β–1,3葡聚糖酶基因转化提高苹果对斑点落叶病的抗性

为提高苹果抗病性,利用根癌农杆菌介导的叶片转化法,将带有NPTⅡ筛选基因、GUS标记基因及β–1,3葡聚糖酶目的基因质粒导入‘富士’和‘嘎拉’苹果。具有卡那霉素(Kan)抗性的转化株系,经GUS组织化学染色及PCR扩增检测,得到17个‘嘎拉’阳性株系和11个‘富士’阳性株系。选取‘嘎拉’和‘富士’各3个阳性株系进行Southern blot检测,结果表明外源目的基因已整合到所试转化株系中。选取‘嘎拉’及‘富士’各5个阳性株系进行半定量RT-PCR检测,除1个‘富士’株系没有条带外,其余株系均有阳性条带,且各条带的明亮清晰程度有差别,说明不同转基因株系中目的基因的表达量有差异。转化植株离体叶片接种苹果斑点落叶病菌进行抗病性表型检测结果证实,‘嘎拉’10个转化株系中8个株系表现抗病,7个‘富士’转化株系中2个株系表现一定的抗病性,抗病性的增强与目的基因的表达水平有关。

Transformation of β-1,3 Glucanase Gene Increased Alternaria mali Disease Resistance in Apple

In order to enhance the disease resistant ability of apple,the plasmid encoding NPTⅡ gene,GUS gene,and f-l,3 glucanase gene was transferred into apple cultivars of Fuji and Gala via Agrobacterium tumefaciens mediated.Seventeen Gala and 11 Fuji transgenic lines succeeded in the Kanamycin resistant screen,GUS stain,and PCR analysis.From those,3 lines of each cultivar were chosen for Southern blot analysis,which confirmed that the exogenous genes had been integrated into the genome of the transgenic lines.Then,five lines of each cultivar were chosen for Semi-quantitative RT-PCR analysis.All the lines except one from Fuji cultivar showed the positive target bands.The bands varied in brightness and sharpness indicated the difference of the gene expression among the transgenic lines.The detached leaves from the lines were inoculated with the spores ofAlternaria mali and incubated.Among 10 Gala lines and 7 Fuji lines tested,8 Gala lines and 2 Fuji lines showed disease resistance at certain extem,and the enhancing of the disease resistance related to the gene expression.