Cloning and Functional Analysis of Mulberry MaNAC90 Gene Under Drought Stress

doi:10.16420/j.issn.0513-353x.2025-0214

Acta Horticulturae Sinica ›› 2026, Vol. 53 ›› Issue (5): 1274-1284.doi: 10.16420/j.issn.0513-353x.2025-0214

• Genetic & Breeding · Germplasm Resources · Molecular Biology • Previous Articles Next Articles

Cloning and Functional Analysis of Mulberry MaNAC90 Gene Under Drought Stress

HUANG Renwei, LI Xiaodong, DENG Hongmei, YANG Yan, ZENG Rui, YANG Cairong^*()

Sichuan Provincial Key Laboratory for Development and Utilization of Characteristic Horticultural Biological Resources， College of Chemistry and Life Sciences，Chengdu Normal University， Chengdu 611130， China

Received:2025-12-15 Revised:2026-02-12 Online:2026-05-25 Published:2026-05-26
Contact: YANG Cairong

Abstract

Abstract:

A NAC transcription factor gene MaNAC90 was cloned from Morus alba‘Deguo 1’. The open reading frame of MaNAC90 was 732 bp，encoding 243 amino acids with molecular weight 27.436 kD and isoelectric point 5.32. MaNAC90 contained one NAC domain and belonged to TERN group，was localized in the nucleus and the cell membrane. Under simulated drought stress（200 mmol · L^-1mannitol），transgenic Arabidopsis expressing MaNAC90 exhibited a significant reduction in germination rate，a notable decrease in root length，an increased severity of seedling wilting，and significantly increased MDA content and electrolyte leakage，along with significantly decreased expression of RD22 and RD29B when compared to wild-type plants. These results preliminary indicated that MaNAC90 plays a negative regulatory role in response to drought stress.

Key words: mulberry, fruit use, MaNAC90, drought stress, function analysis

HUANG Renwei, LI Xiaodong, DENG Hongmei, YANG Yan, ZENG Rui, YANG Cairong. Cloning and Functional Analysis of Mulberry MaNAC90 Gene Under Drought Stress[J]. Acta Horticulturae Sinica, 2026, 53(5): 1274-1284.

Figures/Tables 9

Fig. 1 PCR amplification of MaNAC90 gene 1：MaNAC90 fragment；2：Negative control

Fig. 2 Multiple sequence alignment of MaNAC90 with NAC protein other plants Ma：Morus alba；Me：Manihot esculenta（XP_021629180.1）；Mr：Morella rubra（KAB1200809.1）；Cs：Cannabis sativa（XP_030503399.2）；Hb：Hevea brasiliensis（XP_021678053.1）；Pa：Parasponia andersonii（PON65421.1）. A-E：Subdomain

Fig. 3 Phylogenetic tree analysis of MaNAC90 and NAC proteins from Arabidopsis

Fig. 4 Subcellular localization of MaNAC90 protein

Fig. 5 DNA identification（A）and expression level analysis（B）of MaNAC90 transgenic Arabidopsis WT：Wild type；OE：Over expression line. The same below

Fig. 6 Seeds germination rate and root length of wild-type and transgenic Arabidopsis under 200 mmol · L-1 mannitol treatment A：Germination phenotype；B：Germination rate；C：Root length phenotype；D：Root length. * indicates significant differences from wild type（P < 0.05）. The same below

Fig. 7 Phenotype of wild-type and transgenic Arabidopsis under natural drought treatment

Fig. 8 Physiological indicators of wild-type and transgenic Arabidopsis under drought treatment

Fig. 9 The expression analysis of RD22 and RD29B of wild-type and transgenic Arabidopsis under drought treatment

