阅读说明：本技术 转基因絮凝微藻的构建及其制备方法 (Construction and preparation method of transgenic flocculation microalgae ) 是由 郭锁莲 邹蒙 张平 张硕硕 于 2019-01-02 设计创作，主要内容包括：转基因絮凝微藻的构建及其制备方法,涉及一种生物基因构建及其制备方法,具体而言,本发明涉及构建可诱导表达絮凝基因的表达载体,将其转入微藻中,获得转基因自絮凝微藻。本发明所获得的转基因藻表现出正常生长,而在收获期通过热诱导表现出自絮凝现状,利于微藻收集以及下游生物炼制的处理。本发明包括诱导型絮凝微藻的构建及其在微藻采收中的应用。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<I>FLO1</I>重组到不同藻株中,并通过筛选获得基因突变藻株,实现了微藻转化基因工程技术的重点突破。(The invention relates to construction and a preparation method of transgenic flocculation microalgae, in particular to construction of an expression vector capable of inducing expression of a flocculation gene, and the expression vector is transferred into the microalgae to obtain the transgenic self-flocculation microalgae. The transgenic algae obtained by the invention shows normal growth, and shows self-flocculation status through heat induction in the harvest period, thereby being beneficial to microalgae collection and downstream biorefinery treatment. The invention comprises the construction of the induction type flocculation microalgae and the application thereof in microalgae recovery. The invention successfully constructs a microalgae transformation platform. By the invention canEfficient gene for flocculating yeast FLO1 Recombining the strain into different strains, and obtaining the gene mutation strain through screening, thereby realizing key breakthrough of the microalgae transformation gene engineering technology.)

1. Constructing transgenic flocculation microalgae, which is characterized by comprising an expression vector, wherein the expression vector contains a flocculation gene from yeast, and is operatively connected with the flocculation gene and positioned between an inducible promoter and a screening marker gene which are positioned at the upstream of the flocculation gene; the flocculation gene is selected from:

(1) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2; and

(2) a nucleotide sequence which hybridizes with the nucleotide sequence defined in (1) under stringent conditions and encodes a protein having flocculation activity.

2. The construction of the transgenic flocculated microalgae of claim 1 wherein the vector contains the nucleotide sequence shown in SEQ ID NO. 1, HSP70A promoter and chloramphenicol resistance gene.

3. The construction of transgenic flocculated microalgae according to claim 1 wherein said expression vector is selected from the group consisting of:

(1) 3, the nucleotide sequence shown in SEQ ID NO; and

(2) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 3.

4. The construction of transgenic flocculated microalgae according to claim 1, comprising transgenic self-flocculating algal cells stably transformed with expression vectors and having inducible self-settling properties.

5. Construction of transgenic flocculated microalgae according to claim 4, characterized in that said algal cells, algae, are selected from Scenedesmus sp.

6. The construction of the transgenic flocculated microalgae according to claim 4 wherein said algal cells are Chlorella algal cells with the preservation number CGMCC number 5905.

7. A method for preparing transgenic flocculated microalgae, comprising:

(1) transferring the expression vector into the microalgae; and

(2) screening the microalgae with flocculation performance.

8. The method for preparing transgenic flocculated microalgae according to claim 7, wherein said method comprises:

(a) collecting microalgae cells in logarithmic growth phase, and suspending in an osmotic buffer solution;

(b) thermally shocking the cells obtained in step (1) and placing on ice;

(c) mixing the cells treated in (2) with the expression vector of any one of claims 1 to 4 and salmon sperm DNA, and placing on ice;

(d) resuspending the cells, and treating the cells by an ultrasonic wave induction method or an electric shock method; and

(e) and (d) transferring the cells in the step (d) into a freshly prepared DM culture medium for dark culture, then transferring the cells into a DM liquid culture medium containing antibiotics for culture, and screening to obtain the transgenic self-flocculating microalgae.

9. The construction and preparation method of the transgenic flocculated microalgae according to claim 1 or 7, wherein an expression vector of the transgenic flocculated microalgae is used for preparing the transgenic self-flocculating microalgae cells.

Technical Field

The invention relates to a biological gene construction and a preparation method thereof, in particular to a construction and a preparation method of transgenic flocculation microalgae.

