WO2013185388A1

WO2013185388A1 - Construction of transgenic flocculation microalgae and application thereof in microalgae recovery

Info

Publication number: WO2013185388A1
Application number: PCT/CN2012/077832
Authority: WO
Inventors: 白凤武; 赵心清
Original assignee: 大连理工大学
Priority date: 2012-06-12
Filing date: 2012-06-29
Publication date: 2013-12-19
Also published as: CN102690836A; CN102690836B

Abstract

The present invention provides transgenic flocculation microalgae and a preparation method thereof, comprising constructing an expression vector comprising a yeast flocculation gene, a promoter, and an antibiotics resistance gene, and transferring it into the microalgae. The transgenic microalgae provided in the present invention show a self-flocculation property and normally grow and are advantageous to microalgae collection and bio-refinery processing.

Description

转基因絮凝微藻的构建及其在微藻采收中的应用技术领域 Construction of transgenic flocculated microalgae and its application in microalgae harvesting

本发明属于生物基因工程领域，特别涉及微藻转化平台的建立及其在微藻采收中的应用。背景技术 The invention belongs to the field of biological genetic engineering, and particularly relates to the establishment of a microalgae transformation platform and its application in microalgae harvesting. Background technique

微藻多为单细胞或简单多细胞的光合微生物，可以利用空气中的二氧化碳、工业废气中的二氧化碳或者碳酸盐来生产有机物，因此可用于食品、饲料及精细化学品的生产。随着世界经济的快速发展，能源的消耗和供应不足的矛盾日益突出，以石油为基础的化石燃料已经明显不能满足人类日益增长的能源需求，而且化石燃料的使用是全球温室效应中二氧化碳的主要来源，因此，寻求新型清洁能源的开发和利用对于缓解能源危机-保护环境以及经济的可持续发展具有重要的战略意义。而受技术和资源等因素的限制，核能、风能和太阳能等自然能源，还无法替代石油等传统化石能源。 Microalgae are mostly single-cell or simple multi-cell photosynthetic microorganisms. They can use carbon dioxide in the air, carbon dioxide in industrial waste gas or carbonate to produce organic matter, so they can be used in the production of food, feed and fine chemicals. With the rapid development of the world economy, the contradiction between energy consumption and supply shortage has become increasingly prominent. Oil-based fossil fuels have clearly failed to meet the growing energy needs of mankind, and the use of fossil fuels is the main cause of carbon dioxide in the global greenhouse effect. Sources, therefore, the search for new clean energy development and utilization is of strategic importance for mitigating the energy crisis – protecting the environment and economic sustainability. Due to factors such as technology and resources, natural energy such as nuclear energy, wind energy and solar energy cannot replace traditional fossil energy such as oil.

生物柴油作为近年来再生能源的新生力量，因其热值接近石油，对环境友好 (N、 S含量低），因此近年来发展迅速，但是原料不足俨然已经成为国内外生物柴油大规模生产的瓶颈。目前主要以大豆、甘蔗以及玉米淀粉等油料作物为原料来炼制生物柴油，由于油料作物油脂面积产油率不高，生物柴油的大规模生产必然会导致这类可食用原料的大面积种植进而影响粮食生产。 As a new force in renewable energy in recent years, biodiesel is environmentally friendly (low in N and S content) because of its calorific value. It has developed rapidly in recent years, but the shortage of raw materials has become a bottleneck for large-scale production of biodiesel at home and abroad. . At present, the biodiesel is mainly made from oil crops such as soybean, sugar cane and corn starch. Due to the low oil production rate of oil crops, the large-scale production of biodiesel will inevitably lead to the large-scale cultivation of such edible materials. Affect food production.

同油料作物相比，微藻作为大力发展生物柴油的优质原料具有以下几个优点： ( 1 ) 微藻生长速度快，多数藻类油脂含量高，多为低不饱和自然脂肪，如栅藻 {Scenedesmus sp. ) 油脂含量为 20-21% (w/w) ，油脂产率为 41-54 ( mg/1/天），小球藻 ( Chlorella sp. ) 油脂含量为 19-22% (w/w) ，油脂产率在 45 ( mg/1/天）左右；（2 ) 同油料作物相比，微藻培养不需要占用耕地；（3 ) 微藻拥有跟高等植物类似的 CO₂固定***并将其转化成碳水化合物以及脂类，如甘油三酯（TGA) ； ( 4 )微藻能吸收工业生产等排放的二氧化碳同时释放氧气，对于降低温室效应维持环境稳定有重要作用。 Compared with oil crops, microalgae has the following advantages as a high-quality raw material for biodiesel development: (1) Microalgae grows fast, most algae oils are high in content, mostly low-unsaturated natural fats, such as Scenedesmus {Scenedesmus Sp.) oil content 20-21% (w/w), oil yield 41-54 (mg/1/day), Chlorella sp. oil content 19-22% (w/w) ), the oil yield is about 45 (mg/1/day); (2) microalgae cultivation does not need to occupy cultivated land compared with oil crops; (3) microalgae have a CO ₂ fixation system similar to higher plants and It is converted into carbohydrates and lipids, such as triglycerides (TGA); (4) Microalgae can absorb carbon dioxide emitted from industrial production and release oxygen, which plays an important role in reducing environmental effects and maintaining environmental stability.

除了能作为生物柴油的原料外，微藻本身还含有高附值产品。栅藻 (Scenedesmus sp. )和小球藻（C z/ore//a sp. )蛋白干重含量分别为 50-56%、 51-58%。微藻还可以用来发酵生产沼气，丙酮和丁醇；从微藻体内提取纯化的 ω -3 脂肪酸是高价值的食品添加剂；有报道称小球藻内含有大量的色素，而藻株内的色素能作为药膏内的螯合剂以及溃疡和损伤组织的恢复起着重要作用，同时也是食品颜色的添加剂的天然原料；由于藻株本身含有蛋白等多种营养物质，可以作为鱼饵、家禽家畜饲料以及农作物的肥料等。 In addition to being used as a raw material for biodiesel, microalgae itself also contain high value products. The dry weight contents of Scenedesmus sp. and Chlorella (Cz/ore//a sp.) were 50-56% and 51-58%, respectively. Microalgae can also be used to ferment biogas, acetone and butanol; extracting purified omega-3 fatty acids from microalgae is a high-value food additive; it has been reported that chlorella contains a large amount of pigment, while algae Pigment can play an important role as a chelating agent in ointments and recovery of ulcers and damaged tissues. It is also a natural raw material for food color additives. Because algae itself contains various nutrients such as protein, it can be used as bait, poultry and livestock feed. Fertilizers for crops, etc.

微藻不仅可以作为生物柴油炼制的优质原料，还是多种高附值产品的加工厂。但目前存在的问题是微藻产品的生产成本过高。其中，微藻采收的成本占生产成本的比重很大，限制了微藻能源及精细化学品的工业化生产。 Microalgae can be used not only as a high-quality raw material for biodiesel refining, but also as a processing plant for a variety of high-value products. However, the current problem is that the production cost of microalgae products is too high. Among them, the cost of microalgae harvesting accounts for a large proportion of production costs, which limits the industrial production of microalgae energy and fine chemicals.

由于微藻自身特点，如大小约为 5到 50 μ m，并且细胞密度低，为 15到 108g/L，含水量很大，目前微藻采收的方法主要有气浮法，过滤法，离心法及化学絮凝法。气浮法耗能大成本高；过滤法依赖于藻细胞大小，不适用大规模微藻收集；而基于藻密度不同于培养液采用的离心法，沉淀中会含有大量水分，增加下游处理费用；虽然目前化学絮凝能达到较好的微藻收集效果，而且有些已经工业化，但是大量金属离子以及难降解高聚合物的添加无疑是对环境的二次污染；虽然有报道微生物絮凝剂在微藻采收中应用，但生物安全性未知。因此，开发微藻低成本采收方法是进行微藻生物炼制及大规模工业化生产微藻能源的重要保证。 Due to the characteristics of microalgae, such as the size of 5 to 50 μ m, and the low cell density, 15 to 108 g / L, the water content is very large. At present, the methods of microalgae harvesting mainly include air flotation, filtration, and centrifugation. Method and chemical flocculation method. The air floatation method consumes a large amount of energy; the filtration method depends on the size of the algae cells, and is not suitable for large-scale microalgae collection; and based on the centrifugation method in which the algae density is different from that of the culture solution, the precipitate contains a large amount of water, which increases the downstream treatment cost; Although chemical flocculation can achieve better microalgae collection, and some have been industrialized, the addition of large amounts of metal ions and refractory high polymers is undoubtedly secondary pollution to the environment; although microbial flocculants have been reported in microalgae. The application is closed, but the biosafety is unknown. Therefore, the development of a low-cost harvesting method for microalgae is an important guarantee for microalgae biorefinery and large-scale industrial production of microalgae energy.

某些微藻具有细胞自絮凝的能力，推测是在培养过程中，藻体合成分泌某种物质从而使自身沉降。同传统絮凝方法相比，微藻细胞的自絮凝无需外源物质加入，无能耗无污染，环境友好。微藻多以非絮凝种类为主，天然絮凝藻株并不多，通过基因工程的方法改造现有的非絮凝藻株让其表现出絮凝形状从而达到微藻收集的目的。利用微藻自絮凝进行微藻采收是有应用前途的微藻收集方法，目前还没有关于将絮凝基因转入非絮凝微藻的报道。发明内容 Some microalgae have the ability to self-flocculate cells, presumably during the cultivation process, the algae synthesizes a substance to precipitate itself. Compared with the traditional flocculation method, the self-flocculation of microalgae cells does not require the addition of exogenous substances, no energy consumption, no pollution, and environmental friendliness. Most of the microalgae are non-flocculated species, and there are not many natural flocculated algae strains. The existing non-flocculated algal strains are genetically engineered to show the flocculation shape to achieve the purpose of microalgae collection. The use of microalgae self-flocculation for microalgae harvesting is a promising microalgae collection method. There are no reports on the transfer of flocculating genes into non-flocculated microalgae. Summary of the invention

本发明的目的是建立微藻转化平台及其在微藻采收中的应用。 The object of the present invention is to establish a microalgae transformation platform and its use in microalgae harvesting.

为实现上述目的，本发明首先将酵母絮凝基因 FL01重组到强启动子和报告基因基因下游，然后***含有选择标记基因的质粒中，构建转化用的载体，通过电转的方法转质粒入示例性的非絮凝栅藻 S. obliquus FSP 和普通小球藻 C. vulgaris C^U , 经过抗生素筛选和荧光检测，获得稳定遗传的转化子。 In order to achieve the above object, the present invention firstly recombines the yeast flocculating gene FL01 into a strong promoter and a reporter gene downstream, and then inserts into a plasmid containing a selectable marker gene, constructs a vector for transformation, and converts the plasmid into an exemplary method by electroporation. Non-flocculated S. obliquus FSP and common chlorella C. vulgaris C^U, through antibiotic screening and fluorescence detection, obtain stable genetic transformants.

