花生Δ~9-硬脂酰-ACP脱氢酶基因启动子的克隆及功能分析

发布时间：2019-06-26 18:01

【摘要】：Δ~9-硬脂酰-ACP脱氢酶(SAD)是决定植物体内饱和脂肪酸与不饱和脂肪酸比值的关键酶。以花生品种豫花9326基因组DNA为模板,通过基因组步移技术,克隆到花生Δ~9-硬脂酰-ACP脱氢酶基因(Ah SAD)起始密码子ATG上游720 bp片段,利用5'RACE方法获得了该基因的5'UTR序列,通过序列比对确定720 bp片段为Ah SAD启动子区域。PLACE在线启动子预测分析表明,该序列具有真核生物启动子必需的核心元件TATA-box和CAAT-box,含有多个与光诱导和激素响应相关顺式序列元件。将Ah SAD启动子片段替换pBI121质粒中的CaMV35S启动子驱动下游GUS基因表达,构建植物表达载体pBI-PAh SAD。通过农杆菌介导法转化拟南芥和在花生不同组织中瞬时表达,利用GUS组织化学染色研究其表达特性。表明在拟南芥和花生受体中,AhSAD启动子主要调控下游基因在根、茎、叶片和子叶中表达,在花生的果针中也检测到GUS活性;拟南芥的茎生叶只有叶脉中具有GUS活性,而花生整个叶片中都具有GUS活性。
[Abstract]:螖 ~ 9-stearyl-ACP dehydrogenase (SAD) is the key enzyme to determine the ratio of saturated fatty acids to saturated fatty acids in plants. Using genomic DNA of peanut variety Yuhua 9326 as template, 720 bp fragment upstream of (Ah SAD) initiation codon ATG of peanut 螖 ~ 9-stearyl-ACP dehydrogenase gene was cloned by genomic step technique. The 5'UTR sequence of peanut 螖 ~ 9-stearyl-ACP dehydrogenase gene was obtained by 5'RACE method. 720 bp fragment was identified as Ah SAD promoter region by sequence alignment. The sequence has the core elements TATA-box and CAAT-box, necessary for eukaryotic promoter to contain a number of sequence elements related to light induction and hormone response. The downstream GUS gene expression was driven by replacing the CaMV35S promoter in pBI121 plasmid with Ah SAD promoter fragment, and the plant expression vector pBI-PAh SAD. was constructed. The transient expression of Arabidopsis thaliana and peanut in different tissues was transformed by Agrobacterium tumefaciens. The expression characteristics of Arabidopsis thaliana and peanut were studied by GUS histochemical staining. The results showed that in Arabidopsis thaliana and peanut receptors, AhSAD promoter mainly regulated the expression of downstream genes in roots, stems, leaves and cotyledons, and also detected GUS activity in peanut fruit needles. Arabidopsis thaliana only had GUS activity in leaf veins, while peanut leaves had GUS activity in the whole leaf.
【作者单位】：河南省农业科学院经济作物研究所/农业部黄淮海油料作物重点实验室/河南省油料作物遗传改良重点实验室/花生遗传改良国家地方联合工程实验室;
【基金】：国家高技术研究与发展计划(863计划)项目(2013AA102602-6) 河南省重大科技专项(141100110600) 国家现代农业产业技术体系建设专项(CARS-14) 河南省现代农业产业技术体系建设项目(S2012-5) 河南省农业科学院优秀青年基金(2013YQ11)资助~~
【分类号】：S565.2

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