Background

Microalgae are mostly unicellular or simple multicellular photosynthetic microorganisms, and can produce organic substances by using carbon dioxide in the air, carbon dioxide in industrial waste gas or carbonate, so that the microalgae can be used for producing food, feed and fine chemicals. With the rapid development of the world economy, the contradiction between the consumption and the insufficient supply of energy is increasingly prominent, petroleum-based fossil fuels obviously cannot meet the increasing energy demand of human beings, and the use of fossil fuels is a main source of carbon dioxide in the global greenhouse effect, so that the search for the development and utilization of novel clean energy has important strategic significance for relieving the energy crisis, protecting the environment and realizing the sustainable development of economy. And natural energy sources such as nuclear energy, wind energy and solar energy cannot replace traditional fossil energy sources such as petroleum and the like due to the limitation of factors such as technology, resources and the like.

The biodiesel is taken as the new energy regeneration force in recent years, because the calorific value of the biodiesel is close to petroleum and the biodiesel is environment-friendly (the N, S content is low), the biodiesel is developed rapidly, the proportion of the biodiesel in the energy market is steadily increased, but the shortage of raw materials is the bottleneck of large-scale production of the biodiesel at home and abroad. At present, oil crops such as soybean, sugarcane, corn starch and the like are mainly used as raw materials to refine the biodiesel, and because the oil yield of the oil crop is not high in the oil area, the large-scale production of the biodiesel inevitably causes the large-area planting of the edible raw materials to further influence the grain production.

Compared with oil crops, microalgae as a high-quality raw material for the vigorous development of biodiesel has the following advantages: (1) the microalgae have high growth speed, most of the algae have high oil content and are mostly low unsaturated natural fat, such as Scenedesmus ((A))Scenedesmussp.) oil content of 20-21% (w/w), oil yield of 41-54 (mg/l/day), and Chlorella (Chlorella vulgaris)Chlorella sp.) the oil content is 19-22% (w/w), the oil yield is about 45 (mg/l/day); (2) compared with oil crops, microalgae culture does not occupy cultivated land; (3) microalgae possess CO similar to higher plants₂Immobilising the system and converting it into carbohydrates as well as lipids, such as Triglycerides (TGA); (4) microalgae can absorb carbon dioxide discharged from industrial production and release oxygen, and has effect in reducing oxygenThe greenhouse effect plays an important role in maintaining the environmental stability.

Besides being used as raw material of biodiesel, the microalgae also contains high value products. Scenedesmus (A. Scenedesmus)Scenedesmussp.) and Chlorella (Chlorella vulgarisChlorellasp.) protein content of 50-56% and 51-58% by dry weight, respectively. Microalgae can also be used for fermentation to produce biogas, acetone butanol; the purified omega-3 fatty acid extracted from the microalgae is a high-value food additive; chlorella is reported to contain a large amount of pigment, and the pigment in the chlorella strain can play an important role as a chelating agent in ointment and recovery of ulcer and injured tissues, and is also a natural raw material of an additive for food color; since the algal strain itself contains various nutrients such as protein, it can be used as bait, feed for poultry and livestock, fertilizer for crops, and the like.

The microalgae can be used as a high-quality raw material for refining biodiesel and also can be used as a processing plant for various high-value products. But the problem exists at present that the production cost of the microalgae biodiesel is too high. Wherein, the microalgae harvesting cost accounts for a large proportion of the production cost, and the industrial production of microalgae energy and fine chemicals is limited.

Due to the characteristics of microalgae, such as about 5 to 50 μm in size, low cell density of 15 to 108g/L and high water content, the existing microalgae harvesting methods mainly include an air floatation method, a filtration method, a centrifugation method and a chemical flocculation method. The air floatation method has large energy consumption and high cost; the filtration method depends on the size of algae cells and is not suitable for large-scale microalgae collection; because the density of algae is different from that of the culture solution by adopting a centrifugal method, the sediment contains a large amount of water, and the downstream treatment cost is increased; although the chemical flocculation can achieve a better microalgae collecting effect at present and some microalgae are industrialized, the addition of a large amount of metal ions and refractory high polymers undoubtedly causes secondary pollution to the environment; although microbial flocculants have been reported for use in microalgae harvesting, the biosafety is unknown. Therefore, the development of a low-cost microalgae harvesting method is an important guarantee for performing microalgae biorefinery and large-scale industrial production of microalgae energy.

