本文選題：普通小球藻 + 基因工程��； 參考：《華南理工大學(xué)》2016年博士論文

【摘要】：由溫室氣體導(dǎo)致的全球變暖現(xiàn)象已經(jīng)引起全世界的廣泛關(guān)注。其中CO2是溫室氣體的主要成分。據(jù)世界氣象組織(WMO)估計,現(xiàn)今大氣中的CO2濃度已驚人地超過400ppm,比工業(yè)化之前的280 ppm提高了約43%。因此,發(fā)展低碳經(jīng)濟技術(shù)、實現(xiàn)碳減排已刻不容緩。目前已有多種技術(shù)致力于大氣中CO2的捕獲與封存,其中利用微藻進(jìn)行固碳的生物碳減排技術(shù)逐漸成為當(dāng)今的研究熱點之一。微藻通過光合作用,將CO2轉(zhuǎn)化成油脂、蛋白質(zhì)、淀粉和類胡蘿卜素等生物質(zhì),有助于實現(xiàn)碳減排。同時,微藻具有光合效率高、生長快、培養(yǎng)成本低,以及易工業(yè)化集成等優(yōu)點,因此,通過微藻固定CO2實現(xiàn)碳減排是一項經(jīng)濟有效的技術(shù),具有廣闊的發(fā)展前景。但微藻通過光合作用固定CO2的效率仍然有限,如何實質(zhì)性的提高其內(nèi)在的光合固碳能力成為制約微藻生物固碳技術(shù)發(fā)展的一個難題。本研究以具備良好的CO2固定和耐受能力的普通小球藻(Chlorella vulgaris)為研究對象,首次提出并嘗試通過基因工程技術(shù)過表達(dá)光合作用卡爾文循環(huán)中的關(guān)鍵酶基因來實質(zhì)性的提高普通小球藻光合固碳的能力,從而達(dá)到碳減排的目的。首先利用增強型綠色熒光蛋白報告基因(EGFP)建立了一套穩(wěn)定有效的普通小球藻遺傳轉(zhuǎn)化體系,在此基礎(chǔ)上,將集胞藻(Synechocystis sp.PCC6803)卡爾文循環(huán)中的關(guān)鍵酶——果糖-1,6-二磷酸醛縮酶基因(FBA)導(dǎo)入到普通小球藻基因組中,成功地將其定位到葉綠體中進(jìn)行了表達(dá),有效地提高了普通小球藻的生長速率和光合效率(CO2固定速率)。此外還探究了集胞藻FBA基因?qū)ζ胀ㄐ∏蛟骞夂瞎烫伎赡艿恼{(diào)控機理。本研究結(jié)果為小球藻基因工程和細(xì)胞生物學(xué)研究奠定了良好的基礎(chǔ),同時為今后進(jìn)一步提高其光合固碳效率提供了參考與依據(jù)。本研究的主要結(jié)果如下:(1)確定了普通小球藻外源基因遺傳轉(zhuǎn)化體系的最適篩選標(biāo)記。通過在普通小球藻的固體培養(yǎng)基中分別添加4種不同的抗生素(卡那霉素,G418,壯觀霉素和氯霉素),發(fā)現(xiàn)其對G418最為敏感,半致死率(LC50)可達(dá)11.74μg/m L。之后通過在液體培養(yǎng)基中分別添加不同濃度的G418,發(fā)現(xiàn)30μg/m L的G418可強烈地抑制藻細(xì)胞的生長,抑制率可達(dá)90%以上。因此,30μg/m L的G418可作為普通小球藻遺傳轉(zhuǎn)化體系篩選轉(zhuǎn)化子的有效濃度,同時G418對應(yīng)的抗性基因——新霉素磷酸轉(zhuǎn)移酶基因(npt II)可作為該遺傳轉(zhuǎn)化體系的最適抗性篩選標(biāo)記。(2)利用EGFP報告基因建立了一套穩(wěn)定有效的普通小球藻外源基因遺傳轉(zhuǎn)化體系。通過克隆EGFP基因,利用聚乙二醇(PEG)轉(zhuǎn)化法首次將EGFP基因?qū)氲狡胀ㄐ∏蛟宓幕蚪M中,轉(zhuǎn)化率為356±30 cfu/μg DNA。通過PCR、Southern雜交和反轉(zhuǎn)錄PCR(RT-PCR)和熒光顯微實驗,結(jié)果表明EGFP基因成功整合到了普通小球藻的基因組中,并可在細(xì)胞質(zhì)基質(zhì)中進(jìn)行可見表達(dá)。