Expression of fatty acid synthesis genes and fatty acid accumulation in haematococcus pluvialis under different stressors
不同胁迫条件下雨生红球藻脂肪酸合成基因的表达及脂肪酸积累

Abstract  摘要

Background  背景

Biofuel has been the focus of intensive global research over the past few years. The development of 4^th generation biofuel production (algae-to-biofuels) based on metabolic engineering of algae is still in its infancy, one of the main barriers is our lacking of understanding of microalgal growth, metabolism and biofuel production. Although fatty acid (FA) biosynthesis pathway genes have been all cloned and biosynthesis pathway was built up in some higher plants, the molecular mechanism for its regulation in microalgae is far away from elucidation.
近年来，生物燃料一直是全球研究的重点。基于藻类代谢工程的第四代生物燃料生产（藻类转化为生物燃料）的发展仍处于起步阶段，其主要障碍之一是我们对微藻的生长、代谢和生物燃料生产的了解不足。尽管脂肪酸（FA）生物合成途径的相关基因已在一些高等植物中被克隆并建立了生物合成途径，但微藻中这一过程的分子调控机制仍远未被阐明。

Results  结果

We cloned main key genes for FA biosynthesis in Haematococcus pluvialis, a green microalga as a potential biodiesel feedstock, and investigated the correlations between their expression alternation and FA composition and content detected by GC-MS under different stress treatments, such as nitrogen depletion, salinity, high or low temperature. Our results showed that high temperature, high salinity, and nitrogen depletion treatments played significant roles in promoting microalgal FA synthesis, while FA qualities were not changed much. Correlation analysis showed that acyl carrier protein (ACP), 3-ketoacyl-ACP-synthase (KAS), and acyl-ACP thioesterase (FATA) gene expression had significant correlations with monounsaturated FA (MUFA) synthesis and polyunsaturated FA (PUFA) synthesis.
我们克隆了绿色微藻雨生红球藻中与脂肪酸（FA）生物合成相关的主要关键基因，并研究了这些基因表达变化与脂肪酸组成和含量（通过 GC-MS 检测）在不同胁迫条件（如缺氮、高盐、高温或低温）下的相关性。结果表明，高温、高盐和缺氮处理在促进微藻脂肪酸合成方面起到了重要作用，而脂肪酸的品质变化不大。相关性分析显示，酰基载体蛋白（ACP）、3-酮酰基-ACP-合酶（KAS）和酰基-ACP 硫解酶（FATA）基因的表达与单不饱和脂肪酸（MUFA）和多不饱和脂肪酸（PUFA）的合成存在显著相关性。

Conclusions  结论

We proposed that ACP, KAS, and FATA in H. pluvialis may play an important role in FA synthesis and may be rate limiting genes, which probably could be modified for the further study of metabolic engineering to improve microalgal biofuel quality and production.
我们提出，H. pluvialis 中的 ACP、KAS 和 FATA 可能在脂肪酸（FA）合成中发挥重要作用，并可能是限速基因，这些基因可能通过进一步的代谢工程研究进行改造，以提高微藻生物燃料的质量和产量。

Background  背景

With the economic development, fossil fuels from non-renewable resources will eventually run out. According to BP Statistical Review of World Energy 2010, two main energy resources, crude oil and natural gas, may be used up in only 45.7 and 62.8 years, respectively [1]. Thus there is an urgent need to find alternative new energy. Since biofuel is renewable, environmentally friendly, safe to use, with wide applications, as well as biodegradable, it has become a major focus on intensive global research and development of new energy. Although the composition of biofuel is complex, it includes mainly palmitic acid, stearic acid, oleic acid, linoleic acid and other long-chain fatty acids and esters formed by alcohols [2]. Therefore, raw materials containing higher content of fatty acid (FA) should be chosen for biofuel production. However, the traditional biofuel were mainly derived from soybeans, corn, rapeseed, castor oil and other crops, which inevitably induce more serious food crisis. Microalgae biofuel is believed to be a powerful potential solver to this issue [3–7] and biofuels from metabolic modified microalgae is regarded as the 4th generation of biofuels [8].
随着经济的发展，来自不可再生资源的化石燃料最终将耗尽。根据《BP 世界能源统计年鉴 2010》，两种主要能源资源——原油和天然气——可能分别在 45.7 年和 62.8 年内被耗尽[1]。因此，迫切需要寻找替代的新型能源。由于生物燃料具有可再生、环保、使用安全、应用广泛以及可生物降解的特点，已成为全球研发新型能源的一个重要研究热点。虽然生物燃料的成分较为复杂，但主要包括棕榈酸、硬脂酸、油酸、亚油酸等长链脂肪酸及其与醇形成的酯类[2]。因此，应该选择含有较高脂肪酸（FA）含量的原料用于生物燃料生产。然而，传统生物燃料主要来源于大豆、玉米、油菜籽、蓖麻油等农作物，这不可避免地加剧了粮食危机。微藻生物燃料被认为是解决这一问题的一个强有力的潜在方案[3-7]，而经过代谢改造的微藻生物燃料被认为是第四代生物燃料[8]。

The importance of screening high FA content microalgae species and optimization for large biomass culture conditions were recognized as early as in 1980s [9]. Research in this area mainly focused on comparing FA composition in different microalgae and a variety of stress on content and composition of the FA in microalgae [10–12]. Microalgal biofuels production has gained a renewed interest in recent years but is still not economically feasible due to several limitations related to algal culture, for instance, at present, the cost of microalgal biofuel is still much higher than conventional diesel [13]. It is well known that microalgae biofuels could not make an impact on the fuel market until they are economically feasible. One of the main barriers is the high producing cost due to our lacking of understanding of microalgal growth, metabolism and biofuels production.
早在 20 世纪 80 年代，人们就认识到筛选高脂肪酸含量的微藻物种以及优化大规模培养条件的重要性[9]。该领域的研究主要集中在比较不同微藻中的脂肪酸组成以及各种胁迫对微藻脂肪酸含量和组成的影响[10-12]。近年来，微藻生物燃料的生产重新引起了人们的兴趣，但由于与藻类培养相关的多种限制，目前仍未达到经济可行性。例如，目前微藻生物燃料的成本仍远高于传统柴油[13]。众所周知，只有当微藻生物燃料实现经济可行时，它才能对燃料市场产生影响。主要障碍之一是生产成本过高，这主要是由于我们对微藻的生长、代谢及生物燃料生产的理解不足。

To address this important issue, scientists generally believe that metabolic engineering is an effective solution. In recent years, more emphasis focuses on discovering metabolic engineering methods to improve content of microalgal FAs in vivo [14–17]. However, though FA biosynthesis pathway in higher plants and microalgae have been explored [10, 18], multiple genes expression and their relationship with FA synthesis in microalgae has not been fully reported.
为了解决这一重要问题，科学家们普遍认为代谢工程是一个有效的解决方案。近年来，人们更关注于发现代谢工程方法，以提高微藻体内脂肪酸（FAs）的含量[14–17]。然而，尽管高等植物和微藻中的脂肪酸生物合成途径已经被探索过[10, 18]，但关于多基因表达及其与微藻脂肪酸合成之间关系的研究仍未完全报道。