Some microalgae have the ability of cells to self-flocculate, presumably during culture, and the algal bodies synthesize and secrete substances that settle themselves. Compared with the traditional flocculation method, the self-flocculation of the microalgae cells does not need to add exogenous substances, and has no energy consumption, no pollution and environmental protection. The microalgae is mainly of non-flocculation type, the number of natural flocculation algae strains is small, and the conventional non-flocculation algae strains are transformed by a genetic engineering method to show flocculation shapes, so that the aim of collecting the microalgae is fulfilled. Microalgae harvesting by utilizing microalgae self-flocculation is a microalgae collecting method with application prospect, and at present, there are few reports about transferring flocculation genes into non-flocculation microalgae.

Disclosure of Invention

The invention aims to provide a construction method and a preparation method of transgenic flocculation microalgae. The invention can effectively flocculate the yeastFLO1Recombining the strain into different strains, and obtaining the gene mutation strain through screening, thereby realizing key breakthrough of the microalgae transformation gene engineering technology.

The purpose of the invention is realized by the following technical scheme:

constructing transgenic flocculation microalgae, comprising an expression vector, wherein the expression vector contains a flocculation gene from yeast, and is operatively connected with the flocculation gene and positioned between an inducible promoter and a screening marker gene at the upstream of the flocculation gene; the flocculation gene is selected from:

(1) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2; and

(2) a nucleotide sequence which hybridizes with the nucleotide sequence defined in (1) under stringent conditions and encodes a protein having flocculation activity.

The construction of the transgenic flocculation microalgae is that the vector contains a nucleotide sequence shown in SEQ ID NO. 1, an HSP70A promoter and a chloramphenicol resistance gene.

The construction of the transgenic flocculation microalgae is that the expression vector is selected from:

(1) 3, the nucleotide sequence shown in SEQ ID NO; and

(2) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 3.

The construction of the transgenic flocculation microalgae comprises transgenic self-flocculation algae cells, wherein the cells are stably transformed with expression vectors and have inducible self-sedimentation performance.

The transgenic flocculation microalgae is constructed, and the algae cells and the algae are selected from scenedesmus.

The construction of the transgenic flocculation microalgae is that the algal cells are chlorella cells with the preservation number of CGMCC number 5905.

A method of making a transgenic flocculated microalgae, the method comprising:

(1) transferring the expression vector into the microalgae; and

(2) screening the microalgae with flocculation performance.

The preparation method of the transgenic flocculated microalgae comprises the following steps:

(a) collecting microalgae cells in logarithmic growth phase, and suspending in an osmotic buffer solution;

(b) thermally shocking the cells obtained in step (1) and placing on ice;

(c) mixing the cells treated in (2) with the expression vector of any one of claims 1 to 4 and salmon sperm DNA, and placing on ice;

(d) resuspending the cells, and treating the cells by an ultrasonic wave induction method or an electric shock method; and

(e) and (d) transferring the cells in the step (d) into a freshly prepared DM culture medium for dark culture, then transferring the cells into a DM liquid culture medium containing antibiotics for culture, and screening to obtain the transgenic self-flocculating microalgae.

The construction and preparation method of the transgenic flocculation microalgae are characterized in that the expression vector of the transgenic flocculation microalgae is applied to the preparation of transgenic self-flocculation algae cells.

The invention has the advantages and effects that:

the invention successfully constructs a microalgae transformation platform. The invention can effectively flocculate the yeastFLO1Recombining into different algae strains and obtaining by screeningObtaining the gene mutation strain. Compared with the prior art, the invention realizes key breakthrough of microalgae transformation gene engineering technology, and has the following beneficial effects:

1. the invention proves that the HSP70A promoter can start the expression of an exogenous gene in scenedesmus.

2. In the aspect of screening and marking, experiments prove that the scenedesmus freshwater algae strain is sensitive to chloramphenicol, and the invention has the technical characteristic that a feasible combination of a selectable marker gene and a reporter gene is adopted.

3. The invention constructs a microalgae expression vector pSLG06 of the yeast flocculation gene and realizes the stable expression of the exogenous gene in scenedesmus and chlorella.

Drawings

FIG. 1 shows a schematic representation of the expression vector construction;

FIG. 2 shows cloning of flocculation genes from yeastFLO1；

FIG. 3 shows antibiotic selection of transformants;

wherein, 1: wild-type scenedesmus cells; 2: a transformant transformed into pCAMBIA 1302-HSP; 3: the transformant was transformed with pCAMBIA1302-HSP-FLO 1.

