普通小球藻稳定遗传体系的建立及基因改造其光合固碳效率的研究

发布时间：2018-06-17 12:03

本文选题：普通小球藻 + 基因工程　；参考：《华南理工大学》2016年博士论文

【摘要】：由温室气体导致的全球变暖现象已经引起全世界的广泛关注。其中CO2是温室气体的主要成分。据世界气象组织(WMO)估计,现今大气中的CO2浓度已惊人地超过400ppm,比工业化之前的280 ppm提高了约43%。因此,发展低碳经济技术、实现碳减排已刻不容缓。目前已有多种技术致力于大气中CO2的捕获与封存,其中利用微藻进行固碳的生物碳减排技术逐渐成为当今的研究热点之一。微藻通过光合作用,将CO2转化成油脂、蛋白质、淀粉和类胡萝卜素等生物质,有助于实现碳减排。同时,微藻具有光合效率高、生长快、培养成本低,以及易工业化集成等优点,因此,通过微藻固定CO2实现碳减排是一项经济有效的技术,具有广阔的发展前景。但微藻通过光合作用固定CO2的效率仍然有限,如何实质性的提高其内在的光合固碳能力成为制约微藻生物固碳技术发展的一个难题。本研究以具备良好的CO2固定和耐受能力的普通小球藻(Chlorella vulgaris)为研究对象,首次提出并尝试通过基因工程技术过表达光合作用卡尔文循环中的关键酶基因来实质性的提高普通小球藻光合固碳的能力,从而达到碳减排的目的。首先利用增强型绿色荧光蛋白报告基因(EGFP)建立了一套稳定有效的普通小球藻遗传转化体系,在此基础上,将集胞藻(Synechocystis sp.PCC6803)卡尔文循环中的关键酶——果糖-1,6-二磷酸醛缩酶基因(FBA)导入到普通小球藻基因组中,成功地将其定位到叶绿体中进行了表达,有效地提高了普通小球藻的生长速率和光合效率(CO2固定速率)。此外还探究了集胞藻FBA基因对普通小球藻光合固碳可能的调控机理。本研究结果为小球藻基因工程和细胞生物学研究奠定了良好的基础,同时为今后进一步提高其光合固碳效率提供了参考与依据。本研究的主要结果如下:(1)确定了普通小球藻外源基因遗传转化体系的最适筛选标记。通过在普通小球藻的固体培养基中分别添加4种不同的抗生素(卡那霉素,G418,壮观霉素和氯霉素),发现其对G418最为敏感,半致死率(LC50)可达11.74μg/m L。之后通过在液体培养基中分别添加不同浓度的G418,发现30μg/m L的G418可强烈地抑制藻细胞的生长,抑制率可达90%以上。因此,30μg/m L的G418可作为普通小球藻遗传转化体系筛选转化子的有效浓度,同时G418对应的抗性基因——新霉素磷酸转移酶基因(npt II)可作为该遗传转化体系的最适抗性筛选标记。(2)利用EGFP报告基因建立了一套稳定有效的普通小球藻外源基因遗传转化体系。通过克隆EGFP基因,利用聚乙二醇(PEG)转化法首次将EGFP基因导入到普通小球藻的基因组中,转化率为356±30 cfu/μg DNA。通过PCR、Southern杂交和反转录PCR(RT-PCR)和荧光显微实验,结果表明EGFP基因成功整合到了普通小球藻的基因组中,并可在细胞质基质中进行可见表达。另外,经过细胞传代实验,发现普通小球藻在传代16次后仍能表达出可见荧光。由此建立了一个稳定有效的普通小球藻外源基因遗传转化体系。(3)克隆并验证了rbc S基因叶绿体导肽(c TP)序列的亚细胞定位功能,同时验证了在普通小球藻的叶绿体中表达核基因组编码的外源蛋白的可行性。从普通小球藻中克隆了核酮糖-1,5-二磷酸羧化酶/加氧酶小亚基基因(rbc S)的c TP序列,通过构建一系列中间载体将其融合到EGFP基因的N端(c TP::EGFP),并利用PEG法导入到普通小球藻中,经过荧光亚细胞定位分析,发现c TP::EGFP融合基因在藻细胞的叶绿体中表达出肉眼可见的绿色荧光,而未融合c TP序列的EGFP基因仅在细胞质基质中表达出绿色荧光,由此表明该c TP序列起到了叶绿体定位的功能,且验证了在普通小球藻的叶绿体中表达核基因组编码的外源蛋白的可行性,同时拓展了荧光蛋白技术在微藻分子生物学研究中的应用。(4)通过在普通小球藻叶绿体中过表达FBA酶,有效地提高了普通小球藻的生长和光合效率。从蓝藻的模式生物——集胞藻中克隆了其FBA基因,通过构建融合上述c TP序列的FBA超表达载体,利用PEG法首次将集胞藻FBA基因导入到普通小球藻基因组中,通过PCR、southern杂交、RT-PCR和western杂交共同鉴定,表明集胞藻FBA基因成功整合到了转化藻株的基因组DNA中并进行了表达。另外,通过测定发现转化藻#3和#5的FBA酶活显著高于WT 1.27和1.30倍(p0.05),其生物量在培养中后期也显著高于WT(p0.05),同时,它们的净放氧速率和CO2固定速率也分别是WT的1.18~1.21倍与1.15~1.18倍(p0.05),表明集胞藻FBA基因的过表达能有效地促进藻细胞的生长与光合效率。(5)通过比较转FBA藻(#3和#5)和野生型藻的生理指标,探究了过表达的集胞藻FBA酶对普通小球藻光合固碳可能的调控机理。与野生型藻相比,转FBA藻#3和#5的叶绿素含量显著提高(p0.05)。叶绿素荧光分析结果表明,#3和#5的光化学淬灭系数(q P)和PSII的实际光化学效率(ΦPSII)均有显著提高(p0.05),而非光化学淬灭系数(NPQ)均显著下降(p0.05),表明转FBA藻在光合作用光反应阶段的电子(能量)传递速率有明显提高。此外,通过测定其卡尔文循环关键酶的基因表达量和酶活,发现转FBA藻的Rubisco酶的基因表达量与初始酶活性均有明显提高,而其余关键酶的基因表达量的提高并未引起相应的酶活发生明显变化。上述结果表明,FBA酶在普通小球藻中的过表达,可能通过提高Ru BP的含量、激活更多的Rubisco酶来加速碳流周转速率,同时提高其光系统的能量传递速率,从而使光合效率得到提高。
[Abstract]:Global warming caused by greenhouse gases has aroused widespread concern around the world. CO2 is the main component of greenhouse gases. According to the World Meteorological Organization (WMO), the CO2 concentration in the atmosphere has exceeded 400ppm in the present day, and is about 43% higher than the 280 ppm before industrialization. Therefore, the development of low carbon economic technology to achieve carbon emission reduction has been achieved. There is no delay. At present, many technologies have been devoted to the capture and sequestration of CO2 in the atmosphere, and the technology of carbon reduction by microalgae for carbon sequestration has gradually become one of the hotspots of current research. Through photosynthesis, microalgae transform CO2 into oil, protein, starch and carotene like biomass, which can help to reduce carbon emission. Microalgae have the advantages of high photosynthetic efficiency, fast growth, low culture cost, and easy industrialization