摘要
草莓果实成熟的早晚,直接影响果农的收益。课题组前期研究发现,草莓ARF4基因能够调控草莓开花时间。启动子是基因转录的开关,其转录活性影响着基因表达的强弱。克隆ARF4基因启动子并分析其转录活性,对进一步明确ARF4基因的功能具有重要意义。根据草莓基因组信息设计特异引物,克隆草莓ARF4基因启动子。使用Plant CARE在线软件,预测其序列上的顺式作用元件。将ARF4启动子进行分段缺失克隆,利用农杆菌介导的果实注射法及GUS组织染色,确定GUS基因表达的强弱。通过农杆菌介导的叶盘转化法,获得转proARF4::GUS基因草莓植株,利用GUS组织染色法分析启动子的组织表达特异性。对转基因草莓植株进行非生物胁迫处理,通过qRT-PCR方法确定影响ARF4基因启动子转录活性的因素。结果表明:在ARF4基因启动子序列上,除了存在基本的核心元件外,还包含一些光响应、胁迫响应、厌氧诱导及激素响应等作用元件。GUS组织染色结果显示,随着启动子序列长度逐渐变短,GUS基因表达变弱。在转proARF4::GUS基因草莓植株叶柄、嫩叶及新茎中GUS表达水平较高,且在嫩叶中表达量最高。在非生物胁迫处理下,IAA和GA可使GUS基因表达显著提高。这些结果提供了影响ARF4基因表达的相关因素,为调控草莓开花时间奠定了基础。
The ripening period of strawberry fruit directly affects the income of fruit farmers. A preliminary study by the project team found that the strawberry ARF4 gene could regulate the flowering time of strawberries. A promoter is a switch of gene transcription, and its transcriptional activity affects the strength of gene expression. Cloning of the ARF4 gene promoter and analysis of its transcriptional activity is of great significance for further clarifying the function of the ARF4 gene. Specific primers were designed based on the strawberry genome sequence, and the ARF4 gene promoter was cloned using specific primers. The cis-acting elements on the ARF4 gene promoter sequence were predicted by using Plant CARE online software. The ARF4 promoter was subjected to segmentation deletion cloning, and the expression of GUS gene was determined by Agrobacterium-mediated fruit injection and GUS tissue staining. The Agrobacterium-mediated leaf transformation experiment was used to obtain the proARF4::GUS strawberry plants, and the tissue expression specificity of the promoter was analyzed by GUS tissue staining.Transgenic strawberry plants were subjected to abiotic stress treatment, and factors affecting the transcriptional activity of the ARF4 gene promoter were determined by qRT-PCR. The results showed that the ARF4 gene promoter sequence also contained light response elements, stress response elements, anaerobic induction elements and hormone response elements in addition to the basic core elements. GUS tissue staining results showed that the GUS gene expression became weaker as the promoter sequence length became shorter. The expression level of GUS was higher in petiole, young leaves and new stems of the proARF4::GUS strawberry plants, and the highest expression was found in young leaves. Under abiotic stress treatment, IAA and GA can significantly increase the expression of GUS gene. These results provide relevant factors affecting the expression of ARF4 gene,which lays a foundation for regulating the flowering time o
作者
董向向
王矢乔
关宇涵
张志宏
李贺
DONG Xiang-xiang;WANG Shi-qiao;GUAN Yu-han;ZHANG Zhi-hong;LI He(College of Horticulture/Key Laboratory of Strawberry Breeding and Cultivation,Shenyang Agricultural University,Shenyang 110161,China)
出处
《沈阳农业大学学报》
CAS
CSCD
北大核心
2019年第5期513-521,共9页
Journal of Shenyang Agricultural University
基金
国家自然科学基金项目(31872069)
辽宁省百千万人才工程项目(2016921067)
辽宁省自然科学基金项目(201602659)
关键词
草莓
ARF4
启动子
转录活性分析
strawberry
Auxin response factor 4
promoter
activity analysis
