Cloning and Initial Functional Analysis of ThEIN3 Gene Related to Adventitious Root Development in Taxodium hybrid 'Zhongshanshan'
SUN Xiao-Wei, CUI Luo-Min, XUAN Lei, YANG Ying, WANG Zhi-Quan*, YU Chao-Guang*
Jiangsu Key Laboratory for the Conservation and Utilization of Plant Resources, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (Nanjing Botanical Garden MEM. Sun Yat-Sen), Nanjing 210014, China
Abstract：Taxodium hybrid 'Zhongshanshan' relies on softwood cutting for asexual propagation. Elucidating the molecular mechanism of its adventitious root (AR) development and identifying key genes are of great significance for establishing an efficient genetic transformation system and improving propagation efficiency. To investigate the function of ethylene-insensitive 3 (EIN3), a core transcription factor in the ethylene (ET) signal transduction pathway of T. hybrid 'Zhongshanshan', during AR development, the ThEIN3 gene (GenBank No. PX209540) was cloned in this study. Its function was identified by overexpressing ThEIN3 in tobacco (Nicotiana benthamiana) through an Agrobacterium tumefacien-mediated expression system. The results showed that the full-length ThEIN3 gene was 2 073 bp, encoding 690 amino acids. It contained a typical conserved domain of the EIN3 superfamily and was localized in the nucleus. With the development of AR in T. hybrid 'Zhongshanshan', the expression level of ThEIN3 was gradually down-regulated, and its expression in mature soft shoots was significantly higher than that in rejuvenated soft shoots (P<0.05). Compared with wild-type tobacco (WT), the AR development of ThEIN3-overexpressing plants was inhibited. Hormone content measurements at the base of adventitious roots revealed a significant increase in the content of gibberellin A3 (GA3)(P<0.05), while the contents of indole-3-acetic acid (IAA) and trans-zeatin riboside (TZR) decreased. Further research on the molecular mechanism indicated that ThEIN3 might inhibit the occurrence of AR by coordinating key genes in the related hormone metabolism pathways. This study provides a foundation for further research on the regulatory mechanisms of AR development in T. hybrid 'Zhongshanshan' and offers genetic resources and theoretical guidance for its molecular breeding.
孙晓威, 崔罗敏, 宣磊, 杨颖, 王芝权, 於朝广. 中山杉不定根发育相关基因ThEIN3的克隆及初步功能分析[J]. 农业生物技术学报, 2026, 34(4): 788-800.
SUN Xiao-Wei, CUI Luo-Min, XUAN Lei, YANG Ying, WANG Zhi-Quan, YU Chao-Guang. Cloning and Initial Functional Analysis of ThEIN3 Gene Related to Adventitious Root Development in Taxodium hybrid 'Zhongshanshan'. 农业生物技术学报, 2026, 34(4): 788-800.
[1] 陈永辉, 王名金, 伍寿彭. 1988. 落羽杉属的引种和选育[J]. 江苏林业科技,(2):43-47+49. [2] 陈永辉, 伍寿彭, 王名金, 等. 1989. 中山杉302和401无性系在碱地上的生长和适应性的初步研究[J]. 江苏林业科技, (3):14-18. (Chen Y H, Wu S P, Wang M J, et al. 1989. A preliminary study on the growth and adaptability of Taxodium hybrid 'Zhongshanshan' clones 302 and 401 in alkaline soils[J]. Journal of Jiangsu Forestry Science and Technology, (3):14-18.) [3] 陈永辉, 伍寿彭, 殷云龙, 等. 1996. 江苏滨海盐碱地中山杉造林推广试验[J]. 江苏林业科技, 23(4):18-22. (Chen Y H, Wu S P, Yin Y L, et al.1996. Plantation experiment of Taxodium hybrids on the saline and alkaline soils in the coastal area in Jiangsu province[J]. Journal of Jiangsu Forestry Science and Technology, 23(4):18-22.) [4] 扈嘉鑫, 陈宇熙, 余敏芬, 等. 2024. 