本发明所获得的转基因藻表现出自絮凝现状而且正常生长，利于微藻收集以及下游生物炼制的处理。本发明建立的转化平台转化效率高，适用于不同藻株，而且所获得的基因工程藻株具有遗传稳定性。因此，本发明第一方面提供一种表达载体，所述表达载体含有来自酵母的絮凝基因，与所述絮凝基因操作性连接的、位于所述絮凝基因上游的启动子和抗生素抗性基因。 The transgenic algae obtained by the present invention exhibit self-flocculation status and normal growth, which facilitates the collection of microalgae and the processing of downstream biorefinery. The transformation platform established by the invention has high conversion efficiency, is suitable for different algae strains, and the genetically engineered algae strain obtained has genetic stability. Accordingly, a first aspect of the present invention provides an expression vector comprising a flocculating gene derived from yeast, a promoter operably linked to the flocculating gene, and an antibiotic resistance gene located upstream of the flocculation gene.

在一个具体实施例中，所述来自酵母的絮凝基因是酵母絮凝基因 FL01、 FLO 5, FW9、 FW10 FWU。在一个具体实施例中，所述酵母絮凝基因如 CN 200910200097.X的 SEQ ID ΝΟ: 1所示。 In a specific embodiment, the flocculating genes from yeast are yeast flocculating genes FL01, FLO 5, FW9, FW10 FWU. In a specific embodiment, the yeast flocculating gene is represented by SEQ ID NO: 1 of CN 200910200097.X.

在一个具体实施例中，所述絮凝基因选自： In a specific embodiment, the flocculation gene is selected from the group consisting of:

( 1 ) 编码 SEQ ID NO:2所示的氨基酸序列的核苷酸序列；和 (1) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2;

(2) 在严格条件下与（1 ) 所限定的核苷酸序列杂交且编码具有絮凝活性的蛋白质的核苷酸序列。 (2) A nucleotide sequence which hybridizes to the nucleotide sequence defined in (1) under stringent conditions and encodes a protein having flocculating activity.

在一个具体实施例中，所述载体含有 SEQ ID ΝΟ: 1 所示的核苷酸序列、 CaMV35S启动子和氯霉素抗性基因。 In a specific embodiment, the vector comprises the nucleotide sequence set forth in SEQ ID NO: 1, the CaMV35S promoter, and the chloramphenicol resistance gene.

在一个具体实施例中，所述表达载体选自： In a specific embodiment, the expression vector is selected from the group consisting of:

( 1 ) SEQ ID NO:3所示的核苷酸序列; 和 (1) the nucleotide sequence shown in SEQ ID NO: 3;

(2) 与 SEQ ID NO:3具有至少 80%序列相同性的核苷酸序列。 (2) A nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 3.

本发明第二方面提供一种转基因自絮凝藻类细胞，所述细胞稳定转化了本发明所述的表达载体，并具有自沉降性能。 A second aspect of the present invention provides a transgenic self-flocculating algae cell, which stably transforms the expression vector of the present invention and has self-settling properties.

在一个具体实施例中，所述藻选自栅藻（例如斜生栅藻）和小球藻。 In a specific embodiment, the alga is selected from the group consisting of Scenedesmus (such as Scenedesmus obliquus) and Chlorella.

在一个具体实施例中，所述藻类细胞为保藏编号为 CGMCC No. 5800小球藻细胞或保藏编号为 CGMCC No. 5905的栅藻细胞。 In a specific embodiment, the algal cell is a Scenevine cell having the accession number CGMCC No. 5800 Chlorella cells or the accession number CGMCC No. 5905.

本发明第三方面提供一种制备转基因自絮凝微藻的方法，所述方法包括： A third aspect of the invention provides a method for preparing a transgenic self-flocculated microalgae, the method comprising:

( 1 ) 将本发明所述的表达载体转入所述微藻；和 (1) transferring the expression vector of the present invention into the microalgae;

(2) 筛选具备絮凝性能的微藻。 (2) Screening of microalgae with flocculation properties.

在一个具体实施例中，本发明制备转基因自絮凝微藻的包括： In a specific embodiment, the preparation of the transgenic self-flocculated microalgae comprises:

(a) 收集对数生长期的微藻细胞，重悬在渗透缓冲液中； (a) collecting microalgal cells in the logarithmic growth phase and resuspending in permeation buffer;

(b) 热击 (1)中获得的细胞，然后于冰上放置； (b) Heat the cells obtained in (1) and place them on ice;

(c) 将 (2)中处理后的细胞与本发明所述的表达载体和鲑鱼精 DNA (否）混合，并于冰上放置； (c) mixing the treated cells in (2) with the expression vector of the present invention and salmon sperm DNA (No), and placing on ice;

(d) 将细胞重悬，对其实施转化处理（例如实施超声波介导法或电击法处理），获得转化的细胞；和 (d) resuspending the cells, performing a transformation treatment (for example, performing ultrasonic mediated or electroshock treatment) to obtain transformed cells;

(e) 将步骤（d) 的细胞转入新鲜配制的 DM培养基中暗培养，然后转入含有抗生素的 DM液体培养基中培养，筛选获得所述转基因自絮凝微藻。本发明第四方面提供本发明所述表达载体在制备转基因自絮凝藻类细胞中的应用。 (e) The cells of the step (d) are transferred to freshly prepared DM medium for dark culture, and then transferred to a DM liquid medium containing antibiotics, and the transgenic self-flocculated microalgae are obtained by screening. In a fourth aspect of the invention, the use of the expression vector of the invention in the preparation of a transgenic self-flocculated algae cell is provided.

本发明成功构建了微藻转化平台。通过本发明能够有效的将酵母絮凝基因重组到不同藻株中，并通过筛选获得基因突变藻株。与现有的技术相比，本发明实现了微藻转化基因工程技术的重点突破，具有如下有益效果： The invention successfully constructs a microalgae transformation platform. Through the present invention, the yeast flocculating gene can be efficiently recombined into different strains of the algae, and the mutant strain of the gene is obtained by screening. Compared with the prior art, the present invention realizes a key breakthrough of microalgae transformation genetic engineering technology, and has the following beneficial effects:

1. 本发明证明了 CaMV35S启动子在栅藻中可启动外源基因的表达。 1. The present invention demonstrates that the CaMV35S promoter can initiate expression of a foreign gene in Scenedesmus.

2. 在筛选标记方面，本发明的实验证明栅藻藻株对氯霉素敏感，采用一种可行的选择标记基因和报告基因组合是本发明的一技术特点。 2. In terms of screening markers, the experiments of the present invention demonstrate that S. cerevisiae strains are sensitive to chloramphenicol, and it is a technical feature of the present invention to employ a viable selection marker gene and reporter gene combination.

3. 本发明构建了酵母絮凝基因的微藻表达载体 pSLG01，并实现了外源基因在栅藻和小球藻中的稳定表达。附图说明 3. The present invention constructs a microalgal expression vector pSLG01 of a yeast flocculating gene, and achieves stable expression of the foreign gene in Scenedesmus and Chlorella. DRAWINGS

图 1显示表达载体构建的示意图。 Figure 1 shows a schematic representation of the construction of an expression vector.

图 2显示 In-Fusion cloning示意图。 Figure 2 shows the In-Fusion cloning diagram.

图 3显示克隆获得酵母絮凝基因 FL01。 Figure 3 shows the clone obtained the yeast flocculating gene FL01.

图 4显示抗生素筛选转化子，其中， 1 : 野生型栅藻细胞； 2: 转入 pCAMBIA 1302-CAT的转化子； 3 : 转入 pCAMBIA 1302-CAT-FLO1的转化子。 Figure 4 shows antibiotic-selected transformants, wherein: 1 : wild-type VSG cells; 2: transformants transformed into pCAMBIA 1302-CAT; 3: transformants transformed into pCAMBIA 1302-CAT-FLO1.

图 5显示扫描电镜观察栅藻细胞表型变化。 1,4: 野生型栅藻细胞； 2,5: 转入 pCAMBIA 1302-CAT的转化子； 3,6: 转入 pCAMBIA 1302-CAT- LC 的转化子。 Figure 5 shows the phenotypic changes of Scenedesmus cells observed by scanning electron microscopy. 1,4: wild type Scenedesmus cells; 2,5: transformants transformed into pCAMBIA 1302-CAT; 3,6: transformants transformed into pCAMBIA 1302-CAT-LC.

图 6显示荧光电镜观察栅藻表型变化。其中，左：野生型栅藻细胞；中：转入 pCAMBIA 1302-CAT的转化子；右：转入 pCAMBIA 1302-CAT-FLO1的转化子。 Figure 6 shows the phenotypic changes of the genus Scenedesmus by fluorescence electron microscopy. Among them, left: wild type Scenedesmus cells; medium: transformants transformed into pCAMBIA 1302-CAT; right: transformants transformed into pCAMBIA 1302-CAT-FLO1.

图 7显示流式细胞仪检测转化子的情况。其中， 1 : 野生型栅藻细胞； 2: 转入 pCAMBIA 1302-CAT的转化子； 3：转入 pCAMBIA 1302-CAT-FLO1的转化子。 Figure 7 shows the detection of transformants by flow cytometry. Among them, 1 : wild type Scenedesmus cells; 2: transformants transformed into pCAMBIA 1302-CAT; 3: transformants transformed into pCAMBIA 1302-CAT-FLO1.

图 8显示 PCR和 RT-PCR分析酵母絮凝基因 FL01在转基因栅藻细胞中的表达。其中，左图为 PCR分析酵母絮凝基因 FLO1在转基因栅藻细胞中表达情况；右图为 RT-PCR酵母絮凝基因 FLO1在转基因栅藻细胞中表达情况。图中， M: lkb ladder marker (TaKaRa Bio Inc., Japan); W: 野生型栅藻细胞； T, Τ1 转入 pCAMBIA 1302-CAT-FLO1的栅藻细胞； C: 转入 pCAMBIA 1302-CAT的栅藻细胞； P: 质粒 pCAMBIA 1302-CAT-FLO1。 Figure 8 shows the expression of the yeast flocculating gene FL01 in transgenic G. stratifolia cells by PCR and RT-PCR. Among them, the left picture shows the expression of yeast flocculating gene FLO1 in transgenic G. argyi cells by PCR; the right picture shows the expression of RT-PCR yeast floc gene FLO1 in transgenic griddes. In the figure, M: lkb ladder marker (TaKaRa Bio Inc., Japan); W: wild type Scenedesmus cells; T, Τ1 transferred to pCAMBIA 1302-CAT-FLO1 of Scenedesmus cells; C: transferred to pCAMBIA 1302-CAT Scenedesmus cells; P: plasmid pCAMBIA 1302-CAT-FLO1.