Haematococcus pluvialis, a green microalga with high commercial values that has the ability to synthesize and accumulate large amounts of red carotenoid astaxanthin (ca. 2% of dry weight) under various stress conditions [12], is a good model to study FA accumulation as a potential astanxanthin feedstock with FA as byproducts [19]. In this study, we cloned five main FA synthesis genes from H. pluvialis (Figure 1), and explored their expression patterns using quantitative real-time RT-PCR under different treatments, such as high salt, high/low temperature, and nitrogen depletion. All treatment conditions selected had impact on the accumulation of lipid and astaxanthin [12, 20, 21]. At the same time, FA contents and composition was analyzed by GC-MS to study the correlations between FA synthesis and gene expression patterns.
**血红裸藻**（*Haematococcus pluvialis*）是一种具有高商业价值的绿藻，在各种胁迫条件下能够合成并积累大量红色类胡萝卜素虾青素（约占干重的 2%）[12]，因此是一种研究脂肪酸积累的良好模型，同时也可作为一种潜在的虾青素原料，脂肪酸作为副产品[19]。在本研究中，我们从血红裸藻中克隆了五个主要的脂肪酸合成基因（图 1），并通过实时定量 RT-PCR 分析了它们在不同处理条件下（如高盐、高/低温和氮缺乏）下的表达模式。所有选定的处理条件均对脂质和虾青素的积累有影响[12, 20, 21]。与此同时，通过气相色谱-质谱法（GC-MS）分析了脂肪酸的含量和组成，以研究脂肪酸合成与基因表达模式之间的相关性。

Figure 1  **图 1**

Pathways of lipid biosynthesis and acyl chain desaturation which are known or hypothesized to occur in green microalgae. The assignment of candidate genes encoding enzymes catalyzing the reactions were also shown in the diagramm in this study Abbreviations: ACP, acyl carrier protein; CoA, coenzyme A; DGDG, digalactosyldiacylglycerol; FA, fatty acid; MGDG, monogalactosyldiacylglycerol; SQDG, sulfoquinovosyldiacylglycerol.
绿色微藻中已知或假设发生的脂质生物合成和酰基链去饱和途径。本研究中还在图示中显示了催化这些反应的酶的候选基因分配。缩写：ACP，酰基载体蛋白；CoA，辅酶 A；DGDG，二半乳糖基二酰基甘油；FA，脂肪酸；MGDG，单半乳糖基二酰基甘油；SQDG，磺基奎诺糖基二酰基甘油。

The aims of this research were: i) to determine an environmental stress treatment potential for high FA production and quality in this microalgae; ii) to explore the correlations between the key synthesis genes and the FA accumulation to target rate-limiting genes in the microalgal FA synthesis pathway, and iii) to target the key gene(s) responsible for high FA accumulation and evaluate these candidate genes for metabolic engineering for more biofuel production with high quality and less production cost.
本研究的目标是： i) 确定一种环境胁迫处理方案，以提高该微藻的脂肪酸（FA）产量和质量； ii) 探索关键合成基因与脂肪酸积累之间的相关性，以定位微藻脂肪酸合成途径中的限速基因； iii) 瞄准负责高脂肪酸积累的关键基因，并评估这些候选基因在代谢工程中的应用潜力，从而实现更高质量、更低生产成本的生物燃料生产。

Results  结果

Cloning of algal FA synthesis genes
**藻类脂肪酸合成基因的克隆**

Using degenerated primers (Table 1) and normal RT-PCR, five genes were successfully cloned, verified and submitted to NCBI GenBank. The nucleotide sequences of cDNAs of 3-keto acyl-acyl carrier protein synthase gene (KAS), acyl-acyl carrier protein thioesterase (FATA), ω-3 fatty acid desaturase (FAD), ACP and malonyl-CoA:ACP transacylase (MCTK) have been deposited in the GenBank database under the accession numbers, HM560033, HM560034, HM560035, HM560036, and HM560037, respectively. Together with other two known FA synthesis genes, biotin carboxylase (BC) and stearoyl-ACP-desaturase (SAD), total seven genes were investigated in this study (Figure 1).
使用简并引物（表 1）和常规 RT-PCR 技术，成功克隆了五个基因，并经过验证后提交至 NCBI GenBank。这些基因的 cDNA 核苷酸序列分别为 3-酮酰基-酰基载体蛋白合酶基因（KAS）、酰基-酰基载体蛋白硫酯酶（FATA）、ω-3 脂肪酸去饱和酶（FAD）、ACP 以及丙二酰辅酶 A：ACP 转酰基酶（MCTK），并已在 GenBank 数据库中登记，登录号分别为 HM560033、HM560034、HM560035、HM560036 和 HM560037。再加上另外两个已知的脂肪酸合成基因，生物素羧化酶（BC）和硬脂酰-ACP-去饱和酶（SAD），本研究共分析了七个基因（图 1）。

Table 1 Primers for real time RT-PCR in this study
表 1 本研究中实时 RT-PCR 引物

Expression of FA synthesis related genes
脂肪酸（FA）合成相关基因的表达

Haematocccus cells grown under different stress conditions and were harvested for RNA isolation. cDNA synthesis and gene expression of FA synthesis related genes were explored using quantitative real-time RT-PCR. The results showed that the mRNA levels of most selected genes were significantly or very significantly up-regulated under all stress conditions, with all genes were up-regulated under Fe + AC and HT conditions, and some genes, such as FATA, SAD and FAD, were more sensitive to treatments (Figure 2).
将雨生红球藻（Haematococcus）细胞在不同胁迫条件下培养后收集，用于 RNA 提取。通过定量实时 RT-PCR（qRT-PCR）研究了 cDNA 合成及脂肪酸合成相关基因的表达情况。结果表明，在所有胁迫条件下，大多数选定基因的 mRNA 水平显著或极显著上调，其中在 Fe + AC 和 HT 条件下所有基因均呈上调趋势，而某些基因（如 FATA、SAD 和 FAD）对这些处理更为敏感（图 2）。

Figure 2  图 2

Gene expression detected by real time RT-PCR in control and stress treatment conditions. BC: Biotin carboxylase; ACP: Acyl carrier protein; MCTK: Malonyl-CoA:ACP transacylase; KAS: 3-ketoacyl- ACP synthase; FATA: Acyl-ACP thioesterase; SAD: Stearoyl-ACP-desaturase; FAD: ω-3 fatty acid desaturase.
通过实时 RT-PCR 检测在对照和胁迫处理条件下的基因表达。 BC：生物素羧化酶；ACP：酰基载体蛋白；MCTK：丙二酰 CoA:ACP 转酰酶；KAS：3-酮酰基-ACP 合酶；FATA：酰基-ACP 硫酯酶；SAD：硬脂酰-ACP 去饱和酶；FAD：ω-3 脂肪酸去饱和酶。

BC, as a subunit of acetyl coenzyme A carboxylase biotin carboxylase, involves in catalyzing acetyl-CoA to malonyl-CoA, the first step of de novo FA synthesis (Figure 1). It is intriguing that different conditions had various impacts on BC mRNA level (Figure 2a). For instance, no change of BC mRNA was observed under AC treatment, while 1,626 fold of up-regulation was detected under HT. The change was 4.8 fold under Fe treatment, however, the combined salinity treatment (Fe + AC) resulted in about 10 fold up-regulation, which was almost 2 fold of up-regulation under Fe. Under LT, BC mRNA level was increased up to 4.8 fold, which was far lower than that of HT (1,626), indicating that FA biosynthesis in H. pluvialis is more sensitive to high temperature than low temperature. Depletion of nitrogen (NL) also induced BC expression level at about 1.3 fold. Student's t test analysis showed that NL significantly (p < 0.05), Fe, Fe + AC, LT, and HT treatments very significantly (p < 0.01) increased BC gene expression, while AC alone treatment had no significant effect. Multiple comparison analysis indicated that the gene expression of BC induced by Fe, AC, and Fe + AC were significantly different between the single and combined salinity treatment (data not shown).
BC 作为乙酰辅酶 A 羧化酶（acetyl coenzyme A carboxylase）中的生物素羧化酶（biotin carboxylase）亚基，参与催化乙酰辅酶 A（acetyl-CoA）生成丙二酰辅酶 A（malonyl-CoA），这是新生脂肪酸（de novo FA）合成的第一步（图 1）。有趣的是，不同条件对 BC mRNA 水平的影响各不相同（图 2a）。例如，在 AC 处理下，BC mRNA 水平没有变化，而在 HT 条件下检测到高达 1626 倍的上调。在 Fe 处理下，变化为 4.8 倍，而联合盐度处理（Fe + AC）导致约 10 倍的上调，几乎是单独 Fe 处理上调的 2 倍。在 LT 条件下，BC mRNA 水平增加至 4.8 倍，远低于 HT 条件下的 1626 倍，表明 H. pluvialis 中的脂肪酸合成对高温比低温更为敏感。氮缺乏（NL）也诱导了 BC 表达水平上调约 1.3 倍。学生 t 检验分析表明，NL 显著（p < 0.05），Fe、Fe + AC、LT 和 HT 处理非常显著（p < 0.01）增加了 BC 基因表达，而单独 AC 处理无显著影响。多重比较分析显示，单一和联合盐度处理下 Fe、AC 和 Fe + AC 诱导的 BC 基因表达差异显著（数据未显示）。

