沙吸附DNA的分布及转化活性  

The distribution and transforming ability of sand-absorbed DNA

作　　者：欧剑虹[1] 倪丽娜[1] 叶学成[1] 黄永胜[1] 谢志雄[1] 陈向东[1] 沈萍[1] 

机构地区：[1]武汉大学生命科学学院,武汉430072

出　　处：《生态学报》2005年第8期2019-2024,共6页Acta Ecologica Sinica

基　　金：国家自然科学基金资助项目(20207005;30070408和30070010)~~

摘　　要：尽管自然环境中存在着大量的DNase,大分子体外DNA在土等生态环境中存在并保持转化活性.实验表明DNA被沙土吸附之后会被沙土保护而产生对DNase的抗性.然而对沙中DNA的检测多是通过PCR或者探针杂交的方法来进行,采用转化方法进行来检测的却鲜有报道.为了研究DNA释放进沙之后的分布及转化活性,建立了流过式微型离心沙柱.将20μL pUC18置于1.0g直径介于1.2～2.5mm之间的沙中,1.5mL 0.1mol/L CaCl2洗脱之后再用200μL TEN缓冲液提取.结果表明吸附于沙中的DNA经提取后仍然具有生物转化活性,并且与洗脱液、提取液具有不同的转化活性和对DNaseⅠ抗性,说明DNA与沙粒的结合不只是一种简单的附着,而是3种复杂的复合体结构.相对于没有沙保护而只能在室温下保留7d转化活性的DNA,在被沙吸附保护之后的DNA可以保存转化活性达35d以上.研究结果还表明在存在感受态细胞的情况下,生物释放出来的DNA并非直接与沙粒结合,而是优先与感受态细胞结合.推测认为沙吸附DNA的转化活性同DNA的构象及同感受态细胞接触的机会相关,而与DNA的浓度不直接相关.这些结果为研究环境中存在的DNA库及其与混居的微生物间的相互作用,水平基因转移及其生态效应提供了有价值的信息.In spite of the presence of ubiquitous DNase in the environment, high-molecular-weight extracellular DNA molecules can survive in natural habitats such as soil and maintain the transforming ability. It has been evidenced that DNA molecules, once being absorbed in sand, can be protected from DNase degradation. However, the mechanism of DNA-sand interaction and its effect on transforming ability remain unclear. In the present study, we developed a flow-through system of sand-filled micro Bio-Spin columns to determine the distribution of extracellular DNA in sand and employed E. coli transformation method to directly monitor its transforming ability. 20μL of free plasmid pUC18 DNA was introduced into 1.0 g sand （1.2mm〈 diameter〈2.5mm）, eluted by 1.5 mL of 0. 1 mol/L CaCl2 and extracted by 200μL of TEN buffer. The transforming ability and stability of the plasmid DNA in the eluate, extract and sand were examined with three different transformation protocols. The results indicated that the DNA transforming ability and accessibility to DNase I ineluate, extract and sand were different, suggesting at least three types of DNA-sand complexes. Moreover, the transforming ability of adsorbed DNA in sand was detected over a period of 35 days, whereas the activity of free plasmid without protection of sand only maintained for 7 days. In the presence of competent cells, the DNA molecules, released from other organisms, were uptaken by the competent cells insteadof being absorbed by sand. This result suggested that the transforming ability of sand adsorbed-DNA was affected by the conformation of DNA molecules and the accessibility to competent cells, but not the DNA concentration. Our data showed that there were at least three forms of DNA in the environment, which might make different contributions to the dynamicchange of extracellular DNA pool.

关 键 词：沙 DNA 转化 水平基因转移 

分 类 号：Q522[生物学—生物化学] Q938