CN107058555B - Screening method of microalgae mutant with high cell growth density - Google Patents

Abstract

The invention discloses a screening method of a microalgae mutant with high cell growth density, which comprises the following steps: (1) transforming the resistance gene into microalgae, obtaining single microalgae colonies of the mutant microalgae transformed into the resistance gene sequence through solid plate resistance screening, and collecting each single microalgae colony to obtain a microalgae mutant library; (2) sampling and mixing each single algal colony in the microalgae mutant library, culturing in a liquid culture medium, and continuously carrying out generation transmission; (3) detecting by an RFLP analysis method of sequence specificity of a plurality of alternative resistance genes every time, finishing screening when the detection result shows that only one microalgae mutant remains, and obtaining the microalgae mutant with high cell growth density; and when the detection result shows that 2-5 microalgae mutants remain, performing single-algal colony separation by using a solid culture medium to obtain the high-cell-growth-density microalgae mutant. The screening method of the invention does not need highlight conditions and special detection equipment, and can complete the screening process quickly, simply and efficiently.

Description

Screening method of microalgae mutant with high cell growth density

Technical Field

The invention relates to the technical field of biology, in particular to a screening method of a microalgae mutant with high cell growth density.

Background

Microalgae are considered to be the most potential oil biological energy source due to their characteristics of high growth rate, short growth cycle, high oil content and the like. Chlamydomonas reinhardti (Chlamydomonas reinhardti) has become a potential energy source microalgae suitable for large-scale production as a unicellular model organism for researching microalgae. However, chlamydomonas reinhardtii has a smaller biomass compared to other commercial microalgae, and the lower growth density and biomass limit its potential for large-scale energy production.

Insertional mutagenesis is a commonly used method for screening mutant strains, and has been widely used for screening chlamydomonas reinhardtii mutant strains with special phenotypes. The inserted sequences generally contain a marker gene, for example Aph VIII (Sizova, I., Fuhrmann, M.and Hegemann, P. (2001) A Streptomyces rimosus aphVIII gene coding for a new type of phosphor transfer enzyme variant, genetic to Chlamydomonas reharantii, 277, 221. 229.), blade (Lumbreras, V., Stevens, D.R.and Purton, S. (1998) effective for gene expression in Chlamydomonas reinhardtii modified by strain in Plant, 14, 441. 447) and the like, to facilitate the selection of mutants.

Research has shown that: insertion mutants that are designed to increase biomass, reduce pigments, remove tentacles, etc. can be targeted at high light levels but are not viable at low light levels. This is mainly because under high light conditions, the antennary cells will float on the surface and receive more light than the wild type. But the biomass without tentacles under low light is similar to the wild type because the intensity of the light is now not much different at the surface and underneath (Polle, J.E.W., Kanakagiri, S.D. and Melis, A. (2003) tla1, a DNA inert transform of the green alga Chlamydomonas reinhardtii with a rounded light-modifying chlorophyl antenna size. plant, 217, 49-59.). Although the chlamydomonas biomass can be improved under high light, the high light energy consumption is large, and the large-scale application is not facilitated. Therefore, it becomes more important how to screen microalgae strains that still have higher biomass under ordinary light intensity.

Disclosure of Invention

The invention provides a screening method of a microalgae mutant with high cell growth density, aiming at the problem that a proper method for screening microalgae strains with high biomass under common illumination intensity is lacked in the prior art.

A screening method of microalgae mutants with high cell growth density comprises the following steps:

(1) converting the resistance gene into microalgae, obtaining single colonies of the mutant microalgae transformed with the resistance gene sequence through solid plate resistance screening, and collecting each single colony to obtain a microalgae mutant library;

(2) sampling and mixing each single algal colony in the microalgae mutant library, culturing in a liquid culture medium, and continuously carrying out generation transmission;

(3) detecting by RFLP analysis method of resistance gene sequence specificity every passage for several times, and ending screening when the detection result shows that only one microalgae mutant remains to obtain a high cell growth density microalgae mutant; and when the detection result shows that 2-5 microalgae mutants remain, performing single-algal colony separation by using a solid culture medium to obtain the high-cell-growth-density microalgae mutant.

Screening mutants with higher cell density from mutants obtained by random insertional mutagenesis was not possible by solid plate screening. Because the cells spread on the plate do not grow synchronously and regional nutrient depletion also leads to a lack of correlation between algal colony size and growth density. Therefore, the invention selects the mixed algae liquid of the continuous passage mutant in the liquid culture medium, and the mutant with high density can gradually take advantage in the continuous passage culture process, so that the high cell density mutant can be separated from the algae liquid after effective passage culture after enrichment.

