胡杨<i>WINDs</i>基因克隆及功能初步分析<sup>*</sup>

doi:10.13523/j.cb.2205005

中国生物工程杂志

2022, Vol. 42

Issue (10): 9-20 DOI: 10.13523/j.cb.2205005

研究报告

胡杨WINDs基因克隆及功能初步分析^*

侯思佳,陈静,孟剑桥,杜俊红,王聪,梁丹,邬荣领,郭允倩^**(

)

北京林业大学生物科学与技术学院计算生物学中心林木育种国家工程实验室北京 100083

Cloning and Functional Analysis of WINDs gene in Populus euphratica

Si-jia HOU,Jing CHEN,Jian-qiao MENG,Jun-hong DU,Cong WANG,Dan LIANG,Rong-ling WU,Yun-qian GUO^**(

)

College of Biological Science and Technology, Center for Computational Biology, National Engineering Laboratory for Tree Breeding, Beijing Forestry University, Beijing 100083, China

全文: PDF(5941 KB) HTML

摘要：

目的: WIND(WOUND INDUCED DEDIFFERENTIATION),是属于ERF/AP2 (ETHYLENE RESPONSE FACTOR/ APETALA 2)家族的一种重要转录因子,该类基因最早被发现在拟南芥中可以与乙烯响应元件GCC-BOX和脱水响应元件DRE结合,响应干旱信号和调节乙烯水平。最近的研究发现WIND基因在植物伤口信号回应、愈伤组织形成及不定芽的产生过程中也发挥了关键作用。已有的研究阐述了WIND基因在拟南芥中控制愈伤组织形成及不定芽再生的机制,但其在木本植物中的功能尚不明确,将探究WIND基因在胡杨中与伤口信号响应及不定芽再生相关的功能,同时为在分子水平上解决胡杨再生问题提供理论依据。方法: 采用基因克隆、qRT-PCR、转基因表型分析等方法研究WIND基因在胡杨外植体伤口响应和再生不定芽过程中的作用。结果: 克隆胡杨WIND家族中的基因PeWIND1和PeWIND2,发现其编码区序列长度分别为1 050 bp和1 032 bp,编码349个和343个氨基酸,亚细胞定位均在细胞核中。组织特异性分析显示PeWIND1和PeWIND2在胡杨根、茎、叶、愈伤组织中均有表达,且在愈伤组织中表达量最高。时间表达特异性显示,在经伤口刺激后的24 h内,PeWIND1和PeWIND2基因均呈现先升高后降低的表达趋势,且均在伤口刺激后1 h达到表达量峰值。转基因植株表型统计发现,过表达PeWIND1和PeWIND2基因后转基因植株不定芽再生能力增强。结论: 在胡杨叶片有伤口刺激后,PeWIND1和PeWIND2响应伤口信号,表达量先升高后降低,PeWIND1和PeWIND2能够促进杨树茎段再生不定芽。

关键词： 植物再生; 胡杨; PeWIND; 伤口响应

Abstract:

Objective: WIND (WOUND INDUCED DEDIFFERENTIATION) are important transcription factors belonging to ERF/AP2 (ETHYLENE RESPONSE FACTOR/ APETALA 2) family. The genes were first found to bind to ethylene response element GCC-BOX and dehydration response element DRE in Arabidopsis thaliana to respond to drought signals and regulate ethylene levels. Recent studies have found that WIND genes also play a key role in plant wound signal response, callus formation and adventitious shoot production. Previous studies have explored the mechanisms of the WIND gene controlling callus formation and adventitious shoot regeneration in Arabidopsis thaliana. However, the functions of WIND in woody plants remain unclear. The functions of WIND genes in wound signal response and adventitious shoot regeneration will be explored in Populus euphratica. A theoretical basis for solving the regeneration problem of P. euphratica at the molecular level will be provided. Methods: Gene cloning, qRT-PCR and transgenic phenotype analysis were used. Results: Two WIND genes of P.euphratica were cloned as PeWIND1 and PeWIND2, respectively. The coding regions were 1 050 bp and 1 032 bp, encoding 349 and 343 amino acids, respectively. Subcellular localization analysis showed that both genes were functional in the nucleus. Gene expression analysis showed that PeWIND1 and PeWIND2 expressed in roots, stems, leaves, and calli. The highest expression level was found in calli. The time course expression analysis showed that the expression levels of PeWIND1 and PeWIND2 were first increased and then decreased within 24 h after wound stimulation, and peaked at 1 h after wound stimulation. Phenotypic statistics of transgenic plants showed that the regeneration ability of adventitious shoots was enhanced after overexpression of PeWIND1 and PeWIND2. Conclusion: PeWIND1 and PeWIND2 responded to wound signal and the expression levels of PeWIND1 and PeWIND2 were first increased and then decreased within 24 h after wound stimulation. PeWIND1 and PeWIND2 promoted adventitious shoot regeneration from stems of poplars.