FIG. 4 shows the changes in phenotype of Scenedesmus cells observed by scanning electron microscopy;

1,4: wild-type scenedesmus cells; 2,5: a transformant transformed into pCAMBIA 1302-HSP; 3,6: transfer into pCAMBIA1302-HSP-FLO1The transformant of (1).

FIG. 5 shows fluorescence electron microscopy observation of Scenedesmus phenotypic changes;

wherein, left: wild-type scenedesmus cells; the method comprises the following steps: a transformant transformed into pCAMBIA 1302-HSP; and (3) right: the transformant was transformed with pCAMBIA1302-HSP-FLO 1.

FIG. 6 shows PCR and RT-PCR analysis of yeast flocculation genesFLO1Expression in transgenic Scenedesmus cells.

Wherein, the left picture is the expression condition of the yeast flocculation gene FLO1 in the transgenic scenedesmus cells analyzed by PCR; the right picture shows the expression of RT-PCR yeast flocculation gene FLO1 in transgenic scenedesmus cells. In the figure, M: 1kb ladder marker (TaKaRa Bio Inc., Japan); w: wild-type scenedesmus cells; t, T1 is transferred into Scenedesmus cells of pCAMBIA1302-HSP-FLO 1; c: transferring Scenedesmus cells of pCAMBIA 1302-HSP; p: plasmid pCAMBIA1302-HSP-FLO 1.

Detailed Description

The present invention will be described in detail with reference to examples.

The invention aims to establish a microalgae transformation platform and application thereof in microalgae harvesting.

The invention firstly uses the yeast flocculation geneFLO1Recombination into inducible promoters and reporter genesgfpGenes are inserted into a plasmid containing a selective marker gene to construct a vector for transformation, the plasmid is transformed into the exemplary non-flocculated freshwater chlorella by an electrotransformation method, and a positive transformant is obtained by antibiotic screening and fluorescence detection.

The transgenic algae obtained by the invention shows normal growth, and shows self-flocculation status through heat induction in the harvest period, thereby being beneficial to microalgae collection and downstream biorefinery treatment. The transformation platform established by the invention has high transformation efficiency, is suitable for different algae strains, and the obtained genetic engineering algae strains have genetic stability.

Accordingly, in a first aspect, the present invention provides an inducible expression vector comprising a flocculation gene from yeast, an inducible promoter and a resistance selection marker gene operably linked to the flocculation gene upstream of the flocculation gene.

In a specific embodiment, the flocculation gene from yeast is the yeast flocculation gene FLO1, FLO5, FLO9, or FLO 10. In one embodiment, the yeast flocculation gene is as set forth in SEQ ID NO. 1 of CN 200910200097. X.

In a particular embodiment, the flocculation gene is selected from the group consisting of:

(1) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2; and

(2) a nucleotide sequence which hybridizes with the nucleotide sequence defined in (1) under stringent conditions and encodes a protein having flocculation activity.

In one embodiment, the vector comprises the nucleotide sequence shown in SEQ ID NO. 1, HSP70A promoter and chloramphenicol resistance gene.

In one embodiment, the expression vector is selected from the group consisting of:

(1) 3, the nucleotide sequence shown in SEQ ID NO; and

(2) a nucleotide sequence having at least 80% sequence identity to SEQ ID NO. 3.

In a second aspect, the present invention provides a transgenic self-flocculating algal cell stably transformed with the expression vector of the present invention and having self-settling properties.

In a particular embodiment, the algae is selected from chlorella.

In a specific embodiment, the algae cells are freshwater chlorella cells with the preservation number of CGMCC number 5905.

In a third aspect, the invention provides a method of preparing a transgenic self-flocculating microalgae, the method comprising:

(1) transferring the expression vector into the microalgae; and

(2) screening the microalgae with flocculation performance.

In one embodiment, the method for preparing transgenic self-flocculating microalgae according to the invention comprises:

(a) collecting microalgae cells in logarithmic growth phase, and suspending in an osmotic buffer solution;

(b) thermally shocking the cells obtained in step (1) and placing on ice;

(c) mixing the cells treated in the step (2) with the expression vector and salmon sperm DNA (No), and placing on ice;

(d) resuspending the cells, and treating the cells by an ultrasonic wave induction method or an electric shock method; and

(e) and (d) transferring the cells in the step (d) into a freshly prepared DM culture medium for dark culture, then transferring the cells into a DM liquid culture medium containing antibiotics for culture, and screening to obtain the transgenic self-flocculating microalgae.