ACP is an important component in both FA and polyketide biosynthesis with the growing chain bound during synthesis as a thiol ester at the distal thiol of a 4'-phosphopantethiene moiety (Figure 1). Similar to BC gene, ACP mRNA was shown to react differently under different conditions (Figure 2b). The most significant up-regulation of ACP gene expression was observed under HT with a 8.7 fold rise, while LT induced only 2.6 fold. Fe + AC salinity treatment induced 9 fold of ACP expression, while separately salinity treatment increased less, at 1 and 0.4 fold under Fe and AC, respectively. Obviously, the combined salinity condition (Fe + AC) had higher impacts on ACP gene expression than separately treatments (Fe, or AC), implying a synergistic effect of Fe and AC on FA synthesis in H. pluvialis. LT treatment induced ACP gene expression at 3.6 fold, only 37.6% for the effect of HT treatment, suggesting that ACP gene may also be less sensitive to LT. The other conditions did not induce ACP gene expression significantly.
ACP 是脂肪酸（FA）和聚酮（polyketide）生物合成中的重要组分，在合成过程中，生长链以硫酯的形式结合在 4'-磷酸泛酰巯基乙胺（4'-phosphopantethiene）结构的远端硫醇上（图 1）。与 BC 基因类似，ACP mRNA 在不同条件下显示出不同的反应（图 2b）。ACP 基因表达在高温（HT）条件下显著上调，达到 8.7 倍，而低温（LT）仅诱导了 2.6 倍。铁（Fe）和乙酸铜（AC）共同处理的盐度条件使 ACP 表达提高了 9 倍，而单独的盐度处理诱导效应较低，在 Fe 和 AC 条件下分别仅增加了 1 倍和 0.4 倍。显然，联合盐度条件（Fe + AC）对 ACP 基因表达的影响高于单独处理（Fe 或 AC），这表明 Fe 和 AC 在小球藻（H. pluvialis）脂肪酸合成中具有协同效应。低温（LT）处理诱导了 3.6 倍的 ACP 基因表达，仅为高温（HT）处理效果的 37.6%，表明 ACP 基因对低温可能也不太敏感。其他条件对 ACP 基因表达没有显著诱导作用。

The initiation of the FA elongation step, which extends the length of the growing acyl chain by two carbons, requires MCTK to transfer malonyl moiety from malonyl-CoA onto the acyl carrier protein (Figure 1). Very high induction of MCTK gene expression (2,261 fold) was observed under HT and LT caused about 1 fold up-regulation, while NL and AC conditions did not cause significant change to its expression (Figure 2c). Fe treatment enhanced MCTK gene expression about 2 fold, and combined salinity Fe + AC treatment induced about 5.4 fold. Multiple comparison analysis indicated that there was significantly different between the single (Fe or AC alone) and combined salinity treatment (Fe + AC) (data not shown).
脂肪酸（FA）延长步骤的启动需要 MCTK 将丙二酰基（malonyl moiety）从丙二酰辅酶 A（malonyl-CoA）转移到酰基载体蛋白上（图 1）。在高温（HT）和低温（LT）条件下，MCTK 基因表达呈现非常高的诱导水平（2261 倍），而正常光照（NL）和无碳源（AC）条件下，其表达未发生显著变化（图 2c）。铁（Fe）处理使 MCTK 基因表达提高了约 2 倍，而盐度与铁联合处理（Fe + AC）诱导表达增加了约 5.4 倍。多重比较分析表明，单一处理（仅 Fe 或仅 AC）与联合盐度处理（Fe + AC）之间存在显著差异（数据未显示）。

KAS catalyzes the initial condensing reaction in FA biosynthesis (Figure 1). Consistent with BC, ACP, and MCTK, KAS gene expression was promoted by Fe + AC for about 3.5 fold, while Fe and AC separated treatments induced its gene expression as 81% and 42% of that of control, respectively (Figure 2d). Under NL treatment, 1.7 fold of KAS gene expression increase was also observed. HT and LT treatments up-regulated KAS mRNA levels at about 1.1 fold and 20% only, respectively.
KAS 在脂肪酸生物合成中催化初始缩合反应（图 1）。与 BC、ACP 和 MCTK 一致，Fe + AC 处理使 KAS 基因表达提升了约 3.5 倍，而单独的 Fe 和 AC 处理分别使其基因表达达到对照组的 81%和 42%（图 2d）。在 NL 处理下，KAS 基因表达也增加了 1.7 倍。HT 和 LT 处理分别仅使 KAS mRNA 水平上调了约 1.1 倍和 20%。

FATA is the chain-length-determining enzyme in de novo biosynthesis of plant FAs (Figure 1). FATA mRNA levels were up-regulated significantly or very significantly under all treatments in this study, with 2.0 (NL), 2.9 (Fe + AC combined), 3 (LT), 9.9 (Fe), 13.8 (HT), and 18.8 fold changes (AC), respectively (Figure 2e). Interestingly, different from BC, ACP, MCTK, and KAS, the fold change of FATA gene expression under Fe + AC combined treatment was less than that under Fe or AC treatment alone. This indicated that for FATA, Fe + AC combined treatment had no synergistic but antagonistic effect.
FATA 是植物脂肪酸（FA）从头合成过程中决定链长的酶（图 1）。本研究中，在所有处理条件下，FATA mRNA 的水平均显著或极显著上调，分别为 2.0 倍（NL）、2.9 倍（Fe + AC 联合处理）、3 倍（LT）、9.9 倍（Fe）、13.8 倍（HT）和 18.8 倍（AC）（图 2e）。有趣的是，与 BC、ACP、MCTK 和 KAS 不同，FATA 基因在 Fe + AC 联合处理下的表达倍数变化低于单独处理 Fe 或 AC 的情况。这表明，对于 FATA 而言，Fe + AC 联合处理不是协同效应，而是拮抗效应。

SAD functions to position a single double bond into an acyl-ACP substrate and is best represented by the ubiquitous Δ9 18:0-ACP desaturase (Figure 1). Similar to FATA, SAD gene expression was significantly or very significantly induced by all treatments (Figure 2f). In details, HT induced SAD mRNA levels about 625.0 fold, while LT induced only for 2.5 fold as only 1/180 of that under HT. Fe or AC treatment alone enhanced SAD gene expression 2.4 and 1.4 fold, respectively, compared with 8.6 fold of up-regulation under combined treatment Fe + AC. NL also increased SAD mRNA level at about 2.4 fold.
SAD 的功能是将单个双键引入酰基-ACP 底物中，其最典型的代表是广泛存在的 Δ9 18:0-ACP 去饱和酶（图 1）。与 FATA 类似，SAD 基因表达在所有处理条件下均被显著或极显著诱导（图 2f）。具体而言，高温（HT）诱导了约 625 倍的 SAD mRNA 水平，而低温（LT）仅诱导了 2.5 倍，仅为高温下诱导水平的 1/180。单独 Fe 或 AC 处理分别使 SAD 基因表达增加了 2.4 倍和 1.4 倍，而 Fe + AC 联合处理则使其上调了 8.6 倍。NL 处理同样使 SAD mRNA 水平约增加了 2.4 倍。

