Bates G W, Carle S A, Piastuch W C
Department of Biological Science, Florida State University, Tallahassee 32306.
Plant Mol Biol. 1990 Jun;14(6):899-908. doi: 10.1007/BF00019388.
The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration. This paper provides evidence for the ligation of plasmid fragments by plant cells. Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24 h later. Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression. Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40-50%. Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts. CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression. Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kb Hind III fragment of pCaMVCATM. DNA isolated from nuclei of the protoplasts was used to transform competent cells of Escherichia coli, and colonies were recovered that carried pGEM with Hind III-CaMVCAT inserts. Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transform E. coli yielded an estimate of the frequency of plasmid ligation. A maximum of only 4% of the input linear DNA was recovered as circular molecules. This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms. Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning into E. coli.
通过直接基因转移形成的稳定转化体中的整合DNA常常呈现出复杂的限制性图谱。这些复杂限制性图谱的一个原因可能是质粒片段在整合之前发生了连接。本文提供了植物细胞连接质粒片段的证据。在pCaMVCATM存在的情况下对胡萝卜原生质体进行电穿孔处理，并在24小时后检测氯霉素乙酰转移酶（CAT）活性。pCaMVCATM的线性和超螺旋形式支持相似水平的CAT表达。令人惊讶的是，在CaMV 35S启动子和CAT编码区域之间的位点对质粒进行酶切，表达仅降低了40 - 50%。在分离的质粒片段存在的情况下进行电穿孔表明，这一结果是由于原生质体对线性化质粒的连接。用分离的CaMV 35S启动子和CAT编码区域的混合物获得了CAT表达；单独的任何一个片段都不支持表达。在pGEM线性化产物和pCaMVCATM的1.5 kb Hind III片段的混合物存在的情况下对原生质体进行电穿孔，提供了进一步的连接证据。从原生质体细胞核中分离的DNA被用于转化大肠杆菌感受态细胞，回收得到了携带带有Hind III - CaMVCAT插入片段的pGEM的菌落。在线性和超螺旋pGEM存在的情况下对原生质体进行电穿孔，并使用从细胞核中分离的DNA转化大肠杆菌，得出了质粒连接频率的估计值。最多只有4%的输入线性DNA以环状分子形式回收。这一结果表明连接频率较低，但通过电泳检查植物细胞核中的质粒DNA表明质粒发生了广泛降解，且环状形式优先丢失。因此，连接的质粒可能会转化为线性形式，从而通过克隆到大肠杆菌中无法回收。
