提高苹果基因转化效率的研究

采用根瘤农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ　ｔｕｍｅｆａｃｉｅｎｓ）强株系ＥＨＡ１０５（ｐ３５ｓＧＵＳ－ｉｎｔｒｏｎ）研究了影响‘皇家嘎拉’苹果（Ｍａｉｎｓ ｄｏｍｅｓｔｉｃａ Ｂｏｒｋｈ．）外植体的β-葡萄糖醛酸酶（ＧＵＳ）基因的瞬时、稳定表达水平和转基因植株的再生。证明在培养基生长素（ＩＢＡ、ＮＡＡ）存在的条件下，外植体的ＧＵＳ基因的瞬时表达水平提高了３－４倍，而共培养两周后稳定表达水平提高２倍以上，产生９．８个ＧＵＳ愈伤组织表达区域。白化处理促进外植体的基因转化，白化处理的新梢顶端第一节间外植体ＧＵＳ表达区域比常用的叶片外植体高４倍。在生长素存在的条件下 ２％外植体获得了转基因植株。Ｓｏｕｔｈｅｒｎ　ＢｌｏｔＤＮＡ杂交和组织化学染色分析证明ＧＵＳ基因已整合到苹果的染色体上，并得以表达。

Study on Enhancing Efficiency of Apple Transformation

Transient and stable β-glucuronidase (GUS) expressions and transgenic plant regeneration from the etiolated internode explant of' Royal Cala' apple (Malus domestica Borkh. ) were examined using Agrobacterium tumefaciens supervirulent strains EHA105(p35sGUS intron) during transformation. Explants with auxins (IBA, NAA) yeilded more than twice as many GUS-expressing zones and calli compared with ones on the medium without auxin. All etiolated internodes exhibited GUS expressing zones and yielded 4-fold as many GUS expressing zones compared to commonly used leaf explants from nonetiolated shoots. High efficiency transformation system was established. On regeneration medium with 0. 5 mg.l-1 NAA,transgenic plants with GUS gene were Obtained from 400 internode explants after cocultivation with EHA105. The integration of the GUS gene into apple genome was confirmed and Southern blot analysis. The gene expression was confirmed by GUS-histochem-istry staining.