Molecular Biology Section, Laboratory of Algal Biology, Center of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, India.
Gene. 2012 Dec 15;511(2):248-55. doi: 10.1016/j.gene.2012.09.033. Epub 2012 Sep 21.
This study is the first to demonstrate cloning of alr0882, a hypothetical protein gene of Anabaena PCC7120, its heterologous expression in Escherichia coli strain LN29MG1655 (∆uspA::Kan) and functional complementation of abiotic stress tolerance of E. coli UspA. The recombinant vector pGEX-5X-2-alr0882 was used to transform ∆uspA E. coli strain. The IPTG induced expression of a 56.6kDa GST fusion protein was visualized on SDS-PAGE and attested by immunoblotting. E. coli ∆uspA strain harboring pGEX-5X-2-alr0882 when grown under carbon, nitrogen, phosphorus and sulphur limitation and abiotic stresses e.g. nalidixic acid, cycloserine, CdCl(2), H(2)O(2), UV-B, phenazine methosulphate (PMS), dinitrophenol (DNP), NaCl, heat, carbofuron and CuCl(2) demonstrated about 22.6-51.6% increase in growth over the cells transformed with empty vector. Expression of alr0882 gene in mutant E. coli as measured by semi-quantitative RT-PCR at different time points under selected treatments reaffirmed its role in tolerance against stresses employed in this study. Thus the results of this study vividly demonstrated that the novel protein alr0882, although appreciably different from the known UspA of E. coli, offers tolerance to abiotic stresses hence holds potential for the development of transgenic cyanobacteria.
本研究首次证明了鱼腥藻 PCC7120 的假定蛋白基因 alr0882 的克隆,其在大肠杆菌 LN29MG1655(∆uspA::Kan)中的异源表达,以及对大肠杆菌 UspA 非生物胁迫耐受性的功能互补。使用重组载体 pGEX-5X-2-alr0882 转化 ∆uspA 大肠杆菌菌株。在 SDS-PAGE 上可以观察到 IPTG 诱导表达的 56.6kDa GST 融合蛋白,并通过免疫印迹证实。在碳、氮、磷和硫限制以及非生物胁迫(如萘啶酸、环丝氨酸、CdCl(2)、H(2)O(2)、UV-B、吩嗪甲磺酸盐(PMS)、二硝基苯酚(DNP)、NaCl、热、carbofuron 和 CuCl(2))下生长的携带 pGEX-5X-2-alr0882 的 ∆uspA 大肠杆菌菌株相对于转化空载体的细胞,其生长增加了约 22.6-51.6%。在选定处理下,通过半定量 RT-PCR 在不同时间点测量 alr0882 基因在突变大肠杆菌中的表达,再次证实了其在耐受本研究中使用的应激方面的作用。因此,本研究的结果生动地表明,尽管新型蛋白 alr0882与大肠杆菌的已知 UspA 有明显不同,但它提供了对非生物胁迫的耐受性,因此具有开发转基因蓝藻的潜力。