摘要
将拟南芥 ATL P- 3基因从质粒 p UCTL 亚克隆到植物表达载体 p BI12 1中 ,得到的质粒命名为p BITL。被亚克隆到重组质粒 p BITL 的 ATL P- 3基因通过农杆菌 L BA44 0 4介导法导入烟草 (N icotiana tobacco L.var Gexin No.1)。通过卡那霉素抗性筛选 ,得到 19株再生烟草植株。PCR(用根据 ATL P- 3基因合成的引物 )检测到其中 4株 (A1、A4、B2、C6 )有 ATL P- 3基因的整合。进一步的 PCR(用根据 Ca MV35 s启动子和 NOS终止子序列合成的引物 )分析表明 ,ATL P- 3基因是在 Ca MV35 s启动子和 NOS终止子控制下整合到烟草的基因组 DNA中的。 4株转基因烟草的 Southern blotting检测结果显示 ,只有被整合到 B2和 C6的 ATL P- 3基因稳定地存在于基因组 DNA中 ;Western blotting检测分析表明 ,仅有 C6转基因植株表达了 ATL P-
ATLP 3 gene from plasmid pUCTL was subcloned into plasmid pBI121 and the recombinatant plasmid was designated as pBITL.ATLP 3 gene in pBITL was mobilized into the Agrobacterium tumefactins strain LBA4404 and then introduced into tobacco plants(Nicotiana tobacco L.var Gexin No.1)by the method of agrobacterium tumefactins mediated gene transfer.By the selection of kanamycine on MS plate,19 kanamycine resistant tobacco plants were obtained.Among these plants,4 strains(A1,A4,B2,C6)were identified the integration of ATLP 3 gene into genomic DNA by PCR(primers were synthesized on the basis of the ATLP 3 gene).The further PCR(primers were synthesized on the basis of the CaMV35s promoter and NOS terminator) identified that chimerical ATLP 3 gene was integrated into the genomic DNA under the control of cauliflower mosaic virus 35s promoter and NOS terminator.Among 4 transgenic tobacco plants,Southern blotting analysis showed that the integrated ATLP 3 gene consistently existed in the genomic DNA of B2 and C6 transgenic tobacco plants,and Western blotting analysis showed that only C6 expressed the ATLP 3 protein.
出处
《江苏农业学报》
CSCD
北大核心
2000年第4期217-221,共5页
Jiangsu Journal of Agricultural Sciences
关键词
拟南芥ATLP-3基因
转基因烟草
整合
表达
Agrobacterium tumefaciens
Arabidopsis thalinana ATLP-3 gene
transgenic tobacco
integration
expression
