Chemeresiuk N N, Elanskaia I V
Genetika. 1994 Sep;30(9):1141-5.
The hordein B1 gene of Hordeum vulgare L. (variety Donetskii 4) was cloned in cells of Escherichia coli and cyanobacterium Synechocystis sp. PCC6803. For cloning in E. coli, a 2.3-kb Hpa I/EcoR I DNA fragment carrying the hordein B1 gene was inserted into plasmid pBSM13(-), controlled by a lac promoter. The constructed recombinant plasmid pBSB1 provides hordein B1 gene expression in transformed E. coli cells. To introduce the hordein B1 gene into the genome of Synechocystis sp. PCC6803, an integrative vector, containing a fragment of cyanobacterium chromosomal DNA with inserted Tn5 antibiotic resistance genes (KmR, BleoR, StpR) was constructed. A Pvu II-fragment of pBSB1, containing hordein B1 gene under control of the lac promoter, was cloned in the Sma I-site of gene BleoR. The resulting recombinant plasmid was used to transform cyanobacterial cells. One KmR transformants contained a DNA fragment yielding a positive signal during Southern blotting with a [32P]-labeled DNA fragment carrying hordein B1 gene. Western blotting with polyclonal antibodies to barley hordein revealed hordein B1 gene expression in Synechocystis sp. PCC6803.
将大麦（品种为Donetskii 4）的大麦醇溶蛋白B1基因克隆到大肠杆菌和集胞藻属蓝细菌Synechocystis sp. PCC6803的细胞中。为了在大肠杆菌中进行克隆，将携带大麦醇溶蛋白B1基因的2.3 kb Hpa I/EcoR I DNA片段插入受lac启动子控制的质粒pBSM13(-)中。构建的重组质粒pBSB1可在转化的大肠杆菌细胞中实现大麦醇溶蛋白B1基因的表达。为了将大麦醇溶蛋白B1基因导入集胞藻属蓝细菌Synechocystis sp. PCC6803的基因组中，构建了一个整合载体，该载体包含插入了Tn5抗生素抗性基因（KmR、BleoR、StpR）的蓝细菌染色体DNA片段。将在lac启动子控制下包含大麦醇溶蛋白B1基因的pBSB1的Pvu II片段克隆到基因BleoR的Sma I位点。所得的重组质粒用于转化蓝细菌细胞。一个KmR转化体包含一个DNA片段，在用携带大麦醇溶蛋白B1基因的[32P]标记DNA片段进行Southern印迹杂交时产生阳性信号。用针对大麦醇溶蛋白的多克隆抗体进行Western印迹分析显示，集胞藻属蓝细菌Synechocystis sp. PCC6803中存在大麦醇溶蛋白B1基因的表达。
