【摘要】 以中华结缕草(Zoysia sinica Hance)萌动种子为转化受体,利用新霉素磷酸转移酶基因(neomycinphos photransferaseⅡ,nptⅡ)作为筛选基因,对影响根癌农杆菌(Agrobacterium tumefaciens)介导的遗传转化外部因素进行了实验,通过优化转化条件,建立了农杆菌浸种途径介导的中华结缕草遗传转化体系。结果表明,利用催芽6～7d的种子,加入OD600为0.9的农杆菌浸染菌液(在浸染菌液中添加100μmol/L乙酰丁香酮),附加5min抽真空并在迅速恢复常压后再抽真空30s的处理,振荡浸染48h,共培养3d后用75mg/L卡那霉素筛选能够获得较好的转化效果。通过对卡那霉素抗性苗进行PCR检测和GUS组织化学染色,初步验证nptⅡ和β-葡萄糖苷酶基因(β-glucuronidase,gus)已经整合进植物基因组,阳性转化率为55.21%。更多还原

【Abstract】 A high-frequency transformation system has been established by Agrobacterium tumefaciens soaked seeds transforma-tion technology and useing Zoysia sinica Hance germinating seeds as transformation receptor.External factors that affect the efficiency of genetic transformation on the base of neomycin phosphotransferase Ⅱ(nptⅡ) were studied and optimized.The major results were as follows:seed was germinated for 7 days, then was infected with Agrobacterium tumefaciens which OD600 was 0.9 with 100 μmol/L acetosyringone for 48 h after pumping vacuum for 5 min and then 30 s vacuum treatment again recovered normal press rapidly.Then after co-cultivation for 3 days, the transferred plant selected by 75 mg/L kanamycin.It was originally proved by the PCR and GUS examination that nptⅡand B-glucuronidase(gus) genes had been transferred into the genome.The positive transformation rate was 55.21% by the analysis of PCR.更多还原