桑树MaERF105-Like的克隆及其在干旱胁迫下的表达分析

doi:10.16420/j.issn.0513-353x.2021-0543

摘要/Abstract

摘要：

以桑树‘德果1号’为材料，克隆获得MaERF105-Like及其启动子序列。序列分析发现，MaERF105-Like的cDNA序列长为1 131 bp，包含1个长度为882 bp的开放阅读框，编码293个氨基酸，蛋白质分子量为32.43 kD，等电点为8.71。MaERF105-Like含有1个AP2/ERF保守结构域，与ERF家族B3亚家族成员聚为一类，且与番茄SlERF5亲缘关系较近。亚细胞定位预测显示，MaERF105-Like蛋白可能定位在细胞核。启动子分析显示，MaERF105-Like启动子中含有与ABA、MeJA、乙烯及防御与胁迫相关的顺式作用元件。qRT-PCR结果显示，MaERF105-Like的表达水平受干旱诱导显著上调，复水后MaERF105-Like的表达水平显著下调。本研究结果表明MaERF105-Like可能在桑树抵御干旱的过程中具有重要作用。

关键词: 桑树, MaERF105-Like, 启动子, 干旱, 表达分析

Abstract:

MaERF105-Like gene and its promoter were cloned from‘Deguo 1’. Sequence analysis showed that the length of MaERF105-Like cDNA was 1 131 bp with an ORF of 882 bp，encoding 293 amino acids with molecular weight 32.43 kD and isoelectric point 8.71. MaERF105-Like has one AP2/ERF domain，belongs to B3 group of ERF family and shares a close relationship with tomato SlERF5. Subcellular localization prediction showed that MaERF105-Like might be located in nucleus. Promoter analysis showed that MaERF105-Like promoter possesses cis-acting elements involved in ABA，MeJA，ethylene and defense and stress responsiveness. qRT-PCR result showed that the expression level of MaERF105-Like was up-regulated by drought，and its expression was significantly down-regulated after rewatering treatment. The results of this study showed that MaERF105-Like might play an important role

in the resistance to drought in mulberry.

Key words: mulberry, MaERF105-Like, promoter, drought, expression analysis

中图分类号:

S663.9

黄仁维, 任迎虹, 祁伟亮, 曾睿, 刘欣宇, 邓彬艳. 桑树MaERF105-Like的克隆及其在干旱胁迫下的表达分析[J]. 园艺学报, 2022, 49(11): 2439-2448.

HUANG Renwei, REN Yinghong, QI Weiliang, ZENG Rui, LIU Xinyu, DENG Binyan. Cloning of Mulberry MaERF105-Like Gene and Its Expression Under Drought Stress[J]. Acta Horticulturae Sinica, 2022, 49(11): 2439-2448.

