【作者】 黎明；

【导师】 李明春；

【作者基本信息】 南开大学 ， 微生物学， 2012， 博士

【摘要】 多不饱脂肪酸(Polyunsaturated fatty acids, PUFAs),尤其是超长链多不饱和脂肪酸(Very long chain polyunsaturated fatty acids, VLC-PUFAs)，具有十分重要的生理功能，是维持人体健康所必需的。通常情况下PUFAs主要来源于深海鱼油和海洋藻类，但是由于过度捕捞，海洋污染以及藻类生产PUFAs价格的昂贵，因此迫切需要寻找一种PUFAs的替代来源。随着转基因技术的迅速发展，利用转基因的油料作物生产PUFAs被认为是一种十分有前景的的替代来源。大豆是最主要的油料作物之一，含有非常丰富的亚油酸(Linoleic acid, LA)和α-亚麻酸(α-Linolenic acid,ALA)，它们分别占大豆中总脂肪酸的55%和13%，是合成花生四烯酸(Arachidonic acid, ARA)和二十碳五烯酸(Eicosapentaenoic acid, EPA)的前体。因此，大豆是通过代谢工程生产ARA和EPA的重要宿主。在转基因植物中，ARA和EPA生物合成的Δ6途径已经被广泛研究了，但Δ8途径仅在拟南芥中进行研究并且不是种子特异性表达。由于在转基因植物中ARA和EPA生物合成的Δ6途径存在复杂的底物转换瓶颈，因此，在本研究中，我们分别在酿酒酵母和大豆中重构ARA和EPA生物合成的Δ8途径，构建出产ARA和EPA的酿酒酵母工程菌和无选择标记基因的转基因大豆；同时对BCSP952启动子进行改造，以便提高转基因大豆中ARA和EPA的合成效率。首先，分别从球等鞭金藻H29(Isochrysis galbana H29)、小眼虫藻FH277(Euglena gracilis FH277)和三角褐指藻(Phaeodactylum tricornutum)中克隆出Δ8途径中的三个基因Δ9延长酶基因IgASE2、Δ8脱氢酶基因efd2和Δ5脱氢酶基因ptd5，并在酿酒酵母INVSc1中鉴定它们的功能。IgASE2基因长1653bp，包括一个786bp的ORF (Open reading frame)、一个44bp的5’-UTR(Untranslatedregion)和一个823bp3’-UTR。该基因编码的延长酶IgASE2与已经报道的延长酶IgASE1的氨基酸序列有87%的相似性。IgASE2在酿酒酵母中表达时，能分别将LA和ALA转化成二十碳二烯酸(ω6-eicosadienoic acid, EDA)和二十碳三烯酸(ω3-eicosatrienoic acid, EtrA)，LA和ALA的转化率分别是57.6%和56.1%，表明IgASE2基因是一个新的C18-Δ9专一性的PUFAs延长酶基因。efd2基因ORF长1266bp，编码421个氨基酸，它与已经报道的EFD1的氨基酸序列相似性为96%。efd2基因在酿酒酵母中表达时，催化底物EDA和EtrA转化成二高γ-亚麻酸(dihomo-γ-linolenic acid, DGLA)和二十碳四烯酸(eicosatetraenoic acid, ETA)的转化效率分别约为31.2％和46.3％，表明efd2是一个位置专一性的Δ8脱氢酶基因。ptd5基因ORF长1410bp，编码469个氨基酸。它在酿酒酵母中表达时，催化DGLA和ETA生成ARA和EPA的效率分别约28.7%和37.2%，表明ptd5是一个位置专一性的Δ5脱氢酶基因。然后，利用克隆的IgASE2、efd2和ptd5基因，分别在酿酒酵母和大豆中构建了ARA和EPA生物合成的Δ8(ω6-Δ8, ω3-Δ8)途径，获得了产ARA和EPA的酿酒酵母工程菌和无选择标记基因的转基因大豆。根据ARA和EPA生物合成的Δ8(ω6-Δ8, ω3-Δ8)途径，将该途径中所需要的三个目的基因IgASE2、efd2和ptd5的表达盒有机集合在一起，构建成酿酒酵母共表达载体pYAE5。将该表达载体pYAE5转化至酿酒酵母INVSc1，构建成酿酒酵母工程菌YAE985。该菌在添加外源底物LA和ALA的诱导培养基中诱导表达后，产生的ARA和EPA分别占总脂肪酸含量的1.6%和2.5%，LA转化成ARA以及ALA转化成EPA的最终转化率分别是10.1%和16.9%。这些结果表明，在酿酒酵母中成功地构建了ARA和EPA生物合成的Δ8(ω6-Δ8, ω3-Δ8)途径。利用RT-PCR(Real Time PCR)对工程菌YAE985的遗传稳定性进行了检测，结果表明：工程菌连续转接20次后，IgASE2、efd2和ptd5基因在转录水平仍然保持1：1：1的关系，说明没有发生遗传重组和目的基因部分丢失的情况，因此酿酒酵母工程菌YAE985具有很好的遗传稳定性。用大豆种子特异性启动子BCSP952分别构建基因IgASE2、efd2和ptd5的表达盒，并克隆进表达载体pBX，构建成通过Δ8(ω6-Δ8, ω3-Δ8)途径生物合成ARA和EPA的无选择标记转基因大豆的表达载体pX9AE5。用根瘤农杆菌介导的方法进行大豆转化，筛选出含有Δ8(ω6-Δ8, ω3-Δ8)生物合成途径的转基因大豆。然后对阳性转化植株进行雌二醇诱导，进一步筛选出无选择标记的转基因大豆。转基因大豆种子中ARA和EPA的含量分别占总脂肪酸含量的6.8%和3.6%。这些结果表明，在无选择标记的转基因大豆中成功地构建了ARA和EPA生物合成的Δ8(ω6-Δ8, ω3-Δ8)途径。最后，为了提高转基因大豆中ARA和EPA的合成水平，本文对BCSP952进行了功能分析。在此基础上，对BCSP952进行改造，以提高BCSP952的强度。为了分析BCSP952的功能，分别构建了BCSP952的5-端缺失启动子片段BCSP666、BCSP471、BCSP285和BCSP156。将它们连接到pBI121质粒的GUS基因上游后转化拟南芥并通过GUS组化检测和GUS酶活性测定来鉴定这些启动子的功能。结果表明：BCSP666的活性与BCSP952活性基本相同，达到BCSP952活性的96.5%；BCSP471的活性次之，约为BCSP952活性的69.4%；BCSP285和BCSP156的活性相对较低，仅为BCSP952活性的15.5%和10.1%；除BCSP156片段外，其余5’-端缺失启动子片段都具有种子特异性。说明：种子特异性元件的种类和数量直接影响启动子的强度；并且，在BCSP952启动子的转录起始位点至上游约-594位这个区段内，种子特异性启动子元件的数量越多，启动子的活性越强，但是超过这个区域，增加这些元件的数量，并不能有效增加启动子的强度。