植物不定根生成的生理及分子调控机制研究进展[J]. 分子植物育种, 22(18):6151-6159. (Hu J X, Chen Y X, Yu M F, et al.2024. Research progress on regulation mechanism of plant adventitious root formation[J]. Molecular Plant Breeding, 22(18):6151-6159.) [5] 王紫阳, 徐建华, 李火根, 等. 2015. 中山杉优良无性系302, 118, 405扦插生根能力比较[J]. 浙江农林大学学报, 32(4):648-654. (Wang Z Y, Xu J H, Li H G, et al.2015. Rooting capabilities for Taxodium 'Zhongshanshan' 302, 118, and 405[J]. Journal of Zhejiang A & F University, 32(4):648-654.) [6] 殷云龙, 於朝广, 华建峰, 等. 2014. 重庆万州三峡库区消落带中山杉造林试验[J]. 林业科技开发, 28(2):110-114. (Yin Y L, Yu C G, Hua J F, et al.2014. A trial on the silviculture of Taxodium hybrid 'Zhongshanshan 118' planted in the hydro-fluctuation belt of the Three Gorges Reservoir within the Wanzhou district area of Chongqing city[J]. China Forestry Science and Technology, 28(2):110-114.) [7] 於朝广, 徐建华, 芦志国, 等. 2015. “杂交墨杉”新品种区域试验初报[J]. 南方林业科学, 43(6):27-30. (Yu C G, Xu J H, Lu Z G, et al.2015. A regional experiment of new cultivars of Taxodium mucronatum hybrid[J]. South China Forestry Science, 43(6):27-30.) [8] 於朝广, 殷云龙. 2008. 落羽杉属树木种间杂交选育研究进展[J]. 江苏林业科技, 35(2):39-46. (Yu C G, Yin Y L.2008. Review on cross-breeding between different species of Taxodium[J]. Journal of Jiangsu Forestry Science and Technology, 35(2):39-46.) [9] 於朝广, 殷云龙, 徐建华. 2011. 落羽杉属4个新品种[J]. 林业科学, 47(5):181-182. (Yu C G, Yin Y L, Xu J H.2011. Four hybrid varieties of Taxodium[J]. Scientia Silvae Sinicae, 47(5):181-182.) [10] 张博, 2024. 杨树EIN3.1/2基因的克隆及功能初步研究[D]. 硕士学位论文, 山东农业大学, 导师:刘丽君, pp. 44-51. (Zhang B.2024. Cloning and functional preliminary study of EIN3.1/2 gene in poplar[D]. Thesis for M.S., Shandong Agricultural University, Suppervisor:Liu L J, pp. 44-51.) [11] 张强, 兰飞燕, 李成林, 等. 2024. 毛白杨PtoEIN3基因启动子的克隆及逆境胁迫响应模式分析[J]. 分子植物育种, 1-12. (Zhang Q, Lan F Y, Li C L, et al.2024. Cloning of PtoEIN3 gene promoter and adversity stress response patterns analysis of Populus tomentosa[J]. Molecular Plant Breeding, 1-12.) [12] 赵曾强, 张析, 李潇玲, 等. 2022. GhEIN3基因对棉花枯萎病胁迫响应的功能分析[J]. 棉花学报, 34(3):173-186. (Zhao Z Q, Zhang X, Li X L, et al.2022. Functional analysis of GhEIN3 gene in response to cotton Fusarium wilt stress[J]. Cotton Science, 34(3):173-186.) [13] An F Y, Zhao Q, Ji Y S, et al.2010. Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE 1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis[J]. Plant Cell, 22(7):2384-2401. [14] Chang K N, Zhong S, Weirauch M T, et al.2013. Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis[J]. Elife, 2:e00675. [15] Feng Y, Xu P, Li B S, et al.2017. Ethylene promotes root hair growth through coordinated EIN3/EIL1 and RHD6/RSL1 activity in Arabidopsis[J]. Proceedings of the National Academy of Sciences of the USA, 114(52):13834-13839. [16] Gagne J M, Smalle J, Gingerich D J, et al.2004. Arabidopsis EIN3-binding F-box 1 and 2 form ubiquitin-protein ligases that repress ethylene action and promote growth by directing EIN3 degradation[J]. Proceedings of the National Academy of Sciences of the USA, 101(17):6803-6808. [17] Guo H W, Ecker J R.2003. Plant responses to ethylene gas are mediated by SCFEBF1/EBF2-dependent proteolysis of EIN3 transcription factor[J]. Cell, 115(6):667-677. [18] Guo X K, Zhang Y, Tu Y, et al.2018. Overexpression of an EIN3-binding F-box protein2-like gene caused elongated fruit shape and