In a fourth aspect, the invention provides the use of an expression vector of the invention in the preparation of transgenic self-flocculating algal cells.

The flocculation genes of the invention comprise yeast flocculation genes FLO1 (SEQ ID NO: 1), FLO5, FLO9 and FLO 10. In one embodiment, the yeast flocculation gene is as set forth in SEQ ID NO. 1 of CN 200910200097. X. In a specific embodiment, the flocculation gene of the invention encodes the amino acid sequence shown in SEQ ID NO. 2 of application SEQ ID NO. 2 or CN 200910200097.X, SEQ ID NO. 2.

The invention includes nucleotide sequences that are highly homologous to the flocculation genes of the invention, or that hybridize under stringent conditions to the flocculation genes of the invention. As used herein, "highly homologous" in reference to a nucleotide sequence may refer to a nucleotide sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to the referenced sequence. The nucleotide sequence of the invention may be a degenerate variant of a flocculating gene of the invention (e.g.SEQ ID NO:1 of SEQ ID NO:1 and CN 200910200097.X of the invention). As used herein, "degenerate variant" means in the present invention a variant that encodes a polypeptide comprising SEQ ID NO:2 or CN 200910200097.X, but is identical to SEQ ID NO:1 or CN 200910200097.X, and the nucleotide sequence shown in SEQ ID NO. 1 is different.

Readily available computer programs such as ALIGH, Dayhoff, M.O (Atlas of Protein Sequence and Structure, M.O.Dayhoff editor, 5 Suppl., 3: 353-. Programs for determining nucleotide Sequence homology, such as BESTFIT, FASTA and GAP programs, which also rely on Smith and Waterman algorithms, are available from Wisconsin Sequence Analysis Package (8 th edition, available from Genetics Computer Group, Madison, Wis.). These programs are readily available using the default parameters suggested by the manufacturer and described in the Wisconsin Sequence analysis Package above. For example, the percentage of homology of a nucleotide sequence to a reference sequence can be determined using the default scoring tables of the homology algorithms of Smith and Warerman and a gap penalty (gap penalty) of 6 nucleotide positions.

The invention also relates to variants of the flocculation genes of the invention (e.g. SEQ ID NO:1 of the invention or SEQ ID NO:1 of CN 200910200097. X), the coding of which is identical to the sequence of the flocculation genes of the invention SEQ ID NO:2 or CN 200910200097.X SEQ ID NO 2 fragments, analogues, derivatives and variants of flocculation genes having the same amino acid sequence. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the protein encoded thereby. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. The nucleic acid fragments may be used in nucleic acid amplification techniques (e.g., PCR) to determine and/or isolate a polynucleotide encoding a lipase of the invention.

The invention also includes the flocculation gene of the invention of the RNA sequence, and under stringent conditions with the flocculation gene of the invention or the RNA sequence hybridization molecules.

As used herein, the term "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is 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. For example, the sequence may be a complementary sequence of the sequence defined in (a), or may be a sequence having 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 complementarity to the sequence defined in (a).

A "coding sequence" or a sequence "encoding" a selected polypeptide refers to a nucleic acid molecule that is transcribed (for DNA) and translated (for mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence may be determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. The transcription termination sequence may be located 3' to the coding sequence.

"operably linked" refers to an arrangement of elements wherein the components are arranged in a configuration so as to perform their intended function. Thus, a given promoter operably linked to a coding sequence enables efficient expression of the coding sequence in the presence of the correct transcription factors and the like. The promoter need not be contiguous with the coding sequence, so long as it functions to direct expression of the sequence. Thus, for example, sequences which are not involved in translation but are transcribed may be present between the promoter sequence and the coding sequence, as may the transcribable intron; and the promoter sequence may still be considered "operably linked" to the coding sequence.

The vectors of the invention may contain various control elements in addition to the coding sequences. "control element" refers to a polynucleotide sequence that facilitates expression of a coding sequence to which it is linked. The term includes promoters, transcription termination sequences, upstream regulatory domains, polyadenylation signals, untranslated regions (including 5'-UTR and 3' -UTR), and, where appropriate, leader sequences and enhancers, which collectively provide for the transcription and translation of a coding sequence in a host cell.