图 9显示转基因絮凝微藻自沉降性能进行研究。其中， A: 野生型栅藻细胞； B: 转入 pCAMBIA 1302-CAT的转化子； C: 转入 pCAMBIA 1302-CAT-FLO1的转化子。其中 min代表分钟。 Figure 9 shows the self-settling properties of transgenic flocculated microalgae. Among them, A: wild type Scenedesmus cells; B: transformants transformed into pCAMBIA 1302-CAT; C: transferred to pCAMBIA 1302-CAT-FLO1 Turn. Where min stands for minutes.

图 10显示转基因絮凝微藻的油脂积累。其中，对照为野生型栅藻，细胞为游离状态， T-1302为转入空载体的对照藻细胞， T-FLO1为转入絮凝基因表达载体的絮凝藻。 Figure 10 shows the accumulation of oil in transgenic flocculated microalgae. Among them, the control is wild-type S. cerevisiae, the cells are in a free state, T-1302 is a control algae cell transferred into an empty vector, and T-FLO1 is a flocculating algae transferred into a flocculating gene expression vector.

图 11 显示转基因絮凝小球藻自沉降性能进行研究。其中， A: 野生型小球藻细胞； B: 转入 pCAMBIA 1302-CAT-FLO1的转化子。其中 min代表分钟。具体实施方式 Figure 11 shows the self-settling properties of transgenic flocculated chlorella. Among them, A: wild-type chlorella cells; B: transformants transformed into pCAMBIA 1302-CAT-FLO1. Where min stands for minutes. detailed description

本发明的絮凝基因包括酵母絮凝基因 0? ( SEQ ID NO: l ) 、 FL05、 FL09、 FLOW和 ¾( 77。在一个具体实施例中，所述酵母絮凝基因如 CN 200910200097.X 的 SEQ ID NO: l所示。在一具体实施例中，本发明的絮凝基因编码本申请 SEQ ID NO:2或 CN 200910200097.X的 SEQ ID NO:2所示的氨基酸序列。 The flocculation genes of the present invention include yeast flocculating genes 0 (SEQ ID NO: l), FL05, FL09, FLOW, and 3⁄4 (77. In a specific embodiment, the yeast flocculating gene such as SEQ ID NO of CN 200910200097.X : l. In a specific embodiment, the flocculating gene of the invention encodes the amino acid sequence set forth in SEQ ID NO: 2 of the present application or SEQ ID NO: 2 of CN 200910200097.X.

本发明包括与本发明絮凝基因高度同源的核苷酸序列，或在严格条件下与本发明絮凝基因杂交的核苷酸序列。提到核苷酸序列时，本文所用的 "高度同源" 可以指与所提及序列的相同性为至少约 70%、至少约 75%、至少约 80%、至少约 85%、至少约 90%、至少约 95%、至少约 98%、至少约 99%或 100%的核苷酸序列。本发明的核苷酸序列可以是本发明絮凝基因（例如本发明的 SEQ ID NO: 1和 CN 200910200097.X的 SEQ ID NO: 1 ) 的简并变异体。如本文所用， "简并变异体" 在本发明中是指编码包含本发明 SEQ ID NO: 2或 CN 200910200097.X的 SEQ ID NO:2的氨基酸序列，但与本发明 SEQ ID NO: 1或 CN 200910200097.X的 SEQ ID ΝΟ: 1所示的核苷酸序列有差别的核苷酸序列。 The present invention includes a nucleotide sequence highly homologous to the flocculating gene of the present invention, or a nucleotide sequence which hybridizes under stringent conditions to the flocculating gene of the present invention. When referring to a nucleotide sequence, "highly homologous" as used herein may mean at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90 identical to the sequences mentioned. %, at least about 95%, at least about 98%, at least about 99% or 100% of the nucleotide sequence. The nucleotide sequence of the present invention may be a degenerate variant of the flocculating gene of the present invention (e.g., SEQ ID NO: 1 of the present invention and SEQ ID NO: 1 of CN 200910200097.X). As used herein, "degenerate variant" in the present invention refers to an amino acid sequence encoding SEQ ID NO: 2 comprising SEQ ID NO: 2 or CN 200910200097.X of the present invention, but with SEQ ID NO: 1 of the present invention or The nucleotide sequence shown by SEQ ID NO: 1 of CN 200910200097.X has a nucleotide sequence which differs.

在相同性分析中可辅助使用易于获得的计算机程序，如 ALIGH、 Dayhoff, M.O. (Atlas of Protein Sequence and Structure、 M.O.Dayhoff编辑， 5 Suppl. , 3： 353-358， National Biomedical Research Foundation, Washington, DC)，它适用于 Smith 和 Waterman 分析肽用的局部同源性算法 (Advances in Appl. Math. , 2： 482-489, 1981)。可从 Wisconsin Sequence Analysis Package (第 8版，从 Genetics Computer Group, Madison, WI获得)获得测定核苷酸序列同源性的程序，例如， BESTFIT、 FASTA和 GAP程序，这些程序也依赖于 Smith和 Waterman算法。使用制造者建议的和上述 Wisconsin Sequence Analysis Package所述的默认参数可容易地使用这些程序。例如，可使用 Smith和 Warerman的同源性算法的默认计分表和 6个核苷酸位置的间隔罚分 (gap penalty)测定的核苷酸序列与参比序列的同源性百分数。本发明还涉及本发明絮凝基因（如本发明 SEQ ID ΝΟ: 1或 CN 200910200097.X 的 SEQ ID NO: 1 )的变异体，其编码与本发明 SEQ ID NO: 2或 CN 200910200097.X 的 SEQ ID NO:2有相同的氨基酸序列的絮凝基因的片段、类似物、衍生物和变异形式。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和***变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或***，但不会从实质上改变其编码的蛋白的功能。如本文所用， "核酸片段" 的长度至少含 15个核苷酸，较好是至少 30个核苷酸，更好是至少 50 个核苷酸，最好是至少 100个核苷酸以上。核酸片段可用于核酸的扩增技术 (如 PCR)以确定和 /或分离编码本发明脂肪酶的多核苷酸。 An easy-to-obtain computer program can be assisted in the identity analysis, such as ALIGH, Dayhoff, MO (Atlas of Protein Sequence and Structure, MODayhoff, ed., 5 Suppl., 3: 353-358, National Biomedical Research Foundation, Washington, DC) It is applicable to the local homology algorithm used by Smith and Waterman to analyze peptides (Advances in Appl. Math., 2: 482-489, 1981). Procedures for determining nucleotide sequence homology, such as BESTFIT, FASTA, and GAP programs, are also available from the Wisconsin Sequence Analysis Package (version 8, available from Genetics Computer Group, Madison, WI), which also rely on Smith and Waterman. algorithm. These programs can be easily used using the default parameters suggested by the manufacturer and described in the Wisconsin Sequence Analysis Package above. For example, the percentile homology of the nucleotide sequence to the reference sequence can be determined using the default score table for the homology algorithm of Smith and Warerman and the gap penalty for 6 nucleotide positions. The invention also relates to variants of the flocculating gene of the invention (such as SEQ ID ΝΟ: 1 of the invention or SEQ ID NO: 1 of CN 200910200097.X) encoding a SEQ of SEQ ID NO: 2 or CN 200910200097.X of the invention ID NO: 2 Fragments, analogs, derivatives and variant forms of the flocculation gene having the same amino acid sequence. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the protein encoded thereby. . As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate polynucleotides encoding the lipases of the invention.

本发明还包括与本发明絮凝基因对应的 RNA序列，和在严格条件下与本发明絮凝基因或所述 RNA序列杂交的分子。 The present invention also encompasses an RNA sequence corresponding to the flocculating gene of the present invention, and a molecule which hybridizes under stringent conditions to the flocculating gene of the present invention or the RNA sequence.

如本文所用，术语 "严格条件"是指：（1)在较低离子强度和较高温度下的杂交和洗脱，如 0.2 X SSC, 0.1%SDS， 60 °C _; 或 (2)杂交时加有变性剂，如 50%(v/v) 甲酰胺， 0.1%小牛血清 /0.1%Ficoll， 42°C等；或 (3)仅在两条序列之间的相同性至少在 50%，优选 55%以上、 60%以上、 65%以上、 70%以上、 75%以上、 80%以上、 85%以上或 90%以上，更优选是 95%以上时才发生杂交。例如，所述序列可为 (a) 中所限定序列的互补序列，或者可以是与 (a)中所限定序列的互补性为至少 50%、优选 55%以上、 60%以上、 65%以上、 70%以上、 75%以上、 80%以上、 85%以上或 90%以上，更优选是 95%以上的序列。 As used herein, the term "stringent conditions" means: (1) hybridization and elution at lower ionic strengths and higher temperatures, such as 0.2 X SSC, 0.1% SDS, 60 °C _; or (2) hybridization Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 50% identity between the two sequences, Hybridization occurs preferably at 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, and more preferably 95% or more. For example, the sequence may be a complementary sequence of the sequence defined in (a), or may have a complementarity to the sequence defined in (a) of at least 50%, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, more preferably 95% or more.

"编码序列"或 "编码"选定多肽的序列，是指当处于适当的调节序列的控制下时，一种核酸分子在体外或体内被转录 (；对于 DNA)和翻译 (；对于 mRNA)成一种多肽。该编码序列的界限可由 5' (氨基；)末端的起始密码子和 3' (羧基；)末端的翻译终止密码子来确定。转录终止序列可以位于该编码序列的 3'端。 "Coding sequence" or "encoding" a sequence of a selected polypeptide means that a nucleic acid molecule is transcribed (for DNA) and translated (for mRNA) in vitro or in vivo when under the control of appropriate regulatory sequences. a polypeptide. The boundaries of the coding sequence can be determined by the start codon at the 5' (amino;) terminus and the translation stop codon at the 3' (carboxy;) terminus. The transcription termination sequence can be located at the 3' end of the coding sequence.

"可操作性连接"指元件的排列，其中所述成分被排成一定的形状，以便执行它们所需的功能。因而，可操作性连接于编码序列的给定的启动子，在正确的转录因子等存在时，能使该编码序列有效表达。该启动子不需要与该编码序列邻接，只要它起到指导该序列表达的功能即可。因此，例如不参与翻译但转录的序列可存在于启动子序列和编码序列之间，与可转录的内含子一样；并且仍可认为该启动子序列 "可操作性连接"于该编码序列。 "Operative connection" refers to the arrangement of elements in which the components are arranged in a shape to perform their desired functions. Thus, a given promoter operably linked to a coding sequence enables efficient expression of the coding sequence in the presence of a correct transcription factor or the like. The promoter need not be contiguous with the coding sequence as long as it functions to direct expression of the sequence. Thus, for example, a sequence that is not involved in translation but is transcribed may be present between the promoter sequence and the coding sequence, as is the transcribed intron; and the promoter sequence is still considered "operably linked" to the coding sequence.