The enzyme of FAD converts linoleic to alpha-linolenic acid (C18:3n3) (Figure 1). It seemed that FAD was the most sensitive among all FA biosynthesis genes selected in this study, since each treatment highly increased FAD gene expression in our study (Figure 2g). HT treatment still had the highest enhance (5,947 fold), and NL had the lowest impact with 5.0 fold of up-regulation. Fe up regulated SAD gene for 35.5 fold and AC enhanced 17.9 fold of FAD mRNA level. Interestingly, Fe + AC combined treatment only caused 12.8 fold increase (p < 0.01), as only 36% and 71.5% of those under Fe or AC separately treatment, respectively. LT treatment increased FAD mRNA levels at 8.90 fold.
FAD 酶将亚油酸转化为α-亚麻酸（C18:3n3）（图 1）。在本研究中，FAD 似乎是所有选择的脂肪酸生物合成基因中最敏感的，因为每种处理都显著提高了 FAD 基因的表达（图 2g）。HT 处理的增强效果最高（提高了 5,947 倍），而 NL 的影响最小，上调了 5.0 倍。Fe 使 SAD 基因表达上调了 35.5 倍，AC 使 FAD mRNA 水平提高了 17.9 倍。有趣的是，Fe + AC 联合处理仅导致 12.8 倍的增加（p < 0.01），分别仅为单独 Fe 或 AC 处理效果的 36%和 71.5%。LT 处理使 FAD mRNA 水平增加了 8.90 倍。

FA content and composition
FA 含量和组成

With internal standard, our FA extraction efficiency was 87.5% (data now shown). The FA profiles under treatments were listed on Table 2. We detected 24 individual FAs in H. pluvialis under different treatments. The overall FA profile in H. pluvialis was similar under control and stress conditions, and palmitic, stearic, oleic, linoleic acids were the major components (Table 2), among which linoleic acid (C18:2n6) had the highest content under most conditions in H. pluvialis. Under NL, AC and HT, the total FA (TFA) content was, 77.2, 71.39, and 72.04 mg g^-1, respectively, which were considerably higher than that observed under control conditions (58.4) (Table 2). Moreover, no significant differences were found under the other stress treatments. The percentage of saturated fatty acids (SFA) was significantly higher in cultures grown under NL (23.99%) and AC (31.06%) conditions compared to the control (20.2%).
在使用内标的情况下，我们的脂肪酸（FA）提取效率为 87.5%（数据未显示）。不同处理条件下的脂肪酸分布列于表 2 中。我们在不同处理条件下的**雨生红球藻**中检测到 24 种单一脂肪酸。控制条件和胁迫条件下，**雨生红球藻**的整体脂肪酸分布相似，棕榈酸、硬脂酸、油酸、亚油酸是主要成分（表 2）。其中，在大多数条件下，亚油酸（C18:2n6）含量最高。在 NL、AC 和 HT 条件下，总脂肪酸（TFA）含量分别为 77.2、71.39 和 72.04 mg g ^-1 ，显著高于控制条件下的 58.4（表 2）。此外，在其他胁迫处理下未发现显著差异。在 NL（23.99%）和 AC（31.06%）条件下培养的样品中，饱和脂肪酸（SFA）的百分比显著高于控制组（20.2%）。

Table 2 Fatty acid profile (mg/g dry weight) in control (C) and stress conditions (NL, Fe, AC, Fe + AC, LT and HT)
表 2 控制组（C）和胁迫条件（NL、Fe、AC、Fe + AC、LT 和 HT）下的脂肪酸组成（mg/g 干重）

The total monounsaturated fatty acid (MUFA) content showed no significant differences between control and stress conditions, except that contents of most MUFA were significantly or very significantly increased under NL (C15:1, C16:1, C20:1, and C22:1) and HT (C16:1, C18:1, C20:1, C22:1 and C24:1). Similarly, percentage of MUFA in total FA indicated no significant differences between control and stress conditions, except for a lower percentage in algae grown under AC condition (Table 2). Regarding polyunsaturated fatty acids (PUFA), there were significant alternation (increase or reduction) under stress conditions compared to the control (Table 2). For example, most PUFAs were significantly or very significantly increased under NL condition. This increase may be attributed to higher proportions of linoleic (C18:2n6), eicosapentaenoic acid (EPA, C20:5n3) and docosahexanoic acid (DHA, C22:6n3). Similar increase of EPA was observed under Fe + AC, LT and HT conditions.
总单不饱和脂肪酸（MUFA）含量在对照组和胁迫条件下没有显著差异，但在 NL 条件下（C15:1、C16:1、C20:1 和 C22:1）和 HT 条件下（C16:1、C18:1、C20:1、C22:1 和 C24:1），大多数 MUFA 的含量显著或非常显著增加。同样，MUFA 在总脂肪酸中的比例在对照组和胁迫条件下也没有显著差异，但在 AC 条件下培养的藻类中，MUFA 的比例较低（表 2）。关于多不饱和脂肪酸（PUFA），在胁迫条件下与对照组相比，存在显著的变化（增加或减少）（表 2）。例如，在 NL 条件下，大多数 PUFA 显著或非常显著增加。这种增加可能归因于亚油酸（C18:2n6）、二十碳五烯酸（EPA，C20:5n3）和二十二碳六烯酸（DHA，C22:6n3）的比例较高。在 Fe + AC、LT 和 HT 条件下也观察到了类似的 EPA 增加现象。

Fatty acid methyl esters (FAME) quality for biodiesel
生物柴油用脂肪酸甲酯（FAME）的质量

FAs are precursors for biodiesel production. According to recent studies [22], the FA profile of microalgae has a significant influence on the fuel properties of biodiesel, such as cetane number (CN), iodine number (IN) and saponification number (SN). Our analysis indicated that conditions selected in this study had no significant impact on SN, IN and CN (Figure 3).
脂肪酸（FAs）是生物柴油生产的前体。根据最近的研究[22]，微藻的脂肪酸组成对生物柴油的燃料特性（如十六烷值（CN）、碘值（IN）和皂化值（SN））有显著影响。我们的分析表明，本研究中选择的条件对 SN、IN 和 CN 没有显著影响（图 3）。

Figure 3  图 3

The biodiesel quality of Haematococcus. a-c, SN, IN and CN values under different treatments, respectively. Error bars represent standard error (n = 4).
雨生红球藻的生物柴油质量。a-c 分别为不同处理条件下的皂化值 (SN)、碘值 (IN) 和十六烷值 (CN)。误差线表示标准误差 (n = 4)。

As shown in Figure 3, the SN changes were slight under different conditions with a range of 201.9-205.7 (Figure 3a), while little reduction of IN was observed under Fe, AC, Fe + AC and HT, and little increase under NL and LT around 120.5-121.5 (Figure 3b). The CN of the FAME under different conditions were calculated and compared (Figure 3c). The CN values under different conditions were relatively high, ranging from 45.9 to 51.6. However, little difference of CN values was observed under different conditions, with a minimum CN values under NL condition. Based on SN and IN analysis, the increase of MUFA and PUFA was the key to the reduction of CN under NL condition.
如图 3 所示，在不同条件下，SN 的变化较小，范围为 201.9-205.7（图 3a）；在 Fe、AC、Fe + AC 和 HT 条件下，IN 几乎没有减少，而在 NL 和 LT 条件下略有增加，范围约为 120.5-121.5（图 3b）。在不同条件下 FAME 的 CN 值也被计算并比较（图 3c）。在不同条件下，CN 值相对较高，范围在 45.9 到 51.6 之间。然而，在不同条件下 CN 值的差异较小，其中 NL 条件下的 CN 值最低。根据 SN 和 IN 的分析，NL 条件下 CN 值降低的关键在于 MUFA 和 PUFA 的增加。

