茉莉花香气相关基因JsTPS启动子的克隆与活性分析  

Cloning and Activity Analysis of JsTPS Promoter of Jasminum sambac Aroma Related Gene

作　　者：林俊杰 李小婷[1] 陈雪津 崔萌 吉玉婷 叶乃兴[1] 陈桂信[1] LIN Junjie;LI Xiaoting;CHEN Xuejin;CUI Meng;JI Yuting;YE Naixing;CHEN Guixin(College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China)

机构地区：[1]福建农林大学园艺学院,福建福州350002

出　　处：《热带作物学报》2022年第1期43-50,共8页Chinese Journal of Tropical Crops

基　　金：福州茉莉花茶科技与全球重要农业文化遗产联合研究中心专项(No.KH1501920);福州市科技计划项目(No.2019-N-15)。

摘　　要：以茉莉(Jasminum sambac)的花苞为材料,采用染色体步移技术,分离出JsTPS基因的5’端调控序列,对该启动子序列进行顺式作用元件预测。根据茉莉花TPS基因启动子的顺式作用元件分布,扩增出5个不同片段长度的启动子,分别命名为JsTPS-1(494 bp)、JsTPS-2(689 bp)、JsTPS-3(1016 bp)、JsTPS-4(1466 bp)和JsTPS-5(2040 bp),应用GATEWAY技术,构建5个不同长度融合GUS基因的植物表达载体,用农杆菌GV3101侵染烟草叶片,对转化后的烟草叶片进行GUS染色。检测不同片段长度启动子活性,找出该启动子的关键活性区域,对其功能进行初步分析,序列分析结果表明:克隆得到的JsTPS启动子序列长度为2040bp,该序列包含TATA-box、G-box、CAAT-box等启动子核心元件、光响应元件(ACE、ATCT-motif、Box 4、3-AF1 binding site、G-Box、Sp1和GT1-motif)和激素响应元件[茉莉酸甲酯响应元件(TGACG-motif、CGTCA-motif)、ABRE脱落酸响应元件、生长素响应元件(TGA-box、TGA-element)、水杨酸响应元件(TCA-element)、赤霉素响应元件(GARE-motif)]等,说明该基因的表达可能受到光照、激素(ABA、生长素、茉莉酸、茉莉酸甲酯和水杨酸)的诱导。GUS染色结果表明:其JsTPS-1启动子几乎不染色,JsTPS-2染色相对较弱,而JsTPS-3的染色程度高于JsTPS-4、JsTPS-5,且是5个不同缺失片段中染色最深的片段;GUS酶活性检测结果显示,不同缺失片段长度酶活性与染色结果一致,JsTPS-1的GUS酶活性最低,随着启动子片段加长,GUS酶活性增强,在片段长度为JsTPS-3时酶活性最强,在JsTPS-4、JsTPS-5片段长度,GUS酶活下降。JsTPS启动子至少包括–788~0 bp这段区域才能驱动JsTPS起始转录,相比较其他片段,发现在–1016~0 bp区域时启动子的活性表现最强。推测可能在–1016~–689 bp中含有水杨酸响应(TCA-element)、光响应(3-AF1 binding site)等元件增强启动子的活性,而在–1466~–1016bp中含有赤霉素负调控响应元件减弱JsJasmine(Jasminum sambac) bud was used as the material, and the chromosome walking technology was used to isolate the 5’ end regulatory sequence of JsTPS, and predict the promoter cis-acting element for this sequence. According to the cis-acting element distribution of the TPS gene promoter in jasmines, 5 promoters with different fragment lengths were amplified, named JsTPS-1(494 bp) and JsTPS-2(689 bp), JsTPS-3(1016 bp), JsTPS-4(1466 bp) and JsTPS-5(2040 bp), then 5 plant expression vectors with different fragment lengths fused with GUS gene by GATEWAY technology were constructed. Tobacco leaves were infected with GV3101 Agrobacterium to perform Gus staining. Sequence analysis showed that the sequence length of the cloned JsTPS promoter was 2040 bp. It contained the core elements of the promoter such as TATA-box, G-box, CAAT-box and light response elements(ACE,ATCT-motif, Box 4,3-AF1 binding site, G-Box, Sp1 and GT1-motif), hormone-related response elements such as methyl jasmonate response Element(TGACG-motif, CGTCA-motif), ABRE abscisic acid response element, auxin response element(TGA-box,TGA-element), salicylic acid response element(TCA-element), gibberellin response element(GARE-motif), etc., which indicating that the expression of this gene may be induced by light and hormones(ABA, auxin, jasmonic acid, methyl jasmonate and salicylic acid). The results of GUS staining showed that the JsTPS-1 promoter hardly stained, and the staining of JsTPS-2 was relatively weak. The staining degree of JsTPS-3 was higher than that of JsTPS-4 and JsTPS-5,and it was stained in five deepest deletion fragments. According to the GUS enzyme activity test results, the enzyme activity results of different missing fragment lengthswere consistent with the staining results. The GUS enzyme activity of JsTPS-1 was the lowest. As the promoter fragment lengthened, the GUS enzyme activity increased. When the fragment length was JsTPS-3, the activity was the strongest. The JsTPS promoter must include at least –788 bp to 0 bp to drive i

关 键 词：茉莉花 萜类合成酶 启动子 顺式作用元件 启动子活性 

分 类 号：S685.16[农业科学—观赏园艺]