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通过对大麦小孢子进行基因枪轰击获得 4株转绿色荧光蛋白基因 (gfp)的植株 (A、C、D、E) ,以gfp基因为探针进行荧光原位杂交 (FISH)研究转化植株中转基因插入位置和基因表达。 4个株系在染色体 7L(5HL)的不同位置都有一个插入点 ,而E株系在染色体 5S(7HS)还有第 2个插入点。所有的转基因T0 代植株都是半合子并在T1、T2代发生分离。D株系GFP未表达 ,但FISH和PCR分析表明gfp基因已成功插入其染色体。各株系在根尖和花粉中的GFP表达水平不同 :C株系在花粉表达强而在根尖表达中等 ;A株系在花粉中等表达而在根尖表达较淡 ;E株系则在根尖高表达 ,花粉中等表达。A和C株系在根尖和花粉的GFP分离都表现单位点特性 ,而E株系的根尖分离表现重叠作用 (15∶1)特征 ,但在花粉中表达GFP的频率低。PCR结果和 3个分离株系的根尖表达结果一致。D和E株系的GFP表达不正常可能和gfp基因插入位置或基因的结构有关。
Four plants (A, C, D and E) with green fluorescent protein gene (gfp) were obtained by particle bombardment of microspore of barley. FISH analysis of gfp gene was used to probe the transformed plants Transgenic insertions and gene expression. Four lines had an insertion point at different positions on chromosome 7L (5HL), whereas E strain had a second insertion point on chromosome 5S (7HS). All transgenic T0 plants are hemizygous and segregate on T1 and T2 generations. D strain GFP was not expressed, but FISH and PCR analysis showed that the gfp gene has been successfully inserted into its chromosome. The expression level of GFP in root and pollen of each strain was different: the expression of GFP was stronger in pollen and middle apical; A was moderate in pollen and lighter in apical; E was in roots Spike high expression, moderate expression of pollen. A and C lines showed single locus trait in GFP separation of apical pollen and pollen, while the apical separation of E line showed the feature of overlap (15:1), but the frequency of GFP expression in pollen was low. PCR results were consistent with the apical expression of three isolates. GFP expression in D and E lines may be related to the insertion position of gfp gene or the structure of the gene.