References 32

[1]	Baranwal V K, Khurana P. 2016a. Genome-wide analysis,expression dynamics and varietal comparison of NAC gene family at various developmental stages in Morus notabilis. Molecular Genetics and Genomics, 291:1305-1317. doi: 10.1007/s00438-016-1186-z URL
[2]	Baranwal V K, Negi N, Khurana P. 2016b. Genome-wide identification and structural,functional and evolutionary analysis of WRKY components of mulberry. Scientific Reports,6:30794.
[3]	Cui Xiyan, Zhang Yingying, Zhou Ying. 2022. Research progress of plant transcription factors in response to drought stress. Journal of Jilin Agricultural University, 44 (5):505-511. (in Chinese)
	崔喜艳, 张莹莹, 周莹. 2022. 植物响应干旱胁迫转录因子研究进展. 吉林农业大学学报, 44 (5):505-511.
[4]	He L, Bian J, Xu J Y, Yang K J. 2019. Novel maize NAC transcriptional repressor ZmNAC071 confers enhanced sensitivity to ABA and osmotic stress by downregulating stress-responsive genes in transgenic Arabidopsis. Journal of Agricultural and Food Chemistry, 67 (32):8905-8918. doi: 10.1021/acs.jafc.9b02331 URL
[5]	He N J, Zhang C, Qi X W, Zhao S C, Tao Y, Yang G J, Lee T H, Wang X Y, Cai Q L, Li D, Lu M Z, Liao S T, Luo G Q, He R J, Tan X, Xu Y M, Li T, Zhao A C, Jia L, Fu Q, Zeng Q W, Gao C, Ma B, Liang J B, Wang X L, Shang J Z, Song P H, Wu H Y, Fan L, Wang Q, Shuai Q, Zhu J J, Wei C J, Zhu-Salzman K, Jin D C, Wang J P, Liu T, Yu M D, Tang C M, Wang Z J, Dai F W, Chen J F, Liu Y, Zhao S T, Lin T B, Zhang S G, Wang J Y, Wang J, Yang H M, Yang G W, Wang J,H. Paterson A, Xia Q Y, Ji D F, Xiang Z H. 2013. Draft genome sequence of the mulberry tree Morus notabilis. Nature Communications,4:2445.
[6]	Hou Yiyao, Li Xiao, Long Ruicai, Yang Qingchuan, Kang Junmei, Guo Changhong. 2021. Effect of overexpression of the alfalfa MsHB7 gene on drought tolerance of Arabidopsis. Acta Prataculturae Sinica, 30 (4):170-179. (in Chinese) doi: 10.11686/cyxb2020181
	候怡谣, 李霄, 龙瑞才, 杨青川, 康俊梅, 郭长虹. 2021. 过量表达紫花苜蓿MsHB7基因对拟南芥耐旱性的影响. 草业学报, 30 (4):170-179. doi: 10.11686/cyxb2020181
[7]	Huang Y M, Du B S, Yu M X, Cao Y B, Liang K H, Zhang L Y. 2024. Picea wilsonii NAC31 and DREB2A cooperatively activate ERD1 to modulate drought resistance in transgenic Arabidopsis. International Journal of Molecular Sciences, 25 (4):2037. doi: 10.3390/ijms25042037 URL
[8]	Huang Y Y, Shi Y, Hu X H, Zhang X Q, Wang X, Liu S H, He G J, An K L, Guan F Y, Zheng Y Y, Wang X H, Wei S L. 2024. PnNAC2 promotes the biosynthesis of Panax notoginseng saponins and induces early flowering. Plant Cell Reports, 43 (3):73. doi: 10.1007/s00299-024-03152-8
[9]	Jiang G X, Yan H L, Wu F W, Zhang D D, Zeng W, Qu H X, Chen F, Tan L, Duan X W, Jiang Y M. 2017. Litchi fruit LcNAC1 is a target of LcMYC2 and regulator of fruit senescence through its interaction with LcWRKY1. Plant and Cell Physiology, 58 (6):1075-1089. doi: 10.1093/pcp/pcx054 pmid: 28419348
[10]	Jin X X, Chai Q C, Liu C C, Niu X, Li W X, Shang X G, Gu A X, Zhang D Y, Guo W Z. 2024. Cotton GhNAC4 promotes drought tolerance by regulating secondary cell wall biosynthesis and ribosomal protein homeostasis. The Plant Journal, 117 (4):1052-1068. doi: 10.1111/tpj.v117.4 URL