In order to monitor the whole process in real time, the invention adopts an RFLP analysis method to track and test the enrichment effect. RFLP (Restriction Fragment Length Polymorphism) refers to the difference in Restriction Fragment Length between genotypes caused by insertion, deletion, rearrangement or point mutation of bases at Restriction sites. The specific DNA fragment is visualized by hybridization with a radiolabeled probe (Southern hybridization) by cleaving the DNA with restriction enzymes, separating the DNA fragments by gel electrophoresis, transferring the DNA fragments to a filter, and finally analyzing the results. In the present invention, the insertion position of the resistance marker gene is different, and the Southern hybridization detection is carried out using a probe for the resistance marker gene sequence, in which the length of the fragment containing the resistance marker gene differs after treatment with a specific restriction enzyme (which is not generally contained in the resistance marker gene sequence).

Preferably, the microalgae is chlamydomonas reinhardtii. The screening method of the present invention is also applicable to other microalgae that can be genetically transformed, such as Chlorella (Chlorella sp.), Dunaliella salina (Dunaliella salina), Volvox (R) Carteri), Phaeodactylum tricornutum (R) and the like.

Preferably, the resistance gene is aadA gene, ble gene, aphVIII gene or als gene.

Further preferably, the resistance gene is a ble gene. The resistance gene used in the screening method of the present invention is used for resistance screening of a mutant strain on the one hand and for detection by the RFLP analysis method on the other hand, and therefore, gene sequences or fragments satisfying both of these points are suitable for the screening method of the present invention.

Preferably, the resistance gene is transformed into microalgae by using an electric transformation method. Of course, other transformation methods suitable for microalgae may be used. For example, the particle gun method, also known as microparticle bombardment method, is suitable for genetic transformation of various types of microalgae; microalgae that are cell wall deficient may use glass bead transformation. Preferably, the inoculation density at passage is 0.1-0.2 OD 750.

Further preferably, the number of passages is not less than 30. The passage times are combined with the RFLP detection results, and if the RFLP detection results show that the types of the mutant strains are few when the passages are few, the solid culture medium can be used for separation; and if the number of passages is more than 30, when the RFLP detection result shows that the mutant strain is still more, the passage is continued to screen and enrich.

Preferably, in the step (3), the RFLP analysis method specific to the resistance gene sequence is used for detecting 5-15 times per passage. The RFLP detection result may have small variation every passage, so that the detection can be performed once after several successive passages.

Preferably, RFLP analysis is performed by restriction with restriction enzymes followed by Southern detection using DIG-labeled probes for resistance genes.

The screening method adopts resistance genes to carry out random insertion mutation, then all the obtained mutants are mixed to carry out liquid culture and continuous passage, the mutant strains which grow fast in the passage process gradually occupy advantages and are enriched, the RFLP analysis method is used for monitoring the screening effect in the continuous passage process, and finally the microalgae mutants with the growth density higher than that of the wild type can be screened and obtained under the common illumination condition. The screening method of the invention does not need highlight conditions and special detection equipment, and can complete the screening process quickly, simply and efficiently.

Drawings

FIG. 1 is a graph showing the growth of the subcultured mutant strain in example 2.

FIG. 2 is a schematic view of a continuous subculture process.

FIG. 3 is a RFLP analysis result chart in which A, B, C represents 3 replicates, respectively, lane M is DNA marker (same below), lane C is wild type Chlamydomonas reinhardtii CC503 (same below) as a control, and lanes 0, 10, 20, and 30 represent the number of passages, respectively.

FIG. 4 is a RFLP analysis result chart of the mutants screened in A, B, C three groups of experiments after the passage screening in example 4 was completed, wherein lanes i1-i 6 represent single isolates (the same below).

FIG. 5 is a diagram showing the results of RFLP analysis after double digestion of genomic DNA in example 4.

FIG. 6 is a graph showing the results of growth curve detection of 3 mutants obtained by screening in example 5.

FIG. 7 is a graph showing a comparison of growth rates of 3 mutants selected in example 5, in which WT represents wild-type Chlamydomonas reinhardtii CC503 (the same applies below).

FIG. 8 is a graph comparing the results of maximum biomass of the 3 mutants screened in example 5 in HS medium and 2 times HS medium.

FIG. 9 is a graph showing the results of the chlorophyll content of the 3 mutant strains selected in example 5.

Detailed Description

Original strains of Chlamydomonas reinhardtii: chlamydomonas reinhardtii CC503, purchased from Chlamydomonas Resource Center (website: www.chlamy.org).

Plasmid pSP 124S: purchased from the Chlamydomonas resource center, the plasmid contains a ble gene sequence.

The detection of the OD value of the chlamydomonas reinhardtii is carried out under the wavelength of 750 nm.

HS medium: the formulation is described in Sueoka, n. (1960) proc.natl.acad.sci.usa 46,83-91, and the specific components are as follows:

mother liquor 1 (salt solution):

NH₄Cl 100.0g

MgSO₄·7H₂O 4.0g

CaCl₂·2H₂O 2.0g

adding H₂O to 1L

Mother liquor 2 (phosphate buffer):

K₂HPO₄ 288.0g

KH₂PO₄ 144.0g

adding H₂O to 1L

Mother liquor 3 (trace elements):

when the HS medium is used: 5mL of mother liquor 1, 5mL of mother liquor 2, 1mL of mother liquor 3, and adding water to 1L.

