中文摘要：本发明涉及生物基因工程领域，具体地说是花生转录因子AhJ11‑FAR1‑5基因的克隆及功能表达方法。本发明通过材料的准备与处理、RNA的提取和cDNA的合成、RACE克隆技术，合成出了AhJ11‑FAR1‑5。本发明还通过使用荧光定量RT‑PCR对AhJ11‑FAR1‑5基因干旱胁迫下的表达进行分析，并最终得出AhJ11‑FAR1‑5基因在花生对干旱胁迫的适应性中发挥重要作用。本发明将该基因通过转基因手段导入花生中，转基因植株比对照组具有明显的抗旱特性，说明AhJ11‑FAR1‑5基因能显著提高花生抗旱性。
外文摘要：The invention relates to the field of biology gene engineering, in particular to a method for cloning and functional expression of the peanut transcription factor AhJ11-FAR1-5 gene. AhJ11-FAR1-5 is synthesized through material preparation and treatment, RNA extraction, cDNA synthesis and RACE cloning. Expression of the AhJ11-FAR1-5 gene under drought stress is further analyzed through fluorescent quantitation RT-PCR, and it is concluded that the AhJ11-FAR1-5 gene plays an important role in adaptation of peanuts to drought stress. The gene is imported into peanuts through transgenosis, and a transgenic plant has remarkable drought resistance compared with a control group, indicating that the AhJ11-FAR1-5 gene can improve the drought resistance of peanuts remarkably.
主权项：一种花生转录因子AhJ11-FAR1-5基因的克隆方法，其特征在于，包括以下步骤： (a)材料的准备与处理，包括花生种子的萌发、生长以及干旱胁迫处理； (b)花生幼苗的RNA的提取和cDNA的合成； (c)通过RACE进行克隆 RACE所用试剂盒为Clontech的SMART-RACE试剂盒，RACE扩增基因全长所用引物为3-GPS-AhJ11-FAR1-5:5’-CGTGCTGAGGGTGCAGAAATGA-3’、5-1-AhJ11-FAR1-5:5’-TGTTGCCCGCCGAGAAAGATG-3’、5-2-AhJ11-FAR1-5:5’-CGTAGCAACGCCCTTGATCTGTT-3’、5-3-AhJ11-FAR1-5:5’-ATCCGACATCATCTCACCATCCAC-3’和NGPS-AhJ11-FAR1-5：5’-GTATAATCAGTCACTGGGAAG-3’； RACE克隆产物经过1％琼脂糖凝胶电泳分离后，用UNIQ-10 PCRPurification Kit试剂盒进行纯化，纯化产物连接pGEM-T Easy载体后转化至感受态大肠杆菌，于LB培养基培养，随机挑取扩大培养，再进行PCR扩增检测并测序； 将测序的结果进行拼接后设计全长PCR扩增产物，用UNIQ-10PCRPurification Kit试剂盒纯化，纯化产物与pGEM-T Easy载体连接后转化至感受态大肠杆菌中，用LB培养基培养，之后随机挑取10个阳性克隆进行扩大培养，采用菌液PCR扩增预检测是否有插入片段并测序，扩增引物为AhJ11-FAR1-5-S1：5’-CGCAGTGGTTTCCAATGGATTT-3’和AhJ11-FAR1-5-S2：5’-GCCACACCTGGGTTGGTGGACCCC-3’。
申请号：CN201611019808.X
专利号：CN107058334A
公开/公告号：CN107058334A
申请/专利权人：山东省花生研究所;
发明/设计人：赵小波; 李春娟; 单世华; 张廷婷; 闫彩霞; 王娟
分类号：C12N15/29; C12N15/10; C12Q1/68;
主分类号：C12N15/29
法律状态：实质审查
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