论文部分内容阅读
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。
Δ ~ 9-stearoyl-ACP dehydrogenase (SAD) is the key enzyme that determines the ratio of saturated fatty acids to unsaturated fatty acids in plants. The genomic DNA of Yuhua 9326, a peanut variety, was used as a template to generate a 720 bp fragment upstream of ATG, a start codon of Ah SAD, by the genome walking technique. The 5’UTR sequence of this gene was obtained by RACE method. The 720 bp fragment was identified as Ah SAD promoter region by sequence alignment. PLACE online promoter prediction analysis showed that this sequence contains the essential elements TATA-box and CAAT-box, which are necessary for eukaryotic promoter, and contain multiple cis-elements related to light-induced and hormone responses. The Ah SAD promoter fragment was substituted for the CaMV35S promoter in pBI121 plasmid to drive downstream GUS gene expression to construct plant expression vector pBI-PAh SAD. Arabidopsis thaliana was transformed by Agrobacterium tumefaciens method and transiently expressed in different tissues of peanut. GUS histochemical staining was used to study its expression characteristics. The results showed that AhSAD promoter mainly regulated downstream genes in roots, stems, leaves and cotyledons of Arabidopsis thaliana and peanut receptors, and GUS activity was also detected in peanut fruit needles. The stem leaves of Arabidopsis thaliana had only veins GUS activity in the entire leaf of peanut.