图/表 6

参考文献 27

[1]	Cui Bo, Hao Ping’an, Liang Fang, Zhang Yan, Wang Ximeng,Li Junlin,Jiang Suhua,Xu Shenping. 2020. Cloning and expression analysis of AP2/ERF family gene from Phalaenopsis under low temperature. Acta Horticulturae Sinica, 47 (1):85-97. (in Chinese)
	崔波, 郝平安, 梁芳, 张燕, 王喜蒙, 李俊霖, 蒋素华, 许申平. 2020. 蝴蝶兰AP2/ERF 家族基因的克隆及在低温下表达特性分析. 园艺学报,2020, 47 (1):85-97.
[2]	Fischer U, Droge-Laser W. 2004. Overexpression of NtERF5,a new member of the tobacco ethylene response transcription factor family enhances resistance to Tobacco mosaic virus. Molecular Plant-microbe Interactions, 17 (10):1162-1171. doi: 10.1094/MPMI.2004.17.10.1162 pmid: 15497409
[3]	Gou Yan-li, Zhang Le, Guo Huan, Ma Hong-ping, Bao Ai-ke. 2020. Research progress on the AP2/ERF transcription factor in plants. Pratacultural Science, 37 (6):1150-1159. (in Chinese)
	苟艳丽, 张乐, 郭欢, 马红萍, 包爱科. 2020. 植物AP2/ERF类转录因子研究进展. 草业科学, 37 (6):1150-1159.
[4]	Hao Di-qiu, Zhao Lin, Chen Li-miao, Li Wen-bin. 2010. Comparison and application of promoter clone methods. Journal of Northeast Agricultural University, 41 (3):154-160. (in Chinese)
	郝迪萩, 赵琳, 陈李淼, 李文滨. 2010. 克隆启动子方法的比较及应用. 东北农业大学学报, 41 (3):154-160.
[5]	Hennig L. 2012. Plant gene regulation in response to abiotic stress. Biochimica et Biophysica Acta, 1819 (2):85-194. doi: 10.1016/j.bbagrm.2012.01.005 pmid: 22309844
[6]	Huang R W, Liu D F, Huang M, Ma J, Li Z N, Li M Y, Sui S Z. 2019. CpWRKY71,a WRKY transcription factor gene of wintersweet (Chimonanthus praecox),promotes flowering and leaf senescence in Arabidopsis. International Journal of Molecular Sciences, 20:5325. doi: 10.3390/ijms20215325 URL
[7]	Ji Xian-ling, Gai Ying-ping, Mu Zhi-mei, Liu Xun-li, Wang Hong-li. 2004. Effect of water stress on physiological and biochemical character of mulberry. Acta Sericologica Sinica, 30 (2):117-122. (in Chinese)
	冀宪领, 盖英萍, 牟志美, 刘训理, 王洪利. 2004. 干旱胁迫对桑树生理生化特性的影响. 蚕业科学, 30 (2):117-122.
[8]	Jin L G, Liu J Y. 2008. Molecular cloning,expression profile and promoter analysis of a novel ethylene responsive transcription factor gene GhERF4 from cotton(Gossypium hirstum). Plant Physiology and Biochemistry, 46 (1):46-53. doi: 10.1016/j.plaphy.2007.10.004 URL
[9]	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. doi: 10.3389/fpls.2018.00346 pmid: 29616059
[10]	Lan Bi-zhong, Teng Wei-guo, Bin Rong-pei, Huang Ruo-yu, Luo Jian, Huang Yi, Dong Gui-qing, Tang Qing-kun, Huang Hong-yan. 2020. Experiments on different planting patterns of 5 mulberry varieties in rocky desertification areas. Guangxi Sericulture, 57 (1):1-7. (in Chinese)
	蓝必忠, 滕伟国, 宾荣佩, 黄若雨, 罗坚, 黄艺, 董桂清, 汤庆坤, 黄红燕. 2020. 5个桑树品种在石漠化地区不同种植模式试验. 广西蚕业, 57 (1):1-7.
[11]	Li Jidong, Ni Jing, Ye Xia, Chen Peng, Tan Bin, Zhang Mengyang, Zheng Xianbo, Zhang Yu, Feng Jiancan. 2020. Genomic identification of jujube AP2/ERF transcription factors and their expression pattern during jujube witches’ broom pathogenesis process. Acta Horticulturae Sinica, 47 (8):1463-1474. (in Chinese)
	李继东, 倪静, 叶霞, 陈鹏, 谭彬, 张梦洋, 郑先波, 张玉, 冯建灿. 2020. 枣AP2/ERF 转录因子鉴定及其响应枣疯病植原体表达分析. 园艺学报, 47 (8):1463-1474.
[12]	Li J J, Guo X, Zhang M H, Wang X, Zhao Y, Yin Z G, Zhang Z Y, Wang Y M, Xiong H Y, Zhang H L, Todorovska E, Li Z C. 2018a. OsERF71 confers drought tolerance via modulating ABA signaling and proline biosynthesis. Plant Science, 270:131-139. doi: 10.1016/j.plantsci.2018.01.017 URL
[13]	Li Z J, Tian Y S, Xu J, Fu X Y, Gao J J, Wang B, Han H J, Wang L J, Peng R H, Yao Q H. 2018b. A tomato ERF transcription factor,SlERF84,confers enhanced tolerance to drought and salt stress but negatively regulates immunity against Pseudomonas syringae pv. tomato DC3000. Plant Physiology and Biochemistry, 132:683-695. doi: 10.1016/j.plaphy.2018.08.022 URL
