裸藻高产生物活性物质突变株的筛选及光暗条件下转录组初步分析

发布时间：2018-04-25 04:01

本文选题：纤细裸藻 + 诱变　；参考：《深圳大学》2017年硕士论文

【摘要】：随着化石燃料的枯竭,各国纷纷把发展可再生能源作为国家能源发展的重要组成部分。微藻生物能源作为新型的可再生能源,受到广泛的重视。本研究中的纤细裸藻(Euglena gracilis)在一定的条件下能够产生大量的油脂,是很好的生物柴油原料,同时因其具备丰富的活性成分,因此被作为食品和营养添加剂的良好来源。本研究结合纤细裸藻既可自养又可异养的特性,在光照条件下于有机培养基中,采用紫外线(UV)照射和梯度浓度的亚硝基胍(NTG)对纤细裸藻野生型进行诱变,以期获得生长较快且活性成分(如脂肪酸、裸藻副淀粉等)含量较高的突变藻株。此外,为了深入了解光合作用机理,通过将处于异养培养的绿色纤细裸藻转换到自养培养后,比较纤细裸藻野生型在光照和黑暗培养时的叶绿体转录组差异表达,进一步深入绿色裸藻叶绿体转录组与光照条件的关联机制。结果如下:(1)本研究经过上述两种诱变方法处理纤细裸藻野生型后总共成功得到34株突变藻株。其中,经过NTG诱变所得的突变株的生长速度相对于UV处理所得的突变株高。在浓度为0.8mg/mL的NTG的诱变条件下,能够得到生长速率和生物量较野生型纤细裸藻高的突变藻株;而在UV诱变中所获得的突变藻株生长速率不及野生型高。(2)通过生长曲线的绘制及最高细胞浓度的对比后,筛选得出6株突变株与野生型藻株作后续的活性成分含量对比实验,取样时间为第4天(对数期)和第9天(细胞顶峰期)。(3)本研究在脂肪酸的测定实验中检测了第4天和第9天的脂肪酸含量及种类。总体来说,第9天的总脂肪酸(TFA)比第4天高。其中,突变株A4的TFA含量最高,达171.34μg/mg dw;其他突变株的TFA含量在122.57~140.07μg/mg dw,野生型含量最低,为97.91μg/mg dw。而不论是野生型还是突变株,其脂肪酸种类当中都是饱和脂肪酸(SFA)含量最高,多不饱和脂肪酸(PUFA)其次,单不饱和脂肪酸(MUFA)最低。其中,A4的SFA和PUFA含量高于其他藻株,而MUFA含量与其他藻株相当,而PUFA中含量高的主要是C18:2ω6、C18:3ω6、C20:4、C20:5ω3和C22:6ω3(DHA)。(4)在裸藻副淀粉(paramylon,Pm)含量测定中发现,大多数藻株在第4天的Pm含量高于第9天的含量。当培养至第4天时,突变株A4、A11和野生型的Pm含量最高,分别占自身干重的60.64%、56.57%和55.02%。(5)对异养培养转换到自养培养的纤细裸藻进行转录组测序和分析后,得到有75个转录本,总共注释得到48个叶绿体基因,涉及到光合作用的基因有13个,主要是光合系统I和光合系统II的基因。其中,差异表达显著(差异表达倍数≥2,p≤0.05)的基因总有2个,为核糖体相关蛋白基因rpl5和光合作用相关蛋白基因psbH,前者上调,后者下调。纤细裸藻叶绿体转录组分析表明其调控可能主要在于转录后水平上。综上所述,本研究摸索纤细裸藻在NTG和紫外线的处理下能够得到突变株的适合条件,并且经过最高细胞浓度的对比后得到的6株突变藻株,而后进行了脂肪酸和Pm的含量测定,并利用GC-MS技术进行了脂肪酸分析初步探究了脂肪酸在不同的培养时期的积累量规律,为纤细裸藻脂质的探究和大规模工业化提供一个可能的新思路。此外,还初步分析了绿色纤细裸藻在两种不同光照培养条件下的基因表达差异性,为绿色裸藻叶绿体转录组与光照条件的关联机制探究提供基础。
[Abstract]:With the depletion of fossil fuels, countries have taken the development of renewable energy as an important part of national energy development. Microalgae bioenergy, as a new renewable energy, has received extensive attention. The Euglena gracilis in this study can produce a large amount of oil and oil under certain conditions. It is a very good biodiesel. As a good source of food and nutritional additives, the raw material is a good source of food and nutritional additives. This study combines with the characteristics of both autotrophic and heterotrophic bacteria. Under light conditions, ultraviolet (UV) irradiation and gradient concentration of nitroguanidine (NTG) are used to mutagenesis the wild type of fine algae. In order to obtain faster growth and active ingredients (such as fatty acid, naked alga starch and other starch, etc.), the mutant algae with higher content are obtained. In addition, in order to understand the photosynthesis mechanism, the chloroplast transcriptome differences of the wild algae in the light and dark culture are compared by converting the green fine green algae in the heterotrophic culture to the autotrophic culture. The results are as follows: (1) a total of 34 mutant algae were successfully obtained after the two mutagenesis methods mentioned above, of which the growth rate of the mutant strain obtained by NTG mutation was higher than that of the UV treated mutant. Under the condition