At the same time , phacl gene was modified by pcr . three plant expression vectors containing kanamycin photransferase gene : pc3cl ( containing phacl ) , pc3cl ( containing phac2 ) , pc3c ! c2 ( containing phacl and phact ) were constructed 同时利用套叠pcr技术对phac1基因进行了改造，经过基因拼接构建了带有卡那霉素抗性基因的植物表达载体pc3c1 (嵌合phac1 ) 、 pc3c2 (嵌合phac2 )和pc3c1c2 (嵌合phac1和phac2双基因) 。
The high performance two - gene expression vector pc3c ! c2 was transformed to nicotiana tobacum honghuadajinyuan plants mediated by agrobacterium tumefaciens eh a105 according to the leaf disc procedure . transformed shoots were selected on solidified medium containing l00mg / l kanamycin 将含有phac1和phac2双基因的植物高效表达载体pc3c1c2 ，用冻融法，构建了pc3c1c2根癌土壤杆菌( agrobacteriumtumefacienseha105 )的工程菌。
Promoter - probe vector psupv4 was used to clone promoters of pseudomonas pseudoalcaligenes in escherichia coli . nine kanamycin resistant recombinants were obtained and designated as ppal - ppa9 . the ppa7 , which has the highest kanamycin resistance , was chose for further characterization 利用启动子探针型载体psupv4直接在大肠杆菌中克隆类产碱假单胞菌( pseudomonaspseudoalcaligenes )基因启动子片段，获得具有强启动子的重组子ppa7 。
The growing graphs of e . coli bl21 containing pbv221 and pbv221svhb according to the od600 values showed that the e . coli vgb + grew faster 4 the highly - efficient bivalent vectors which are named pgbif4abcvhb and pgbi4asvhbbt respectively , havfs been . su ? essful ] y constructed . the tobacco leave discs were transformed with agrobacterium tumefaciens carrying the plant expression vectors pgbif4abcvhb and pgbi4asvhbbt . 38 and 36 transgenic tobacco plants have been obtained respectively after kanamycin screening , pcr detection , southern blot analysiss showed that the foreign genes have been inserted into the tocacco plant genome . the expression of 16kd vhb protein in transgenic plants was proved by western blot 4构建了双价基因高效植物表达载体pgbif4abcvhb和pgbi4asvhbbt 。通过农杆菌介导法，将这两个双价基因高效表达载体导入烟草，分别获得了38 、 36株阳性转基因植株。经pcr 、 southernblot分子生物学方法分析，证明vgbm ， bt基因、 bt - cpti基因已整合到烟草基因组中， westernblot证明vgbm成功地表达出16kd的vhb蛋白。
One to offspring ( t1 ) was selected for further genetic analysis from two types of self - pollinated transgenic plants and screened for the presence of the recombinant gene by the pcr respectively . the result suggested that the resistance to kanamycin can be a stable heredity by genetic analysis , separation ratio of t1 was 3 : 1 and t1 kanamycin - resistant tomato plants can carry vp7 gene 此外，从两种转基因植株中分别选取一自交株进行t _ 1代遗传分析，其kan抗性基因分离比皆为3 : 1 ， t _ 1代抗性番茄植株的pcr结果也呈阳性。
3 . potato stems and agrobacterium fumefaciens containing recombinant vector were co - cultured at 28cfor 48 hours and transplanted onto callus - inducing medium at 24c for 7 days . and then , the explants were transplanted onto differentiation medium and cultured at 24c for 21 days . resistant buds rooted and grew into plants in medium with kanamycin for 20 days , and 83 plants were obtained 3将含外源基因的根癌农杆菌与马铃薯茎段共培养后在愈伤诱导培养基上培养7天，转接到分化培养基上分化出抗性芽，抗性芽在生根培养基上生根长成完整植株，共获83株。