[11]	Jin Z L, Wang W N, Nan Q, Liu J W, Ju Y L, Fang Y L. 2025. VvNAC17,a grape NAC transcription factor,regulates plant response to drought-tolerance and anthocyanin synthesis. Plant Physiology and Biochemistry,219:109379.
[12]	Karanja B K, Xu L, Wang Y, Muleke E M, Jabir B M, Xie Y, Zhu X W, Cheng W W, Liu L W. 2017. Genome-wide characterization and expression profiling of NAC transcription factor genes under abiotic stresses in radish(Raphanus sativus L.). PeerJ,5:e4172.
[13]	Kiranmai K, Rao G L, Pandurangaiah M, Nareshkumar A, Reddy V A, Lokesh U, Venkatesh B, Johnson A M A, Sudhakar C. 2018. A novel WRKY transcription factor,MuWRKY3(Macrotyloma uniflorum Lam. Verdc.)enhances drought stress tolerance in transgenic groundnut(Arachis hypogaea L.)plants. Frontiers in Plant Science,9:346.
[14]	Li M R, Li Y, Li H Q, Wu G J, Näsholm T. 2011. Overexpression of AtNHX5 improves tolerance to both salt and drought stress in Broussonetia papyrifera(L.)Vent. Tree Physiology, 31 (3):349-357. doi: 10.1093/treephys/tpr003 URL
[15]	Li S, Jing X L, Tan Q P, Wen B B, Fu X L, Li D M, Chen X D, Xiao W, Li L. 2023. The NAC transcription factor MdNAC29 negatively regulates drought tolerance in apple. Frontiers in Plant Science, 14:1173107.
[16]	Liu X Q, Zhu J J, Wei C J, Guo Q, Bian C K, Xiang Z H, Zhao A C. 2015. Genome-wide identification and characterization of the DREB transcription factor gene family in mulberry. Biologia Plantarum, 59,253-265. doi: 10.1007/s10535-015-0498-x URL
[17]	Ma Xueqi, Yin Yanhong, Feng Jingxian, Chen Wansheng, Sun Lianna, Xiao Ying. 2021. Research progress of NAC transcription factors in plant. Plant Physiology Journal, 57 (12):2225-2234. (in Chinese)
	马雪祺, 阴艳红, 冯婧娴, 陈万生, 孙连娜, 肖莹. 2021. 植物NAC转录因子研究进展. 植物生理学报, 57 (12):2225-2234.
[18]	Ooka H, Satoh K, Doi K, Nagata T, Otomo Y, Murakami K, Matsubara K, Osato N, Kawai J, Carninci P, Hayashizaki Y, Suzuki K, Kojima K, Takahara Y, Yamamoto K, Kikuchi S. 2003. Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana. DNA Research, 10 (6):239-247. doi: 10.1093/dnares/10.6.239 URL
[19]	Ren Yinghong, Liu Songqing, Qi Weiliang, Luo Cuihua, Gong Biran, Ou Liping. 2017. Changes of super oxygen anion in mulberry varieties under water stress. Guihaia, 37 (9):1122-1129. (in Chinese)
	任迎虹, 刘松青, 祁伟亮, 罗翠华, 龚壁燃, 欧莉萍. 2017. 干旱胁迫下桑树叶片中超氧阴离子的变化规律. 广西植物, 37 (9):1122-1129.
[20]	Shah F A, Chen Z, Ni F, Kamal K A, Zhang J M, Chen J H, Ren J. 2024. ArNAC 148 induces Acer rubrum leaf senescence by activating the transcription of the ABA receptor gene ArPYR13. International Journal of Biological Macromolecules,279 (Pt 2):134950.
[21]	Shang X G, Yu Y J, Zhu L J, Liu H Q, Chai Q C, Guo W Z. 2020. A cotton NAC transcription factor GhirNAC2 plays positive roles in drought tolerance via regulating ABA biosynthesis. Plant Science, 296:110498. doi: 10.1016/j.plantsci.2020.110498 URL
[22]	Shen J B, Lv B, Luo L Q, He J M, Mao C J, Xi D D, Ming F. 2017. The NAC-type transcription factor OsNAC2 regulates ABA-dependent genes and abiotic stress tolerance in rice. Scientific Reports,7:40641.
