摘要
miRNA是一类内源非编码单链小RNA,广泛存在于动植物中,主要通过切割靶基因或者抑制靶基因表达参与细胞分化、生长和发育等多种生物学调控。目前缺少一种快速简便验证miRNA靶基因的方法。本研究基于前期筛选到的小麦特有的miRNA——Tae-miR9666a,合成其前体及前体突变体,并连接到表达载体上,构建含有GFP(绿色荧光蛋白)标记的靶基因过表达载体,共转化烟草,通过观察GFP的荧光亮度判断靶基因的切割情况,以此来验证miRNA与靶基因的相互作用。结果发现,共转miRNA前体的烟草叶片的荧光亮度较低,表明靶基因被miRNA切割,其后的GFP无法正常表达;共转miRNA前体突变体的烟草叶片的荧光亮度与单转靶基因的相当,表明miRNA前体突变体未切割靶基因,导致GFP正常表达。本实验有效证明了miRNA的靶基因切割情况,也表明利用烟草验证miRNA与靶基因相互作用是可行的。
miRNAs are a class of endogenous non-coding small single-stranded RNA,which are widely found in the plants and animals.They are involved in various biological regulations such as cell differentiation,growth and development mainly by cutting target genes or inhibiting target gene expression.At present,it is short of a quick and simple method for verification of miRNA target genes.Based on the pre-screened wheat specific miRNA—Tae-miR9666a,this study was conducted to synthesize its precursor and precursor mutant,connect them to the expression vector,construct the target gene overexpression vector containing GFP(green fluorescent protein) label and co-transform it into tobacco,and verify the interaction between miRNA and the target gene by determining the cleavage of target gene through observing the fluorescence brightness of GFP.The results showed that the fluorescence brightness of the tobacco leaves with co-transmutation miRNA precursors was lower,indicating that the target gene was cut by miRNA,and the subsequent GFP could not be expressed normally.The fluorescence brightness of tobacco leaves of co-transmutation miRNA precursor mutants was comparable to that of single transmutation target genes,indicating that miRNA precursor mutants did not cut the target genes,resulting in normal GFP expression.This experiment effectively demonstrated the cleavage of miRNA target genes,and also indicated that it was feasible to verify the interaction between miRNA and target genes by using Nicotiana benthamiana.
作者
房春豪
陈志浩
王开
汪晓璐
徐文竞
祁广
马朋涛
韩冉
刘成
Fang Chunhao;Chen Zhihao;Wang Kai;Wang Xiaolu;Xu Wenjing;Qi Guang;Ma Pengtao;Han Ran;Liu Cheng(College of Life Sciences,Yantai University,Yantai 264000,China;Crop Research Institute,Shandong Academy of Agricultural Sciences/Key Laboratory of Biology and Genetics Breeding of Wheat in Northern HuangHuai Region,Ministry of Agriculture/National Engineering Laboratory of Wheat and Maize,Jinan 250100,China)
出处
《山东农业科学》
北大核心
2024年第2期1-7,共7页
Shandong Agricultural Sciences
基金
山东省自然科学基金项目(ZR2020MC098)
国家自然科学基金项目(32001544)
小麦玉米国家工程实验室开放课题(2018LYZWS05)
山东省重点研发计划(重大科技创新工程)项目(2021LZGC009)。
作者简介
房春豪(1997-),男,硕士研究生,主要从事小麦新品种培育工作,E-mail:fangchunhao0213@163.com;通信作者:韩冉(1982-),女,博士,助理研究员,主要从事小麦新品种培育工作,E-mail:hr022cn@aliyun.com;通信作者:刘成(1979-),男,博士,研究员,研究方向为小麦细胞遗传学,E-mail:lch6688407@163.com。
