本文關(guān)鍵詞： 萊茵衣藻 脂肪酸代謝 珠磨法 lpaat基因 gpd1基因　出處：《深圳大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：經(jīng)濟(jì)全球化和人口數(shù)量的急劇擴(kuò)增,使我們對(duì)能源的需求越來(lái)越大,與此同時(shí),石油等不可再生能源正面臨枯竭,由此引發(fā)研究人員對(duì)可再生能源產(chǎn)生濃厚的研究興趣。目前,生物柴油已被認(rèn)為是最理想的可再生能源之一,其主要成分是脂肪酸甲酯(FAME),是以可再生資源為原料通過(guò)酯交換制備而成,具備與石化柴油相近的性能。微藻制備生物柴油因具有不占用耕地,易收易養(yǎng),含油量高等特點(diǎn)而成為近年來(lái)的研究熱點(diǎn)。利用基因工程可以實(shí)現(xiàn)定向、效果顯著的改變物種性狀的目的,是常用的改造基因的方法。在萊茵衣藻脂肪酸合成路徑中,甘油醛-3-磷酸脫氫酶(glyceraldehyde-3-phosphate dehydrogenase,G3PDH)和溶血磷脂酸�；D(zhuǎn)移酶(Lyso-Phosphatidic Acid Acyltransferase,LPAAT)是三酰甘油組裝路徑上的催化酶,在調(diào)控脂肪酸含量方面具有重要作用。本研究分別將活性較高的外源基因,即油菜(Brassica napus)溶血磷脂酸�；D(zhuǎn)移酶基因(lpaat)和酵母(Saccharomyces cerevisiae)的甘油3-磷酸酰基轉(zhuǎn)移酶基因(gpd1)轉(zhuǎn)入萊茵衣藻CC-849基因組中并進(jìn)行表達(dá),進(jìn)而改變?nèi)R茵衣藻脂肪酸的含量。具體研究結(jié)果如下:1、從NCBI上獲得lpaat和gpd1基因的表達(dá)序列,根據(jù)萊茵衣藻密碼子優(yōu)先性進(jìn)行改造得到c-lpaat和c-gpd1基因,經(jīng)人工合成獲得改造好的基因序列,再連接到p H124載體中,構(gòu)建衣藻表達(dá)載體p H124-c-lpaat和p H124-c-gpd1,通過(guò)珠磨法轉(zhuǎn)化到萊茵衣藻中,經(jīng)過(guò)抗性篩選、PCR檢測(cè)和GC-MS分析,最終篩選得到高效表達(dá)目的基因的轉(zhuǎn)基因藻株,成功轉(zhuǎn)入c-lpaat基因的轉(zhuǎn)化子有Tranc-lpaat-J、K、L、M、N、Q、1、2、3、4、5、14、15,成功轉(zhuǎn)入c-gpd1基因的轉(zhuǎn)化子有Tranc-gpd1-P、T、C、O、N、10、15、16;2、利用RT-PCR、熒光定量PCR等技術(shù)對(duì)轉(zhuǎn)基因藻中c-lpaat和c-gpd1基因的表達(dá)情況進(jìn)行分析。半定量RT-PCR結(jié)果顯示Tranc-lpaat-15和Tranc-gpd1-T的目的基因表達(dá)水平最高,進(jìn)一步的熒光定量PCR結(jié)果表明:在熱激誘導(dǎo)的條件下,外源基因c-lpaat和c-gpd1表達(dá)量明顯上升,熱激一次后c-lpaat基因表達(dá)量上升了1.93倍,c-gpd1基因表達(dá)量上升了2.98倍。連續(xù)熱激三次后,c-lpaat基因表達(dá)量分別為原來(lái)的3.01倍,4.46倍和5.30倍;c-gpd1基因的表達(dá)量分別為原來(lái)的3.6倍,5.42倍和8.58倍;3、利用GC-MS分析轉(zhuǎn)基因藻的總脂肪酸含量及組分,得到結(jié)果如下:(1)對(duì)所挑選的轉(zhuǎn)化子進(jìn)行篩選,分別獲得脂肪酸含量最高的轉(zhuǎn)化株,即含有c-lpaat基因的Tranc-lpaat-15和c-gpd1基因的Tranc-gpd1-T轉(zhuǎn)化子;(2)在熱激一次后,轉(zhuǎn)基因藻Tranc-lpaat-15的脂肪酸含量提高了16.8%,其中提高最多的是C18:1t,較對(duì)照組增加了177.27%;轉(zhuǎn)基因藻Tranc-gpd1-T脂肪酸含量提高了26.7%,其中提高最多的是C18:1t,較對(duì)照組增加了270.91%;(3)連續(xù)熱激三次后,轉(zhuǎn)基因藻Tranc-lpaat-15脂肪酸含量提高了44.5%,其中提高最多的是C18:0,C18:1t,較對(duì)照組提高了355.29%和220.11%。轉(zhuǎn)基因藻Tranc-gpd1-T脂肪酸含量提高了67.5%,其中提高最多的是C18:0,C18:1t,較對(duì)照組提高了428.24%和394.18%。在轉(zhuǎn)基因藻Tranc-lpaat-15和Tranc-gpd1-T中,C16:0,C18:0,C18:1t、C18:2t的含量提高最多。通過(guò)遺傳重組的方法將促進(jìn)油脂合成的外源基因?qū)肴R茵衣藻核基因組,經(jīng)整合后可獲得性狀能穩(wěn)定遺傳的轉(zhuǎn)基因藻株。本研究轉(zhuǎn)入酶活性較高的外源基因lpaat和gpd1,采用可誘導(dǎo)的熱休克蛋白啟動(dòng)子表達(dá)目的基因,實(shí)現(xiàn)目的基因的可控表達(dá),最終調(diào)控三酰甘油TAG的合成代謝,使藻細(xì)胞的油脂含量增加,以上研究為獲得油脂含量高的轉(zhuǎn)基因藻株用于生物柴油的生產(chǎn)奠定實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:The number of the economic globalization and the rapid population expansion, so we demand more and more energy, at the same time, oil and other non renewable energy is exhausted, which caused the researchers generated strong interest in the study of renewable energy. At present, biodiesel has been considered to be one of the most ideal renewable energy, which is the main component of fatty acid methyl ester (FAME), is based on renewable resources as raw materials prepared by ester exchange, have similar performance with diesel. Microalgae biodiesel preparation has no land occupation, easy collection and easy maintenance, high oil content and become the research hotspot in recent years. The use of genetic engineering can achieve directional, significant effect the change of species traits, gene transformation method is commonly used. In the synthesis of fatty acids in the path of Rhine Chlamydomonas, glyceraldehyde dehydrogenase (glyceraldehyde-3-phosphate dehydrog, -3- Enase, G3PDH) and lysophosphatidic acid acyltransferase (Lyso-Phosphatidic Acid, Acyltransferase, LPAAT) is the three TG assembly path of the catalytic enzyme, which plays an important role in the regulation of fatty acid content. This study will be exogenous gene high