Correlations between gene expression and FA profile
基因表达与脂肪酸组成之间的相关性

We determined both expression of key FA biosynthesis genes and FA profile under different treatments. To study the relationship between gene expression and FA profile, Pearson Correlation analysis (SPSS13.0) was carried out and specific results were summarized in Table 3.
我们测定了在不同处理条件下关键脂肪酸（FA）合成基因的表达情况及脂肪酸组成特征。为了研究基因表达与脂肪酸组成之间的关系，我们使用 SPSS 13.0 进行 Pearson 相关性分析，具体结果总结在表 3 中。

Table 3 The correlations between gene expression and fatty acid synthesis (cofactors, Pearson Correlation in SPSS13
表 3 基因表达与脂肪酸合成的相关性（协变量，SPSS13 中的 Pearson 相关性）

Based on the summary on Table 3, the correlations between different FAs and gene expression were different. ACP, KAS, and FATA shared close correlations with FAs, while the other did not. For instance, C12:0 had significant positive correlations with all selected genes (Table 3). ACP gene expression shared negative correlations with C15:0, C17:1, C18:3n6, C18:2n6, C20:4n6, C20:5n3, and positive correlation with C18:1n9, C20:2, C20:1, C22:1n9, and C24:1. KAS gene expression had negative correlations with C15:0, C18:3n6 and C20:4n6, while it shared positive correlations with C18:3n3, C18:0, C20:2, C20:1, C22:1n9, and C24:1. FATA gene expression was observed negatively correlated with synthesis of C17:1, C18:3n6, C20:4n6 and C22:6n3, while it positively correlated with synthesis of C18:3n3, C18:0, C20:1, C20:0, C22:1n9 and C24:1. The correlations between other genes and FA synthesis were found not significant.
根据表 3 的总结，不同脂肪酸（FAs）与基因表达之间的相关性有所不同。ACP、KAS 和 FATA 与脂肪酸的相关性较为密切，而其他基因则相关性不显著。例如，C12:0 与所有选定基因均呈显著正相关（表 3）。ACP 基因表达与 C15:0、C17:1、C18:3n6、C18:2n6、C20:4n6、C20:5n3 呈负相关，与 C18:1n9、C20:2、C20:1、C22:1n9 和 C24:1 呈正相关。KAS 基因表达与 C15:0、C18:3n6 和 C20:4n6 呈负相关，而与 C18:3n3、C18:0、C20:2、C20:1、C22:1n9 和 C24:1 呈正相关。FATA 基因表达被观察到与 C17:1、C18:3n6、C20:4n6 和 C22:6n3 的合成呈负相关，而与 C18:3n3、C18:0、C20:1、C20:0、C22:1n9 和 C24:1 的合成呈正相关。其他基因与脂肪酸合成之间的相关性未发现显著性。

Discussion  讨论

Extreme environmental conditions, such as nitrogen depletion [11, 19], high salinity [23], high light intensity [19, 24] as well as extreme temperatures [25], were intensively reported to induce the FA accumulation in several microalgae. Thus, we were interested in evaluating the correlations between FA accumulation and these stress conditions in H. pluvialis cultures.
极端环境条件（如氮缺乏[11, 19]、高盐度[23]、高光强[19, 24]以及极端温度[25]）被广泛报道能够诱导若干微藻中的脂肪酸（FA）积累。因此，我们对 *H. pluvialis*（雨生红球藻）培养中脂肪酸积累与这些胁迫条件之间的相关性进行了评估。

Our results indicated that all treatments selected in this study increased FA contents in H. pluvialis, which is highly consistent with previous reports [23, 26]. The analysis of FA profile suggested that saturated FAs in H. pluvialis mainly included C16:0 and C18:0, content percentage of TFA was 19.2-23.5% and 6.9-13.6%, respectively. Unsaturated FAs were C18:1n9, C18:2n6, C18:3n3, C18:3n6, C20:4n6 and C20:5n3, with content percentage of 35.5-52.9%, which was in accordance with what [27] reported. Previous study indicated that NL could increase both TFA and the proportion of unsaturated FAs, our study once again verified this conclusion. What's more, we noticed that DHA content was increased from 0 to 4.4% (dry weight of algal cells), indicating that DHA could be highly induced by NL. Fe treatment increased EPA content of algal by 48%, and the proportion of other PUFAs was also significantly increased, indicating that Fe also could increase the content of unsaturated FAs [28]. Many previous studies pointed out that temperature could affect the FA content, with higher TFA content under lower temperature [29] and low temperature induced the accumulation of PUFAs [30]. In this study, we found that high temperature was more inductive for accumulation of FAs, with 24% more of TFA than that at low temperature, which is different from previous findings [29]. Treating cells with the maximum temperature of 28°C may have not stressed microalgae cells enough in that report [31]. Our HT treatment was under 42°C, under this condition growth of algal cells was significantly inhibited. However, consistent with the previous report [31], we detected that HT treatment decreased unsaturated FA content and increased significantly saturated FA content, such as C18:3n6, C20:4n6, C20:5n3, C22:6n3 under HT were only 51.9%, 65.6%, 57.8%, 23.8% of low-temperature treatment.
我们的研究结果表明，本研究中选定的所有处理均提高了雨生红球藻的脂肪酸含量，这与之前的报道[23, 26]高度一致。脂肪酸组成分析显示，雨生红球藻中的饱和脂肪酸主要包括 C16:0 和 C18:0，其在总脂肪酸（TFA）中的含量百分比分别为 19.2%-23.5% 和 6.9%-13.6%。不饱和脂肪酸包括 C18:1n9、C18:2n6、C18:3n3、C18:3n6、C20:4n6 和 C20:5n3，其含量百分比为 35.5%-52.9%，这与 [27] 的报道一致。先前的研究表明，氮缺乏（NL）能够增加总脂肪酸（TFA）含量以及不饱和脂肪酸比例，我们的研究再次验证了这一结论。此外，我们注意到 DHA（C22:6n3）含量从 0 增加到 4.4%（以藻细胞干重计），这表明 DHA 在氮缺乏条件下可被高度诱导。铁（Fe）处理使藻细胞的 EPA（C20:5n3）含量增加了 48%，其他多不饱和脂肪酸（PUFAs）的比例也显著增加，表明铁处理同样能够提高不饱和脂肪酸的含量[28]。许多先前研究指出，温度会影响脂肪酸含量，较低温度下总脂肪酸含量较高[29]，且低温会诱导多不饱和脂肪酸的积累[30]。但在本研究中，我们发现高温更有利于脂肪酸的积累，总脂肪酸含量比低温条件高 24%，这一结果与之前的研究结果[29]不同。在那项研究中，将细胞暴露于 28°C 的最高温度可能不足以对微藻细胞造成足够的胁迫[31]。我们的高温处理（HT）设置为 42°C，在此条件下，藻细胞的生长受到显著抑制。然而，与之前的研究[31]一致，我们检测到高温处理降低了不饱和脂肪酸的含量，同时显著增加了饱和脂肪酸的含量。例如，在高温条件下，C18:3n6、C20:4n6、C20:5n3 和 C22:6n3 的含量分别仅为低温处理的 51.9%、65.6%、57.8% 和 23.8%。