Key words: Plant regeneration Populus euphratica PeWIND Wound response

收稿日期: 2022-05-04 出版日期: 2022-11-04

ZTFLH:

Q812

基金资助: * 国家自然科学基金(31370669)

通讯作者: 郭允倩 E-mail: guoyunqian@bjfu.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	侯思佳
	陈静
	孟剑桥
	杜俊红
	王聪
	梁丹
	邬荣领
	郭允倩

引用本文:

侯思佳,陈静,孟剑桥,杜俊红,王聪,梁丹,邬荣领,郭允倩. 胡杨WINDs基因克隆及功能初步分析^*[J]. 中国生物工程杂志, 2022, 42(10): 9-20.

Si-jia HOU,Jing CHEN,Jian-qiao MENG,Jun-hong DU,Cong WANG,Dan LIANG,Rong-ling WU,Yun-qian GUO. Cloning and Functional Analysis of WINDs gene in Populus euphratica. China Biotechnology, 2022, 42(10): 9-20.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2205005 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I10/9

表1 PCR扩增目的基因引物序列

表2 PCR扩增带有BamHI和SalI 酶切位点的目的基因引物序列

表3 qRT-PCR基因引物序列

图1 PeWIND1、PeWIND2基因克隆电泳图

图2 PeWIND基因图谱

图3 PeWIND与PtrRAP2-4、PalRAP2-13-like、PdeKAH8508800.1和PtoKAG6774166.1蛋白氨基酸序列比对结果图

图4 PeWIND1、PeWIND2蛋白结构域预测

图5 PeWIND1、PeWIND2蛋白三维结构预测

图6 PeWIND1、PeWIND2与其他植物WIND蛋白的系统进化树

图7 转基因烟草叶片激光共聚焦显微镜镜检

图8 qRT-PCR检测胡杨PeWIND基因的组织表达特异性

图9 qRT-PCR检测PeWIND的时间表达特异性

图10 qRT-PCR检测转基因毛白杨WIND基因相对表达量

图11 WIND基因体外促进不定芽再生

[1]	李志军, 刘建平, 于军, 等. 胡杨、灰叶胡杨生物生态学特性调查. 西北植物学报, 2003, 23(7): 236-240.
	Li Z J, Liu J P, Yu J, et al. Investigation on the characteristics of biology and ecology of Populus euphratica and Populus pruinosa. Acta Botanica Boreali-Occidentalia Sinica, 2003, 23(7): 236-240.
[2]	张平冬, 康向阳, 高鹏, 等. 胡杨离体培养分化增殖途径的比较研究. 北京林业大学学报, 2003, 25(6): 50-54.
	Zhang P D, Kang X Y, Gao P, et al. Comparison of different proliferations of Populus euphratica under in vitro culture. Journal of Beijing Forestry University, 2003, 25(6): 50-54.
[3]	Parsons T J, Sinkar V P, Stettler R F, et al. Transformation of poplar by Agrobacterium tumefaciens. Bio/Technology, 1986, 4(6): 533-536.
[4]	Nilsson Q, Moritz T, Sundberg B, et al. Expression of the Agrobacterium rhizogenes rolC gene in a deciduous forest tree alters growth and development and leads to stem fasciation. Plant Physiology, 1996, 112(2): 493-502. pmid: 12226405
[5]	Li L G, Zhou Y H, Cheng X F, et al. Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(8): 4939-4944.
[6]	Levée V, Major I, Levasseur C, et al. Expression profiling and functional analysis of Populus WRKY 23 reveals a regulatory role in defense. New Phytologist, 2009, 184(1): 48-70. doi: 10.1111/j.1469-8137.2009.02955.x
[7]	金慧, 栾雨时. 转录因子在植物抗病基因工程中的研究进展. 中国生物工程杂志, 2010, 30(10): 94-99.
	Jin H, Luan Y S. Progress on transcription factor in gene engineering of diseases resistances in plants. China Biotechnology, 2010, 30(10): 94-99.
[8]	Wang H Z, Xue Y X, Chen Y J, et al. Lignin modification improves the biofuel production potential in transgenic Populus tomentosa. Industrial Crops and Products, 2012, 37(1): 170-177. doi: 10.1016/j.indcrop.2011.12.014
[9]	Han K H, Meilan R, Ma C, et al. An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus). Plant Cell Reports, 2000, 19(3): 315-320. doi: 10.1007/s002990050019 pmid: 30754915
[10]	Ikeuchi M, Iwase A, Rymen B, et al. Wounding triggers callus formation via dynamic hormonal and transcriptional changes. Plant Physiology, 2017, 175(3): 1158-1174. doi: 10.1104/pp.17.01035 pmid: 28904073