delayed fruit development and ripening in tomato[J]. Plant Science, 272:131-141. [19] Hammatt N, Grant N J.1993. Apparent rejuvenation of mature wild cherry (Prunus avium L.) during micropropagation[J]. Journal of Plant Physiology, 141(3):341-346. [20] Li H, Yao L L, Sun L L, et al.2021. Correction:ETHYLENE INSENSITIVE 3 suppresses plant de novo root regeneration from leaf explants and mediates age-regulated regeneration decline[J]. Development, 148(9):dev199635. [21] Li W Y, Ma M D, Feng Y, et al.2015. EIN2-directed translational regulation of ethylene signaling in Arabidopsis[J]. Cell, 163(3):670-683. [22] Li Z H, Peng J Y, Wen X, et al.2013. ETHYLENE-INSENSITIVE3 is a senescence-associated gene that accelerates age-dependent leaf senescence by directly repressing miR164 transcription in Arabidopsis[J]. Plant Cell, 25(9):3311-3328. [23] Liu H, Gao Y, Song X B, et al.2018. A novel rejuvenation approach to induce endohormones and improve rhizogenesis in mature juglans tree[J]. Plant Methods, 14(1):13. [24] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J]. Methods, 25(4):402-408. [25] Potuschak T, Lechner E, Parmentier Y, et al.2003. EIN3-dependent regulation of plant ethylene hormone signaling by two Arabidopsis F box proteins:EBF1 and EBF2[J]. Cell, 115(6):679-689. [26] Qiao H, Shen Z X, Huang S S C, et al.2012. Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas[J]. Science, 338(6105):390-393. [27] Qiu K, Li Z P, Yang Z, et al.2015. EIN3 and ORE1 accelerate degreening during ethylene-mediated leaf senescence by directly activating chlorophyll catabolic genes in Arabidopsis[J]. PLOS Genetics, 11(7):e1005399. [28] Salih H, He S P, Li H G, et al.2020. Investigation of the EIL/EIN3 transcription factor gene family members and their expression levels in the early stage of cotton fiber development[J]. Plants, 9(1):128. [29] Solano R, Stepanova A, Chao Q, et al.1998. Nuclear events in ethylene signaling:A transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1[J]. Genes and Development, 12(23):3703-3714. [30] Sun L L, Zhu Z Q.2021. The molecular basis of age-modulated plant de novo root regeneration decline in Arabidopsis thaliana[J]. Plant and Cell Physiology, 62(1):3-7. [31] Wang Z Q, Shi Q, Chen P P, et al.2023. Grafting causes physiological changes and promotes adventitious root formation in rejuvenated soft shoots of Taxodium hybrid 'Zhongshanshan'[J]. Plants, 12(1):201. [32] Wang Z Q, Hua J F, Yin Y L, et al.2019. An integrated transcriptome and proteome analysis reveals putative regulators of adventitious root formation in Taxodium 'zhongshanshan'[J]. International Journal of Molecular Sciences, 20(5):1225. [33] Wen X, Zhang C L, Ji Y S, et al.2012. Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus[J]. Cell Research, 22(11):1613-1616. [34] Wendling I, Trueman S J, Xavier A.2014. Maturation and related aspects in clonal forestry-part Ⅱ:Reinvigoration, rejuvenation and juvenility maintenance[J]. New Forests, 45(4):473-486. [35] Yoo S D, Sheen J.2008. MAPK signaling in plant hormone ethylene signal transduction[J]. Plant Signaling and Behavior, 3(10):848-849. [36] Yang Y, Yu C G, Hua J F, et al.2025. The baldcypress genome provides insights into the adaptive evolution of flooding stress tolerance[J]. New Phytologist, 247(2):979-997.
