转基因大麦中gfp基因的染色体位置及其表达

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表达不正常可能和加基因插入位置或基因的结构有关。

Chromosomal Location and Expression of Green Fluorescent Protein (gfp) Gene in Microspore Derived Transgenic Barley (Hordeum vulgare L.)

Four doubled haploid barley lines (A,C,D,E) derived from gfp (green fluorescent protein) transformation and selection following particle bombardment of microspores were studied for gene expression pattern and the location of genome inserts.The integration sites were detected by fluorescence in situ hybridization (FISH) using the gfp plasmid DNA as a probe.Plants from events A,C,D and E all have a single insert site on chromosome 7L(5HL) at different locations while line E has a second insert site on chromosome 5S(7HS).All original transgenic plants were hemizygous for the transgenes and segregated in the T1 and T2 generations.Although line D had no GFP expression,FISH and PCR could detect gfp gene on its chromosome in transformed plants.Expression levels of GFP varied with lines and tissues examined.Plants from line C showed good expression in pollen and an intermediate level in root tips.Plants from A have intermediate expression of GFP in the pollen and light expression in the root tips.Line E showed strong expression in the root-tips and an intermediate level of GFP in the pollen.Lines A and C segregated as a single Mendelian locus while E segregated in a duplicate loci ratio (15∶1) on seedling root tips but had low expression frequency in the pollen.PCR results were consistent with GFP expression on root tips in the three segregating lines.The expression of GFP for lines D and E was abnormal and may be related to the physical location of the transgene or the gene construct used.