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杂交鹅掌楸LhFB1基因启动子的克隆及活性分析
刘立盘, 钟永达, 杨爱红,等
江西省科学院 生物资源研究所,江西省观赏植物遗传改良重点实验室
摘要:
【目的】研究杂交鹅掌楸LhFB1基因启动子(pLhFB1)的活性,为研究该基因功能及相关机制提供参考。【方法】利用染色体步移法(Genome Walking)克隆LhFB1基因上游5′侧翼调控区序列,利用生物信息学软件PlantCARE分析其包含的顺式作用调控元件。构建pCAMBIA1300-pLhFB1重组载体,对本氏烟草幼苗期叶片进行瞬转注射和GUS染色表达。花序浸染法将重组载体转化至野生型拟南芥,GUS组织染色分析其在T2代转基因植株开花期根、茎、叶和花组织中的表达量,并用qRT-PCR对GUS组织染色进行活性验证。【结果】pLhFB1启动子序列长1 780 bp,含有多个TATA-box和CAAT-box及压力响应、激素响应、光信号转导、代谢循环元件。瞬转结果表明,烟草叶片注射部位有蓝色斑点,说明启动子有活性。遗传转化结果表明,转pLhFB1启动子拟南芥根、茎、叶和花组织中都有不同程度的蓝色,且根和茎中的蓝色斑块较深。qRT-PCR结果说明,pLhFB1启动子在拟南芥根、茎、叶和花组织都有表达,与GUS染色结果基本相符。【结论】克隆得到pLhFB1启动子序列,其在转拟南芥植株各组织中都有活性,但以根和茎中较强。
关键词:  杂交鹅掌楸  pLhFB1启动子  转基因拟南芥  GUS染色
DOI:
分类号:
基金项目:国家自然科学基金项目(31260188);江西省优势科技创新团队计划项目(20161BCB24008);江西省科学院重点项目(2018-YZD2-16);江西省科学院博士基金项目(2016-YYB-01)
Cloning and activity analysis of LhFB1 gene promoter in Liriodendron hybrids
LIU Lipan, ZHONG Yongda, YANG Aihong,et al
Abstract:
【Objective】This research studied the activity of LhFB1 gene promoter in Liriodendron hybrids to provide reference for studying function and related mechanism of this gene.【Method】Genome Walking was used to clone the upstream 5′ flanking regulatory region sequence of LhFB1 gene,and the bioinformatics software PlantCARE was used to analyze the cis-acting regulatory elements.The pCAMBIA1300-pLhFB1 recombinant vector was constructed,and tobacco leaves were instantaneously injected for GUS staining at the seedling stage.The recombinant vector was transformed to wild Arabidopsis thaliana by inflorescence impregnation for testing tissue expression.The histochemical stain of GUS was used to analyze expression quantity of root,stem,leaf and flower in T2 transgenic plant at flowering period,and the activity was verified by qRT-PCR.【Result】The length of pLhFB1 promoter sequence was 1 780 bp,and it contained multiple TATA-box and CAAT-box,stress response,hormone response,optical signal transduction and metabolic cycle elements.The instantaneous injection results showed that the injection sites on tobacco leaf had blue spots,indicating its activity.Genetic transformation results showed that root,stem,leaf and flower of transgenic A.thaliana with pLhFB1 promoter had blue color at different degrees,and root and stem had very dark blue patches.The qRT-PCR indicated that pLhFB1 promoter had activity in root,stem,leaf and flower of A.thaliana,which was consistent with GUS staining results.【Conclusion】The promoter of pLhFB1 was cloned successfully and it had activity in all tissues of A.thaliana with the strongest in root and stem.
Key words:  Liriodendron hybrids  pLhFB1 promoter  transgenic Arabidopsis thaliana  GUS staining