花生Δ~9-硬脂酰-ACP脱氢酶基因启动子的克隆及功能分析  

Cloning and Functional Analysis of Peanut Δ~9-Stearoyl-Acyl Carrier Protein Desaturase Promoter

作　　者：石磊[1] 苗利娟[1] 齐飞艳[1] 张忠信[1] 高伟 孙子淇[1] 黄冰艳[1] 董文召[1] 汤丰收[1] 张新友[1] SHI Lei MIAO Li-Juan QI Fei-Yan ZHANG Zhong-Xin GAO Wei SUN Zi-Qi HUANG Bing-Yan DONG Wen-Zhao TANG Feng-Shou ZHANG Xin-You(Industrial Crops Research Institute, Henan Academy of Agricultural Sciences / Key Laboratory of Oil Crops in Huanghuaihai Plains, Ministry of Agriculture / Henan Provincial Key Laboratory for Oil Crops Improvement, Zhengzhou 450002, Chin)

机构地区：[1]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,河南郑州450002

出　　处：《作物学报》2016年第11期1629-1637,共9页Acta Agronomica Sinica

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~~

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。The Δ~9-stearoyl-acyl carrier protein desaturase（SAD） plays key roles in determining the ratio of saturated to unsaturated fatty acids in higher plants. The 720 bp 5′ flanking sequence of peanut SAD（Ah SAD） gene was isolated from the genomic DNA of peanut cultivar Yuhua 9326 by nested PCR using genomic walking method. The fragment of 5′ UTR was obtained using 5′ RACE（Rapid Amplification of c DNA End）, confirming that the 720 bp 5′ flanking sequence is promoter fragment based on sequence alignments. Bioinformatics analysis indicated that Ah SAD promoter contained several light, hormone responsive elements and enhancer-like elements as well as CAAT box and TATA box. To study the function of this promoter, we constructed a binary expression vector p BI-PAh SAD by replacing Ca MV35 S promoter of p BI121 with the Ah SAD promoter, which was introduced into Arabidopsis and transiently expressed in peanut, respectively, by Agrobacterium-mediated transformation. Histochemical staining analysis showed that the GUS gene mainly expressed in roots, stem, leaves, cotyledon and peanut pegs. The histochemical staining was observed in peanut whole leaves, while only in Arabidopsis veins of cauline leaves.

关 键 词：花生 Δ9-硬脂酰-ACP脱氢酶基因(SAD) 启动子 基因组步移 GUS报告基因 

分 类 号：S565.2[农业科学—作物学]