. Plant Physiology and Biochemistry, 205: 107964. [23] Wu Y, Deng Z, Lai J, et al.2009. Dual function of Arabidopsis ATAF1 in abiotic and biotic stress responses[J]. Cell Research. 19(11): 1279-1290. [24] Xu P, Ma W, Hu J, et al.2022. The nitrate-inducible NAC transcription factor NAC056 controls nitrate assimilation and promotes lateral root growth in Arabidopsis thaliana[J]. PLOS Genetics, 18(3): e1010090. [25] Yan R, Wang Z, Ren Y, et al.2019. Establishment of efficient genetic transformation systems and application of CRISPR/Cas9 genome editing technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven[J]. International Journal of Molecular Sciences, 20(12): 2920. [26] Zhang J, Xue B, Gai M, et al.2017. Small RNA and transcriptome sequencing reveal a potential miRNA-mediated interaction network that functions during somatic embryogenesis in Lilium pumilum DC. Fisch[J]. Frontiers in Plant Science, 8: 566. [27] Zhang Q, Luo F, Zhong Y, et al.2020. Modulation of NAC transcription factor NST1 activity by XYLEM NAC DOMAIN1 regulates secondary cell wall formation in Arabidopsis[J]. Journal of Experimental Botany, 71(4): 1449-1458. [28] Zhang W, Zhi W, Qiao H, et al.2023. H2O2-dependent oxidation of the endoplasmic reticulum-associated transcription factor GmNTL1 triggers translocation to the nucleus and salt tolerance in soybean[J]. The Plant Cell, 36(1): 112-135.