"promoter" as used herein refers to a DNA regulatory region capable of binding RNA polymerase in a host cell and initiating transcription of a downstream (3' direction) coding sequence to which it is operably linked. Promoter sequences useful 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 the promoter sequence is a transcription initiation site and a protein binding domain (consensus sequence) responsible for RNA polymerase binding. Eukaryotic promoters often (but not always) contain "TATA" boxes and "CAT" boxes. The promoter of the expression vector used in the present invention may be HSP, and its use allows inducible expression of the gene of interest in microalgae.

The control sequence "directs the transcription" of a coding sequence in a cell when RNA polymerase binds to the promoter sequence and transcribes the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.

Herein, an "expression cassette" or "expression construct" refers to an assembly capable of directing the expression of a sequence or gene of interest. The expression cassette includes the control elements described above, such as a promoter operably linked to (to direct transcription of) the sequence or gene of interest, and often also includes a polyadenylation sequence. In certain embodiments of the invention, the expression cassettes described herein may be contained in an expression vector (i.e., a plasmid construct) of the invention. In addition to the components of the expression cassette, the plasmid construct contains one or more selectable markers, a signal to allow the plasmid construct to exist as a single stranded DNA (e.g., M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., SV40 or adenovirus origin of replication).

"transformation" refers herein to the insertion of an exogenous polynucleotide (e.g., an expression vector of the invention) into a host cell. The nucleic acid molecule comprising the nucleotide sequence of interest can be stably integrated into the host cell genome or maintained on stable episomal elements in a suitable host cell by a variety of gene delivery methods well known in the art. See, e.g., U.S. Pat. No.5,399,346. Methods for introducing the expression vector into the recipient microalgae cell may include electric shock methods, ultrasonic methods, and the like.

A "host cell" is a cell that has been transformed, or that can be transformed with a foreign DNA sequence. Various algal cells may be used in the present invention, including, but not limited to, microalgae such as Chlorella pyrenoidosa (Chlorella pyrenoidosa), Chlorella vulgaris (Chlorella vulgaris), Chlorella ellipsoidea (Chlorella ellipsospoidea), Chlorella emersonii, Chlorella sokinensis, Chlorella saccharophila, Chlorella regularis, Chlorella minutissima, Chlorella proutothecoides, Chlorella fragilis, and Chlorella succinogenes, Chlorella abortus, Chlorella microaspirifera, Chlorella succinogenes, Haematococculus, Bradysarum, Chlorella vulgaris, Chlorella viridans, Chlorella succinogenes, Tetracoccus, Terminalia micella vulgaris; cylindrocatheca fusiformis, Nitzschia laevis, Nitzschia alba, Nitzschia fonticola, Navicula incerta, Navicula pellicularilosa, of the phylum Diatoma; anabaena variabilis of the cyanobacteria phylum; poterioochromanemalmamelanensis of the phylum chrysophyceae; amphiinium carterie, Crypthecodinium cohnii, of the phylum Dinophyceae; euglena gricilis of Euglena; and Galdieria subpluraria of the Rhodophyta. Microorganisms suitable for use in the present invention also include Mortierella (Mortierella), Thraustochytrium (Thraustochytrium), Schizochytrium (Labyrinthulomycetes), Ukenella (Ulkenia), and the like. Particularly preferred in the present invention are Scenedesmus sp and Chlorella sp.

In the present invention, once the coding sequences are 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 selection of an appropriate cloning vector is a matter of choice. Suitable carriers include (but are not limited to): plasmids, phages, transposons, cosmids, chromosomes or viruses which replicate when combined with appropriate control elements.

The cloned sequence is then placed under the control of appropriate control elements, depending on the system used for expression. Thus, the coding sequence may be placed under the control of a promoter, ribosome binding site (for bacterial expression) and optionally an operator, so that the DNA sequence of interest is transcribed into RNA by a suitable transformant. The coding sequence may or may not include a signal peptide or leader sequence (which may then be removed by post-translational processing by the host).

In addition to control sequences, regulatory sequences may be added to allow for expression of the regulatory sequences relative to the growth of the host cell. Regulatory sequences are known to those skilled in the art, examples of which include those that result in the turning on or off of gene expression in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector. For example, enhancer elements may be used to increase the expression level of the construct. Examples include the SV40 early gene enhancer (Dijkema et al (1985) EMBO J.4: 761); 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. Pat. No.5,688,688). Also included in the expression cassette are an origin of replication for autonomous replication in a suitable host cell, one or more selectable markers, one or more restriction sites, a high copy number potential, and a strong promoter.