integration. Therefore, it is an economical and effective technology to achieve carbon emission reduction by microalgae fixed CO2. However, the efficiency of microalgae in fixing CO2 through photosynthesis is still limited. How to substantially improve its intrinsic photosynthetic carbon energy Force has become a difficult problem to restrict the development of carbon sequestration technology in microalgae. In this study, the Chlorella vulgaris, which has good CO2 fixation and tolerance, is the research object. The key enzyme gene in the Calvin cycle of photosynthesis was first proposed and tried to improve the essence of the microalgae by gene engineering technology. On the basis of the enhanced green fluorescent protein reporter gene (EGFP), a stable and effective genetic transformation system of Chlorella vulgaris was established by using the enhanced green fluorescent protein reporter gene (EGFP). On this basis, the key enzyme in the Synechocystis sp.PCC6803 cycle, the fructose -1,6- two phosphate aldolase The gene (FBA) was introduced into the genome of Chlorella vulgaris and was successfully expressed in the chloroplast. The growth rate and photosynthetic efficiency (CO2 fixation rate) of Chlorella vulgaris were effectively improved. In addition, the regulation mechanism of the FBA gene on the photosynthetic carbon fixation of Chlorella vulgaris was also explored. The main results of this study are as follows: (1) the optimum screening markers for the genetic transformation system of the exogenous gene of Chlorella vulgaris were determined. With 4 different antibiotics (kanamycin, G418, splendin and chloramphenicol), they were found to be most sensitive to G418, and the semi lethal rate (LC50) could reach 11.74 g/m L. by adding different concentrations of G418 in the liquid medium. It was found that G418 of 30 mu g/m L could strongly inhibit the growth of algae cells, and the inhibition rate could be over 90%. Therefore, 30 micron g/m. The G418 of L can be used to screen the effective concentration of transformants as the genetic transformation system of Chlorella vulgaris, and the corresponding resistance gene of G418, neomycin phosphate transferase gene (NPT II), can be used as the optimum screening marker for the genetic transformation system. (2) a set of stable and effective foreign gene of Chlorella vulgaris is established by using EGFP reporter gene. Genetic transformation system. By cloning EGFP gene and using polyethylene glycol (PEG) transformation method, the EGFP gene was first introduced into the genome of Chlorella vulgaris for the first time. The conversion rate was 356 + 30 cfu/ mu g DNA. through PCR, Southern hybridization and reverse transcriptional PCR (RT-PCR) and fluorescence microscopy. The results showed that the EGFP gene was successfully integrated into the gene of Chlorella vulgaris. In the group, it can be expressed in the cytoplasm matrix. In addition, after the cell passage experiment, it was found that the common Chlorella could still express the visible fluorescence after 16 times of passage. Thus, a stable and effective genetic transformation system for the exogenous gene of Chlorella vulgaris was established. (3) the subunit of the RBC S gene (C TP) sequence was cloned and verified. The feasibility of expressing the exogenous protein encoded by the nuclear genome in chloroplasts of Chlorella vulgaris was verified. The C TP sequence of the -1,5- two phosphate carboxylase / oxygenase small subunit gene (RBC S) was cloned from Chlorella vulgaris, and it was fused to the N end of the EGFP gene by constructing a series of intermediate vectors (C TP:: EGFP) and the PEG method was introduced into the Chlorella vulgaris. After the fluorescence subcellular localization analysis, it was found that the C TP:: EGFP fusion gene expressed the green fluorescence in the chloroplast of the algal cells, and the EGFP gene that did not fuse the C TP sequence was only expressed in the cytoplasm matrix, which showed that the C TP sequence was a chloroplast. The function of localization and the feasibility of expressing the exogenous protein encoded by the nuclear genome in chloroplasts of Chlorella vulgaris was verified, and the application of fluorescent protein technology in microalgae molecular biology was expanded. (4) the growth and photosynthetic efficiency of Chlorella vulgaris were effectively improved by overexpressing FBA enzyme in chloroplasts of Chlorella vulgaris. The FBA gene was cloned from the model organism of the cyanobacteria. By constructing the FBA overexpression vector that fused the C TP sequence, the FBA gene was introduced into the genome of Chlorella vulgaris by PEG method for the first time. It was identified by PCR, Southern hybridization, and RT-PCR and Western heterozygosity. It showed that the FBA gene of the alga was integrated successfully. In addition, the FBA enzyme activity of transforming algae #3 and #5 was found to be significantly higher than that of WT 1.27 and 1.30 times (P0.05), and their biomass was also significantly higher than WT (P0.05) in the middle and late stages of culture, and their net oxygen rate and CO2 fixed rate were also 1.18~1.21 times and 1.15~1.18 times of WT, respectively, by determination. The overexpression of FBA gene could effectively promote the growth and photosynthetic efficiency of the algal cells. (5) by comparing the physiological indexes of FBA algae (#3 and #5) and wild type algae, the regulation mechanism of the overexpressed FBA enzyme in the photosynthetic carbon of Chlorella vulgaris was explored. Compared with the wild type algae, the chlorophyll content of #3 and #5 of FBA algae was displayed. The results of P0.05. The results of chlorophyll fluorescence analysis showed that the photochemical quenching coefficient of #3 and #5 (Q P) and the actual photochemical efficiency of PSII (P0.05) were significantly improved (P0.05), but the non photochemical quenching coefficient (NPQ) decreased significantly (P0.05), indicating that the transfer rate of electron (energy) in the light reaction phase of the transgenic FBA algae was obviously improved. In addition, by measuring the gene expression and enzyme activity of the key enzyme of the Calvin cycle, it was found that the gene expression of Rubisco enzyme and the activity of the initial enzyme were obviously improved, while the increase of the gene expression of the other key enzymes did not cause significant changes in the corresponding enzyme activity. The results showed that the FBA enzyme was in the Chlorella vulgaris of FBA. Over expression, by increasing the content of Ru BP, more Rubisco enzymes can be activated to accelerate the rate of carbon flow turnover and increase the energy transfer rate of the optical system, thus improving the photosynthetic efficiency.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X173;X51

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