为了提高BCSP952的强度，通过在BCSP952启动子的-140位插入ABRE和Sph元件，构建成启动子BCSP952-aa、 BCSP952-as和BCSP952-ss，并将它们转化至拟南芥中进行功能鉴定。GUS组化检测和GUS活性测定表明：GUS基因只在种子中表达，说明改造后的启动子是种子特异性的启动子；BCSP952-as控制的GUS活性最高，约为BCSP952的180%、BCSP952-aa控制的GUS活性次之，约为BCSP952的112%、BCSP952-ss控制的GUS活性反而降低，约为BCSP952的88%。然后，从每种类型的启动子中选择GUS活性最高的4个株系进行southern杂交和RT-PCR分析。Southern杂交表明，含有BCSP952-ss的一个株系的GUS基因是双拷贝，其余启动子的株系中GUS基因都是单拷贝。RT-PCR分析表明：BCSP952-as控制的GUS基因转录水平最高，BCSP952-aa次之，BCSP952-ss最低，这与测定的GUS活性是一致的。这些结果说明：通过对BCSP952进行改造，提高了BCSP952的强度；并且BCSP952的-140位插入一个ABRE和一个Sph元件时的BCSP952-as最强，其强度比BCSP952提高了80%，在BCSP952的-140位插入两个ABRE元件时的BCSP952-aa次之，其强度比BCSP952提高了12%，在BCSP952的-140位插入两个Sph元件时的BCSP952-ss的强度反而降低。更多还原

【Abstract】 Polyunsaturated fatty acids (PUFAs), especially very long chain polyunsaturatedfatty acids (VLC-PUFAs) with20carbons or more in length, are of great importancefor the normal development and metabolism of all organisms, and are essential formaintaining human health. Currently, the most available sources of PUFAs are marinefishes and marine algae. However, owing to long-term over-fishing andenvironmental pollution of the marine ecosystems as well as expensive cost to obtainPUFAs from marine algae, it is urgent for scientists to find an alternative source ofPUFAs for a sustainable source for VLC-PUFAs.With the rapid development of transgenic technologies, producing PUFAs bytransgenic oilseed crops has been demonstrated to be a promising alternative sourcefor PUFAs. Soybean is one of the most important oilseed crops as well as linoleicacid (LA) and α-linolenic acid (ALA) which are substrates of the synthesis of PUFAsin soybean oil account for up to55%and13%of total fatty acids, respectively. So,soybean has been an important host producing PUFAs by metabolic engineering. Thetwo pathways, conventional Δ6pathway and alternative Δ8pathway, for PUFAsbiosynthesis in transgenic plants have been described so far and genes encodingelongases and desaturases involved have been identified. Thanks to the bottlenecks ofcomplex substrate conversion in Δ6pathway, in present study, Δ8pathways ofarachidonic acid (ARA) and Eicosapentaenoic acid (EPA) biosynthsis werereconstituted in Saccharomyces cerevisiae and soybean to obtain marker-freetransgenic soybean producing ARA and EPA and soybean seed-specific promoterBCSP952was modified to improve the level of ARA and EPA biosynthsis in thetransgenic soybean.First, three genes presented in Δ8pathway, Δ9elongase gene (IgASE2), Δ8desaturase gene (efd2) and Δ5desaturase gene (ptd5) were cloned from Isochrysisgalbana H29, Euglena gracilis and Phaeodactylum tricornutum, repectively, andcharacterized by their heterologous expression in S. cerevisiae INVSc1. The IgASE2gene was1653bp in length, contained a786bp open reading frame (ORF) encoding a protein of261amino acids that shared87%identity with the reported Δ9elongase,IgASE1, and possessed