Expression vectors are constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, and the coding sequence associated with the control sequence is positioned and oriented so that the coding sequence is transcribed under the "control" of the control sequence (i.e., the coding sequence is transcribed by the RNA polymerase associated with the DNA molecule in the control sequence). Modifications to the sequence encoding the molecule of interest may be required to achieve this. For example, in some cases it may be desirable to modify the sequence so that it is linked to a control sequence of suitable orientation, i.e., to maintain the reading frame. Control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into the vector. Alternatively, the coding sequence may be cloned directly into an expression vector which already contains the control sequences and appropriate restriction sites.

In other examples, flocculation genes suitable for use in the yeast of the present invention may include FLO1 (SEQ ID NO: 1), FLO5, FLO9, FLO10 (ZhaoXQ, Li Q, He LY, Li F, Que WW, Bai F, expression of flocculation genes from microorganisms and improved flocculation expression of flocculation genes encoding flocculation biological assays, Process Biochemistry, 2011 in press, Franziska Heine, Frastahl, Heike ä uber, Claudia Wiacek, Dirk Bennella, Frankmidt, Martinus Martinctoria, flocculation genes having similar or similar functional sequences to those of flocculation genes expressed in flocculation vectors such as the flocculation protein found in Bacillus strain, flocculation protein found in Bioflocculation protein, flocculation protein found in DNA, flocculation protein found in Bacillus strain, flocculation protein found in SEQ ID NO. 25, or flocculation protein found in Bacillus strain.

Accordingly, the present application provides a method of obtaining transgenic self-flocculating microalgae, the method comprising: (1) cloning flocculation genes from flocculation yeast, and constructing an expression vector by using the flocculation genes; and (2) transforming the expression vector (by, e.g., shock treatment) into the microalgae cells; thereby obtaining the transgenic self-flocculation microalgae.

Further, the method of the present invention may further comprise: (3) recovering and confirming the transgenic flocculated microalgae; and optionally, (4) harvesting cells of the transgenic flocculated microalgae.

The cloning of flocculation gene includes the amplification of flocculation gene with cell self-flocculation function from yeast or other biological cells, and the invention uses FLO1 gene cloned from flocculation yeast as example to construct flocculation gene expression vector (see CN 200910200097.X, herein incorporated by reference).

The expression vector can comprise plant genetic engineering expression vectors such as pCAMBIA1301 and the like, and other expression vectors which can be used in microalgae, wherein necessary genetic elements of the expression vector comprise a promoter, a resistance gene, a multiple cloning site and the like. The invention uses an expression vector which is modified based on pCAMBIA1301 and contains HSP inducible promoter, GFP reporter gene and chloramphenicol resistance gene CAT.

Methods for introducing the expression vector into the recipient microalgae cell may include electric shock methods, ultrasonic methods, and the like. The invention provides a genetic transformation method of microalgae (such as chlorella) or a preparation method of transgenic self-flocculating microalgae, which comprises the following specific steps:

(a) collecting microalgae cells in logarithmic growth phase, and suspending in an osmotic buffer solution;

(b) thermally shocking the cells obtained in step (1) and placing on ice;

(c) mixing the cells treated in the step (2) with the expression vector and salmon sperm DNA (No), and placing on ice;

(d) resuspending the cells, and treating the cells by an ultrasonic wave induction method or an electric shock method; and

(e) and (d) transferring the cells in the step (d) into a freshly prepared DM culture medium for dark culture, then transferring the cells into a DM liquid culture medium containing antibiotics for culture, and screening to obtain the transgenic self-flocculating microalgae.

The permeation buffer used in the present invention may be one commonly used in the art. An exemplary osmotic buffer contains, for example, 0.2M to 0.3M mannitol, 0.2M to 0.3M sorbitol, and 10 to 15% glycerol. The microalgae cells are typically resuspended at a cell density of about 107-108 cells per ml.

The heat shock is generally carried out at a temperature of, for example, 38 to 45 ℃. In one embodiment, the heat shock is performed at 42 ℃. The heat shock time is generally controlled to be about 1 to 10 minutes. The heat shock is followed by a few minutes (e.g. 3-8 minutes, e.g. 5 minutes) of resting on ice.