[14]	Liu Xue-qin, Ding Tian-long, Wei Cong-jin, Long Ding-pei, Guo Qing, Zhu Fang-rong, He Ning-jia, Yu Mao-de, Xiang Zhong-huai, Zhao Ai-chun. 2014. An evaluation on salt and drought tolerance of F₁generations from 13 mulberry hybrid combinations. Acta Sericologica Sinica, 40 (5):764-773. (in Chinese)
	刘雪琴, 丁天龙, 魏从进, 龙定沛, 郭庆, 朱方容, 何宁佳, 余茂德, 向仲怀, 赵爱春. 2014. 13份桑树杂交组合F₁代的耐盐性和耐旱性鉴定. 蚕业科学, 40 (5):764-773.
[15]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method. Methods, 25 (4):402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[16]	Pan Y, Seymour G B, Lu C, Hu Z, Chen X, Chen G. 2012. An ethylene response factor(ERF5)promoting adaptation to drought and salt tolerance in tomato. Plant Cell Reports, 31 (2):349-360. doi: 10.1007/s00299-011-1170-3 pmid: 22038370
[17]	Qi Jingjing, Dou Wanfu, Zhang Qingwen, Hu Anhua, Chen Shanchun, Lei Tiangang, Peng Aihong, Xu Lanzhen, Yao Lixiao, He Yongrui, Li Qiang. 2020. Interacting protein screening and analysis of CsAP2-09—a citrus bacterial canker related transcription factor. Acta Horticulturae Sinica, 47 (3):432-444. (in Chinese)
	祁静静, 窦万福, 张庆雯, 胡安华, 陈善春, 雷天刚, 彭爱红, 许兰珍, 姚利晓, 何永睿, 李强. 2020. 柑橘抗溃疡病转录因子CsAP2-09 互作蛋白筛选与分析. 园艺学报, 47 (3):432-444.
[18]	Qiao Z X, Huang B, Liu J Y. 2008. Molecular cloning and functional analysis of an ERF gene from cotton(Gossypium hirsutum). Biochimica et Biophysica Acta, 1779 (2):122-127.
[19]	Ren Ying-hong, Liu Song-qing, Qi Wei-liang, Luo Cui-hua, Gong Bi-ran, Ou Li-ping. 2017. Changes of super oxygen anion in mulberryvarieties 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]	Rong W, Qi L, Wang A Y, Ye X G, Du L P, Liang H X, Xin Z Y, Zhang Z Y. 2014. The ERF transcription factor TaERF 3 promotes tolerance to salt and drought stresses in wheat. Plant Biotechnology Journal, 12 (4):468-479. doi: 10.1111/pbi.12153 pmid: 24393105
[21]	Shao Wen-jing, Ao Tegenbaiyin, Lang Ming-lin. 2020. Research advances on the mechanism of AP2/ERF transcriptional factors in response to abiotic stresses in plants. Molecular Plant Breeding, 18 (5):4981-4988. (in Chinese)
	邵文靖, 熬特根白音, 郎明林. 2020. AP2/ERF转录因子对植物非生物胁迫的应答机制研究进展. 分子植物育种, 18 (5):4981-4988.
[22]	Siegel C S, Stevenson F O, Zimmer E A. 2017. Evaluation and comparison of FTA card and CTAB DNA extraction methods for non-agricultural taxa. Applications in Plant Sciences, 5 (2):1600109. doi: 10.3732/apps.1600109 URL
[23]	Xu Yun-gang, Zhan Ya-guang. 2009. Progress of the research on plant drought-resistant mechanism and related genes. Biotechnology Bulletin,(2):11-17. (in Chinese)
	徐云刚, 詹亚光. 2009. 植物抗旱机理及相关基因研究进展. 生物技术通报,(2):11-17.
[24]	Xu Zhixuan, Ren Zhonghai. 2020. Re-identification of tomato AP2/ERF transcription factor superfamily and phenotypic analysis of the overexpressing SlERF.D.3 lines. Acta Horticulturae Sinica, 47 (4):653-664. (in Chinese)
	徐志璇, 任仲海. 2020. 番茄AP2/ERF超家族重鉴定及过表达SlERF.D.3株系表型分析. 园艺学报, 47 (4):653-664.
[25]	Yang Xin, Lu Hong-mei, Yang Shuang-quan, Yang Hua-lian, Chen Li. 2019. Research progress on mulberry and mulberry wine. Food and Fermentation Industries, 45 (4):257-262. (in Chinese)
	杨新, 卢红梅, 杨双全, 杨华连, 陈莉. 2019. 桑葚及桑葚果酒的研究进展. 食品与发酵工业, 45 (4):257-262.
[26]	Yang Y Q, Dong C, Li X, Du J C, Qian M, Sun X D, Yang Y P. 2016. A novel Ap2/ERF transcription factor from Stipa purpurea leads to enhanced drought tolerance in Arabidopsis thaliana. Plant Cell Reports, 35:2227-2239. doi: 10.1007/s00299-016-2030-y URL
[27]	Zhuo C L, Liang L, Zhao Y Q, Guo Z F, Lu S Y. 2018. A cold responsive ethylene responsive factor from Medicago falcata confers cold tolerance by up-regulation of polyamine turnover,antioxidant protection,and proline accumulation. Plant Cell and Environment, 41 (9):2021-2032.