of NTG concentration of 0.8mg/mL, the mutant algae with higher growth rate and biomass higher than that of wild type was obtained, and the growth rate of mutant algae obtained in UV mutagenesis was not as high as that of the wild type. (2) 6 mutant strains and wild types were screened by drawing the growth curve and comparing the maximum cell concentration. The results were fourth days (logarithmic phase) and ninth days (cell peak period). (3) the fatty acid content and type of fourth days and ninth days were detected in the test of fatty acid. Overall, the total fatty acid (TFA) of the ninth days was higher than the fourth day. The TFA content of the mutant A4 was the highest, Up to 171.34 mu g/mg DW; the TFA content of other mutant strains is 122.57~140.07 mu g/mg DW, the wild type content is the lowest, which is 97.91 mu g/mg dw. and the species of fatty acids are the highest of saturated fatty acids (SFA), followed by polyunsaturated fatty acids (PUFA), and the lowest of monounsaturated fatty acids (MUFA). The content of UFA was higher than that of other algae, while the content of MUFA was equal to that of other algae, while the high content of PUFA was C18:2 Omega 6, C18:3 Omega 6, C20:4, C20:5 Omega 3 and C22:6 Omega 3 (DHA). (4) in the determination of the starch (paramylon, Pm) content of the algal alga (paramylon, Pm), the majority of the algae were higher than ninth days in the fourth days. When the culture was fourth days, the mutant strain The content of Pm and wild type was the highest, which accounted for 60.64% of its own dry weight, 56.57% and 55.02%. (5). After the transcriptional group was sequenced and analyzed, 75 transcripts were obtained and 48 chloroplast genes were annotated. There were 13 genes involved in light cooperation, mainly photosynthetic system I and photosynthesis. The gene of system II, among which there were 2 genes with significant difference expression (difference expression multiple > 2, P < 0.05), were ribosome related protein gene rpl5 and photosynthesis related protein gene psbH, the former was up-regulated, and the latter down regulated. The study explored the suitable conditions for the mutant plants under the treatment of NTG and ultraviolet light, and 6 mutant algae were obtained by comparison of the highest cell concentration. Then the content of fatty acids and Pm was measured. The fatty acid analysis was carried out by GC-MS technology to investigate the fatty acid in different culture period. The regularity of accumulation provides a possible new idea for the exploration and large-scale industrialization of the lipid of naked algae. In addition, the difference of gene expression of green fine algae under two different illumination conditions is also preliminarily analyzed, which provides a basis for exploring the association mechanism between chloroplast chloroplast transcriptional group and light strip.

【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;Q949.2

【参考文献】