Biolistic transformation, expression, and inheritance of bovine β-casein in soybean (Glycine max)
Biolistic transformation, expression, and inheritance of bovine β-casein in soybean (Glycine max)
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大豆 (Glycine max) 中牛 β-酪蛋白的基因枪转化、表达和遗传
DOI：
10.1007/s11627-999-0046-5
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
L. Vodkin
中科院分区：
文献类型：
--
作者：
P. J. Maughan;R. Philip;M. Cho;J. Widholm;L. Vodkin
将编码牛乳蛋白β-酪蛋白（β-casein）的630 bp的DNA片段克隆到种子特异性凝集素启动子表达盒中，并通过基因枪法将其导入大豆体细胞胚。筛选出4个潮霉素抗性胚性细胞系。通过Southern印迹分析，所有四个品系均为β-酪蛋白基因阳性;然而，只有一个品系未显示出β-酪蛋白基因和/或凝集素启动子的显著重排。通过Southern印迹杂交分析证实了从该培养物再生的四株植物的基因组中β-酪蛋白基因的存在。DNA印迹实验和后代遗传分析表明，这些植株含有4 ~ 8个拷贝的β-酪蛋白基因，插入发生在一个遗传位点上。牛β-酪蛋白mRNA在发育中的子叶中高表达。虽然在同一样品中未发现内源凝集素转录物，但在叶组织中发现极低水平的β-酪蛋白mRNA。牛β-酪蛋白在转化植物的子叶中表达，但通过免疫印迹在转化植物的叶组织中检测不到。在第一代转基因植物的单个发育种子中产生的牛β-酪蛋白迁移为两条紧密间隔的条带，与纯牛β-酪蛋白的条带非常相似。本实验代表了在大豆中表达乳蛋白的第一份报告，并为通过种子特异性启动子指导大豆蛋白质质量的全面改善开辟了道路。
SummaryA 630-bp DNA fragment encoding a bovine milk protein, β-casein, was cloned into a seed-specific lectin promoter expression cassette and introduced into soybean somatic embryos via particle bombardment. Four hygromycin-resistant embryogenic cell lines were selected. All four lines were positive for the β-casein gene via Southern blot analysis; however, only one line did not show significant rearrangement of the β-casein gene and/or lectin promoter. The presence of the β-casein gene in the genome of the four plants regenerated from this culture was confirmed via Southern blot hybridization analysis. DNA blot experiments and progeny inheritance analysis indicated that the plants contain four to eight copies of the β-casein gene and that the insertion occurred at a single genetic locus. Bovine β-casein mRNA was highly expressed in developing cotyledons. A very low level of β-casein mRNA was found in leaf tissues although endogenous lectin transcripts were not found in the same samples. Bovine β-casein protein was expressed in cotyledons of transformed plants but was not detectable in leaf tissues of transformed plants by immunoblotting. Bovine β-casein produced in the individual developing seed of the first generation of transgenic plants migrated as two closely spaced bands very similar to those of pure bovine β-casein. This experiment represents the first report of the expression of a milk protein in soybean and opens the way for the general improvement of protein quality in soybean directed by seed-specific promoters.