另外,經(jīng)過細(xì)胞傳代實驗,發(fā)現(xiàn)普通小球藻在傳代16次后仍能表達(dá)出可見熒光。由此建立了一個穩(wěn)定有效的普通小球藻外源基因遺傳轉(zhuǎn)化體系。(3)克隆并驗證了rbc S基因葉綠體導(dǎo)肽(c TP)序列的亞細(xì)胞定位功能,同時驗證了在普通小球藻的葉綠體中表達(dá)核基因組編碼的外源蛋白的可行性。從普通小球藻中克隆了核酮糖-1,5-二磷酸羧化酶/加氧酶小亞基基因(rbc S)的c TP序列,通過構(gòu)建一系列中間載體將其融合到EGFP基因的N端(c TP::EGFP),并利用PEG法導(dǎo)入到普通小球藻中,經(jīng)過熒光亞細(xì)胞定位分析,發(fā)現(xiàn)c TP::EGFP融合基因在藻細(xì)胞的葉綠體中表達(dá)出肉眼可見的綠色熒光,而未融合c TP序列的EGFP基因僅在細(xì)胞質(zhì)基質(zhì)中表達(dá)出綠色熒光,由此表明該c TP序列起到了葉綠體定位的功能,且驗證了在普通小球藻的葉綠體中表達(dá)核基因組編碼的外源蛋白的可行性,同時拓展了熒光蛋白技術(shù)在微藻分子生物學(xué)研究中的應(yīng)用。(4)通過在普通小球藻葉綠體中過表達(dá)FBA酶,有效地提高了普通小球藻的生長和光合效率。從藍(lán)藻的模式生物——集胞藻中克隆了其FBA基因,通過構(gòu)建融合上述c TP序列的FBA超表達(dá)載體,利用PEG法首次將集胞藻FBA基因?qū)氲狡胀ㄐ∏蛟寤蚪M中,通過PCR、southern雜交、RT-PCR和western雜交共同鑒定,表明集胞藻FBA基因成功整合到了轉(zhuǎn)化藻株的基因組DNA中并進(jìn)行了表達(dá)。另外,通過測定發(fā)現(xiàn)轉(zhuǎn)化藻#3和#5的FBA酶活顯著高于WT 1.27和1.30倍(p0.05),其生物量在培養(yǎng)中后期也顯著高于WT(p0.05),同時,它們的凈放氧速率和CO2固定速率也分別是WT的1.18~1.21倍與1.15~1.18倍(p0.05),表明集胞藻FBA基因的過表達(dá)能有效地促進(jìn)藻細(xì)胞的生長與光合效率。(5)通過比較轉(zhuǎn)FBA藻(#3和#5)和野生型藻的生理指標(biāo),探究了過表達(dá)的集胞藻FBA酶對普通小球藻光合固碳可能的調(diào)控機理。與野生型藻相比,轉(zhuǎn)FBA藻#3和#5的葉綠素含量顯著提高(p0.05)。葉綠素?zé)晒夥治鼋Y(jié)果表明,#3和#5的光化學(xué)淬滅系數(shù)(q P)和PSII的實際光化學(xué)效率(ΦPSII)均有顯著提高(p0.05),而非光化學(xué)淬滅系數(shù)(NPQ)均顯著下降(p0.05),表明轉(zhuǎn)FBA藻在光合作用光反應(yīng)階段的電子(能量)傳遞速率有明顯提高。此外,通過測定其卡爾文循環(huán)關(guān)鍵酶的基因表達(dá)量和酶活,發(fā)現(xiàn)轉(zhuǎn)FBA藻的Rubisco酶的基因表達(dá)量與初始酶活性均有明顯提高,而其余關(guān)鍵酶的基因表達(dá)量的提高并未引起相應(yīng)的酶活發(fā)生明顯變化。上述結(jié)果表明,FBA酶在普通小球藻中的過表達(dá),可能通過提高Ru BP的含量、激活更多的Rubisco酶來加速碳流周轉(zhuǎn)速率,同時提高其光系統(tǒng)的能量傳遞速率,從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.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：X173;X51

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