CONSTRUCTION OF OIL-RICH MUTANTS IN NANNOCHLOROPSIS LIMNETICA
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摘要: 研究旨在建立湖泊微拟球藻(Nannochloropsis)的遗传转化体系, 然后根据外源基因随机插入的特性, 挑取抗性转化子构建突变体库, 并从突变体库中筛选高含油突变株。以湖泊微拟球藻自身β-tublin基因启动子和三角褐指藻fcpA终止子驱动和终止来源于细菌的sh ble抗性选择基因, 构建了一个转化载体pPha-T1-TUB。将线性化后的质粒以电穿孔的方法转化湖泊微拟球藻, 通过1 μg/mL zeocin的抗性平板筛选, 并经液体培养基连续传代后, 得到了可以稳定遗传的转化子。我们从这些转化子中筛选得到了数株油含量高于野生型, 且生长也优于野生型的突变株。其中, 2个突变株K26和G5的总脂中多不饱和脂肪酸含量更低, 其脂肪酸组成更符合作为生物柴油原料的标准。研究通过随机插入构建突变体库的方法为快速获取优良目的性状的高产油突变株提供了一个有效手段。Abstract: Nannochloropsis are model species for investigating biofuel production by algae. Many marine Nannochloropsis genomes have been sequenced recently and their transformation systems have been established. N. limnetica is the only freshwater species, and its genome and transformation system are not yet available. In this study, a genetic transformation method for N. limnetica is established and a zeocin-resistance gene is introduced into the genome to obtain mutant library, then high oil-yield mutants are screened from the library. A vector pPha-T1-TUB that has a sh ble gene derived by Nannochloropsis β-tublin promoter and Phaeodactylum tricornutum fcpA terminator was constructed, and the linearized plasmid was introduced to N. limnetica by electroporation. Transformants were able to grow on 1 μg/mL zeocin. PCR detection indicated that 100% of the selected colonies were positive transformants and all resistant colonies contained sh ble gene after half a year. Several mutants with higher triacylglycerols content and biomass were obtained. Among them, K26 and G5 showed lower polyunsaturated fatty acid contents than wild type, which meets the requirements of the European Standard EN 14214 (2004) for biodiesel production. This study provides an efficient method to obtain advantageous mutants via generating a library of random resistant gene insertion mutations in N. limnetica.
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图 1 野生型和突变株培养10d(上)和12d(下)后提取总脂的硅胶板薄层层析图
薄层层析板上的数字表示突变株中三油酸甘油酯相对于野生型(设为1.00)的量; M. 三油酸甘油酯, Triolein; WT. 野生型, wild type; 其他为抗性藻株, others are resistant algal colonies
Figure 1. Lipid composition and content in mutants and wild type (WT) of Nannochloropsis limnetica at day 10 and 12
The values above the TLC panel indicate the relative triacylglycerols normalized to the WT, which was set as 1
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图 2 野生型和突变株培养10d和12d后相对生物量(OD730)的比较
WT. 野生型, wild type; 其他为抗性藻株, others are resistant algal colonies
Figure 2. The relative biomass (OD730) in mutants and wild type (WT) of Nannochloropsis limnetica at day 10 and 12
表 1 野生型湖泊微拟球藻与富油突变株K26和G5的总脂与三酰基甘油脂肪酸组成及含量
Table 1 Fatty acid composition (mol %) in total lipids and triacylglycerol (TAG) of mutants (strains K26 and G5) and wild type (WT) of Nannochloropsis limnetica
脂肪酸Fatty acids 野生型WT 野生型WT 突变株K26 突变株K26 突变株G5 突变株G5 总脂Total lipids 三酰基甘油TAG 总脂Total lipids 三酰基甘油TAG 总脂Total lipids 三酰基甘油TAG C14﹕0 8.34±0.45 12.15±1.86 6.97±0.88 11.31±0.78 8.26±0.2 10.36±0.45 C14﹕1 0.03±0.01 0.15±0.01 0.02±0 0.06±0.07 0.02±0.01 0.02±0 C16﹕0 25.96±1.42 42.23±0.34 26.09±0.53 39.02±0.33 27.35±1.87 39.85±1.38 C16﹕1 28.43±0.49 24.6±2.27 32.39±0.18 31.31±2.08 33.84±0.37 30.52±1.13 C18﹕0 2.03±0.71 5.4±1.17 1.74±1.14 4.14±1.91 0.01±0.01 8.9±1.02 C18﹕1 4.3±0.61 6.11±0.82 5.29±1.64 6±1.65 10.18±1.27 5.21±2.18 C18﹕2 3.09±0.36 1.1±0.1 2.91±0.09 0.69±0.01 4.81±0.55 1.2±0.03 γ-C18﹕3 0.08±0 2.74±0.42 0.08±0 1.52±1.93 0.11±0 0.04±0.03 α-C18﹕3 0.59±0.11 0.67±0.13 0.76±0.02 0.09±0.09 0.65±0.06 0.23±0.16 C18﹕4 0.03±0.03 0.23±0.01 0.12±0.09 1.04±0.63 0.05±0.07 0.44±0.04 C20﹕0 0.17±0 0.26±0.06 0.1±0.01 0.29±0.11 0.07±0.01 0.28±0.01 C20﹕4 4.58±0.49 0.68±0.14 4.67±0.3 0.14±0.15 5.46±0.73 0.02±0 C20﹕5 22.2±1.09 2.2±1.91 18.49±1.9 2.79±0.89 8.69±1.16 2.15±0.17 C22﹕0 0.01±0.01 0.95±0.04 0.08±0.1 0.94±0.03 0.04±0.04 0.45±0.31 C22﹕4 0.02±0.02 0.05±0.01 0.03±0.03 0.2±0.08 0.02±0.03 0.05±0.04 C22﹕5 0.14±0.20 0.26±0.29 0.22±0.25 0.24±0.07 0.05±0.04 0.15±0.02 C22﹕6 0±0 0.2±0.25 0.03±0.04 0.23±0.22 0.38±0.11 0.13±0.08 占干重百分比 % dry cell weight 22.0 11.0 24.1 12.2 25.5 12.3 
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