甘蓝型油菜脂肪酸脱氢酶2兔源多克隆抗体的制备与应用  

Preparation and application of rabbit-derived polyclonal antibody against fatty acid desaturase 2 in Brassica napus

作　　者：熊丹 周霆 田方艳 饶力群[1] 陈松[3] 汪启明[1] XIONG Dan;ZHOU Ting;TIAN Fang-yan;RAO Li-qun;CHEN Song;WANG Qi-ming(College of Bioscience and Biotechnology, Hunan Agricultural university, Changsha 410128, China;Hunan Forevertech Biotechnology Co., Ltd., Changsha 410000, China;Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Cotton and Rapeseed, Ministry of Agriculture, Nanjing 210014, China)

机构地区：[1]湖南农业大学生物科学技术学院,湖南长沙410128 [2]湖南远泰生物技术有限公司,湖南长沙410000 [3]江苏省农业科学院经济作物研究所/农业部长江下游棉花与油菜重点实验室,江苏南京210014

出　　处：《江苏农业学报》2020年第4期851-857,共7页Jiangsu Journal of Agricultural Sciences

基　　金：国家转基因生物新品种培育科技重大专项(2018ZX08011-01B);湖南省重点领域研发计划项目(2019DK2011)。

摘　　要：脂肪酸脱氢酶2(FAD2)是调控油菜种子油酸含量的关键酶。为建立其灵敏、便捷的免疫学检测方法,本研究合成了BnFAD2(UniProtKB:C3W518)中2个多肽片段,与KLH载体蛋白偶联,混合免疫新西兰大白兔,分离纯化得到了油菜BnFAD2兔源多克隆抗体。应用于FAD2-RNAi高油酸转基因油菜品系W-4不同世代果荚及成熟叶片总蛋白质的免疫印迹检测,结果表明,该兔源多克隆抗体,可在油菜内质网成分中识别FAD2,特征性条带大小为95000。在果荚总蛋白质样本中,WT的FAD2含量高于转基因材料W-4,而在叶片总蛋白质样本中WT和W-4的FAD2含量相同。本研究成功制备出BnFAD2兔源多克隆抗体,并首次验证出油菜内源性FAD2以多聚体形式行使功能,为BnFAD2快速检测以及进一步研究BnFAD2对油脂稳定性影响提供研究工具。Fatty acid desaturase 2(FAD2)is the key enzyme regulating oleic acid content.The immunological methods for its rapid detection are limited by the lack of antibodies.In this study,the Brassica napus FAD2 polyclonal antibodies were successfully prepared with the rabbit immunized by two synthesized polypeptides from BnFAD2(UniProtKB:C3W518).The pods or mature leaves extracts of different generations of W-4 which was the RNAi transgenic material were detected by western blot.The results showed that the rabbit-derived polyclonal antibody could recognize the FAD2 in the endoplasmic reticulum,and the size of characteristic band was 95000.The content of FAD2 in WT was higher than that in transgenic material W-4 in pods extracts,but the content of FAD2 in WT was the same as that in W-4 in leaves extracts.For the first time,the endogenous BnFAD2 is verified to exercise function in the form of polymer,providing a research tool for rapid detection of BnFAD2 and further study of the effect of BnFAD2 on oil stability.

关 键 词：脂肪酸脱氢酶2 多克隆抗体 油酸 甘蓝型油菜 

分 类 号：Q786[生物学—分子生物学]