圆叶葡萄‘Noble’的芪合成酶STS启动子的功能分析

Functional analysis of the stilbene synthetase gene promoter from the Muscadine rotundifolia‘Noble'

【目的】分析圆叶葡萄‘Noble’的芪合成酶(stilbene synthetase,STS)基因上游调控序列的顺式作用元件及其功能,为研究白藜芦醇在葡萄抗病机制中的作用提供理论基础。【方法】在前期通过染色体步移法分离得到圆叶葡萄‘Noble’芪合成酶Mr STS基因上游调控序列的基础上,利用Plant CARE在线启动子预测工具对其顺式作用元件进行初步预测,构建该启动子5’端侧翼序列缺失表达载体,启动报告基因绿色荧光蛋白(green fluorescent protein,GFP)表达,并通过农杆菌介导法真空瞬时转化离体葡萄叶片,研究激素和霜霉菌诱导条件下GFP蛋白的活性差异。【结果】PlantCARE在线预测结果表明,Mr STS基因上游调控序列含有启动子的特定结构,如TATA-box、CAAT-box,另外也含有一些与逆境相关的顺式作用元件,如ABA响应元件ABRE、Me JA响应元件CGTCA-motif、赤霉素响应元件P-box、激发子响应元件Box-W1和胁迫响应元件TC-rich repeats等;不同诱导条件下,5’端侧翼序列缺失表达载体启动GFP蛋白的活性存在显著差异。【结论】Mr STS基因上游调控序列含有启动子核心区域和与逆境相关的顺式作用元件,并受ABA、Me JA和霜霉菌的诱导。

【Objective】This paper analyzes the cis-regulation elements and functions of the stilbene synthe-tase gene promoter from the Muscadine rotundifolia‘Noble＇and provides a theoretical basis to study theimportant function of resveratrol in grape resistant mechanisms. Muscadine rotundifolia, a special germ-plasm resource of grapevines, is immune to many kinds of grape fungal pathogens. It contained 20 pairs ofchromosomes which represents one more pair than found in other grapevines. Resveratrol, as a phytoalex-in, plays an important role as a grape resistant mechanism. Stilbene synthase（STS） is the key enzyme forresveratrol synthesis. Therefore, it＇s critical to study the regulation and expression of the stilbene synthase（STS） gene.【Methods】The upstream region sequence of the Mr STS gene, 1 653 bp, was obtained by using aTAKARA genome-walking kit in our lab. The cis-regulation elements of the Mr STS promoter were predict-ed by using Plant CARE online. Based on the vector p HGWFS7 and the Gateway method, four Mr STS 5＇end flanking sequence definition expression vectors were constructed to induce the expression of the greenfluorescent protein（GFP）. The GFP expression in the control vector was induced by using the Ca MV 35 S promoter. The 2-4 top leaves of the Vitis vinifera‘Thompson Seedless＇ tissue culture plantlets were infect-ed in vitro with Agrobacterium tumefaciens, and water was used as the blank control. Four A. tumefaciens stains were cultured until the OD600 of 0.4-0.6 strains were collected and suspended with an induced buf-fer, and were further cultured for 3 hours. These strains were then used to infect the grape leaves. The infection process was carried out through applying a vacuum transient transformation. After that, the infected leaves were first cultured on wet filter papers under dark conditions for 24 h and then light conditions for24 h. The stress treatments of the infected leaves were performed for 24 h using 1 mmol·L- 1hormones（ABA, SA, Me JA, GA and JA）, 105spores·m L-1Plasmopara viticola and water as the blank control. The activity of the GFP protein was detected with a Spectrum Max M5 ELISA after induction by hormones and Plasmopara viticola.【Results】The results of using Plant CARE online showed that the upstream regions of Mr STS contained some specific structures related to promoter（TATA-box and CAAT-box）, some hormone responsive cis-regulation elements（ABA responsive element ABRE, Me JA responsive element CGTCAmotif, and gibberellins responsive element P-box）, some biotic stress-related cis-regulation elements（elicitor responsive element Box-W1, defense and stress responsive element TC-rich repeats）, and some abiotic stress-related cis-regulation elements（MYBHv1 binding site CCAAT-box and MYB binding site which are involved in the drought-inducible MBS element）. Based on the Gateway vector p HGWFS7, five vectors were constructed as p HGWFS7/P35S：：GFP, p HGWFS7/Pmr STS1：：GFP, p HGWFS7/Pmr STS2：：GFP, p HGWFS7/Pmr STS3：：GFP, and p HGWFS7/Pmr STS4：：GFP vectors with the GFP gene expression promoted by35 S, Mr STS1（whole Mr STS promoter sequence）, Mr STS2（Mr STS promoter lock of one P- box and one Box-W1 in 5＇end flanking sequence）, Mr STS3（Mr STS2 promoter lock of one ABRE element in 5＇end flanking sequence）, and Mr STS4（Mr STS3 promoter lock of one Box-W1 and one HSE element in 5＇end flanking sequence） promoter, respectively. The GFP data showed that the intensity of the green fluorescent lights for the p HGWFS7/P35S：：GFP- infected leaves was stronger than that in the p HGWFS7/Pmr STS：：GFP-infected leaves. Green fluorescent lights were hardly observed in the induced buffer-infected leaves.Under different stress induction conditions, significant differences for GFP activity in infected leaves occurred for different vectors. For leaves infected with p HGWFS7/P35S：：GFP, slight differences occurred through using many different types of treatments, which showed that the 35 S promoter was the constitutive promoter. The relative intensity of the GFP protein in the leaves infected by p HGWFS7/Pmr STS3：：GFP,and p HGWFS7/Pmr STS4：：GFP were obviously lower than that in p HGWFS7/Pmr STS1：：GFP and p HGWFS7/Pmr STS2：： GFP under 1 mmol·L-1ABA treatment. This result showed that the ABRE element plays an important role in Mr STS induction by ABA. However, none significant differences in leaves infected by the four p HGWFS7/Pmr STS：：GFPs occurred under 1 mmol·L- 1SA and JA treatments, which meant that the Mr STS promoter in our study was not induced by SA and JA. These results agreed with the prediction from using Plant CARE online. Though there were no significant differences in leaves infected by the four p HGWFS7/Pmr STS：：GFPs under 1 mmol·L- 1Me JA, the relative fluorescent intensity of GFP was very strong, which indicated that the Mr STS was affected by Me JA because of the CGTCA- motif. Under 1mmol·L-1GA and Plasmopara viticola treatment, the relative fluorescent intensity was stronger in leaves infected by p HGWFS7/Pmr STS1：：GFP, but lower in leaves infected by p HGWFS7/Pmr STS4：：GFP, which showed that Box-W1 was important for Mr STS expression during pathogen infection.【Conclusion】These results confirmed that the increased upstream regions of Mr STS were due to the promoter sequence of the gene Mr STS which not only contained specific structures as a promoter but also some stress-related cisregulation elements. These elements can be induced by ABA, Me JA and Plasmopara viticola.