摘要
为了研究巴西固氮螺菌(Azospirillum brasilense)在植物体内的定殖,利用酶切连接的方法,以增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因与表达载体p VLT-33为基本元件,构建了重组表达载体p VLT-EGFP,电转巴西固氮螺菌R7细胞,并利用实时荧光定量PCR(q PCR)研究了不同温度、不同时间EGFP m RNA的表达情况。酶切及测序结果表明,成功构建了p VLT-EGFP载体,并在荧光显微镜下观察到绿色荧光蛋白表达;q PCR结果显示:30℃,诱导9 h EGFP基因的表达水平最高。本研究成功构建了重组表达载体p VLT-EGFP,为实现p VLT-EGFP的可控表达及研究固氮菌在植物体内的定殖规律及促生长机理提供了一种有效的途径。
In order to study the colonization of Azospirillum brasilense in plants,an Escherichia coli-A.brasilense shuttle vector p VLT-EGFP expressed in A.brasilense was constructed by integrating the enhancedgreen fluorescent protein（EGFP） gene of pc DNA3.1-EGFP into the broad host range expression plasmidp VLT-33.The expression vector was transformed into A.brasilense R7 by electroporation methods and theexpression of EGFP gene was observed under fluorescent microscope.Using IPTG as an inducing agent,theeffect of different time and temperature of IPTG on the expression of p VLT-EGFP was analyzed and the resultsindicated that the optimal condition for expression was at the temperature of 30℃.The highest expression levelof EGFP gene was detected 9 h after inducing.This study enriched A.brasilense genetic transformation systemand provided a robust tool to study the colonization of A.brasilense and growth-promoting effect on plants.
出处
《中国农学通报》
2015年第5期96-101,共6页
Chinese Agricultural Science Bulletin
基金
2013年河南省基础前沿项目"巴西固氮螺菌定殖及其促生长机理研究"(132300413219)
2012年河南省重大科技专项"新型复合微生物肥料关键技术研究及产业化"(121100110100)
2013年省级科技基础平台建设专项经费项目"新型农业微生物菌剂的研制及应用"[豫财政(2013)435号]
2014年河南省科学院预研项目"固氮芽孢杆菌的筛选及其竞争适应能力研究"
