利用lpaat和gpd1基因提高莱茵衣藻脂肪酸含量的研究

发布时间：2018-02-12 16:19

本文关键词： 莱茵衣藻脂肪酸代谢珠磨法 lpaat基因 gpd1基因　出处：《深圳大学》2015年硕士论文　论文类型：学位论文

【摘要】：经济全球化和人口数量的急剧扩增,使我们对能源的需求越来越大,与此同时,石油等不可再生能源正面临枯竭,由此引发研究人员对可再生能源产生浓厚的研究兴趣。目前,生物柴油已被认为是最理想的可再生能源之一,其主要成分是脂肪酸甲酯(FAME),是以可再生资源为原料通过酯交换制备而成,具备与石化柴油相近的性能。微藻制备生物柴油因具有不占用耕地,易收易养,含油量高等特点而成为近年来的研究热点。利用基因工程可以实现定向、效果显著的改变物种性状的目的,是常用的改造基因的方法。在莱茵衣藻脂肪酸合成路径中,甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,G3PDH)和溶血磷脂酸酰基转移酶(Lyso-Phosphatidic Acid Acyltransferase,LPAAT)是三酰甘油组装路径上的催化酶,在调控脂肪酸含量方面具有重要作用。本研究分别将活性较高的外源基因,即油菜(Brassica napus)溶血磷脂酸酰基转移酶基因(lpaat)和酵母(Saccharomyces cerevisiae)的甘油3-磷酸酰基转移酶基因(gpd1)转入莱茵衣藻CC-849基因组中并进行表达,进而改变莱茵衣藻脂肪酸的含量。具体研究结果如下:1、从NCBI上获得lpaat和gpd1基因的表达序列,根据莱茵衣藻密码子优先性进行改造得到c-lpaat和c-gpd1基因,经人工合成获得改造好的基因序列,再连接到p H124载体中,构建衣藻表达载体p H124-c-lpaat和p H124-c-gpd1,通过珠磨法转化到莱茵衣藻中,经过抗性筛选、PCR检测和GC-MS分析,最终筛选得到高效表达目的基因的转基因藻株,成功转入c-lpaat基因的转化子有Tranc-lpaat-J、K、L、M、N、Q、1、2、3、4、5、14、15,成功转入c-gpd1基因的转化子有Tranc-gpd1-P、T、C、O、N、10、15、16;2、利用RT-PCR、荧光定量PCR等技术对转基因藻中c-lpaat和c-gpd1基因的表达情况进行分析。半定量RT-PCR结果显示Tranc-lpaat-15和Tranc-gpd1-T的目的基因表达水平最高,进一步的荧光定量PCR结果表明:在热激诱导的条件下,外源基因c-lpaat和c-gpd1表达量明显上升,热激一次后c-lpaat基因表达量上升了1.93倍,c-gpd1基因表达量上升了2.98倍。连续热激三次后,c-lpaat基因表达量分别为原来的3.01倍,4.46倍和5.30倍;c-gpd1基因的表达量分别为原来的3.6倍,5.42倍和8.58倍;3、利用GC-MS分析转基因藻的总脂肪酸含量及组分,得到结果如下:(1)对所挑选的转化子进行筛选,分别获得脂肪酸含量最高的转化株,即含有c-lpaat基因的Tranc-lpaat-15和c-gpd1基因的Tranc-gpd1-T转化子;(2)在热激一次后,转基因藻Tranc-lpaat-15的脂肪酸含量提高了16.8%,其中提高最多的是C18:1t,较对照组增加了177.27%;转基因藻Tranc-gpd1-T脂肪酸含量提高了26.7%,其中提高最多的是C18:1t,较对照组增加了270.91%;(3)连续热激三次后,转基因藻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,较对照组提高了355.29%和220.11%。转基因藻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,较对照组提高了428.24%和394.18%。在转基因藻Tranc-lpaat-15和Tranc-gpd1-T中,C16:0,C18:0,C18:1t、C18:2t的含量提高最多。通过遗传重组的方法将促进油脂合成的外源基因导入莱茵衣藻核基因组,经整合后可获得性状能稳定遗传的转基因藻株。本研究转入酶活性较高的外源基因lpaat和gpd1,采用可诱导的热休克蛋白启动子表达目的基因,实现目的基因的可控表达,最终调控三酰甘油TAG的合成代谢,使藻细胞的油脂含量增加,以上研究为获得油脂含量高的转基因藻株用于生物柴油的生产奠定实验基础。
[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.

【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q949.2;TE667

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