[23]	Sun L N, Xu H Q, Song J, Yang X Y, Wang X Y, Liu H Y, Pang M Z, Hu Y C, Yang Q, Ning X T, Liang S S, Zhang S J, Luan W J. 2024. OsNAC103,a NAC transcription factor,positively regulates leaf senescence and plant architecture in rice. Rice, 17 (1):15. doi: 10.1186/s12284-024-00690-3
[24]	Wang J L, Zhang X X, Yang H Q, Li S R, Hu Y, Wei D Y, Tang Q L, Yang Y, Tian S B, Wang Z M. 2024. Eggplant NAC domain transcription factor SmNST1 as an activator promotes secondary cell wall thickening. Plant,Cell & Environment, 47 (11):4293-4304. doi: 10.1111/pce.v47.11 URL
[25]	Wang W N, Yuan Y L, Yang C, Geng S P, Sun Q, Long L, Cai C W, Chu Z Y, Liu X, Wang G H, Du X M, Miao C, Zhang X, Cai Y F. 2016. Characterization,expression,and functional analysis of a novel NAC gene associated with resistance to Verticillium wilt and abiotic stress in Cotton. Genes Genomes Genetics, 6 (12):3951-3961.
[26]	Wang Haoran, Shao Zhilong, Zhu Yanyu, Kuang Hualin, Huang Minren, Chen Ying. 2015. Cloning and sub-cellular localization of Pd NAC1 in poplar‘Nanlin895’. Journal of Nanjing Forestry University(Natural Sciences Edition), 39 (3):50-54. (in Chinese)
	王浩然, 邵志龙, 朱燕宇, 匡华琳, 黄敏仁, 陈英. 2015. ‘南林895’杨PdNAC1基因克隆及蛋白的亚细胞定位. 南京林业大学学报(自然科学版), 39 (3):50-54. doi: 10.3969/j.issn.1000-2006.2015.03.010
[27]	Yang Rui, Yan Chaojun, Hu Shuaiya, Liu Shenhui, Chen Xiaochou, Chen Faxing. 2018. Physiological responses and tolerance of varieties of Anthurium andraeanum to low temperature. Fujian Journal of Agricultural Sciences, 33 (11):1136-1144. (in Chinese)
	杨芮, 颜超君, 户帅雅, 柳沈辉, 陈孝丑, 陈发兴. 2018. 红掌对低温胁迫的生理响应及耐冷性评价. 福建农业学报, 33 (11):1136-1144.
[28]	Yang Di, Zhang Kaixuan, Zhao Hui, Wu Xiaofang, Yang Keli, Zhou Meiliang, Zhang Jinlin. 2021. Molecular cloning and functional analysis of FtNAC11 from tartary buckwheat[Fagopyrum tataricum(L.)Gaertn.]. Journal of Plant Genetic Resources, 22 (5):1430-1441. (in Chinese) doi: 10.13430/j.cnki.jpgr.20210218004
	杨迪, 张凯旋, 赵辉, 伍小方, 杨克理, 周美亮, 张金林. 2021. 苦荞转录因子FtNAC11的克隆及其功能分析. 植物遗传资源学报, 22 (5):1430-1441. doi: 10.13430/j.cnki.jpgr.20210218004
[29]	Zhang S J, Li N, Gao F, Yang A F, Zhang J R. 2010. Over-expression of TsCBF1 gene confers improved drought tolerance in transgenic maize. Molecular Breeding,26:455-465.
[30]	Zhang X M, Yu H J, Sun C, Deng J, Zhang X, Liu P, Li Y Y, Li Q, Jiang W J. 2017. Genome-wide characterization and expression profiling of the NAC genes under abiotic stresses in Cucumis sativus. Plant Physiology and Biochemistry, 113:98-109. doi: S0981-9428(17)30035-9 pmid: 28193581
[31]	Zeng Rui, Ren Yinghong, Qi Weiliang, Huang Renwei, Li Lianlong, Li Xinxin, Wang Fei, Zhao Bihei. 2022. Comparative transcriptome analysis of drought stress responses in mulberries of differing drought resistances. Journal of Southern Agriculture, 53 (3):684-692. (in Chinese)
	曾睿, 任迎虹, 祁伟亮, 黄仁维, 李恋龙, 李鑫鑫, 王飞, 赵比黑. 2022. 不同桑树品种响应干旱胁迫的比较转录组学分析. 南方农业学报, 53 (3):684-692.
[32]	Zhu D, Che Y M, Xiao P L, Hou L X, Guo Y, Liu X. 2018. Functional analysis of a grape WRKY30 gene in drought resistance. Plant Cell,Tissue and Organ Culture,132:449-459.