用途 Use	引物名称 Primer name	引物序列（5′-3′） Primer sequence	T_m/℃
基因克隆Gene cloning	MaERF105-Like-F	TCCTCTACTTTCGAACTTCTCAAC	57.1
	MaERF105-Like-R	CCTCAATCCCTTTGACTTTTCTTAT	59.9
启动子克隆Promoter cloning	pMaERF105-Like-F	CAACGGTATCACCCTCGCCAC	64.8
	pMaERF105-Like-R	TTCACTTCACCTTTACTCAAGTCCT	58.8
qRT-PCR	qMaERF105-Like-F	AAGGCGTTGAAGAGTTCGGTAG	60.6
	qMaERF105-Like-R	GAGATAACGGTGACAGTGGCG	60.4
	qMaβactin-F	TCCGATGCCCAGAAGTCCTC	62.1
	qMaβactin-R	TGATCTCCTTGCTCATCCTGTCT	60.4

用途 Use	引物名称 Primer name	引物序列（5′-3′） Primer sequence	T_m/℃
基因克隆Gene cloning	MaERF105-Like-F	TCCTCTACTTTCGAACTTCTCAAC	57.1
	MaERF105-Like-R	CCTCAATCCCTTTGACTTTTCTTAT	59.9
启动子克隆Promoter cloning	pMaERF105-Like-F	CAACGGTATCACCCTCGCCAC	64.8
	pMaERF105-Like-R	TTCACTTCACCTTTACTCAAGTCCT	58.8
qRT-PCR	qMaERF105-Like-F	AAGGCGTTGAAGAGTTCGGTAG	60.6
	qMaERF105-Like-R	GAGATAACGGTGACAGTGGCG	60.4
	qMaβactin-F	TCCGATGCCCAGAAGTCCTC	62.1
	qMaβactin-R	TGATCTCCTTGCTCATCCTGTCT	60.4

