烟草BtCry1Ac基因叶绿体转化载体的构建与验证  

Construction and Validation of Chloroplast Transformation Vector of BtCry1Ac Gene in Tobacco

作　　者：杨润蕾[1,2] 董研[1] 于晓月 杨敏生[1] 

机构地区：[1]河北农业大学林学院,保定071000 [2]河北大学生命科学学院,保定071000

出　　处：《分子植物育种》2018年第2期445-451,共7页Molecular Plant Breeding

基　　金：国家高技术研究发展计划(863计划)项目(2013AA102703)资助

摘　　要：叶绿体是外源基因表达的理想场所。为了探究植物叶绿体转化技术,本研究成功构建了BtCry1Ac基因的烟草叶绿体转化载体,其中以aad A基因作为筛选标记,叶绿体基因组同源片段选用rbcL基因与acc D基因。采用基因枪轰击法进行叶绿体转化,以壮观霉素300 mg/L作为初筛浓度,并逐步提高筛选压力,经过3轮筛选后,通过PCR检测,有6个株系检测到了外源Bt基因;同时进行的毒蛋白测定中,有5个株系可检测到Bt毒蛋白,但表达量普遍偏低,T-4的毒蛋白含量最高,为19.32 ng/mL,说明烟草叶绿体基因组中还有大量拷贝没有完全同质化。通过对烟草叶绿体载体构建及转化技术的摸索,将为未来开展其它植物的叶绿体转化研究提供参考。Chloroplast is an ideal place for foreign gene expression.In order to study the technology of plant chloroplast transformation,the chloroplast transformation vector of BtCry1Ac gene in tobacco was successfully constructed.The aad A gene was used as the screening marker,and rbc L gene and acc D gene were the homologous fragments of chloroplast genome of tobacco.Chloroplast transformation was carried out by gene gun bombardment.The initial screening concentration of Spectinomycin was 300 mg/L,and the screening pressure was gradually improved.After 3 rounds screening,the exogenous Bt gene was detected by PCR in 6 lines.At the same time,the BtCry1Ac toxic protein content was determined.There were 5 transgenic lines that can detect Bt toxin protein,but the expression of foreign proteins was generally low.T-4 was the highest,which was 19.32 ng/mL,indicating that there were a large number of copies in the chloroplast genome of tobacco without homogenization.Based on the research of chloroplast transformation technology in tobacco,it might provide reference for future studies on chloroplast transformation of other plants.

关 键 词：烟草 叶绿体转化 BtCry1Ac基因 同质化 

分 类 号：Q943.2[生物学—植物学]