本发明的载体除含有编码序列外，还可含有各种控制元件。 "控制元件"指帮助与其连接的编码序列表达的多核苷酸序列。该术语包括启动子、转录终止序列、上游调节结构域、聚腺苷酸化信号、非翻译区域 (包括 5'-UTR和 3'-UTR)，适当时还有前导序列和增强子，这些序列共同地为宿主细胞中的编码序列的转录和翻译创造条件。 The vector of the present invention may contain various control elements in addition to the coding sequence. "Control element" refers to a polynucleotide sequence that facilitates expression of a coding sequence to which it is ligated. The term includes promoter, transcription termination Columns, upstream regulatory domains, polyadenylation signals, untranslated regions (including 5'-UTR and 3'-UTR), and, where appropriate, leader sequences and enhancers, which are collectively coding sequences in host cells Create conditions for transcription and translation.

"启动子"在本文中指能结合宿主细胞中的 RNA聚合酶并启动与之操作性连接的下游 (3'方向)编码序列的转录的 DNA调节区域。用于本发明的启动子序列包括以高于背景值的可检测的水平启动感兴趣的基因的转录所需的最小数量的碱基或元件。在该启动子序列中有转录启始位点以及负责 RNA聚合酶结合的蛋白质结合结构域 (共有序列；)。真核细胞启动子常常 (但不总是;)含有 " TATA"框和 " CAT " 框。本发明使用的表达载体的启动子可为 CaMV35S，也可以使用其它在微藻中可启动基因表达的启动子，包括在特定的诱导条件下才表达的启动子，如磷枯竭诱导启动子等。 "Promoter" as used herein refers to a region of DNA regulation that binds to RNA polymerase in a host cell and initiates transcription of a downstream (3' direction) coding sequence operably linked thereto. Promoter sequences for use in the present invention include the minimum number of bases or elements required to initiate transcription of a gene of interest at a detectable level above background values. Within this promoter sequence are transcription initiation sites and protein binding domains responsible for RNA polymerase binding (consensus sequences;). Eukaryotic promoters often (but not always;) contain a "TATA" box and a "CAT" box. The promoter of the expression vector used in the present invention may be CaMV35S, and other promoters which can initiate gene expression in microalgae, including promoters expressed under specific induction conditions, such as a phosphorus depletion-inducing promoter and the like, may be used.

控制序列在 RNA聚合酶结合启动子序列时 "指导转录"细胞中的编码序列，并将该编码序列转录成 mRNA，之后该 mRNA被翻译成由该编码序列编码的多肽。 The control sequence "directs transcription" of the coding sequence in the cell when the RNA polymerase binds to the promoter sequence and transcribes the coding sequence into mRNA, after which the mRNA is translated into the polypeptide encoded by the coding sequence.

本文中， "表达盒"或 "表达构建物"指能指导感兴趣的序列或基因的表达的装配。该表达盒包括上述控制元件，如操作性连接于感兴趣的序列或基因 (以指导其转录;)的启动子，该表达盒还常常包括多腺苷酸化序列。在本发明的某些实施例中，在此所述的表达盒可被包含在本发明的表达载体（即质粒构建物）中。除了该表达盒的组分外，该质粒构建物还含有一种或多种可选择的标记物、使该质粒构建物以单链 DNA (；如 M13复制源;)存在的信号、至少一个多克隆位点和 "哺乳动物"复制源 (；如 SV40或腺病毒复制源；)。 As used herein, an "expression cassette" or "expression construct" refers to an assembly that directs the expression of a sequence or gene of interest. The expression cassette includes the above-described control elements, such as a promoter operably linked to a sequence or gene of interest (to direct its transcription;), which often also includes polyadenylation sequences. In certain embodiments of the invention, an expression cassette as described herein can be included in an expression vector (i.e., a plasmid construct) of the invention. In addition to the components of the expression cassette, the plasmid construct also contains one or more selectable markers, a signal that allows the plasmid construct to be present as a single-stranded DNA (such as a M13 replication source), at least one more Cloning sites and "mammalian" replication sources (such as SV40 or adenoviral replication sources;).

"转化"在本文中指将外源多核苷酸（例如本发明所述的表达载体） ***宿主细胞中。可用本领域熟知的各种基因送递的方法，将包含感兴趣的核苷酸序列的核酸分子稳定地整合入宿主细胞基因组中或在合适的宿主细胞中的稳定的附加型元件上维持。参见如美国专利 No.5,399,346。表达载体导入受体微藻细胞的方法可包括电击法、超声法等方法。 "Transformation" as used herein refers to the insertion of an exogenous polynucleotide (e.g., an expression vector of the invention) into a host cell. A nucleic acid molecule comprising a nucleotide sequence of interest can be stably integrated into the host cell genome or maintained on a stable add-on element in a suitable host cell by various methods of gene delivery well known in the art. See, e.g., U.S. Patent No. 5,399,346. The method of introducing the expression vector into the recipient microalgae cell may include an electric shock method, an ultrasonic method, or the like.

"宿主细胞"是已被转化的细胞，或者能被外源 DNA序列转化的细胞。各种藻类细胞可用于本发明，包括但不限于微藻类，如绿藻门小球藻属中的蛋白核小球藻 { Chlorella pyrenoidosa ) ，普通小球藻 ( Chlorella vulgaris ) ， #1圆小球藻 ( Chlorella ellipsoidea ) ， Chlorella emersonii , Chlorella sorokiniana， Chlorella saccharophila, Chlorella regularis e Chlorella minutissima, Chlorella protothecoides , Chlorella zofingiensis , 以及绿藻 |、中的 Brachiomonas submarina， Chlamydobonas reinhardtii， Chlamydomonas acidophil a, Haematococcus pluvial is , Haematococcus lacustris, Scenedesmus obliquus , Spongiococcum exetriccium , Tetraselmis suecica, Tetraselmis chuii， Tetraselmis tetrathele， Tetraselmis verrucosa， Micractinium pusillum；娃藻 |、的 Cylindrotheca fusiformis， Nitzschia laevis， Nitzschia alba， Nitzschia fonticola, Navicula incerta, Navicula pelliculosa-, ί^,^Π^ Anabaena variabilis -, 金藻门的 Poterioochromonas malhamensis；甲藻门的 Amphidinium carterae， Crypthecodinium cohnii；裸藻门的 Euglena gricilis；禾口红藻门的 Galdieria sulph丽 ia。适用于本发明的微生物还包括被孢霉属（Mortierella) ，破囊壶菌属 ( Thraustochytrium ) ，裂殖壶菌属（网粘菌纲） ( Schizochytrium (Labyrinthulomycetes ) ) ，吾肯氏壶藻属（Ulkenia) 等。本发明特别优选的是栅藻 (.Scenedesmus sp. ) 禾口小球藻 ( Chlorella sp. ) 。 A "host cell" is a cell that has been transformed, or a cell that can be transformed by a foreign DNA sequence. Various algae cells can be used in the present invention, including but not limited to microalgae, such as Chlorella pyrenoidosa in the genus Chlorella, Chlorella vulgaris, #1 round ball Chlorella ellipsoidea, Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regularis e Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, and Chlorella |, Brachiomonas submarina, Chlamydobonas reinhardtii, Chlamydomonas acidophil a, Haematococcus pluvial is , Haematococcus Lacustris, Scenedesmus obliquus, Spongiococcum exetriccium, Tetraselmis suecica, Tetraselmis chuii, Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum; cyanobacteria|, Cylindrotheca fusiformis, Nitzschia laevis, Nitzschia alba, Nitzschia fonticola, Navicula incerta, Navicula pelliculosa-, ί^, ^Π^ Anabaena variabilis -, Poterioochromonas malhamensis of the cyanobacteria; Amphidinium carterae, Crypthecodinium cohnii of the genus Dinophyllum; Euglena gricilis of the cyanophyta; Galdieria sulph ia of the red algae. Microorganisms suitable for use in the present invention also include Mortierella, Thraustochytrium, Schizochytrium (Labyrinthulomycetes), and Mykonium Ulkenia) and so on. Particularly preferred in the present invention are Scenedesmus sp. and Chlorella sp.

本发明中，一旦制备或分离了编码序列，就可将这些序列克隆入任何合适的载体或复制子中。对本领域技术人员而言，各种克隆载体是已知的，且适当的克隆载体的筛选只是选择问题。合适的载体包括 (但并非限制于质粒、噬菌体、转座子、粘粒、染色体或当与适当的控制元件结合时能复制的病毒。 In the present invention, once the coding sequences have been prepared or isolated, these sequences can be cloned into any suitable vector or replicon. Various cloning vectors are known to those skilled in the art, and screening of suitable cloning vectors is only a matter of choice. Suitable vectors include, but are not limited to, plasmids, phage, transposons, cosmids, chromosomes or viruses that replicate when combined with appropriate control elements.

然后将克隆序列置于合适的控制元件的控制下，这取决于用于表达的***。因此，可以将编码序列置于启动子、核糖体结合位点 (用于细菌表达;)和任选地操纵子的控制下，从而由合适的转化体将感兴趣的 DNA序列转录到 RNA中。该编码序列可以包含或不包含信号肽或前导序列 (随后可由宿主在翻译后加工除去；)。 The cloned sequence is then placed under the control of a suitable control element, depending on the system used for expression. Thus, the coding sequence can be placed under the control of a promoter, a ribosome binding site (for bacterial expression;) and optionally an operon to transcribe the DNA sequence of interest into RNA from a suitable transformant. The coding sequence may or may not contain a signal peptide or leader sequence (which may then be removed by post-translation processing by the host;).

除了控制序列外，可以添加调节序列，从而可以相对于宿主细胞的生长调节序列的表达。调节序列是本领域技术人员已知的，其实例包括那些能导致应答化学或物理剌激 (包括调节化合物的存在;)启动或关闭基因表达的调节序列。载体中还可存在其它类型的调节元件。例如，可以使用增强子元件以增加构建物的表达水平。实例包括 SV40早期基因增强子 (Dijkema等 (1985)EMBO 14:761)；从 Rous肉瘤病毒的长末端重复序列（LTR)衍生的增强子 /启动子（Gorman 等 (1982)Proc.Natl.Acad.Sci.USA 79:6777)；和从人 CMV 衍生的元件 (Boshart 等， (1985)Cell 41 :521)，如 CMV内含子 A序列中包括的元件 (美国专利 No.5,688,688)。表达盒中还可包括在合适的宿主细胞中自主复制的复制起点、一种或多种可选择的标记物、一个或多个限制酶切位点、高拷贝数量的势能和强启动子。 In addition to the control sequences, regulatory sequences can be added to regulate expression of the sequences relative to the growth of the host cell. Regulatory sequences are known to those skilled in the art, examples of which include regulatory sequences that result in responsive chemical or physical stimulation (including the regulation of the presence of a compound;) initiating or shutting down gene expression. Other types of conditioning elements may also be present in the carrier. For example, enhancer elements can be used to increase the expression level of the construct. Examples include the SV40 early gene enhancer (Dijkema et al. (1985) EMBO 14:761); an enhancer/promoter derived from the long terminal repeat (LTR) of Rous sarcoma virus (Gorman et al. (1982) Proc. Natl. Acad. Sci. USA 79:6777); and elements derived from human CMV (Boshart et al. (1985) Cell 41:521), such as those included in the CMV intron A sequence (U.S. Patent No. 5,688,688). The expression cassette may also include an origin of replication autonomously replicating in a suitable host cell, one or more selectable markers, one or more restriction sites, a high copy number of potential energy, and a strong promoter.

