摘要
根据烟草叶绿体高频同源重组片段的已知序列(GenBank Z00044.1)设计引物,用PCR的方法克隆到1个3.663 kb的番茄叶绿体DNA片段(psbD/trnG),命名为ctDNA。该片段与GenBank中烟草的相应片段有96.7%同源性,与本文所用的烟草的相应片段有95.8%同源性。以其为外源多顺反子定点整合介导的同源重组片段,与来自烟草叶绿体的强启动子Prrn和终止子psbA3′,以及甘露聚糖酶基因man、绿荧光蛋白基因gfp、氨基糖苷3′-腺苷酰基转移酶基因aadA构建番茄质体多顺反子定点整合表达载体pLM2(-psbD-Prrn-RBS-man-RBS-gfp-RBS-aadA-psbA3′t-rnG-)。将该载体用基因枪轰击烟草叶片,用添加了壮观霉素的选择分化培养基筛选,获得质体转基因烟草3株。用PCR、激光扫描、Western blot、RFLP等方法检测都证实man、gfp、aadA基因已整合到烟草质体基因组中,且均得到表达。用番茄质体多顺反子定点整合表达载体成功实现在烟草质体中的表达。
According to the published DNA sequence （GenBank Z00044.1 ） of tobacco chloroplast high-frequency homologous recombination fragment, primers were designed to amplify a 3.663 kb DNA fragment named ctDNA from chloroplast genome of tomato by PCR. The fragment was not only 96.7 % homology compared with the tobacco fragment reported in GenBank, but also 95.8% homology compared with the tobacco fragment used in this paper. The tomato chloroplast mnlticistron expression vector pLM2 （ Fig. 1 ） （ -psbD-Prrn-RBS-man-RBS-gfp-RBS-aadA-psbA3＇-trnG- ） was constructed with ctDNA, Prrn, man, gfp, aadA and psbA3＇. And the tobacco leaves were bombarded with golden particles coated with the vector pLM2. Three transgenic tobacco shoots were obtained on the screening medium. The function of aadA gene was identified by screening medium（Fig.3）, and the function of gfp gene was confirmed by laser scanner （Fig. 4）. The expression of man gene was identified by Western blot（ Fig. 5）. The genes man, gfp and aadA being in the chloroplast of tobacco were confirmed by PCR （ Fig.6 ） . And the tobacco chloroplast mnlticistron expression cassette integrating in tobacco chloroplast genome DNA was confirmed by RFLP（Fig.7）. The results were showed that the three genes（ man, gfp, aadA ）, which were in the tomato chloroplast muhicistron expression vector pLM2, were expressed in tobacco chloroplasts.
出处
《作物学报》
CAS
CSCD
北大核心
2006年第5期755-761,共7页
Acta Agronomica Sinica
基金
国家高新技术研究发展计划(863计划)(2002AA227011)
国家"十五"科技攻关计划(2001BA70BB
2004BA7BB)
湖北省自然科学基金(2003ABA118)项目资助
关键词
多顺反子
番茄质体载体构建
烟草质体转化
基因枪法
Mnlticistron
Construction of Tomato chloroplast expression vector
Tobacco plastid genetic transformation
Bombardment microprojectiles
作者简介
陆云华（1968-）,男,江西丰城人,博士,副教授,主要从事植物分子生物学研究.
通讯作者（Corresponding author）：马立新.E-mail：malixin9@hotmail.com;Tel：027-88666349,027-58865627
