烟草花叶病毒外壳蛋白的基因导入和转化烟株的再生

THE INTRODUCTION OF CHIMERIC COAT PROTEIN GENE OF TOBACCO MOSAIC VIRUS AND THE REGENERATION OF TRANSGENIC TOBACCO PLANT

用T-DNA区携有嵌合的烟草花叶病毒外壳蛋白基因和卡那霉素抗性基因(NPTⅡ)的土壤农杆菌株pACK403和pACK404与烟草品种SRl和斯佩特G-28单倍体无菌菌叶碟片进行共培养转化。转化后的叶碟片在含有头孢噻肟钠500毫克/升和卡那霉索300毫克/升的培养基上诱导芽,在含有头孢噻肟钠500毫克/升和卡那霉素100毫克/升的培养基上诱导生根。Nopaline测定,烟草花叶病毒外壳蛋白基因的表达检测、转化烟株对烟草花叶病毒侵染抗性的检测结果证明:用这种方法能可靠地将外源基因导入烟草,并能在转化烟株中表达。再生得到的转化烟株在烟草花叶病毒强感染情况下能延迟病症表现4—25天。

The Agrobacierium strains pACK403 and pACK404, in which the chimcric coat protein gene of tobacco mosaic virus and NPT Ⅱ gene were inserted into the T-DNA region, were used to transform the leaf discs of tobacco special SRI and special G28 (haploid) with cocultivation method. The transformed discs were cultured on the medium containing sodium cefotaximatc 500mg/l and kanamycin 300mg/l to induce shoots, then the shoots were cut and cultured on the medium containing sodium cefotaximatc 500 mg/1 and kanamycin 100mg/l to induce roots. The results of Nopalinc detection, the gene expression determination of TMV coat protein, and the resistant test of transgenic tobacco plant against TMV infection showed that this method can be reliably used to introduce foreign genes into tobacco plant and that the TMV coat protein genes have been expressed in transgenie tobacco plant. Under the strong infection condition, fhe transgenic tobacco plant could delay the presence of disease symptom of TMV for 4 to 25 days.