构建表达载体，使具体的编码序列位于该具有适合调节序列的载体中，与控制序列有关的编码序列的位置和取向使编码序列在控制序列的"控制"下转录卩，结合于控制序列中 DNA分子的 RNA聚合酶转录该编码序列；)。可能需要对编码感兴趣分子的序列进行修饰来实现此目的。例如，在一些情况中可能需要修饰该序列，从而使其连接于适合取向的控制序列，即维持读框。在***到载体之前，控制序列和其它调节序列可能连接于编码序列。或者，可以将编码序列直接克隆入已包含控制序列和合适的限制酶切位点的表达载体中。 Constructing an expression vector such that the specific coding sequence is located in the vector having the appropriate regulatory sequence, and the position and orientation of the coding sequence associated with the control sequence transcribes the coding sequence under the "control" of the control sequence, and binds to the DNA in the control sequence The molecular RNA polymerase transcribes the coding sequence; It may be necessary to modify the sequence encoding the molecule of interest to accomplish this. For example, in some cases it may be necessary to modify the order The column is thus connected to a control sequence that is suitable for orientation, i.e., the reading frame is maintained. Control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into the vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains a control sequence and a suitable restriction enzyme cleavage site.

在其它实施例中，适用于本发明的酵母菌的絮凝基因可包括 ( SEQ ID NO: 1 ) 、 FLO 5, FW9、 FLOW和 FWll (参考 ZhaoXQ, Li Q, He LY, Li F, Que WW, Bai F, Exploration of a natural reservoir of flocculating genes from various Saccharomyces cerevisiae strains and improved ethanol fermentation using stable genetically engineered flocculating yeast strains, Process Biochemistry, 2012, doi: 10.1016/j.procbio.2011.06.009 ) 等基因或者序列相似的功能基因，例如，如前文所述具有一定序列相同性或在严格条件下与这些絮凝基因杂交的核苷酸序列，这些基因编码可糖基化的絮凝蛋白。可用这些絮凝基因来构建本发明的表达载体，然后转入藻类细胞中，筛选得到稳定表达絮凝蛋白、具有絮凝性能的藻类细胞。 In other embodiments, the flocculating genes suitable for use in the yeast of the invention may include (SEQ ID NO: 1), FLO 5, FW9, FLOW, and FWll (refer to ZhaoXQ, Li Q, He LY, Li F, Que WW, Bai F, Exploration of a natural reservoir of flocculating genes from various Saccharomyces cerevisiae strains and improved ethanol fermentation using stable genetically engineered flocculating yeast strains, Process Biochemistry, 2012, doi: 10.1016/j.procbio.2011.06.009 ) Isogenic or sequence similar A functional gene, for example, a nucleotide sequence having a certain sequence identity or hybridizing to these flocculation genes under stringent conditions as described above, these genes encoding a glycosylated flocculating protein. These flocculating genes can be used to construct the expression vector of the present invention, and then transferred into algae cells to obtain algal cells stably expressing floc protein and having flocculation properties.

因此，本申请提供一种获得转基因自絮凝微藻的方法，该方法包括：（1 )从絮凝酵母克隆絮凝基因，并用所述絮凝基因构建表达载体；和（2 )将表达载体（通过例如电击或超声处理）转化入微藻细胞中；从而可获得转基因自絮凝微藻。 Accordingly, the present application provides a method of obtaining a transgenic self-flocculating microalgae, the method comprising: (1) cloning a flocculating gene from a flocculating yeast, and constructing an expression vector using the flocculating gene; and (2) expressing the expression vector (by, for example, an electric shock) Or sonication) is transformed into microalgae cells; thus, transgenic self-flocculated microalgae can be obtained.

进一步的，本发明的方法还可包括：（3 ) 复苏和确认转基因絮凝微藻；和任选的，（4 ) 采收转基因絮凝微藻的细胞。 Further, the method of the present invention may further comprise: (3) resuscitating and confirming transgenic flocculated microalgae; and, optionally, (4) harvesting cells of the transgenic flocculated microalgae.

絮凝基因的克隆包括从酵母菌或者其他生物细胞中扩增具有细胞自絮凝功能的絮凝基因，本发明以从絮凝酵母中克隆的 FL01 基因为例进行絮凝基因表达载体的构建（具体可参见 CN 200910200097.X,本文以引用的方式将该文纳入本文）。 Cloning of the flocculation gene includes amplifying a flocculating gene having a cell self-flocculation function from yeast or other biological cells. The present invention uses a FL01 gene cloned from flocculating yeast as an example to construct a flocculating gene expression vector (see CN 200910200097 for details). .X, this article is incorporated herein by reference.

表达载体可包括 PCAMBIA1301等植物基因工程表达载体，以及其他可在微藻中使用的表达载体，表达载体的必要遗传学元件包括启动子，抗性基因，多克隆位点等。本发明使用的为含有以 pCAMBIA1301为基础改造的含有 GFP报告基因，氯霉素抗性基因 CAT的表达载体。 The expression vector may include a plant genetic engineering expression vector such as PCAMBIA1301, and other expression vectors which can be used in microalgae, and the necessary genetic elements of the expression vector include a promoter, a resistance gene, a cloning site and the like. The present invention uses an expression vector containing a GFP reporter gene and a chloramphenicol resistance gene CAT engineered based on pCAMBIA1301.

表达载体导入受体微藻细胞的方法可包括电击法、超声法等方法。 The method of introducing the expression vector into the recipient microalgae cell may include a method such as electroporation, sonication or the like.

本发明提供一种微藻（例如栅藻或小球藻）的遗传转化方法或转基因自絮凝微藻的制备方法，具体步骤包括： The invention provides a genetic transformation method of microalgae (such as Scenedesmus or Chlorella) or a preparation method of transgenic self-flocculated microalgae, and the specific steps include:

(c) 将 (2)中处理后的细胞与本发明所述的表达载体和鲑鱼精 DNA混合，并于冰上放置； (c) mixing the treated cells in (2) with the expression vector of the present invention and salmon sperm DNA, and placing on ice;

(d) 将细胞重悬，对其实施转化处理，如超声波介导法或电击法处理，获得转化的细胞；和 (d) Resuspend the cells and perform transformation treatment, such as ultrasonic-mediated or electric shock treatment, to obtain Transformed cells; and

(e) 将步骤（d) 的细胞转入新鲜配制的 DM培养基中暗培养，然后转入含有抗生素的 DM液体培养基中培养，筛选获得所述转基因自絮凝微藻。 (e) The cells of the step (d) are transferred to freshly prepared DM medium for dark culture, and then transferred to a DM liquid medium containing antibiotics, and the transgenic self-flocculated microalgae are obtained by screening.

本发明使用的渗透缓冲液可以是本领域常用的渗透缓冲液。示例性的渗透缓冲液含有例如 0.2M-0.3M的甘露醇， 0.2M-0.3M的山梨醇和 10-15%甘油。通常以大约 10⁷-10⁸个细胞每毫升的细胞密度重悬微藻细胞。 The permeation buffer used in the present invention may be a permeation buffer commonly used in the art. An exemplary permeation buffer contains, for example, 0.2 M to 0.3 M mannitol, 0.2 M to 0.3 M sorbitol, and 10-15% glycerol. The microalgae cells are typically resuspended at a cell density of about 10 ⁷ - 10 ⁸ cells per ml.

热击通常在例如 38-45 °C的温度下进行。在一具体实施例中，在 42°C进行热击。热击时间通常控制在大约 1-10分钟左右。热击后于冰上放置数分钟（例如 3-8 分钟，例如 5分钟）。 The heat shock is usually carried out at a temperature of, for example, 38 to 45 °C. In a specific embodiment, the heat shock is performed at 42 °C. The heat shock time is usually controlled to be about 1-10 minutes. Place the ice on the ice for a few minutes (for example, 3-8 minutes, for example 5 minutes).

然后，将冰上放置数分钟的细胞与本发明的表达载体和鲑鱼精 DNA混合。混合的量可根据实际情况选择。例如，通常每 100微升的细胞溶液（通常可含有 10³— 10⁵个细胞）可与大约 5-20 w g/mL的本发明表达载体以及大约 15-35 μ g/mL 的鲑鱼精 DNA混合。在一具体实施例中，将大约 400微升的细胞溶液（含有大约 4 X 10³— 4 X 10⁵个细胞）与大约 10 g/mL的本发明表达载体以及大约 25 μ g/mL 的鲑鱼精 DNA混合，混合后通常置于冰上数分钟（例如 3-8分钟，例如 5分钟）。 Then, cells placed on ice for several minutes were mixed with the expression vector of the present invention and salmon sperm DNA. The amount of mixing can be selected according to the actual situation. For example, typically every 100 microliters of cell solution (usually containing 10 ³ - 10 ⁵ cells) can be mixed with about 5-20 wg/mL of the expression vector of the invention and about 15-35 μg/mL of salmon sperm DNA. . In a specific embodiment, about 400 microliters of cell solution (containing about 4 X 10 ³ - 4 X 10 ⁵ cells) and about 10 g/mL of the expression vector of the invention and about 25 μg/mL of salmon The fine DNA is mixed and usually mixed on ice for a few minutes (for example, 3-8 minutes, for example 5 minutes).

然后，将细胞重悬，对其实施超声波介导法或电击法处理。对于超声波介导处理，可吸取适量（例如 0.4-lmL)重悬的细胞放入含有超纯水的烧杯中，进行超声，超声时间分别为 30-300秒不等。对于电击处理，可吸取适量（例如 0.4-lmL ) 重悬的细胞放入预冷电击小杯中，按照设置的各种条件组合进行电击.在其它条件不变的情况下，分别梯度改变高渗缓冲液浓度、表达载体、脉冲电压和脉冲持续时间这 4个参数，对电击参数进行优化。一种优化的电击转换条件为：表达载体浓度 50 μ g/mL,高渗缓冲液浓度 0.2 mol/L，脉冲距离为 2 mm,脉冲电压为 2 Kv，脉冲持续时间为 3ms。 Then, the cells are resuspended and subjected to ultrasonic mediated or electroshock treatment. For ultrasonic-mediated treatment, an appropriate amount (for example, 0.4-lmL) of resuspended cells can be aspirated into a beaker containing ultrapure water for ultrasonication, with ultrasonic times ranging from 30 to 300 seconds. For the shock treatment, an appropriate amount (for example, 0.4-lmL) of the resuspended cells can be taken into the pre-cooled electric shock cuvette, and the electric shock is combined according to various conditions set. Under the other conditions, the gradient changes the hypertonicity separately. The parameters of buffer concentration, expression vector, pulse voltage and pulse duration are optimized for the shock parameters. An optimized shock switching condition is: expression vector concentration 50 μg/mL, hypertonic buffer concentration 0.2 mol/L, pulse distance 2 mm, pulse voltage 2 Kv, pulse duration 3 ms.

