Kirubakaran Sundar Isaac, Sakthivel Natarajan
Department of Biotechnology, Pondicherry University, Kalapet, Puducherry 605 014, India.
Curr Microbiol. 2008 Jan;56(1):42-7. doi: 10.1007/s00284-007-9036-1. Epub 2007 Sep 26.
Site-directed mutagenesis on a recombinant plasmid, pUC8, that contained the cah gene, was conducted and confirmed by sequence analysis. Single base substitution, G to A at nucleotide position 81 or T to C at nucleotide position 84 of cah gene does not change the amino acid sequence of cah enzyme but eliminates the HindIII site. The wild-type cah and its mutants were cloned and overexpressed in pQE-60 Escherichia coli expression system. Western blot analysis confirmed the production of 27.7-kDa cah enzyme by all the recombinants. The mutated cah gene devoid of HindIII site was used to generate a recombinant plant transformation vector (pCAMBIA-cah). Agrobacterium-mediated transformation was performed in Nicotiana tabaccum cv. Samsun plants by employing the leaf-disc method. The integration and expression of cah gene in transgenic plants were confirmed by polymerase chain reaction, Southern and Western blot analyses. Antimicrobial activity of cyanamide against phytopathogenic fungi and bacteria was determined. Cyanamide can be used as fertilizer as well as an antimicrobial salt against phytopathogenic fungi and bacteria. The present investigation reports the heterologous expression of the cah marker gene. Due to its innate ability to convert cyanamide to urea and the broad-spectrum antimicrobial activity of cyanamide, the cah gene can be used to facilitate plant growth promotion and biocontrol of phytopathogens.
对含有cah基因的重组质粒pUC8进行了定点诱变，并通过序列分析进行了确认。cah基因核苷酸位置81处的单碱基替换，即从G到A，或核苷酸位置84处从T到C，不会改变cah酶的氨基酸序列，但会消除HindIII位点。野生型cah及其突变体在pQE - 60大肠杆菌表达系统中进行克隆和过表达。蛋白质免疫印迹分析证实所有重组体均产生了27.7 kDa的cah酶。不含HindIII位点的突变cah基因用于构建重组植物转化载体（pCAMBIA - cah）。采用叶盘法在烟草品种Samsun植株中进行了农杆菌介导的转化。通过聚合酶链反应、Southern印迹和蛋白质免疫印迹分析证实了cah基因在转基因植物中的整合和表达。测定了氨基氰对植物病原真菌和细菌的抗菌活性。氨基氰既可以用作肥料，也可以作为对抗植物病原真菌和细菌的抗菌盐。本研究报道了cah标记基因的异源表达。由于其将氨基氰转化为尿素的固有能力以及氨基氰的广谱抗菌活性，cah基因可用于促进植物生长和对植物病原体的生物防治。