元件名称 Motif name	序列 Sequence	数量Number	功能 Function
A-box	CCGTCC	1	顺式调控元件 cis-Acting regulatory element
ABRE	ACGTG	2	脱落酸酸响应的顺式调控元件 cis-Acting element involved in the abscisic acid responsiveness
ARE	AAACCA	2	厌氧诱导必需元件 cis-Acting regulatory element essential for the anaerobic induction
ATC-motif	AGTAATCT	1	光应答中保守DNA模块的部分序列 Part of a conserved DNA module involved in light responsiveness
ATCT-motif	AATCTAATCC	1	光应答中保守DNA模块的部分序列 Part of a conserved DNA module involved in light responsiveness
Box 4	ATTAAT	2	光应答中保守DNA模块的部分序列 Part of a conserved DNA module involved in light responsiveness
CAAT-box	CAAT/CAAAT/CCAAT	28	启动子和增强子区域普通顺式作用元件 Common cis-Acting element in promoter and enhancer regions
CAT-box	GCCACT	1	与分生组织表达有关的顺式作用调控元件 cis-Acting regulatory element related to meristem expression
CGTCA-motif	CGTCA	1	茉莉酸甲酯响应的顺式调控元件 cis-Acting regulatory element involved in the MeJA-responsiveness
ERE	ATTTTAAA	4	乙烯响应元件 cis-Acting regulatory element involved in ethylene-responsiveness
G-Box	CACGTT	2	光响应元件 cis-Acting regulatory element involved in light responsiveness
MRE	AACCTAA	1	参与光响应的MYB结合位点 MYB binding site involved in light responsiveness
TATA-box	TATA/ATTATA/TATAA/TATAAATA/TATAAAT/TATAAA/TAAAGATT	26	启动子核心元件 Core promoter element around -30 of transcription start
TC-rich repeats	GTTTTCTTAC/ATTCTCTAAC	2	参与防御与胁迫应答的顺式调控元件 cis-Acting element involved in defense and stress responsiveness
TCT-motif	TCTTAC	1	部分光响应元件Part of a light responsive element
TGACG-motif	TGACG	1	茉莉酸甲酯响应的顺式调控元件 cis-Acting regulatory element involved in the MeJA-responsiveness

元件名称 Motif name	序列 Sequence	数量Number	功能 Function
A-box	CCGTCC	1	顺式调控元件 cis-Acting regulatory element
ABRE	ACGTG	2	脱落酸酸响应的顺式调控元件 cis-Acting element involved in the abscisic acid responsiveness
ARE	AAACCA	2	厌氧诱导必需元件 cis-Acting regulatory element essential for the anaerobic induction
ATC-motif	AGTAATCT	1	光应答中保守DNA模块的部分序列 Part of a conserved DNA module involved in light responsiveness
ATCT-motif	AATCTAATCC	1	光应答中保守DNA模块的部分序列 Part of a conserved DNA module involved in light responsiveness
Box 4	ATTAAT	2	光应答中保守DNA模块的部分序列 Part of a conserved DNA module involved in light responsiveness
CAAT-box	CAAT/CAAAT/CCAAT	28	启动子和增强子区域普通顺式作用元件 Common cis-Acting element in promoter and enhancer regions
CAT-box	GCCACT	1	与分生组织表达有关的顺式作用调控元件 cis-Acting regulatory element related to meristem expression
CGTCA-motif	CGTCA	1	茉莉酸甲酯响应的顺式调控元件 cis-Acting regulatory element involved in the MeJA-responsiveness
ERE	ATTTTAAA	4	乙烯响应元件 cis-Acting regulatory element involved in ethylene-responsiveness
G-Box	CACGTT	2	光响应元件 cis-Acting regulatory element involved in light responsiveness
MRE	AACCTAA	1	参与光响应的MYB结合位点 MYB binding site involved in light responsiveness
TATA-box	TATA/ATTATA/TATAA/TATAAATA/TATAAAT/TATAAA/TAAAGATT	26	启动子核心元件 Core promoter element around -30 of transcription start
TC-rich repeats	GTTTTCTTAC/ATTCTCTAAC	2	参与防御与胁迫应答的顺式调控元件 cis-Acting element involved in defense and stress responsiveness
TCT-motif	TCTTAC	1	部分光响应元件Part of a light responsive element
TGACG-motif	TGACG	1	茉莉酸甲酯响应的顺式调控元件 cis-Acting regulatory element involved in the MeJA-responsiveness