超声波介导法或电击法处理后，将细胞转入适量（例如 10-30 毫升）新鲜配制的 DM培养基中，室温（例如 25 ) 下进行暗培养，培养时间大约 18-36 (例如 24小时）小时，然后转入含有抗生素例如（50 μ g/mL氯霉素）的 DM液体培养基中，于室温下（例如 28°C ) 培养。 After ultrasonic-mediated or electroporation, transfer the cells to an appropriate amount (for example, 10-30 ml) of freshly prepared DM medium, and perform dark culture at room temperature (for example, 25) for about 18-36 (for example, 24 hours). The hour is then transferred to a DM liquid medium containing an antibiotic such as (50 μg/mL chloramphenicol) and cultured at room temperature (for example, 28 ° C).

示例性的 DM培养基的成分为 (克 /升)： Ca(NO₃)₂-4H₂O, 1.00; KH₂PO₄, 0.26; MgSO₄*7H₂O, 0.55; KCl, 0.25; FeSO₄*7H₂O, 0.02; EDTA«2Na, 0.2; H₃BO₃, 0.0029; ZnCl₂, 0.00011; MnCl₂*4H₂O, 0.00181; (ΝΗ4)₆Μο₇Ο₂₄·4Η₂Ο, 0.000018; CuSO₄*5H₂O, 0.00008。 The composition of the exemplary DM medium is (g/L): Ca(NO ₃ ) ₂ -4H ₂ O, 1.00; KH ₂ PO ₄ , 0.26; MgSO ₄ *7H ₂ O, 0.55; KCl, 0.25; FeSO ₄ *7H ₂ O, 0.02; EDTA «2Na, 0.2; H ₃ BO ₃ , 0.0029; ZnCl ₂ , 0.00011; MnCl ₂ *4H ₂ O, 0.00181; (ΝΗ4) ₆ Μο ₇ Ο ₂₄ ·4Η ₂ Ο, 0.000018; CuSO ₄ *5H ₂ O, 0.00008.

进一步地，本发明方法还包括转基因絮凝微藻的复苏和确认。所述复苏和确认包括将转化后的微藻涂布在含有氯霉素的平板上，调取单克隆，在含有氯霉素的液体培养基中培养 7-14天，此时细胞为绿白色，然后转入不含有抗生素的液体培养基中继续培养 7-14天，细胞生长状态可逐渐转变成活跃，细胞转绿，并可观察到絮凝性状，此时再更换培养基，转化子的絮凝性状逐渐增强，可明显观察到絮凝细胞团（可见图 4) 。由于报告基因可监测转化效率，在荧光显微镜下可观察到，含有绿色荧光蛋白的空载体在栅藻细胞中发出绿色荧光，证明转化成功，此外，可观察到絮凝细胞团内的绿色荧光（图 6) ，证明酵母菌絮凝基因的表达。 Further, the methods of the invention also include resuscitation and confirmation of transgenic flocculated microalgae. The recovery and indeed It is considered that the transformed microalgae is coated on a plate containing chloramphenicol, and the monoclonal is taken up and cultured in a liquid medium containing chloramphenicol for 7-14 days, at which time the cells are greenish white, and then transferred. After continuing to culture for 7-14 days in liquid medium without antibiotics, the cell growth state can be gradually changed into active, the cells turn green, and flocculation traits can be observed. At this time, the medium is changed, and the flocculation property of the transformants is gradually enhanced. Flocculation cell clusters were clearly observed (see Figure 4). Since the reporter gene can monitor the transformation efficiency, it can be observed under the fluorescence microscope that the empty vector containing the green fluorescent protein emits green fluorescence in the Scenedesmus cells, which proves that the transformation is successful, and in addition, green fluorescence in the flocculated cell cluster can be observed (Fig. 6), to prove the expression of yeast flocculating genes.

微藻的自絮凝采收包括将含有自絮凝基因的微藻细胞自然放置，细胞可在 10 分钟内迅速自沉降（图 9) ，而对照游离藻种在放置 12小时以后仍然无法自沉降，因此转基因絮凝藻可通过细胞自沉降采收，方便而简单。下文将以具体实施例的方式描述本发明。对于实施例中涉及的方法、步骤、条件、试剂、用量等，除非另有说明，否则为现有技术的方法、步骤、条件、试剂和用量。实施例 1 : 絮凝基因的克隆和表达载体的构建 The self-flocculation of microalgae involves the natural placement of microalgae cells containing self-flocculation genes, which can rapidly self-settle within 10 minutes (Fig. 9), while the control free algae species still fail to self-settle after 12 hours of placement. Transgenic flocculated algae can be harvested by cell self-settling, which is convenient and simple. The invention will be described below in the context of specific embodiments. The methods, procedures, conditions, reagents, amounts, and the like, which are referred to in the examples, are prior art methods, procedures, conditions, reagents, and amounts unless otherwise indicated. Example 1 : Cloning of flocculation gene and construction of expression vector

( 1 ) 目的基因克隆（可参见 CN 200910200097.X中实施例 1和 3的方法）根据实验室絮凝酵母 SPSC01的絮凝基因 FL01序列设计引物： (1) Cloning of the target gene (see the methods of Examples 1 and 3 in CN 200910200097.X) Primer designed according to the flocculating gene FL01 sequence of the laboratory flocculating yeast SPSC01:

Infusion3_F: Infusion3_F:

(SEQ ID NO:4) ；和 (SEQ ID NO: 4); and

Infusion3 R: Infusion3 R:

PCR反应程序如下： 95°C变性 5 min, 95°C变性 30, 55°C退火 30 s, 72°C延伸 5min， 10 个循环，随后进行 20个如下的循环 95°C变性 30 s, 60°C退火 30 s72°C延伸 5min, 最后 72°C延伸 10 min,用的是全式金的 Taq High Fidelity (HiFi) PCR SuperMix II (TransGenBiotech, Beijing, China)。如图 3所示克隆获得 5.2 Kb酵母絮凝基因 FLOl ( SEQ ID NO: l ) 。其氨基酸序列如 CN 200910200097.X的 SEQ ID NO:4或本申请的 SEQ ID NO:2所示。 (2) 表达载体的构建 The PCR reaction procedure was as follows: denaturation at 95 °C for 5 min, denaturation at 95 °C for 30, annealing at 55 °C for 30 s, extension at 72 °C for 5 min, 10 cycles, followed by 20 cycles of 95 °C denaturation for 30 s, 60 °C annealing at 30 s72 °C for 5 min, and finally at 72 °C for 10 min, using a full-form gold Taq High Fidelity (HiFi) PCR SuperMix II (TransGenBiotech, Beijing, China). The 5.2 Kb yeast floc gene FLO1 (SEQ ID NO: l) was cloned as shown in Figure 3. Its amino acid sequence is shown in SEQ ID NO: 4 of CN 200910200097.X or SEQ ID NO: 2 of the present application. (2) Construction of expression vector

目前在真核微藻的研究中，载体大多数是来自于高等植物基因工程，如 pBI121、 pBI221、 pCAMBIA1302、 pCAT-ControK pSV-LacZ等；作为真核微藻基因工程的启动元件主要有强启动子 CaMV35S、 SV40及 Ubiquitin。 At present, in the study of eukaryotic microalgae, most of the vectors are derived from higher plant genetic engineering, such as pBI121, pBI221, pCAMBIA1302, pCAT-ControK pSV-LacZ, etc.; as eukaryotic microalgae The genetically engineered promoters are mainly the strong promoters CaMV35S, SV40 and Ubiquitin.

本研究采用植物中广泛应用的表达载体 PCAMBIA1302作为载体骨架，其基因序列中含有强启动子 CaMV35S及报告基因 gfp，在原核生物中的筛选标记卡那抗性基因，真核中的筛选标记潮霉素抗性基因。经过栅藻的敏感性测试，发现其对氯霉素敏感，因此通过 Infusion方法将 pCAMBIA1302载体上潮霉素抗性基因替换成氯霉素抗性基因，随后又将酵母的絮凝基因连接到载体强启动子 CaMV35S 下游，报告基因 gfp上游，具体流程如图 2所示。 In this study, the expression vector PCAMBIA1302, which is widely used in plants, was used as a vector backbone. The gene sequence contained the strong promoter CaMV35S and the reporter gene gfp, the marker marker Kana resistance gene in prokaryotes, and the selection marker tide mold in eukaryote. Gene resistance gene. After being sensitive to chloramphenicol, it was found to be sensitive to chloramphenicol. Therefore, the hygromycin resistance gene on the pCAMBIA1302 vector was replaced with the chloramphenicol resistance gene by the Infusion method, and then the flocculating gene of the yeast was ligated to the carrier. The promoter is downstream of CaMV35S, and the reporter gene gfp is upstream. The specific process is shown in Figure 2.

由于目的基因 ¾07总长度 5.2 Kb，且内部存在 3Kb的重复序列，很难找到合适的酶切位点，为表达载体的构建带来了很大的困难。因此本实验采用 In-Fusion cloning的方法进行表达载体的构建工作，其原理是通过 PCR反应在目的片段两端加上 15 bp的碱基，此处的 15 bp碱基序列与线性化的载体（可通过单酶切得到，也可用双酶切获得）两端 15 bp碱基的序列一致，通过同源重组，将目的基因与线性的载体相连，进而获得重组载体。具体操作见图 2。实施例 2: 表达载体通过电击或超声处理转化入微藻细胞 Since the target gene 3⁄407 has a total length of 5.2 Kb and a 3Kb repeat sequence exists inside, it is difficult to find a suitable restriction site, which brings great difficulties for the construction of the expression vector. Therefore, this experiment uses In-Fusion cloning method to construct the expression vector. The principle is to add 15 bp base to both ends of the target fragment by PCR reaction, where the 15 bp base sequence and the linearized vector ( It can be obtained by single enzyme digestion or by double enzyme digestion. The sequence of 15 bp bases at both ends is identical, and the target gene is ligated to a linear vector by homologous recombination, thereby obtaining a recombinant vector. The specific operation is shown in Figure 2. Example 2: Expression vector was transformed into microalgae cells by electric shock or sonication

目前应用于微藻转化方法有激光微束穿孔法、电击法、基因枪法、农杆菌侵染法、微玻璃珠研磨法等，本研究分别使用了超声波介导法和电击法对微藻进行了转化。 At present, microalgae transformation methods include laser microbeam perforation, electric shock, gene gun, Agrobacterium infection, microglass bead milling, etc. In this study, ultrasonically mediated and electroporation were used to microalgae. Conversion.