In this study, the highest FA content was obtained in H. pluvialis growing under NL. Whereas the FA profile was qualitatively similar in NL, AC, and HT stress conditions tested in H. pluvialis, some quantitative differences should be highlighted: i) a significant increase of C18:3n6 and a decline of C18:3n3 content were observed in the microalgae cultured with NL, while opposite observations were revealed under AC and HT; ii) The percentage of cis-10heptadecenoic acid (C17:1) was only detectable in cultures growing under NL conditions, and it was not detected or detected as trace in previous studies of H. pluvialis [19]. The FA content was high under stress conditions tested in our study, but not high enough to meet the reported amounts (30-40% dry weight) in other cysts [19]. Using the internal FAME standards, the percentage of recovery in our FA analysis was 85%, indicating that it will be necessary to further optimize our culture conditions for higher FA accumulation in this organism. For example, either using CO₂ supplementation [32] or a two-phase culture strategy could be implemented to obtain high biomass productivity and FA content.
在本研究中，H. pluvialis 在正常光照（NL）条件下获得了最高的脂肪酸（FA）含量。尽管在 H. pluvialis 的 NL、AC 和 HT 应激条件下，脂肪酸的种类组成相似，但仍需强调一些定量差异： i) 在 NL 条件下培养的微藻中，观察到 C18:3n6 含量显著增加，而 C18:3n3 含量下降；而在 AC 和 HT 条件下则观察到相反的现象； ii) cis-10-十七碳烯酸（C17:1）的比例仅在 NL 条件下培养的样品中检测到，而在以往对 H. pluvialis 的研究中未检测到或仅检测到痕量 [19]。 尽管本研究中测试的应激条件下脂肪酸含量较高，但仍未达到其他研究中报告的孢囊（30-40% 干重）中的脂肪酸含量 [19]。通过使用内部 FAME 标准，本研究中脂肪酸分析的回收率为 85%，表明需进一步优化培养条件以在该生物中积累更高的脂肪酸含量。例如，可以通过补充 CO₂ [32] 或采用两阶段培养策略，以获得高生物量生产率和脂肪酸含量。

H. pluvialis FA was proposed compatible with the engines used today [19]. The most important properties of biofuel, such as SN, IN, CN were evaluated in this study. Usually, SN and IN could be used to characterize FA or FAME quality for biodiesel. SN has a negative correlation with the FA chain length while IN is positive to the extent of unsaturation in FA, while CN is a prime indicator presenting the biodiesel quality. It could be used to justify the biodiesel ignition quality which has an effect on the startability and combustion process of the diesel engine [33]. According to the FA profile observed in H. pluvialis, we could infer some of the features of the biodiesel that we would obtain from this alga. Since the standard ASTM D6751 for biodiesel requires a minimum CN of 47, below which it will cause a delay, incomplete combustion and followed low engine power. In comparison, CN values under NL and LT were slightly lower than those in the control, and other treatment presented slightly higher CN values compared to the control (48.3). The calculated CN values under different conditions were relatively high, ranging from 45.9 to 51.6, suggesting FAME derived from H. pluvialis may be satisfactory as biodiesel.
H. pluvialis FA 被认为与当前使用的发动机兼容[19]。本研究评估了生物燃料最重要的特性，如皂化值（SN）、碘值（IN）和十六烷值（CN）。通常，SN 和 IN 可用于表征生物柴油中脂肪酸或脂肪酸甲酯（FAME）的质量。SN 与脂肪酸链长呈负相关，而 IN 与脂肪酸的不饱和程度呈正相关；CN 是衡量生物柴油质量的主要指标。CN 可用于评估生物柴油的点火质量，这会影响柴油发动机的启动性和燃烧过程[33]。根据观察到的 H. pluvialis 的脂肪酸组成，可以推断从这种藻类中获得的生物柴油的一些特性。由于生物柴油的 ASTM D6751 标准要求 CN 最低值为 47，低于此值会导致延迟、不完全燃烧以及较低的发动机功率。相比之下，NL 和 LT 条件下的 CN 值略低于对照组，而其他处理的 CN 值则略高于对照组（48.3）。计算得出的不同条件下的 CN 值相对较高，范围为 45.9 至 51.6，表明从 H. pluvialis 提取的 FAME 作为生物柴油可能是令人满意的。

Because of the high potential of microalgae as a biodiesel feedstock, detailed characterization of genes crucial in FA biosynthesis is of particular importance (for further information refer to [16]). In this study, we cloned five key genes involved in FA biosynthesis and investigated their gene expression pattern under different stress treatments, and evaluated their relationship with the FA profile under treatments. The correlations between gene expression in FA synthesis pathway and FA profiling were often reported in higher plants [34–37]. The FA profile under different treatments in microalgae were reported but without investigating detailed connection with FA synthesis genes [19, 26, 38]. In this study, under our stress conditions, all selected genes were up-regulated with differeces in the extent for different stresses, however, the extent of upregulation is not reflected in the FA profile. This indicates that the FA biosynthesis regulation occurs at different levels in the cell. Some further studies on regulatory aspects could throw light on regulatory mechanism of FA syntheis in H. pluvialis. The present study evaluated both FA profile and expression patterns of genes involved in FA biosynthesis, and their correlation analysis indicated that there were different correlations between different FAs and genes. Our gene expression analysis showed that all treatments could alter mRNA levels of all seven selected key genes. It was found that under LT treatment, FAD gene expression was 8.87 fold up-regulated, with higher content of C18:3n6 content (1.45 fold of the control), indicating that LT could induce unsaturated FAD gene expression and improve the content of linolenic acid, which was consistent with [35]. Results from further analysis showed that C12:0 had a significant or very significant positive correlation with all selected genes, indicating that induced expression of FA synthesis genes could significantly affect C12:0 levels. Since C12:0 is the shortest carbon chain FAs and other longer-chain FAs are synthesized from C12:0 as the backbone, the changes of expression of FA synthesis genes could be very crucial.
由于微藻作为生物柴油原料具有巨大的潜力，详细表征在脂肪酸（FA）生物合成中起重要作用的基因尤为重要（更多信息请参见[16]）。在本研究中，我们克隆了参与脂肪酸生物合成的五个关键基因，并研究了它们在不同胁迫处理下的基因表达模式，同时评估了它们与胁迫处理下脂肪酸组成的关系。在高等植物中，脂肪酸合成途径中基因表达与脂肪酸组成之间的相关性经常被报道[34–37]。尽管在微藻中已有关于不同处理下脂肪酸组成的报道，但尚未详细探讨其与脂肪酸合成基因之间的联系[19, 26, 38]。在本研究中，在我们的胁迫条件下，所有选定的基因均被上调，但不同胁迫条件下的上调程度不同，然而，这种上调程度并未反映在脂肪酸组成中。这表明脂肪酸生物合成的调控发生在细胞的不同水平。对调控机制的进一步研究可能有助于揭示雨生红球藻中脂肪酸生物合成的调控机制。本研究同时评估了脂肪酸组成和脂肪酸生物合成相关基因的表达模式，其相关性分析表明，不同脂肪酸与基因之间存在不同的相关性。基因表达分析显示，所有处理均可改变七个选定关键基因的 mRNA 水平。研究发现，在低温（LT）处理下，FAD 基因表达上调了 8.87 倍，同时 C18:3n6 的含量增加至对照的 1.45 倍，表明低温可诱导不饱和 FAD 基因的表达并提高亚麻酸的含量，这与[35]的研究结果一致。进一步分析结果显示，C12:0 与所有选定基因均具有显著或极显著的正相关性，表明脂肪酸合成基因的诱导表达可以显著影响 C12:0 水平。由于 C12:0 是碳链最短的脂肪酸，而其他更长链的脂肪酸是以 C12:0 为骨架合成的，因此脂肪酸合成基因表达的变化可能至关重要。

