山杏种子PsMATE40基因启动子的克隆及驱动表达分析  

Cloning of the Promoter of MATE40 Gene from Prunus sibirica Seeds and Analysis of Gene Expression Driven by Promoter

作　　者：王紫睿 刘潇菡 修宇 林善枝 WANG Zi-rui;LIU Xiao-han;XIU Yu;LIN Shan-zhi(National Engineering Laboratory for Tree Breeding,Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants,Ministry of Education,the Tree and Ornamental Plant Breeding and Biotechnology Laboratory of National Forestry and Grassland Administration,Beijing Forestry University,Beijing 100083)

机构地区：[1]北京林业大学生物科学与技术学院林木育种国家工程实验室林木、花卉遗传育种教育部重点实验室树木花卉育种生物工程国家林业和草原局重点实验室,北京100083

出　　处：《生物技术通报》2024年第11期192-201,共10页Biotechnology Bulletin

基　　金：“十四五”国家重点研发计划(2022YFD2200400);国家自然科学基金面上项目(31972592)。

摘　　要：【目的】转运蛋白PsMATE40在山杏种子苦杏仁苷转运中起到重要作用。克隆PsMATE40基因特异启动子并分析其驱动基因表达活性,为PsMATE40基因及其启动子的应用奠定基础。【方法】依据前期克隆得到的PsMATE40基因,以山杏种子基因组DNA为模板,通过染色体步移克隆得到PsMATE40基因特异启动子全长序列(命名为ProPsMATE40),预测分析其顺式调控元件,采用GUS组织化学染色分析检测启动子活性;利用无缝克隆方法分别构建组成型启动子CaMV35S和特异启动子ProPsMATE40驱动的PsMATE40基因的植物表达载体,开展农杆菌介导的烟草瞬时转化,利用RT-qPCR技术比较分析它们驱动PsMATE40基因的转录表达。【结果】山杏种子PsMATE40基因启动子全长序列为1964 bp,含有2种启动子核心元件(CAAT-box和TATA-box)、多种激素信号(生长素、茉莉酸和水杨酸等)响应元件和胁迫(光、干旱和损伤等)响应元件以及种子发育调控元件等,自身特异启动子ProPsMATE40驱动的PsMATE40基因表达水平明显高于CaMV35S。【结论】山杏种子PsMATE4基因表达可能受植物激素以及生物与非生物胁迫等多种因素的复杂调控,而自身启动子可有效促进PsMATE40基因的转录表达。【Objective】The transporter protein PsMATE40 has been identified with an important role in the transport of seed amygdalin of Prunus sibirica.The aim of this study is to clone the specific promoter of PsMATE40 gene from P.sibirica seeds and to explore PsMATE40 expression driven by its native promoter,which should provide an important foundation for the application of PsMATE40 and its specific promoter.【Method】Based on our recent obtained sequence of PsMATE40 gene from the P.sibirica seeds,the full-length promoter fragment(designated as ProPsMATE40)of PsMATE40 gene was cloned by genome walking technique with genomic DNA of P.sibirica seeds as a template,and the cis-acting elements were identified within its promoter region by online programs.The GUS staining analysis was conducted to examine transcriptional activity of PsMATE40 gene promoter.Importantly,the plant expression vectors ofPsMATE40 driven self-specific promoter(ProPsMATE40)and constitutive promoter(CaMV 35S)were constructed by the seamless cloning.Then it was transiently transformed into tobacco leaves by Agrobacterium-mediated method,and the transcriptional expression of PsMATE40 gene driven by them was comparatively analyzed.【Result】The full-length promoter sequence of PsMATE40 gene from P.sibirica seeds was 1964 bp in length,containing two core promoter regulatory elements(CAAT-box and TATA-box)and several cis-acting regulatory elements in response to the hormone(auxin,jasmone acid and salicylic acid),stress(light,drought and wounding)and seed development,and importantly,the transcriptional level of PsMATE40 driven by its self-specific promoter(ProPsMATE40)was significantly higher compared with under the constitutive promoter(CaMV35S).【Conclusion】The expression of PsMATE40 gene from P.sibirica seeds is regulated by the complicated factors involved in plant hormones and biotic and abiotic stresses,and the self-specific promoter could effectively promote transcriptional expression of PsMATE40 gene.

关 键 词：山杏种子 苦杏仁苷 PsMATE40基因 特异启动子 克隆 顺式调控元件 驱动表达分析 

分 类 号：S662.2[农业科学—果树学] Q943.2[农业科学—园艺学]