( 1 ) 超声波介导法转化栅藻 (1) Ultrasound-mediated transformation of Scenedesmus

I.受体材料的准备 I. Preparation of acceptor materials

取培养到指数期的非絮凝栅藻培养液 150 mL， 5000 rpm离心 10 min，收集藻细胞。用预冷的高渗缓冲液 (0.2 mol/L甘露醇， 0.2 mol/L山梨醇， 10%甘油)洗藻体 3次，最后一次冰上放置 l h， 5000 rpm离心 10 min，收集藻细胞，加电击缓冲液调细胞数至 10⁸个 /ml， 42 °C热击 10 min,冰浴 5 min后，加入终浓度为 50 g/mL 的实施例 1所构建的表达载体与 25 μ g/mL的鲑精 DNA混合均匀，冰上放置 5-10 min, 待超声或电击处理。 150 mL of non-flocculated Scenedesmus culture solution cultured to the exponential phase was centrifuged at 5000 rpm for 10 min to collect algal cells. The algae were washed 3 times with pre-cooled hypertonic buffer (0.2 mol/L mannitol, 0.2 mol/L sorbitol, 10% glycerol), and the last time was placed on ice for 1 h, centrifuged at 5000 rpm for 10 min, and algae cells were collected. Add the electroporation buffer to adjust the cell number to 10 ⁸ /ml, heat shock at 42 °C for 10 min, and after ice bath for 5 min, add the expression vector constructed in Example 1 with a final concentration of 50 g/mL and 25 μg/ The mL of sputum DNA is evenly mixed and placed on ice for 5-10 min, subject to ultrasound or electric shock.

II.超声处理 II. Ultrasonic treatment

吸取 0.4 mL混合好的培养细胞放入含有超纯水的烧杯中，进行超声，超声时间分别为 30 s、 60 s、 90 s、 120 s、 200 s每种处理重复三次。 Aspirate 0.4 mL of the mixed culture cells into a beaker containing ultrapure water for sonication, and the ultrasonication time was 30 s, 60 s, 90 s, 120 s, and 200 s, respectively, three times for each treatment.

(2) 电击法转化栅藻 I.受体材料的准备方法同超声波介导法； (2) Electric shock method for transforming Scenedesmus I. The preparation method of the acceptor material is the same as the ultrasonic mediated method;

II.电击处理 II. Electric shock treatment

吸取 0.4 mL混合好的培养细胞放入预冷电击小杯中，按照设置的各种条件组合进行电击，每种处理重复三次。 Pipette 0.4 mL of the mixed culture cells into a pre-cooled electric shock cuvette and combine them according to the various conditions set. Each treatment was repeated three times.

电击参数的优化实验：在其它条件不变的情况下，分别梯度改变高渗缓冲液浓度、表达载体、脉冲电压和脉冲持续时间这 4个参数。 Optimization experiment of electric shock parameters: Under the same conditions, the gradients were changed by hypertonic buffer concentration, expression vector, pulse voltage and pulse duration.

优化后，电击转化条件的各种处理组合为：实施例 1制备的表达载体浓度 50 g/mL，高渗缓冲液浓度 0.2 mol/L，脉冲距离为 2 mm，脉冲电压为 2 Kv，脉冲持续时间为 3 ms。实施例 3 : 转基因絮凝微藻的复苏和确认 After optimization, the various treatment combinations of electroporation transformation conditions were as follows: The expression vector prepared in Example 1 was 50 g/mL, the hypertonic buffer concentration was 0.2 mol/L, the pulse distance was 2 mm, the pulse voltage was 2 Kv, and the pulse was continuous. The time is 3 ms. Example 3: Resuscitation and confirmation of transgenic flocculated microalgae

(1) 转基因絮凝微藻的复苏 (1) Recovery of transgenic flocculated microalgae

转化完毕，用培养液清洗两次，取 0.4 mL混合细胞吸入内含新鲜液体培养基的锥形瓶中，其中一瓶置于适宜条件下连续光照培养 18-24 h，另一瓶置于适宜条件下黑暗培养 18-24 h。 After the transformation is completed, wash twice with the culture solution, and take 0.4 mL of mixed cells into the conical flask containing the fresh liquid medium. One bottle is placed under suitable conditions for continuous illumination for 18-24 h, and the other bottle is placed in a suitable one. Incubate in the dark for 18-24 h.

(2) 转基因絮凝微藻的筛选 (2) Screening of transgenic flocculated microalgae

将复苏培养的部分藻液转入含适合浓度抗生素的新鲜液体培养基中，另取少量藻液离心后涂布于含适合浓度的抗生素的固体培养基；一个月后挑取第一次在抗性平板上长出的转化子藻落，转接于含有适合浓度的液体培养基扩增培养后，接种于无抗生素的固体培养基上，每过一个月挑取单克隆，把单克隆的一半在无抗生素的液体培养基中扩大培养，另一半接种于无抗生素的固体培养基上，筛选稳定的转基因栅藻。 Transfer some of the reconstituted cultured algae solution to a fresh liquid medium containing the appropriate concentration of antibiotics, and take a small amount of algae solution and centrifuge it to apply to the solid medium containing the appropriate concentration of antibiotics; pick up the first time after one month. The transformant algae grown on the slab is transferred to a liquid medium containing the appropriate concentration and then inoculated on a solid medium without antibiotics. Each month, the monoclonal is picked and half of the monoclonal is picked. The culture was expanded in a liquid medium without antibiotics, and the other half was inoculated on a solid medium without antibiotics to screen a stable transgenic griddes.

从图 4中可以看出野生型的栅藻不能在含有抗生素的培养基中生长，转基因栅藻可以在含有抗生素的培养基正常生长；转入 pCAMBIA 1302-CAT-FLOl的转化子出现了细胞絮凝性状，而转入 pCAMBIA 1302-CAT的转化子并没有此现象，说明酵母絮凝基因 FLO1在 pCAMBIA 1302-CAT-FLO1的转化子中表达，并引起絮凝这一现象产生。 (3 ) 转基因絮凝微藻的确认 It can be seen from Fig. 4 that wild-type Scenedesmus can not grow in the medium containing antibiotics, and the transgenic Hybrids can grow normally in the medium containing antibiotics; the cells transformed into pCAMBIA 1302-CAT-FLO1 exhibit cell flocculation The trait, and the transformant transformed into pCAMBIA 1302-CAT did not, indicating that the yeast flocculating gene FLO1 was expressed in the transformant of pCAMBIA 1302-CAT-FLO1 and caused flocculation. (3) Confirmation of transgenic flocculated microalgae

①扫描电镜观察转基因絮凝微藻的表型变化 1 Scanning electron microscopic observation of phenotypic changes of transgenic flocculated microalgae

从图 5中可以看出野生型的栅藻和转入 pCAMBIA 1302-CAT的转化子藻细胞呈分散状态存在，而转入 pCAMBIA 1302-CAT-FLOl的转化子细胞聚集在一起形成一个较大颗粒与肉眼可见的细胞絮凝性状相一致，从放大倍数较大的图片中可以看出，在细胞表面有层物质，推测这种物质与细胞絮凝性状有关。 ②荧光显微镜观察转基因絮凝微藻情况 Figure 5 shows wild-type Scenedesmus and transformant algae cells transferred to pCAMBIA 1302-CAT It exists in a dispersed state, and the transformant cells transferred into pCAMBIA 1302-CAT-FLO1 aggregate to form a larger particle which is consistent with the macroscopic flocculation trait of the naked eye. It can be seen from the larger magnification picture in the cell. There is a layer of material on the surface, which is presumed to be related to cell flocculation traits. 2 Fluorescence microscopy observation of transgenic flocculated microalgae

利用荧光显微镜进行观察，在蓝光激发下，野生型未转化的藻细胞由于自身叶绿体的荧光呈红色；转基因微藻由于报告基因 gfp的表达呈绿色，图 6中 3的现象是由于未转化的藻细胞粘附在成功表达絮凝基因的转化子上，这种现象与宏观肉眼看到的以及扫描电镜观察到的结果完全一致，进一步证明了絮凝基因的表达。 Using fluorescence microscopy, the wild-type untransformed algae cells showed red color due to the fluorescence of their own chloroplasts under blue light excitation; the transgenic microalgae showed green color due to the expression of the reporter gene gfp, and the phenomenon of 3 in Figure 6 was due to untransformed algae. The cell adhered to the transformant that successfully expressed the flocculating gene. This phenomenon was completely consistent with the macroscopic observation and the results observed by scanning electron microscopy, further demonstrating the expression of the flocculating gene.

③流式细胞仪检测转基因絮凝微藻情况 3 flow cytometry detection of transgenic flocculated microalgae

用荧光显微镜定性的观察了转化子中报告基因 gfp 的表达情况，此外还采用 FACSCantoTM flow cytometer (BD Biosciences, SanJose, CA, USA)流式细胞仪转化子中报告基因 gfp的表达情况进行了定量的检测。在 488 nm荧光激发下，野生型栅藻细胞只有自身叶绿体飞本底水平的荧光信号，而转入 pCAMBIA 1302-CAT 和 pCAMBIA 1302-CAT-FLO1 的转基因微藻，既有本底水平的荧光还有报告基因的荧光。转入 pCAMBIA 1302-CAT 的阳性转化率可达 1.8%，略高于转 pCAMBIA 1302-CAT-FLO1 的 1.4%。而通过荧光强度的比较发现，转入 pCAMBIA 1302-CAT-FLO的藻细胞 GFP-A值可达 117866，转入 pCAMBIA 1302-CAT 的能达到 119810，二者的差异并不明显。由于在质粒 pCAMBIA 1302-CAT-FLO 1中报告基因 gfp 与酵母絮凝基因共用强启动子 CaMV35S，且位于的 FLO1下游，因此通过检测报告基因的表达，进而证明目的基因的表达。 ④分子鉴定絮凝基因的表达 The expression of the reporter gene gfp in the transformants was qualitatively observed by fluorescence microscopy. The expression of the reporter gene gfp in the flow cytometry transformants was also quantified using a FACSCantoTM flow cytometer (BD Biosciences, SanJose, CA, USA). Detection. Under 488 nm fluorescence excitation, wild-type Scenedesmus cells only have fluorescence signals at the background level of their own chloroplasts, while transgenic microalgae transferred to pCAMBIA 1302-CAT and pCAMBIA 1302-CAT-FLO1 have both background fluorescence and background fluorescence. There is fluorescence of the reporter gene. The positive conversion rate to pCAMBIA 1302-CAT was 1.8%, slightly higher than 1.4% for pCAMBIA 1302-CAT-FLO1. By comparison of fluorescence intensity, the GFP-A value of algae cells transferred to pCAMBIA 1302-CAT-FLO was 117866, and that of pCAMBIA 1302-CAT was 119810. The difference was not obvious. Since the gene gfp was reported in the plasmid pCAMBIA 1302-CAT-FLO 1 and the strong promoter CaMV35S was shared with the yeast flocculating gene, and located downstream of FLO1, the expression of the target gene was confirmed by detecting the expression of the reporter gene. 4 molecular identification of flocculation gene expression

