桑树成花素基因MaFT的启动子克隆和功能位点分析

Cloning and Functional Site Analysis of the Promoter of Mulberry Flowering Locus T Gene

FT（flowering locus T）是高等植物开花过程中关键的信号整合因子,主要在叶片维管束中合成,通过运输抵达茎顶端分生组织发挥作用。为了研究桑树FT基因的转录调控机制,以白桑种（Morus alba Linn.）品种新一之幼嫩叶片的基因组DNA为模板,根据NCBI数据库公布川桑（Morus notabilis）全基因组中FT同源序列的上游序列设计引物,PCR扩增获得FT基因的启动子片段,其长度为921 bp,命名为MaF TP。通过PLACE和PlantC ARE在线启动子预测工具分析,该序列中除含有TATA-box、CAAT-box等真核生物启动子的核心元件外,还存在脱落酸的响应元件ABRE,生长素响应元件AuxRR-core,水杨酸响应元件TCA-element,生理节律相关顺式作用元件circadian,光响应元件I-box、G-box、MNF1等,以及胚乳表达必需的顺式作用元件Skn-1-motif和抗逆性相关的顺式作用元件W-box、MBS、TC-rich repeats,表明FT基因的转录表达可能受光照、干旱、节律性、脱落酸、生长素、水杨酸等因素的调控,并参与胚乳的形成。将5＇端缺失的5段FT启动子序列（从大到小依次为MaF TP903、MaF TP762、MaF TP542、MaF TP289、MaF TP162）分别与报告基因GUS融合构建表达载体,利用农杆菌转化烟草（Nicotiana benthamiana）植株,通过GUS染色对不同长度的启动子活性进行分析,结果表明：除了MaF TP903的活性较弱外,其余启动子缺失片段均能驱动GUS基因高效表达,且表达量相近。依据研究结果推测：MaF TP启动子的762~903 bp区段可能存在MaF T基因转录表达的负调控元件。

FT（ flowering locus T） is a crucial signal integration factor in flowering process of higher plants. Synthesized mainly in leaf vascular bundle,FT protein is then transported to shoot apical meristem and acts on target genes there. In order to understand the transcriptional regulation mechanism of FT gene in mulberry,genome DNA of young leaves in a white mulberry（ Morus alba Linn.） variety named Shin Ichinose was used as the template,and PCR primers were designed based on the upstream sequence of Morus notabilis FT gene deposited in NCBI database. FT promoter with 921bp was amplified and designated as MaF TP. Promoter analyses using online PLACE and PlantC ARE tools showed that this sequence not only contained TATA-box,CAAT-box and other core components of eukaryotic promoters,but also had abscisic acid responsive element ABRE,auxin responsive element AuxRR-core, salicylic acid responsive element TCA-element,circadian rhythm related cis-element Circadian,light responsive elements such as I-box,G-box and MNF1,endosperm expression essential cis-element Skn-1-motif,and stress-related cis-elements such as W-box,MBS and TC-rich repeats. These results suggested that the transcription of FT gene is regulated by light,drought,circadian rhythm,ABA,auxin,salicylic acid and other factors,and its expressed product participates in the formation of endosperm. Five FT promoter fragments with 5＇ deletion（ named as MaF TP903,MaF TP762,MaF TP542,MaF TP289,MaF TP162 according to length） were fused with GUS reporter gene and their corresponding expression vectors were transformed into the tobacco（ Nicotiana benthamiana） by Agrobacterium. GUS staining showed that except MaF TP903 whose activity was very low,other promoter fragments could drive the expression of GUS gene efficiently,and the expression levels were similar,inferring that a negative regulatory element of FT transcription exists in region from 762 to 903 base pairs in MaF TP promoter.