a44bp5’-untranslated region and a823bp3’-untranslatedregion. IgASE2expressed in S. cerevisiae, elongated LA to eicosadienoic acid (EDA)and ALA to eicosatrienoic acid (EtrA) with57.6%(LA to EDA) and56.1%(ALA toEtrA) conversion ratio, respectively, confirming that IgASE2gene was a novelC18-Δ9-specific PUFAs elongase gene. The ORF of efd2was1266bp and encodeda protein of431amino acids that shared96%identity with the reported Δ8desaturase,EFD1. EFD2expressed in S. cerevisiae converted EDA to dihomo-γ-linolenic acid(DGLA) and EtrA to eicosatetraenoic acid (ETA) with substrate conversion ratio31.2%and46.3%, respectively, confirming that efd2was a Δ8-specific PUFAsdesaturase gene. Ptd5has an ORF of1410bp that encodes469amino acids. PTD5expressed in S. cerevisiae specifically catalyzed DGLA to ARA and ETA to EPA withsubstrate conversion ratio28.7%and37.2%, respectively, confirming that ptd5wasaΔ5-specific PUFAs desaturase gene.Then, using the cloned IgASE2, efd2and ptd5genes, we constructed thealternative Δ8pathway of ARA and EPA biosynthsis in S. cerevisiae and soybean andobtained engineered strain of S. cerevisiae producing ARA and EPA and free-markertransgenic soybean producing ARA and EPA. Based on the Δ8(ω6-Δ8, ω3-Δ8)pathway of ARA and EPA biosynthesis, the co-expression vector pYAE5of S.cerevisiae INVSc1was constructed by cloning the expression cassettes of IgASE2,efd2and ptd5into pYES2and transformed into INVSc1to obtain engineered strainYAE985producing ARA and EPA. When YAE985was cultivated in the inductionmedium with exogenous substrates LA and ALA, YAE985converted LA to ARA andALA to EPA with substrate conversion ratio10.1%and16.9%, respectively, as wellas ARA and EPA in YAE985accounted for up to1.6%and2.5%of total fatty acids,respectively. These results showed that the Δ8(ω6-Δ8, ω3-Δ8) pathway of ARA andEPA biosynthesis was successfully constructed in S. cerevisiae INVSc1.Genetic stability of YAE985was identified by determining the transcription ofIgASE2, efd2and ptd5in YAE985using real-time PCR (RT-PCR). IgASE2, efd2andptd5remained1:1:1at the transcription level when YAE985was continuouslyinoculated and cultured for20generations, confirming that the genes were not lost. The results verified that the engineered strain YAE985was stable.The expression cassettes of IgASE2, efd2and ptd5containing BCSP952wereconstructed by ligating them to the downstream of BCSP952, respectively. They werecloned into the vector pBX to construct the marker-free expression vector pX9AE5inthe transgenic soybean. pX9AE5was transformed into soybean by Agrobacteriumtumefaciens-mediated transformation (ATMT) and the transgenic soybean harboringpX9AE5was identified by kanamycin resistence and PCR. In order to delete theselectable marker gene in the transgenic soybean, the transgenic soybean was inducedwith β-estradiol and the marker-free