Our detailed analysis of correlations between genes and individual FAs provided some interesting hints for metabolic engineering of microalgal biofuel. For instance, One of particular notes is SAD gene expression and its relation with contents of C18:0 and C18:1n9. SAD gene expression shared a certain degree of negative correlation with C18:0 (correlation coefficient -0.216), and had a positive correlation with C18:1n9 (correlation coefficient 0.295). This finding verified the function of this gene. Similarly, FAD gene was proved a negative correlation with C18:2n6 (-0.079) and a positive correlation with C18:3n3 (0.290). Thus, using correlation analysis between FA profile and gene expression may detect some new important genes. According to this, we may further use DNA recombination techniques to identify these potential genes associated with enhanced quality and production of desired FAs (i.e., EPA and DHA) and total FA in green microalgae.
我们对基因与单个脂肪酸（FA）之间相关性的详细分析为微藻生物燃料的代谢工程提供了一些有趣的线索。例如，值得注意的是 SAD 基因的表达及其与 C18:0 和 C18:1n9 含量的关系。SAD 基因的表达与 C18:0 呈一定程度的负相关（相关系数为 -0.216），与 C18:1n9 呈正相关（相关系数为 0.295）。这一发现验证了该基因的功能。同样，FAD 基因被证明与 C18:2n6 呈负相关（-0.079），与 C18:3n3 呈正相关（0.290）。因此，通过脂肪酸谱和基因表达之间的相关性分析，可以发现一些新的重要基因。基于此，我们可以进一步利用 DNA 重组技术识别这些潜在基因，以提高绿色微藻中目标脂肪酸（如 EPA 和 DHA）及总脂肪酸的质量和产量。

Conclusions  结论

In this study, we successfully cloned five key genes of FA synthesis in a green microalga H. pluvialis and correlations of gene expression and FA composition and production were investigated under different environmental stressors. These results expand our understanding of the genes and underlying molecular mechanisms that are involved in FA accumulation and response to the multiple stresses. According to our results, we proposed that the key rate-limiting genes of FA synthesis may include ACP, KAS and FATA because their expression showed linear relationships with synthesis of FAs in H. pluvialis. These genes could be potential candidates for better quality and higher production of FAs for value-added products and biofuel using metabolic engineering techniques.
在本研究中，我们成功克隆了绿色微藻雨生红球藻中脂肪酸合成的五个关键基因，并研究了基因表达与脂肪酸组成及产量在不同环境胁迫下的相关性。这些结果拓展了我们对脂肪酸积累及其应对多重胁迫的基因及分子机制的理解。根据我们的研究结果，我们提出脂肪酸合成的关键限速基因可能包括 ACP、KAS 和 FATA，因为它们的表达与雨生红球藻中脂肪酸的合成呈线性关系。这些基因可能成为通过代谢工程技术生产更高质量和更高产量的脂肪酸以用于增值产品和生物燃料的潜在候选目标。

Methods  方法

Organism, growth medium and culture conditions
**生物体、生长培养基及培养条件**

H. pluvialis strain 797 was obtained from Freshwater Algae Culture Collection of the Institute of Hydrobiology and maintained at the College of Life Sciences, Shenzhen University, China. Algae were incubated in 250 mL flasks, each containing 100 mL BBM [31], at light density of 20 μmol m ^-2 s^-1 with a diurnal cycle of 12 h light and 12 h dark at temperature of 22 ± 1°C. Cultures were continuously aerated with 0.2 μm filtered air through a mechanical pump.
H. pluvialis 797 菌株来源于中国科学院水生生物研究所的淡水藻类培养库，并保存在中国深圳大学生命科学学院。藻类在 250 mL 锥形瓶中培养，每瓶含有 100 mL BBM 培养基 [31]，光照强度为 20 μmol m⁻² s⁻¹，光周期为 12 小时光照和 12 小时黑暗，培养温度为 22 ± 1°C。通过机械泵持续通入 0.2 μm 过滤的空气对培养物进行通气。

The exponentially growing cultures (cell density approximately 5 × 10⁵ cells ml^-1) were treated with various stress conditions, such as high salinity, 450 μM FeSO₄ (Fe) and 45 mM NaAC (AC), separately (Fe, AC) or combined (Fe + AC), high temperature (HT) (42°C), low temperature (LT) (4°C), and nitrogen depletion (NL) for four days. Nitrogen depletion was achieved as harvesting and transferring cells to nitrogen depletion medium. Collected algal cells were rinsed with PBS and divided into replicate parts, one for RNA analysis and gene cloning, and the other for FA profiling, stored at -80°C if not immediately used. All experimental chemicals and reagents were analytical grade.
对处于指数生长期的培养物（细胞密度约为 5 × 10⁶ cells/ml）分别施加各种胁迫条件，包括高盐、450 μM FeSO₄（Fe）和 45 mM 醋酸钠（AC），单独处理（Fe 或 AC）或联合处理（Fe + AC）、高温（HT，42°C）、低温（LT，4°C）以及氮饥饿（NL），处理时间为 4 天。氮饥饿通过收集并转移细胞至无氮培养基实现。收集的藻细胞用 PBS 清洗，并分为两部分，一部分用于 RNA 分析和基因克隆，另一部分用于脂肪酸（FA）分析，若不立即使用，则储存于-80°C。所有实验所用化学试剂均为分析纯级别。

RNA isolation and cloning of FA synthesis pathway genes
RNA 分离和脂肪酸合成途径基因的克隆

RNA was isolated according to the miniprep RNA extraction procedure [39] with minor modifications. Briefly, only 25 mL of cells were applied as the starters with 10 μL DEPC treated water as the RNA solution in this sduty. Nuclear acids were quantified by Nano-Drop 3.0 (Coleman Technologies Inc., USA). Both DNA and RNA solutions were aliquoted and stored at -80°C, if not immediately used.
RNA 按照微量 RNA 提取方法（参考文献[39]）进行提取，并进行了轻微修改。简而言之，本研究中仅使用了 25 mL 的细胞作为起始样本，并用 10 μL 经过 DEPC 处理的水作为 RNA 溶液。核酸浓度使用 Nano-Drop 3.0（Coleman Technologies Inc., USA）进行定量测定。DNA 和 RNA 溶液在未立即使用时，均分装后储存在-80°C。

Using gene sequences retrieved from NCBI databases, such as those from green microalgae (i.e., Chlamydomonas reinhardtii), higher plants (i.e., Arabidopsis, cabbage, and cotton) and other organisms, degenerated primers (Additional file 1: Table S1) were designed and used for FA synthesis gene cloning. The first-strand cDNA synthesis was carried out using the Taqman Reverse Transcription system according to manufacturer's instruction (Applied Biosystems, USA) and the protocol previously described [39]. The RT-PCR amplicons with the expected sizes were purified with the Wizard™ PCR Preps DNA Purification System (Promega, USA). In addition to the five genes successfully cloned in this study, another two genes available in Genbank, BC and SAD, were also employed for gene expression test.
利用从 NCBI 数据库中获取的基因序列（如来自绿色小型藻类（例如，单细胞绿藻 *Chlamydomonas reinhardtii*）、高等植物（例如，拟南芥、卷心菜和棉花）及其他生物的基因序列），设计了简并引物（附加文件 1：表 S1），并用于 FA 合成相关基因的克隆。按照生产商说明（Applied Biosystems, USA）和之前描述的方法（参考文献[39]），使用 Taqman 逆转录系统进行第一链 cDNA 的合成。RT-PCR 扩增产物经 Wizard™ PCR Preps DNA 纯化系统（Promega, USA）纯化。本研究成功克隆了五个基因，此外，还使用了 GenBank 中可获得的两个基因（BC 和 SAD）进行基因表达测试。

