甘蓝型油菜3个AP3重复基因植物反义表达载体的构建及遗传转化

Construction and transfer of the plant antisense-expression vector carrying three AP3 homologous genes of Brassica napus

利用pFGC5941构建了甘蓝型油菜3个AP3重复基因的植物反义表达载体pFGC5941-BnAP3-2、pFGC5941-BnAP3-3和pFGC5941-BnAP3-4.采用快速冻融法,将载体导入农杆菌EHA105,转化甘蓝型油菜下胚轴.在5mg/L PPT的MS培养基中筛选,获得反义BnAP3-2基因的抗性再生植株31株,反义BnAP3-3基因的抗性再生植株20株,反义BnAP3-4基因的抗性再生植株42株.PCR鉴定结果显示,获得BnAP3-2反义的转基因植株12株,BnAP3-3反义的转基因植株7株,BnAP3-4反义的转基因植株16株.半定量PCR分析结果显示,三个外源AP3基因的反义链已在油菜叶片中表达.

Three AP3 homologous sequences,BnAP3- 2, BnAP3- 3 and BnAP3- 4, were reverse inserted into plasmid pFGC5941, constituted the anti-expression vectors pFGC5941-BnAP3-2, pFGC5941- BnAP3-3 and pFGC5941-BnAP3-4. The vectors were introduced into the grobacteriun strain EHA105 by means of rapid frozen thaw method and transferred into hypocotyls of Brassica napus. Thirty one re- sistant plants with antisense BnAP3-2 gene, twenty resistant plants with antisense BnAP3-3 gene and forty two resistant plants with antisense BnAP3-4 gene were obtained from the MS medium with 5 mg/L PPT. Twelve transgenic plants with antisense BnAP3-2 gene, seven transgenic plants with antisense BnAP3-3 gene and sixteen transgenic plants with antisense BnAP3-4 gene were identified by PCR. Semi-quantitative PCR analysis confirmed that antisense fragment of the there foreign AP3 homologous genes had been expressed in the B. napus plants.