摘要
微小RNA(miRNA)是一类内源性非编码的单链小RNA,对植物的生长发育起着重要调控作用。建立高效的桑树瞬时表达体系是研究桑树miRNA及其靶基因功能的有效手段。将川桑(Morus notabilis)基因组中克隆的miRn51前体序列连接到PLGNL载体上,构建了PLGNL-35S-miRn51重组质粒,通过农杆菌介导其在桑树叶片中瞬时表达。采用GUS组织化学染色、多酚氧化酶活性检测以及荧光定量PCR等方法,分析不同缓冲液、重组农杆菌注射菌液浓度、转化时间、桑树品种等试验条件对农杆菌介导的桑树叶片瞬时表达体系转化效率、miRn51及靶基因表达水平的影响,当采用1号缓冲液和菌液D(600 nm)=0.6的重组农杆菌进行瞬时转化时,转化桑树叶片3 d后miRn51的表达量最高,其靶基因Mn PPO2的表达水平受到明显抑制,不同桑树品种对瞬时表达体系的瞬时表达效率有一定影响。以上结果表明,建立的农杆菌介导的桑树瞬时表达体系能简便、有效、快捷地验证桑树miRNA对其靶基因的调控作用。
MicroRNAs（ miRNA） are a class of non-coding endogenous single-strand small RNAs. They play important roles in regulation of plant growth and development. Establishing an efficient transient expression system is an effective method to study the functions of miRNA and its target genes in mulberry. In present study,miRn51 precursor sequence was cloned from Morus notabilis genome and ligated with vector PLGNL to construct recombinant plasmid PLGNL-35SmiRn51,and then PLGNL-35S-miRn51 was transformed into mulberry leaves via agrobacterium. By using the methods of GUS histochemical staining,polyphenol oxidase activity assay and fluorescence quantitative PCR,the effect of experimental conditions, such as different injection buffer,recombinant agrobacterium concentration,transformation time and mulberry variety,on transformation efficiency of mulberry leaf transient expression system mediated by agrobacterium and on the expression level of miRn51 and its target gene were analyzed. The results indicated that after injection with recombinant agrobacterium at concentrations of D（ 600 nm） = 0. 6 mixed with No. 1 injection buffer for three days,the expression level of miRn51 inmulberry leaf was the highest and its target gene Mn PPO2 was significantly inhibited. Different mulberry varieties has a certain impact on transformation efficiency. This established mulberry transient expression system mediated by agrobacterium provides a simple,effective,and instant approach to study the regulatory function of mulberry miRNA on its target gene.
出处
《蚕业科学》
CAS
CSCD
北大核心
2015年第1期10-17,共8页
ACTA SERICOLOGICA SINICA
基金
国家高科技研究发展计划"863"项目(No.2013AA10060-5)
商务部市场运行司茧丝绸产业公共服务体系建设项目(No.3)
关键词
桑树
根瘤农杆菌
瞬时表达
微小RNA
靶基因
Morus L.
Agrobacterium tumefaciens
Transient expression
MicroRNA
Target gene
