發(fā)布時間：2018-04-25 04:01

  本文選題：纖細(xì)裸藻 + 誘變　； 參考：《深圳大學(xué)》2017年碩士論文

【摘要】：隨著化石燃料的枯竭,各國紛紛把發(fā)展可再生能源作為國家能源發(fā)展的重要組成部分。微藻生物能源作為新型的可再生能源,受到廣泛的重視。本研究中的纖細(xì)裸藻(Euglena gracilis)在一定的條件下能夠產(chǎn)生大量的油脂,是很好的生物柴油原料,同時因其具備豐富的活性成分,因此被作為食品和營養(yǎng)添加劑的良好來源。本研究結(jié)合纖細(xì)裸藻既可自養(yǎng)又可異養(yǎng)的特性,在光照條件下于有機(jī)培養(yǎng)基中,采用紫外線(UV)照射和梯度濃度的亞硝基胍(NTG)對纖細(xì)裸藻野生型進(jìn)行誘變,以期獲得生長較快且活性成分(如脂肪酸、裸藻副淀粉等)含量較高的突變藻株。此外,為了深入了解光合作用機(jī)理,通過將處于異養(yǎng)培養(yǎng)的綠色纖細(xì)裸藻轉(zhuǎn)換到自養(yǎng)培養(yǎng)后,比較纖細(xì)裸藻野生型在光照和黑暗培養(yǎng)時的葉綠體轉(zhuǎn)錄組差異表達(dá),進(jìn)一步深入綠色裸藻葉綠體轉(zhuǎn)錄組與光照條件的關(guān)聯(lián)機(jī)制。結(jié)果如下:(1)本研究經(jīng)過上述兩種誘變方法處理纖細(xì)裸藻野生型后總共成功得到34株突變藻株。其中,經(jīng)過NTG誘變所得的突變株的生長速度相對于UV處理所得的突變株高。在濃度為0.8mg/mL的NTG的誘變條件下,能夠得到生長速率和生物量較野生型纖細(xì)裸藻高的突變藻株;而在UV誘變中所獲得的突變藻株生長速率不及野生型高。(2)通過生長曲線的繪制及最高細(xì)胞濃度的對比后,篩選得出6株突變株與野生型藻株作后續(xù)的活性成分含量對比實驗,取樣時間為第4天(對數(shù)期)和第9天(細(xì)胞頂峰期)。(3)本研究在脂肪酸的測定實驗中檢測了第4天和第9天的脂肪酸含量及種類�？傮w來說,第9天的總脂肪酸(TFA)比第4天高。其中,突變株A4的TFA含量最高,達(dá)171.34μg/mg dw;其他突變株的TFA含量在122.57~140.07μg/mg dw,野生型含量最低,為97.91μg/mg dw。而不論是野生型還是突變株,其脂肪酸種類當(dāng)中都是飽和脂肪酸(SFA)含量最高,多不飽和脂肪酸(PUFA)其次,單不飽和脂肪酸(MUFA)最低。其中,A4的SFA和PUFA含量高于其他藻株,而MUFA含量與其他藻株相當(dāng),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)含量測定中發(fā)現(xiàn),大多數(shù)藻株在第4天的Pm含量高于第9天的含量。當(dāng)培養(yǎng)至第4天時,突變株A4、A11和野生型的Pm含量最高,分別占自身干重的60.64%、56.57%和55.02%。(5)對異養(yǎng)培養(yǎng)轉(zhuǎn)換到自養(yǎng)培養(yǎng)的纖細(xì)裸藻進(jìn)行轉(zhuǎn)錄組測序和分析后,得到有75個轉(zhuǎn)錄本,總共注釋得到48個葉綠體基因,涉及到光合作用的基因有13個,主要是光合系統(tǒng)I和光合系統(tǒng)II的基因。其中,差異表達(dá)顯著(差異表達(dá)倍數(shù)≥2,p≤0.05)的基因總有2個,為核糖體相關(guān)蛋白基因rpl5和光合作用相關(guān)蛋白基因psbH,前者上調(diào),后者下調(diào)。纖細(xì)裸藻葉綠體轉(zhuǎn)錄組分析表明其調(diào)控可能主要在于轉(zhuǎn)錄后水平上。綜上所述,本研究摸索纖細(xì)裸藻在NTG和紫外線的處理下能夠得到突變株的適合條件,并且經(jīng)過最高細(xì)胞濃度的對比后得到的6株突變藻株,而后進(jìn)行了脂肪酸和Pm的含量測定,并利用GC-MS技術(shù)進(jìn)行了脂肪酸分析初步探究了脂肪酸在不同的培養(yǎng)時期的積累量規(guī)律,為纖細(xì)裸藻脂質(zhì)的探究和大規(guī)模工業(yè)化提供一個可能的新思路。此外,還初步分析了綠色纖細(xì)裸藻在兩種不同光照培養(yǎng)條件下的基因表達(dá)差異性,為綠色裸藻葉綠體轉(zhuǎn)錄組與光照條件的關(guān)聯(lián)機(jī)制探究提供基礎(chǔ)。
[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.

【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;Q949.2

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