棉纤维蔗糖合酶基因SS3上游调控序列的克隆及其表达分析

Cloning and Analysis of the Upstream Regulated Sequence of a Cotton Fiber Sucrose Synthase Gene

棉纤维蔗糖合酶基因SS3在棉纤维发育过程中起着重要作用.采用YADE技术克隆了该基因5′上游1717bp的调控区,该调控区含有典型的启动子核心元件TATA box ,以及TATC box、G box、GCN4 -motif、Prolamin box、Skn 1 likemotif、TCA element、HSE和O2 site等各种顺式调控元件和其他一些反应元件.将此序列和报告基因GUS融合在烟草、棉花中表达.组织化学分析结果显示棉花SuSyR序列启动GUS基因在烟草的子房、胎座、种子以及在棉花花蕾与棉铃中表达.在棉花花蕾蕾长为3mm、6mm、9mm和15mm花蕾中表达主要存在于雄蕊及雄蕊管、胎座等器官;在棉铃中,1DPA棉铃的花柱、花药、子房及胚珠中出现了蓝色,6DPA棉铃的子房及胚珠被染成蓝色,在2 0DPA的棉铃中蓝色只出现在胚珠及其纤维中、在胚珠中只有珠心被染成蓝色,在4 0DPA胚珠中只有纤维呈蓝色.研究结果揭示,棉花的SuSyR调控序列启动GUS基因主要在子房、胚珠和纤维等器官和主叶脉、茎微管束等输导组织中表达,在棉花中尤为明显,表明棉纤维蔗糖合酶基因SS3除参与棉花蕾铃发育、纤维素的合成外,还参与了光合产物的运输与分配过程.

A cotton sucrose synthase (EC 2.4.1.13) gene SS3 had been suggested to express preferentially in developing ovules and seedlings,and play an important role in fiber initiation and carbon partition in developing cotton seeds.According to the tissue- and cell-specific expression pattern of SS3 gene,we envisioned that the promoter of SS3 gene was potentially useful in cotton improvement and the research of fiber molecular biology.To this end,5′-flanking sequence of 1717 bp SS3 was amplified by using YADE (Y-shaped adaptor dependent extension) according to the SS3 cDNA.By searching in PlantCARE,69 regulatory elements or motifs were found in the 1 605-bp sequence upstream to the initiation codon.These elements mainly included endosperm elements(GCN4-motif, Skn-1 motif and O2-site),MYB binding site(MRE),light response elements(I-box,G-box),auxin response elements(TGA-box),GA response elements(P-box),and wounding response elements(wound motif). The SS3 promoter was fused to the reporter gene GUS and introduced into tobacco and cotton. The expression pattern of the SS3 promoter was determined by GUS histochemical staining.It was demonstrated that GUS gene expressed mainly in developing and germinating seeds.The GUS activity could also be detected in placenta and ovary wall in the early stage of seed development,but not in the other parts of flower,roots,leaves and stems.In transgenic cotton plants,the GUS activity was detected mostly in leaf vein and vascular bundle of shoot buds,ovules and fibers at various stages.