Cloning and Functional Analysis of Mulberry MaNAC90 Gene Under Drought Stress

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 9

References 32

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LI Na, WANG Lili, XUE Dandan, SUN Ze, ZHANG Jing. A New Cold-Hardy Fruit Mulberry Cultivar‘Zhuyu 1’ [J]. Acta Horticulturae Sinica, 2026, 53(S1): 55-56.
[2]	ZHANG Wei, YIN Xilong, FENG Zengwei, LIU Xiaodi, ZHOU Yang, ZHU Honghui, YAO Qing. Arbuscular Mycorrhizal Fungi-Specific Lipid Allocation Patterns in Citrus Mycorrhizae in Response to Drought Stress [J]. Acta Horticulturae Sinica, 2026, 53(4): 1013-1024.
[3]	WANG Yanqiu, BAN Jingjie, ZENG Yuhan, LAI Chunwang, HUANG Xiaoling, ZHOU Jianjin, WAN Kui, LAI Zhongxiong, LIN Yuling. Identification of the DREB Gene Family in Polygonatum cyrtonema and Analysis of Its Response to Salt Stress [J]. Acta Horticulturae Sinica, 2026, 53(1): 159-173.
[4]	ZHU Cunlan, FAN Junliang, WANG Kaitong, WEI Meng, YANG Liang, LIU Haotian, SI Huaijun, ZHANG Ning. Function Analysis ofStRGLG1in Regulating Potato to Drought Stress Response [J]. Acta Horticulturae Sinica, 2025, 52(9): 2329-2342.
[5]	GUO Xinmiao, TIAN Luyao, CAO Jiaqi, XIE Yan, GAO Yanxia, and SUN Zhichao. Preliminary Research on the Molecular Mechanism of Color Loss in White Mulberry Fruit [J]. Acta Horticulturae Sinica, 2025, 52(6): 1451-1462.
[6]	XIAO Zhihao, ZHENG Hankai, ZHANG Mannan, TANG Huaiqian, WANG Jiaying, ZHANG Yuyang, ZHANG Junhong, YE Zhibiao, and YE Jie. Effects of Potassium on Growth and Development of Tomato Seedlings Under Abiotic Stress [J]. Acta Horticulturae Sinica, 2025, 52(6): 1599-1618.
[7]	ZHANG Xiaoting, ZHUANG Yun, XIAO Kaiting, and ZHOU Biyan. Cloning of Litchi LcICE1 Gene and Its Functional Analysis Under Low Temperature Stress [J]. Acta Horticulturae Sinica, 2025, 52(11): 2861-2875.
[8]	MA Bo, LI Lei, HU Zenghui, LENG Pingsheng, WANG Jinxuan, LENG Zhuo, YANG Yihui, JIA Liming, WU Jing. Functional Analysis of SoF3′H in the Synthesis of Anthocyanidin in Syringa oblata Petals [J]. Acta Horticulturae Sinica, 2024, 51(8): 1833-1843.
[9]	SUN Zhichao, GUO Xinmiao, WANG Hui, XIE Yan, GAO Yanxia, LI Jisheng, GAO Yujun. Mechanism of miR408-MnBBP Module Regulating Anthocyanin Biosynthesis in Mulberry [J]. Acta Horticulturae Sinica, 2024, 51(6): 1216-1226.
[10]	WU Xiangqi, SUN Li, YU Zheping, YU Qinpei, LIANG Senmiao, ZHENG Xiliang, QI Xingjiang, ZHANG Shuwen. Functional Study of MrSPL4 Gene in Response to Drought and Low Temperature Stress in Chinese Bayberry [J]. Acta Horticulturae Sinica, 2024, 51(5): 927-938.
[11]	LUO Jiandong, QIU Mengqing, ZHOU Huimin, XIE Weiwei, Huang Haixin, LIU Jieqi, ZHANG Zimin, XU Jian, CHEN Chengjie, HE Yehua, LIU Chaoyang. Cloning and Functional Analysis of Pineapple AcZFP1 Gene Under Low Temperature Stress [J]. Acta Horticulturae Sinica, 2024, 51(3): 495-508.
[12]	LIU Jinhong, WANG Zheng, YU Hao, XIN Yirui, QI Guoning, LIU Shenkui, REN Huimin. Identification of SLAC Gene Family in Phyllostachys edulis and Characterization of PheSLAC1 [J]. Acta Horticulturae Sinica, 2024, 51(3): 545-559.
[13]	CHEN Xin, WU Xiaolong, LIU Shengrui, HU Xianchun, LIU Chunyan. Effects of AMF on Photosynthetic Characteristics and Gene Expressions of Tea Plants Under Drought Stress [J]. Acta Horticulturae Sinica, 2024, 51(10): 2358-2370.
[14]	TIAN Jie, ZHOU Qianyi, TIE Yuanyu, SUN Haihong, and HUANG Sijie. Metabolomic Analysis of Garlic Seedling Under Drought Stress [J]. Acta Horticulturae Sinica, 2024, 51(1): 133-144.
[15]	WU Xiulan, LI Guihua, TANG Wenwu. Cloning and Function Analysis of Ubiquitin-conjugating Enzyme Gene CrUBC2 in Citrus reticulata‘Wuzi Shatangju’ [J]. Acta Horticulturae Sinica, 2023, 50(10): 2069-2078.