TA Cloning Kit with One Shot TOP chemically competent E. coli (Invitrogen, USA) was used for cloning of PCR products. For sequence verification, both strands were sequenced with an overlapping scheme throughout the whole cDNA fragment. Sequences were analyzed using DNAclub (Xiongfong Chen, Cornell Univ., Ithaca), and homology searching was carried out with the translated query against protein database (BlastX) and Nucleotide-nucleotide BLAST (BlastN) in GenBank database.
使用 TA 克隆试剂盒和 One Shot TOP 化学感受态大肠杆菌（Invitrogen，美国）对 PCR 产物进行克隆。为了验证序列，对整个 cDNA 片段采用重叠策略对双链进行测序。序列分析使用 DNAclub（Xiongfong Chen，康奈尔大学，伊萨卡），同源性搜索通过将翻译的查询序列与蛋白质数据库（BlastX）以及 GenBank 数据库中的核酸-核酸 BLAST（BlastN）进行比对完成。

Gene expression profiling: Real-time RT-PCR
基因表达分析：实时 RT-PCR

Real-time RT-PCR analysis was performed on an ABI Prism 7900 Sequence Detection System (Applied Biosystems, USA) following the protocol previously described [12] using actin gene as the internal control.
实时 RT-PCR 分析在 ABI Prism 7900 序列检测系统（美国应用生物系统公司）上进行，按照之前描述的协议[12]操作，并使用 actin 基因作为内参。

Fatty acid methyl esters (FAME) transformation and FAME analysis
脂肪酸甲酯（FAME）转化与 FAME 分析

Total lipid extraction was performed as described by Lu et al. [40] with slightly modifications. Briefly, 20 mg lyophilized cell was suspended in 1 mL 2 M NaOH-CH₃OH solution and shaken (100 rpm) for 1 h at room temperature (RT) and incubated at 75°C for 15 min. After cooled down, the mixture was spiked with 1 mL 4 M HCl-CH₃OH and pH was adjusted to below 2.0 with HCl, followed by incubation at 75°C for 15 min. After that, FAMEs were extracted with 1 mL hexane, shaking by hand for 30s and then centrifuged at 4,000 g for 2 min. The hexane phase was collected and stored at -20°C for further GC-MS analysis. 100 μg of C19 FA was added before extraction to estimate the recovery rate.
总脂质提取按照 Lu 等人[40]的方法进行，并稍作修改。简而言之，将 20 mg 冷冻干燥的细胞悬浮于 1 mL 2 M NaOH-CH ₃ OH 溶液中，在室温（RT）下以 100 rpm 摇动 1 小时后，于 75°C 孵育 15 分钟。冷却后，加入 1 mL 4 M HCl-CH ₃ OH，并用 HCl 将 pH 调至小于 2.0，然后再次于 75°C 孵育 15 分钟。随后，用 1 mL 己烷提取 FAMEs，手动摇晃 30 秒后以 4,000 g 离心 2 分钟。收集己烷相，并在-20°C 保存以备进一步 GC-MS 分析。在提取前加入 100 μg 的 C19 FA 以估算回收率。

Qualification and quantification of FAMEs were performed on a Thermo Trace GC Ultra gas chromatograph coupled to Thermo Polaris Q mass spectrometry which was equipped with a HP-5MS column (30 m × 0.25-mm id, film thickness 0.25 μm). The temperature of the injector was maintained at 250°C. Helium was used as the carrier gas and ions were generated by a 70 eV electron beam and the mass range scanned was 50 to 650 m/z at a rate of 2 scan s^-1. The oven temperature for FAME analysis was initially maintained at 70°C for 5 min followed by a temperature rate of 5°C min^-1 to 200°C and then held for 5 min, followed 5°C min^-1 to 204°C and then held for 2 min, 5°C min^-1 to 220°C and then held for 3 min, and 5°C min^-1 to 255°C and then held for 5 min. Peak identification was performed by matching the mass spectra of each compound with the National Institute of Standards and Technology mass spectral library. Automatic peak deconvolution was processed with Masslynx software (V4.1, Waters Corp., USA) [41]. The datasets of FAME profiling for further analysis were obtained by normalized with the internal standards in the same chromatograms, respectively.
FAMEs 的鉴定和定量是在 Thermo Trace GC Ultra 气相色谱仪上进行的，该仪器配备 Thermo Polaris Q 质谱仪和 HP-5MS 色谱柱（30 m × 0.25 mm 内径，膜厚 0.25 μm）。进样器的温度保持在 250°C。使用氦气作为载气，离子通过 70 eV 的电子束产生，扫描的质量范围为 50 至 650 m/z，扫描速率为 2 次/秒。FAME 分析的柱温程序为：初始温度 70°C 保持 5 分钟，然后以 5°C/分钟的升温速率升至 200°C 并保持 5 分钟，再以 5°C/分钟的升温速率升至 204°C 并保持 2 分钟，再以 5°C/分钟的升温速率升至 220°C 并保持 3 分钟，最后以 5°C/分钟的升温速率升至 255°C 并保持 5 分钟。化合物的峰识别通过将每个化合物的质谱与国家标准与技术研究院（NIST）质谱库匹配完成。峰的自动解析由 Masslynx 软件（V4.1，Waters 公司，美国）处理 [41]。用于进一步分析的 FAME 表征数据集通过与同一色谱图中的内标进行归一化处理获得。

The analysis of microalgae biofuel quality
微藻生物燃料质量分析

The saponification number (SN), iodine number (IN) and cetane number (CN) were estimated by empirical equations according to [42, 43]. Those index factors were predicted according to equations (1-3).
皂化值（SN）、碘值（IN）和十六烷值（CN）是根据文献[42, 43]中的经验公式估算的。这些指标是通过公式（1-3）预测的。

$$\mathsf{\text{SN}}=\left(\mathsf{\text{56}}0\times \mathsf{\text{P}}i\right)/\mathsf{\text{MW}}i$$

(1)

$$\mathsf{\text{IN}}=\left(\mathsf{\text{254}}\times \mathsf{\text{D}}\times \mathsf{\text{P}}i\right)/\mathsf{\text{MW}}i$$

(2)

$$\mathsf{\text{CN}}=\mathsf{\text{46}}.\mathsf{\text{3}}+\mathsf{\text{5458}}/\mathsf{\text{SN}}-0.\mathsf{\text{225}}\times \mathsf{\text{IN}}$$

(3)

Where SN is saponification number, IN is iodine number, CN is cetane number, Pi is the weight percentage of each FAME, MWi is the molecular mass of individual FAME, D is the number of the double bonds in each FAME.
其中，SN 为皂化值，IN 为碘值，CN 为十六烷值，**Pₐ**为每种 FAME（脂肪酸甲酯）的质量百分比，**MWₐ**为各个 FAME 的分子量，**D**为每种 FAME 中的双键数量。

Statistical analysis  统计分析

All exposure experiments were repeated three times independently, and data were recorded as the mean with standard deviation (SD). For gene expression experiments, quantitative real-time PCR analysis was performed using the BioRAD iQ5 software. For each gene, the fold change expressed as the mean ± SD (% control) was calculated using the (standard curve) approximation corrected for primer efficiency and normalized to housekeeping gene actin expression values. Statistical analyses were performed using the Student's t test and Pearson Correlation correlation analysis (SPSS13.0). For all of the data analysis, a p-value < 0.05 was considered statistically significant.
所有暴露实验均独立重复三次，数据以平均值加标准差（SD）的形式记录。对于基因表达实验，使用 BioRAD iQ5 软件进行定量实时 PCR 分析。对于每个基因，利用（标准曲线）方法进行校正以考虑引物效率，并归一化至管家基因 actin 的表达值后，计算其倍数变化，以平均值±SD（%对照）的形式表示。统计分析采用 Student's t 检验和 Pearson 相关性分析（SPSS13.0）。对于所有数据分析，p 值<0.05 被认为具有统计学意义。