[11]	Reymond P, Weber H, Damond M, et al. Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. The Plant Cell, 2000, 12(5): 707-720. doi: 10.1105/tpc.12.5.707
[12]	Ikeuchi M, Ogawa Y, Iwase A, et al. Plant regeneration: cellular origins and molecular mechanisms. Development (Cambridge, England), 2016, 143(9): 1442-1451. doi: 10.1242/dev.134668
[13]	李俊, 范世航, 刘婧琳, 等. 外植体再生分子调控机理. 中国油料作物学报, 2021, 43(3): 376-382.
	Li J, Fan S H, Liu J L, et al. Molecular regulatory mechanisms of explant regeneration. Chinese Journal of Oil Crop Sciences, 2021, 43(3): 376-382.
[14]	Skoog F, Miller C O. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symposia of the Society for Experimental Biology, 1957, 11: 118-130.
[15]	Iwase A, Ohme-Takagi M, Sugimoto K. WIND1: a key molecular switch for plant cell dedifferentiation. Plant Signaling & Behavior, 2011, 6(12): 1943-1945.
[16]	Iwase A, Harashima H, Ikeuchi M, et al. WIND1 promotes shoot regeneration through transcriptional activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis. The Plant Cell, 2017, 29(1): 54-69. doi: 10.1105/tpc.16.00623
[17]	李新宇. 水曲柳愈伤组织不定芽再生与FmWIND1基因克隆和表达研究. 哈尔滨: 东北林业大学, 2019.
	Li X Y. A study on adventitious bud regeneration and FmWIND1 gene cloning and expression in callus of Fraxinus mandshurica. Harbin: Northeast Forestry University, 2019.
[18]	Zhou C, Guo J S, Feng Z H, et al. Molecular characterization of a novel AP 2 transcription factor ThWIND1-L from Thellungiella halophila. Plant Cell, Tissue and Organ Culture (PCTOC), 2012, 110(3): 423-433. doi: 10.1007/s11240-012-0163-4
[19]	Regier N, Frey B. Experimental comparison of relative RT-qPCR quantification approaches for gene expression studies in poplar. BMC Molecular Biology, 2010, 11(1): 57. doi: 10.1186/1471-2199-11-57
[20]	梁机, 陈晓阳, 林善枝, 等. 发根农杆菌Ri质粒rol基因研究进展及在林木改良上的应用. 植物学通报, 2002, 37(6): 650-658.
	Liang J, Chen X Y, Lin S Z, et al. Advance of studies on Agrobacterium rhizogenes ri plasmid rol genes and their applications for forest tree genetic improvement. Chinese Bulletin of Botany, 2002, 37(6): 650-658.
[21]	陆万香, 李名扬, 裴炎, 等. 玉米几丁质酶基因导入甘蓝型油菜的研究. 西南农业大学学报, 2001, 23(2): 130-133.
	Lu W X, Li M Y, Pei Y, et al. Incorporating maize chitinase into rapeseed (Brassica napus) by Agrobacterium tumefaciens. Journal of Southwest Agricultural University, 2001, 23(2): 130-133.
[22]	马智艳. 玉米内切β-1, 3-葡聚糖苷酶基因ZmEGP的克及功能分析. 郑州: 河南农业大学, 2015.
	Ma Z Y. The cloning and functional analysis of glucan endo-β-1, 3-glucosidase gene ZmEGP in maize. Zhengzhou: Henan Agricultural University, 2015.
[23]	Iwase A, Mitsuda N, Koyama T, et al. The AP2/ERF transcription factor WIND 1 controls cell dedifferentiation in Arabidopsis. Current Biology, 2011, 21(6): 508-514. doi: 10.1016/j.cub.2011.02.020
[24]	Ikeuchi M, Favero D S, Sakamoto Y, et al. Molecular mechanisms of plant regeneration. Annual Review of Plant Biology, 2019, 70: 377-406. doi: 10.1146/annurev-arplant-050718-100434 pmid: 30786238
[25]	侯思佳, 张倩倩, 孙振美, 等. WIND转录因子在植物响应伤口胁迫和器官生长发育中的研究进展. 中国生物工程杂志, 2022, 42(4): 85-92.
	Hou S J, Zhang Q Q, Sun Z M, et al. Research progress of WIND transcription factor responsing to wound stress and organ growth in plants. China Biotechnology, 2022, 42(4): 85-92.

[1]	岳宁郑彩霞白雪郝建卿. 胡杨异形叶的蛋白质组学研究[J]. 中国生物工程杂志, 2009, 29(09): 0-0.

Viewed

Full text

Abstract

Cited

Shared

Discussed