Then, the cells left 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. In one embodiment, about 400 microliters of the cell solution is mixed with about 10. mu.g/mL of the expression vector of the invention and about 25. mu.g/mL of salmon sperm DNA, and after mixing, is typically placed on ice for several minutes (e.g., 3-8 minutes, e.g., 5 minutes).

Then, the cells were resuspended, and the cells were treated by electroporation. For shock treatment, a suitable amount (e.g., 0.4-1 mL) of resuspended cells can be aspirated and placed into a pre-cooled shock cuvette, and shock is performed according to various set condition combinations. One optimized shock transition condition is: the concentration of the expression vector is 50g/mL, the concentration of the hypertonic buffer solution is 0.2 mol/L, the pulse distance is 2 mm, the pulse voltage is 2 Kv, and the pulse duration is 3 ms.

After electroporation, the cells are transferred to a suitable volume (e.g., 10-30 mL) of freshly prepared DM medium, dark cultured at 25 ℃ for about 18-36 (e.g., 24 hours) hours, and then transferred to DM broth containing an antibiotic, e.g., 50. mu.g/mL chloramphenicol.

Exemplary medium DM media compositions are (g/l): ca (NO3) 2.4H 2O, 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, (NH4)6 Mo7O24.4H2O, 0.000018, CuSO4.5H2O, 0.00008.

The recovery and confirmation of the transgenic flocculation microalgae comprise the steps of coating the transformed microalgae on a flat plate containing chloramphenicol, taking monoclonal antibody, culturing for 7-14 days in a liquid culture medium containing chloramphenicol, wherein the cells are green-white, then transferring into a liquid culture medium containing no antibiotics, and continuing culturing for 7-14 days, wherein the growth state of the cells can be gradually changed into active state, the cells can be turned green, the flocculation character can be observed, the culture medium is replaced, the flocculation character of a transformant is gradually enhanced, and a flocculation cell group can be obviously observed (see figure 4). Since the reporter gene can monitor the transformation efficiency, it can be observed under a fluorescence microscope that the empty vector containing green fluorescent protein emits green fluorescence in scenedesmus cells, which proves the success of transformation, and in addition, the green fluorescence in the flocculation cell mass (fig. 6) can be observed, which proves the expression of the flocculation gene of the yeast.

The self-flocculation harvesting of the microalgae comprises the step of treating microalgae cells containing self-flocculation genes at 45 ℃ for 30min to form clusters, and the cells are rapidly self-settled, while the control free algae species do not have self-settlement under the same treatment conditions, so that the construction of the transgenic flocculation algae for induction expression can be conveniently and simply carried out through cell self-settlement harvesting.

Example 1: cloning of flocculation Gene and construction of expression vector

(1) Cloning of the Gene of interest (see methods of examples 1 and 3 in CN 200910200097. X)

Designing a primer according to a flocculation gene FLO1 sequence of a flocculation yeast SPSC01 in a laboratory:

infusion3_ F: GGACTCTTGACCATGGATGACAATGCCTCATCGCTATATG (SEQ ID NO: 4); and

Infusion3_R: CTCACATCTACCATGGTTAAATAATTGCCAGCAATAAGGAC（SEQ ID NO:5）。

the PCR reaction procedure was 95 ℃ denaturation for 5min, 95 ℃ denaturation for 30, 55 ℃ annealing for 30 s, 72 ℃ extension for 5min, 10 cycles followed by 20 cycles of 95 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s72 ℃ extension for 5min, and finally 72 ℃ extension for 10 min using a full-scale gold Taq High Fidelity (High Fi) PCRsuper mix II (TransGenBiotech, Beijing, China). The 5.2 Kb yeast flocculation gene FLO1 (SEQ ID NO: 1) was cloned as shown in FIG. 3.

(2) Construction of expression vectors

The research adopts an expression vector pCAMBIA1302 widely applied in plants as a vector framework, the gene sequence of the expression vector pCAMBIA1302 contains a strong promoter HSP70A and a reporter gene gfp, and a screening marker kanamycin resistance gene in prokaryotes and a screening marker hygromycin resistance gene in eukaryotes. The sensitivity test of chlorella shows that the chlorella is sensitive to chloramphenicol, so the hygromycin resistance gene on the pCAMBIA1302 vector is replaced by the chloramphenicol resistance gene by an Infusion method, and then the flocculation gene of yeast is connected to the downstream of a vector strong promoter HSP70A and the upstream of a reporter gene gfp, and the specific flow is shown in figure 1.