拟南芥液泡H^(+)-ATP酶E1亚基基因AtTUF启动子的克隆及表达分析  

Cloning and Expression Analysis of the Promoter of Vacuolar H^(+)-ATPase E1 Subunit AtTUF Gene in Arabidopsis thaliana

作　　者：孙海丽[1,2,3] 梁静 王文佳 丁位华 王妮娜[1,2,3] 柳凯恒 李成伟 SUN Haili;LIANG Jing;WANG Wenjia;DING Weihua;WANG Nina;LIU Kaiheng;LI Chengwei(College of Life Science and Technology,Henan Institute of Science and Technology,Xinxiang 453003,China;Henan Engineering Research Center of Crop Genome Editing,Xinxiang 453003,China;Collaborative Innovation Center of Mordern Bilogical Breeding of Henan Province,Xinxiang 453003,China)

机构地区：[1]河南科技学院生命科技学院,河南新乡453003 [2]河南省粮食作物基因组编辑工程技术研究中心,河南新乡453003 [3]河南省现代生物育种协同创新中心,河南新乡453003

出　　处：《华北农学报》2022年第6期82-90,共9页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金青年基金项目(31700232);河南省青年骨干教师资助计划项目(2019GGJS164);河南省农业科技攻关项目(222102110193);河南省大学生创新基金项目(202010467014)。

摘　　要：AtTUF基因编码液泡膜H^(+)-ATP酶的E1亚基,与跨液泡膜的物质运输密切相关。为了探明拟南芥AtTUF基因的组织表达特征及基因表达调控的分子机制,为AtTUF基因启动子的利用及AtTUF基因表达调控特征的深入研究提供理论依据,以哥伦比亚野生型拟南芥为材料,通过PCR扩增AtTUF的启动子(p AtTUF)序列,利用网站PlantCARE和PLACE预测其顺式作用元件,构建由其驱动半乳糖苷酶基因(Glucuronidase,GUS)的植物表达载体pBI121-p AtTUF,转化拟南芥获得转基因株系,通过GUS染色分析AtTUF启动子的组织表达模式及其对激素与逆境胁迫的响应特征,并对AtTUF在不同组织中的表达进行定量分析。结果表明,成功克隆到AtTUF基因开放阅读框上游2000 bp的启动子序列,其中含有多个响应光信号、脱落酸(ABA)、生长素、茉莉酸甲酯(MeJA)及干旱信号的顺式作用元件;转基因株系的GUS染色结果显示,AtTUF启动子驱动的GUS在拟南芥幼苗的叶与根中,成苗期的莲座叶、主茎、茎生叶、萼片、花瓣、花药、花粉粒、柱头、幼嫩角果及发育期种子中均有表达,而在幼苗期胚轴、成苗期的莲座叶侧枝、成熟期角果及种子中未检测到表达;且其表达受ABA、吲哚乙酸(IAA)、盐、暗处理及PEG的抑制,这与其含有多种光调控元件、激素调控元件及非生物胁迫响应元件相一致。实时荧光定量PCR结果显示,AtTUF在根、茎、叶、花、角果及种子中均有表达,尤以发育期角果中表达量最高,在花与干种子中表达量也较高,根中表达量最低。综上可知,AtTUF基因在植物各器官(尤其是生殖系统)的生长发育及响应ABA、IAA、盐、光及干旱信号的过程中均发挥着重要作用。AtTUF gene encodes E1 subunit of vacuolar H^(+)-ATP enzyme,and is closely related to the material transport across the tonoplast.In order to explore the tissue expression characteristics and molecular regulatory mechanism of AtTUF,and provide a theoretical basis for the utilization of AtTUF gene promoter and the in-depth study of the regulation characteristics of AtTUF gene expression,the promoter of AtTUF(p AtTUF)was cloned by PCR amplification with Columbia wild-type Arabidopsis as material,the cis-acting elements were predicted through PlantCARE and PLACE,the plant expression vector pBI121-p AtTUF,in which the galactosidase gene(GUS)was driven by p AtTUF,was constructed and transformed into wild-type Arabidopsis to obtain transgenic plants.Through GUS staining,the tissue expression pattern of p AtTUF and its response to hormone and stress were analyzed,and the expression of AtTUF in different tissues was quantitatively analyzed.The results showed that 2000 bp promoter sequence upstream of the open reading frame of AtTUF gene was successfully cloned.Sequence analysis results showed that p AtTUF contained several cis-acting elements in response to light,abscisic acid(ABA),auxin,methyl jasmonate(MeJA)and drought.The staining results of transgenic lines showed that GUS driven by p AtTUF mainly expressed in the leaves and roots of Arabidopsis seedlings,and in rosette leaves,stems,cauline leaves,sepals,petals,anthers,stigmas,young siliques and immature seeds of adult plants.However,no GUS expression was detected in the hypocotyl at seedling stage,neither in the lateral branches of rosette leaves,mature pods or seeds at adult plant stage.GUS expression intensity was inhibited by ABA,indole acetic acid(IAA),salt,dark treatment and PEG treatment,which was consistent with its characteristics containing a variety of light regulatory elements,hormone regulatory elements and abiotic stress response elements.The Real-time quantitative RT-PCR results showed that AtTUF expressed in roots,stems,leaves,flowers,siliques and seed

关 键 词：拟南芥 AtTUF 启动子 顺式作用元件 组织表达 

分 类 号：Q78[生物学—分子生物学]