胡杨WINDs基因克隆及功能初步分析  

Cloning and Functional Analysis of WINDs gene in Populus euphratica

作　　者：侯思佳 陈静 孟剑桥 杜俊红 王聪 梁丹 邬荣领 郭允倩 HOU Si-jia;CHEN Jing;MENG Jian-qiao;DU Jun-hong;WANG Cong;LIANG Dan;WU Rong-ling;GUO Yun-qian(College of Biological Science and Technology,Center for Computational Biology,National Engineering Laboratory for Tree Breeding,Beijing Forestry University,Beijingg 100083,China)

机构地区：[1]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

出　　处：《中国生物工程杂志》2022年第10期9-20,共12页China Biotechnology

基　　金：国家自然科学基金(31370669)资助项目。

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1050 bp和1032 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。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 1050 bp and 1032 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 o

关 键 词：植物再生 胡杨 PeWIND 伤口响应 

分 类 号：Q812[生物学—生物工程]