在六个月的培养过程中，每两周进行无抗培养，进而得到稳定的转化子。提取野生型栅藻细胞、转基因藻细胞的 DNA和 RNA，进行 PCR及 RT-PCR分析。得到的结果如图 8 所示，在 PCR 和 RT-PCR 的电泳图中，转入 pCAMBIA 1302-C AT-FLO 1的转化子有和质粒 pCAMBIA 1302-CAT-FLO1同样大小的电泳条带。结果表明酵母基因 FL01只在转入 pCAMBIA 1302-CAT-FLO 1的转化子有表达，而野生型藻细胞和转入 pCAMBIA 1302-CAT的转化子没有表达。本发明转入 pCAMBIA 1302-CAT-FLO 1的斜生栅藻 Scenedesmus obliq画 DUTG-01已与 2012年 3月 15日保藏于中国微生物菌种保藏管理委员会普通微生物中心（北京市朝阳区北辰西路 1号院中科院微生物研究所，邮编： 100101 ) ，保藏号为 CGMCC No. 5905。实施例 4 : 转基因絮凝微藻的性能研究 During the six-month culture period, no anti-culture was performed every two weeks, and a stable transformant was obtained. The DNA and RNA of wild-type Scenedesmus cells and transgenic algae cells were extracted and analyzed by PCR and RT-PCR. The results obtained are shown in Fig. 8. In the electropherograms of PCR and RT-PCR, the transformant transformed into pCAMBIA 1302-C AT-FLO 1 had an electrophoresis band of the same size as plasmid pCAMBIA 1302-CAT-FLO1. The results indicated that the yeast gene FL01 was only expressed in transformants transfected with pCAMBIA 1302-CAT-FLO 1, whereas wild-type algal cells and transformants transformed into pCAMBIA 1302-CAT were not expressed. The present invention was transferred to pCAMBIA 1302-CAT-FLO 1 Scenedesmus obliq painting DUTG-01 has been deposited with the General Microbiology Center of China Microbial Culture Collection Management Committee on March 15, 2012 (Beichen West Road, Chaoyang District, Beijing) Institute of Microbiology, Chinese Academy of Sciences, No. 1 Institute, Zip Code: 100101), and the deposit number is CGMCC No. 5905. Example 4: Study on the properties of transgenic flocculated microalgae

对野生型栅藻与转基因絮凝微藻自沉降性能进行研究，如图 9所示，转入酵母絮凝基因的前述制备的转基因絮凝微藻具有良好的自沉降性能，在 30分钟时已出现自沉降现象，而此时野生型栅藻及转入 pCAMBIA 1302-CAT的转基因微藻并未将沉降现象。 The self-sedimentation properties of wild-type Scenedesmus and transgenic flocculated microalgae were studied. As shown in Fig. 9, the transgenic flocculated microalgae prepared by transferring the yeast floc gene had good self-settling properties, and self-settling occurred at 30 minutes. Phenomenon, while wild type Scenedesmus and transgenic microalgae transferred to pCAMBIA 1302-CAT did not precipitate.

转基因絮凝微藻细胞的生长和油脂的积累未受到明显影响。如图 10所示，空载体和转入絮凝基因的絮凝微藻的油脂积累速率约为 l l mg/L/天，略高于野生型游离的栅藻细胞 (9 mg/L/天；)，转入空载体和絮凝基因的转基因微藻的细胞生长比野生型微藻略慢，可能是油脂积累速率较高的原因。实施例 5 : 转基因絮凝小球藻的制备 The growth of the transgenic flocculated microalgae cells and the accumulation of oil were not significantly affected. As shown in Fig. 10, the oil accumulation rate of the empty carrier and the flocculating microalgae transferred to the flocculation gene was about ll mg/L/day, which was slightly higher than that of wild-type free Scenedesmus cells (9 mg/L/day;). The cell growth of transgenic microalgae transfected with empty vector and flocculation gene is slightly slower than that of wild-type microalgae, which may be the reason for the higher rate of oil accumulation. Example 5: Preparation of transgenic flocculated chlorella

采用与前述实施例 2相同的方法，用实施例 1所制备得到的表达载体转入小球藻 ZRA01 , 如实施例 3所述对转基因絮凝小球藻进行复苏和确认，筛选获得具有絮凝效果的小球藻 i Chlorella vulgaris ) ZRA01 , 该小球藻已与 2012年 2月 27 日保藏于中国微生物菌种保藏管理委员会普通微生物中心（北京市朝阳区北辰西路 1号院中科院微生物研究所，邮编： 100101 ) ，保藏号为 CGMCC No. 5800。实施例 6 : 转基因絮凝小球藻的性能研究 The expression vector prepared in Example 1 was transferred to Chlorella sp. ZRA01 in the same manner as in the above Example 2, and the transgenic floccoliella was resuscitated and confirmed as described in Example 3, and the flocculation effect was obtained by screening. Chlorella vulgaris ) ZRA01 , This chlorella has been deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on February 27, 2012 (Microbiology Institute, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, China) : 100101 ) , the deposit number is CGMCC No. 5800. Example 6: Study on the performance of transgenic floccoliella

对野生型小球藻与实施例 5所述的转基因絮凝小球藻自沉降性能进行研究，如图 1 1所示，转入酵母絮凝基因 ¾07的转基因絮凝微藻（A图）具有良好的自沉降性能，在 9分钟时已出现自沉降现象，而野生型小球藻（B图）在 60分钟时仍沉降不完全。 The self-sedimentation properties of wild-type chlorella and the transgenic flocculated chlorella described in Example 5 were studied. As shown in Fig. 11, the transgenic flocculated microalgae (Fig. A) transferred to the yeast flocculating gene 3⁄407 had a good self. The sedimentation performance showed self-settling at 9 minutes, while wild-type chlorella (Fig. B) still did not settle completely at 60 minutes.

转基因絮凝小球藻细胞的生长和油脂的积累未受到明显影响。野生型小球藻对照和絮凝小球藻的油脂积累速率分别为 3 1.3 mg/L/天和 30.7 mg/L/天，而絮凝小球藻的生长在后期还略好于野生型小球藻，在 30度静止培养 12天时 OD690nm可达 0.5 (对照小球藻为 0.35 ) 。 The growth of the transgenic flocculent chlorella cells and the accumulation of oil were not significantly affected. The oil accumulation rate of wild-type chlorella control and flocculation of chlorella was 3 1.3 mg/L/day and 30.7 mg/L/day, respectively, while the growth of flocculated chlorella was slightly better than that of wild-type chlorella in the later stage. OD690nm can reach 0.5 at 12 ° static culture for 12 days (control chlorella is 0.35).

Claims

1. 一种表达载体，其特征在于，所述表达载体含有来自酵母的絮凝基因，与所述絮凝基因操作性连接的、位于所述絮凝基因上游的启动子和抗生素抗性基因。 An expression vector comprising a flocculating gene derived from yeast, a promoter operably linked to said flocculating gene, and an antibiotic resistance gene located upstream of said flocculation gene.

2. 如权利要求 1所述的表达载体，其特征在于，所述絮凝基因选自： 2. The expression vector according to claim 1, wherein the flocculating gene is selected from the group consisting of:

(2 )在严格条件下与（1 )所限定的核苷酸序列杂交且编码具有絮凝活性的蛋白质的核苷酸序列。 (2) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence defined in (1) and which encodes a protein having flocculating activity.

3.如权利要求 1或 2所述的表达载体，其特征在于，所述载体含有 SEQ ID The expression vector according to claim 1 or 2, wherein the vector contains SEQ ID

ΝΟ: 1所示的核苷酸序列、 CaMV35S启动子和氯霉素抗性基因。 ΝΟ: The nucleotide sequence shown in 1, the CaMV35S promoter, and the chloramphenicol resistance gene.

4. 如权利要求 1-3 中任一项所述的表达载体，其特征在于，所述表达载体选自： The expression vector according to any one of claims 1 to 3, wherein the expression vector is selected from the group consisting of:

( 1 ) SEQ ID NO:3所示的核苷酸序列；和 (1) the nucleotide sequence shown in SEQ ID NO: 3;

(2 ) 与 SEQ ID NO:3具有至少 80%序列相同性的核苷酸序列。 (2) A nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 3.

5. —种转基因自絮凝藻类细胞，其特征在于，所述细胞稳定转化了权利要求 1一 4中任一项所述的表达载体，并具有自沉降性能。 5. A transgenic self-flocculating algae cell, characterized in that the cell stably transforms the expression vector of any one of claims 1 to 4 and has self-settling properties.

6. 如权利要求 5所述的藻类细胞，其特征在于，所述藻选自栅藻和小球藻。 The algal cell according to claim 5, wherein the alga is selected from the group consisting of Scenedesmus and Chlorella.

7. 如权利要求 5所述的藻类细胞，其特征在于，所述藻类细胞为保藏编号为 CGMCC No. 5800小球藻细胞或保藏编号为 CGMCC No. 5905的栅藻细胞。 The algal cell according to claim 5, wherein the algal cell is a C. aeruginosa cell having a CGMCC No. 5800 chlorella cell or a CGMCC No. 5905.

8. 一种制备转基因自絮凝微藻的方法，其特征在于，所述方法包括： ( 1 ) 将如利要求 1一 4中任一项所述的表达载体转入所述微藻；和 A method for producing a transgenic self-flocculating microalgae, the method comprising: (1) transferring the expression vector of any one of claims 1 to 4 into the microalgae;

(2 ) 筛选具备絮凝性能的微藻。 (2) Screening of microalgae with flocculation properties.

9. 如权利要求 8所述的方法，其特征在于，所述方法包括： 9. The method of claim 8, wherein the method comprises:

(b) 热击 (a)中获得的细胞，然后于冰上放置； (b) heat shock the cells obtained in (a) and then place them on ice;

(c) 将 (； b)中处理后的细胞与权利要求 1-4中任一项所述的表达载体和鲑鱼精 DNA混合，并于冰上放置； (c) the treated cell in (b) and the expression vector and squid according to any one of claims 1 to 4 Fine DNA is mixed and placed on ice;

(d) 将 (C)所获得的细胞重悬，对其实施转化处理，获得转化的细胞；和 (d) resuspending the cells obtained in (C), transforming them, and obtaining transformed cells;

(e) 将步骤 (d)的细胞转入新鲜配制的 DM培养基中暗培养，然后转入含有抗生素的 DM液体培养基中培养，筛选获得所述转基因自絮凝微藻。 (e) The cells of the step (d) are transferred to freshly prepared DM medium for dark culture, and then transferred to a DM liquid medium containing antibiotics, and the transgenic self-flocculated microalgae are obtained by screening.

10. 权利要求 1-4中任一项所述的表达载体在制备转基因自絮凝藻类细胞中的应用。 10. Use of an expression vector according to any one of claims 1 to 4 for the preparation of transgenic self-flocculating algae cells.