农杆菌浸润瞬时表达分析番木瓜miR162a的研究(英文)  

Transient Assays of Papaya miR162a Processing by Agro-infiltration

作　　者：彭军[1,2] 杨腊英[1] 王国芬[1] 郭立佳[1] 汪军[1] 黄俊生[1] 

机构地区：[1]中国热带农业科学院环境与植物保护研究所农业部热带农林有害生物入侵监测与控制重点开放实验室海南省热带农业有害生物监测与控制重点实验室,海南儋州571737 [2]中国农业大学,北京100193

出　　处：《热带作物学报》2011年第10期1833-1837,共5页Chinese Journal of Tropical Crops

基　　金：国家科技支撑计划(2008BD02);农业部行业科技项目(200903049);中国热带农业科学院环境与植物保护研究所中央级公益性科研院所基本科研业务费(2009hzs1J007)

摘　　要：农杆菌浸润瞬时表达方法不仅在蛋白互作、鉴定基因沉默抑制子及基因功能研究方面得到广泛应用,目前还应用在小RNA的研究中。在对植物microRNA的研究中,农杆菌浸润方法通过将将转基因导入农杆菌浸润本生烟瞬时表达,可以快速、稳定地对植物microRNA进行检测和鉴定,样品在几天内就可分析完成,比获得稳定遗传的转基因有优势。笔者借鉴国外拟南芥microRNA瞬时表达分析的方法,克隆了番木瓜的miR62a前体序列连接到双元表达载体pSuper1300上构建成35S::cpa-miR162a质粒,导入农杆菌GV3101后浸润本生烟,浸润后48 h提取本生烟叶片总RNA后通过miRNA northern blot检测到miR162a,而本生烟和番木瓜自身则无法检测到低丰度的miR162a。本文介绍的农杆菌浸润瞬时表达方法能够在体外加工产生成熟的miRNA,该技术有助于对microRNA的结构-功能的相关性及靶标验证进一步研究。The most widely used method for small RNA analysis in plants is agro-infiltration, which makes use of Agrobacterium tumefaciens to deliver transgenes into leaf cells of Nicotiana benthamiana. Compared to the traditional method of stably transformed plants, agro-infiltration is more rapid, and samples can be analyzed just a few days after inoculation. Here, a method was described for analysis of papaya mir162a processing using agro- infiltration of N. benthamiana leaves. Cpa-mir162a precursor was amplified by PCR from papaya genomic DNA and ligased to pSuperl300 generating the final binary plasmid and then delivered to Agrobacterium tumefaciens strain GV3101 for agro-infiltration of N. benthamiana leaves. RNA was analyzed by Northern blot after 48 h inoculation, mature miR162a was detected, but not from wild type N. benthamiana leaves and papaya leaves because the low abundance of mir162a. This agro-infiltration method would help to the structure-function analysis and processing mechanism of papaya microRNAs in vitro.

关 键 词：番木瓜 瞬时表达分析 农杆菌浸润 MICRORNA 

分 类 号：Q78[生物学—分子生物学]