activity, namely rape (Brassica napus) lysophosphatidic acid acyltransferase gene (lpaat) and yeast (Saccharomyces cerevisiae 3-) glycerol phosphate acyltransferase (GPD1) gene into the genome of CC-849 Rhine Chlamydomonas and express, and then change the fatty acid content of Rhine Chlamydomonas. The results are as follows: 1. The expression of lpaat and GPD1 gene sequences from NCBI, according to the c-lpaat and c-gpd1 genes by modifying Rhine Chlamydomonas codon preference, by synthetic gene sequences obtained good transformation, then connect to the P H124 vector to construct the expression vector p, Chlamydomonas H124-c-lpaat and P H124-c- GPD1, into Rhine Chlamydomonas through bead milling method, after resistance screening, PCR detection and GC-MS analysis, finally selected high expression of target genes in transgenic strains and transformants of c-lpaat gene was successfully transferred to Tranc-lpaat-J, K, L, M, N, Q, 1,2,3,4,5,14,15, c-gpd1 gene was transferred into the transformants with Tranc-gpd1-P, T, C, O, N, 10,15,16; 2, by RT-PCR, analyzed the expression of fluorescent quantitative PCR technology of c-lpaat and c-gpd1 genes in transgenic Anabaena. Semi quantitative RT-PCR results showed that Tranc-lpaat-15 and Tranc-gpd1-T. The highest expression level, quantitative PCR results further showed that the heat shock induced under the condition of exogenous gene expression of c-lpaat and c-gpd1 was increased after the first heat shock c-lpaat gene expression was increased by 1.93 times, c-gpd1 gene expression was increased by 2.98 times. After three times of continuous heat shock, expression of c-lpaat gene Are the original 3.01 times, 4.46 times and 5.30 times; the expression level of c-gpd1 gene were 3.6 times, 5.42 times and 8.58 times; 3, the use of GC-MS analysis of the total fatty acid content and the group of transgenic algae, the results are as follows: (1) the selected transformants were screened, divided don't get the highest levels of fatty acids of transformants containing Tranc-lpaat-15 and c-gpd1 gene, the c-lpaat gene of Tranc-gpd1-T transformants; (2) in a heat shock, the fatty acid content of transgenic alga Tranc-lpaat-15 increased by 16.8%, which is up to C18:1t, compared with the control group increased by 177.27%; transgenic alga Tranc-gpd1-T fat acid content increased by 26.7%, which is up to C18:1t, compared with the control group increased by 270.91%; (3) continuous heat shock after three times, transgenic algae Tranc-lpaat-15 fatty acid content increased by 44.5%, which is up to C18:0, C18:1t, higher than the control group The Tranc-gpd1-T fatty acid content of 355.29% and 220.11%. transgenic algae increased by 67.5%, which is up to C18:0, C18:1t, compared with the control group increased by 428.24% and 394.18%. in transgenic alga Tranc-lpaat-15 and Tranc-gpd1-T, C16:0, C18:0, C18:1t, C18:2t content increased most. Through genetic recombination method will promote the lipid synthesis of exogenous gene Rhine Chlamydomonas nuclear genome, can be obtained by the characters can be inherited steadily after the integration of the transgenic alga. This research into higher enzyme activity of exogenous gene of lpaat and GPD1, the heat shock protein promoter can induce the expression of target gene, controllable target gene expression, synthesis and metabolism of the regulation three TG TAG,. The oil content of algal cells increased, the above research to obtain transgenic algae with high oil content were used to provide the experimental basis for the production of biodiesel.

【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：Q949.2;TE667

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本文編號(hào)：1506043