Colombo Sergio L, Pollock Steve V, Eger Karla A, Godfrey Ashley C, Adams James E, Mason Catherine B, Moroney James V
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.These authors contributed equally to this work.
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA. These authors contributed equally to this work.
Funct Plant Biol. 2002 Apr;29(3):231-241. doi: 10.1071/PP01189.
Chlamydomonas reinhardtii Dangeard possesses a CO2 concentrating mechanism (CCM) that enables it to grow at very low CO2 concentrations. In previous studies, insertional mutagenesis was successfully used to identify genes required for growth at low CO2 in C. reinhardtii. These earlier studies used the C. reinhardtii genes, Nit1 and Arg7 to complement nit1 or arg7 strains, thereby randomly inserting a second copy of Nit1 or Arg7 into the genome. Because these genes are already present in the C. reinhardtii genome, it was often difficult to identify the location of the inserted DNA and the gene disrupted by the insertion. We have developed a transformation protocol using the Ble gene, which confers resistance to the antibiotic Zeocin. The insertion of this gene allows one to use a variety of existing polymerase chain reaction (PCR) methodologies to identify the disrupted gene. In this study the D66 strain (nit2, cw15, mt) was transformed by electroporation using a plasmid containing the Ble gene. Primary transformants (42 000) were obtained after growth in the dark on acetate plus Zeocin medium. Colonies were then tested for their ability to grow photosynthetically on elevated CO2 or low levels of CO2 (100 ppm). About 120 mutants were identified which grew on elevated CO2 but were unable to grow well at low CO2 concentrations. About 50% of these mutants had low affinities for inorganic carbon as assessed by K0.5(CO2), indicating a potential defect in the CCM. The location of the inserted DNA is being determined using inverse PCR (iPCR) and thermal asymmetric interlaced (TAIL) PCR. Using these methods, one can rapidly locate the inserted DNA in the genome and identify the gene that has been disrupted by the insertion.
莱茵衣藻丹吉尔拥有一种二氧化碳浓缩机制（CCM），这使其能够在极低的二氧化碳浓度下生长。在先前的研究中，插入诱变成功用于鉴定莱茵衣藻在低二氧化碳浓度下生长所需的基因。这些早期研究使用莱茵衣藻基因Nit1和Arg7来互补nit1或arg7菌株，从而将Nit1或Arg7的第二个拷贝随机插入基因组。由于这些基因已存在于莱茵衣藻基因组中，通常很难确定插入DNA的位置以及被插入破坏的基因。我们开发了一种使用Ble基因的转化方案，该基因赋予对博来霉素的抗性。该基因的插入使人们能够使用各种现有的聚合酶链反应（PCR）方法来鉴定被破坏的基因。在本研究中，D66菌株（nit2，cw15，mt）通过电穿孔用含有Ble基因的质粒进行转化。在黑暗中于乙酸盐加博来霉素培养基上生长后获得了初级转化体（42000个）。然后测试菌落光合生长在高二氧化碳或低水平二氧化碳（100 ppm）下的能力。鉴定出约120个突变体，它们在高二氧化碳下生长但在低二氧化碳浓度下不能良好生长。通过K0.5（CO2）评估，这些突变体中约50%对无机碳的亲和力较低，表明CCM存在潜在缺陷。正在使用反向PCR（iPCR）和热不对称交错（TAIL）PCR确定插入DNA的位置。使用这些方法，可以快速在基因组中定位插入的DNA并鉴定被插入破坏的基因。
