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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- 3蛋白
The Arabidopsis ATL P-3 gene was subcloned from the plasmid pUCCTL into the plant expression vector p BI12 1, and the resulting plasmid was named p BITL. The ATL P-3 gene subcloned into the recombinant plasmid pBITL was introduced into tobacco (N icotiana tobacco L. var Gexin No.1) mediated by Agrobacterium tumefaciens L BA44 0 4. Nineteen regenerated tobacco plants were obtained by kanamycin resistance screening. Four (A1, A4, B2, C6) of the ATL P-3 genes were detected by PCR (primers synthesized on the basis of the ATL P-3 gene). Further PCR (with primers synthesized based on the CaMV35S promoter and NOS terminator sequences) showed that the ATL P-3 gene was integrated into the genomic DNA of tobacco under the control of the CaMV35S promoter and NOS terminator. Southern blotting results of four transgenic tobacco plants showed that only ATL P-3 gene integrated into B2 and C6 was stably present in genomic DNA. Western blotting analysis showed that only C6 transgenic plants expressed ATL P-3 protein