transgenic soybean was constructed. In the seedsof the marker-free transgenic soybean, ARA and EPA were about6.8%and3.6%ofof total fatty acids. These results showed that the Δ8(ω6-Δ8, ω3-Δ8) pathway ofARA and EPA biosynthesis was successfully constructed in the marker-freetransgenic soybean.Finally, in order to improve the expression level of ARA and EPA in transgenicsoybean, we undertook the function analysis of BCSP952and further modified theBCSP952to improve its strength according to the function analysis of BCSP952. Inorder to understand the regulatory mechanism of BCSP952, a series of5′-deletedpromoters BCSP666, BCSP471, BCSP285and BCSP156were constructed and fusedto the β-glucuronidase (GUS) gene in pBI121as well as transformed into Arabidopsisthaliana via ATMT. The GUS activities of the promoters were detected in differenttissues. The GUS activities of BCSP666, BCSP471, BCSP285and BCSP156were96.5%,69.4%,15.5%and10.1%of BCSP952GUS activity, and the other promotersonly expressed in seed except BCSP156. The results showed that:(a) The type andamount of the seed-specific promoter elements directly impacted on the strength ofthe promoter.(b) Within the-594region, the more the seed-specific promoterelements, the stronger the promoter. However, beyond this region, the strength of thepromoter was limitedly improved if the elements were increased.In order to improve the strength of BCSP952, BCSP952-aa, BCSP952-as andBCSP952-ss were constructed by inserting ABRE and Sph elements into the-140siteof BCSP952and fused to the GUS gene in pBI121as well as transformed into A.thaliana via ATMT. The histovhemical analysis and fluorometric analysis showed that: GUS genes controlled by the modified BCSP952were only expressed in seed of A.thaliana, confirming that they were all seed-specific promoters, and GUS activities ofBCSP952-as, BCSP952-aa and BCSP952-ss were180%,112%and88%of BCSP952GUS activity, repectively.The southern hybridization and real-time PCR (RT-PCR) on the4lines oftransgenic A. thaliana containing different kinds of promoter in which the GUSactivities were the highest were conducted to further identified the strength of themodified BCSP952. The southern hybridization showed that the GUS genespresented in transgenic A. thaliana were single copy except a line containingBCSP952-ss. RT-PCR showed that at the transcription level, GUS gene controlled byBCSP952-as was expressed at highest level, followed by BCSP952-aa, whereas GUSgene controlled by BCSP952-ss was expressed at the lowest level, which wereconsistent with the GUS activities. The results indicated that the strength of BCSP952was improved by modification, and BCSP952-as inserted a ABRE and a Sph elementwas the strongest, followed by BCSP952-aa inserted two ABREs, whereas the thestrength of BCSP952-ss inserted two Sph elements decreased.更多还原