TAP medium: the formulation is described in Gorman, D.S., and R.P.Levine (1965) Proc.Natl.Acad.Sci.USA 54,1665-1669, and the specific ingredients are as follows:

mother liquor 1(TAP salt):

NH₄Cl 15.0g

MgSO₄·7H₂O 4.0g

CaCl₂·2H₂O 2.0g

adding H₂O to 1L

Mother liquor 2 (phosphate buffer):

K₂HPO₄ 28.8g

KH₂PO₄ 14.4g

adding H₂O to 100mL

Mother liquor 3 (trace elements):

TAP medium when used: 2.42g Tris, 25mL mother liquor 1, 0.375mL mother liquor 2, 1mL mother liquor 31 mL acetic acid (glaciatic acid), plus H₂O to 1L.

Example 1

An upstream primer Ble _ 1F: 5'-TGGAAGCTTAAATGCCAGAAGGAGCGCAGCC-3', respectively;

the downstream primer Ble _ 1160R: 5'-CCGAGCTCAGCTTCAAATACGCCCAGCCCG-3' the flow of the air in the air conditioner,

the plasmid pSP124S was amplified using the above primers to obtain the ble gene sequence, the nucleotide sequence is shown in SEQ ID No.1, and the length is 1.1 kb.

Transforming the ble gene fragments into Chlamydomonas reinhardtii CC503 cells by an electrical transformation method, screening the transformed strains on a TAP (TAP) plate with 2.5 mu g/mL bleomycin (zeocin), wherein Chlamydomonas reinhardtii algae colonies growing on the plate are mutant strains transformed with the ble gene fragments, and storing all the obtained algae colonies into a 96-well plate one by one to construct a Chlamydomonas reinhardtii mutant library. In this example, more than 1000 Chlamydomonas reinhardtii mutants were obtained by the above method.

An electric conversion step:

(1) collecting algae cells by centrifuging algae liquid at early logarithmic growth stage at 2500rpm for 5 min, washing with electrotransformation Reagent (GeneArt MAX Efficiency Transformation Reagent) and centrifuging for 3 times, and resuspending to final concentration of 2 × 10⁸～3×10⁸one/mL.

(2) Adding 2-4 mu g of linearized DNA into each 250 mu L of cell suspension, and incubating for 5 minutes at 2-8 ℃.

(3) 250 μ L of cell suspension containing DNA was added to a pre-cooled cuvette prior to electroporation.

(4) The electrical conversion parameters were: the voltage is 500V, the capacitance is 50 muF, and the resistance is 800 omega. Typically, the electrical pulse duration is 30 ms.

(5) After electrotransformation, the cells were revived for 15 min. Transferring the cells into a 50 mL centrifuge tube containing 10mL TAP-40mM sucrose solution and standing at room temperature; then the algae liquid was transferred to an incubator at 26 ℃ with the light intensity of 50. mu. E m^-2s^-1And (5) incubating for 14-16 h.

(6) The algal cells were collected by centrifugation at 2500rpm for 5 minutes, the supernatant was discarded, and the algal solution was resuspended in 200. mu.L of TAP medium.

(7) Screening of mutants was performed on TAP medium plates containing 2.5. mu.g/mL bleomycin (zeocin), and a total of more than 1000 Chlamydomonas reinhardtii mutants were obtained by multi-plate screening and pooling. These mutants were cultured in liquid medium (liquid medium volume 200. mu.L) in a 96-well plate and stored for later use.

Example 2

mu.L of each of the 1000 mutants obtained in example 1 was collected from a 96-well plate, mixed, centrifuged, suspended in a freshly prepared HS medium containing 2.5. mu.g/mL zeocin, and inoculated into 100mL of a shaking flask containing 2.5. mu.g/mL zeocin and cultured under continuous illumination conditions of 50. mu. mol m^-2s^-1. In this case, the initial cultured mutant strain (about 0.15OD) reached about 0.4OD, about 0.95OD, about 1.35OD, and about 1.45 OD at 1, 2, 3, and 4 days, respectively (FIG. 1). Cells entered plateau at the beginning of the third day, which was selected as the starting point for seeding for serial subculture (FIG. 2). When the initial mixed algae liquid grows to OD750 ═ 1.0, the mutant strain is inoculated to 3 shake flasks at the initial concentration of about 0.15OD to continue culturing, and the three experimental groups A, B and C are divided. Successive passages were performed early in the plateau (3 days, about 1.4OD), and fresh medium (containing 2.5. mu.g/mL zeocin) was re-inoculated (0 days, about 0.15OD) and continued passage in the log phase and plateau phase. The number of serial passages was 30.

Example 3 RFLP detection

In order to monitor the whole process in real time, the invention adopts RFLP to track and test the enrichment effect. Since each insertion mutant strain in the RFLP map has at least one DNA fragment with a unique size, a complicated RFLP map can be seen in the initial culture containing more than 1000 insertion mutants. As the passage continued, the complexity of the RFLP map decreased, indicating a gradual enrichment of HCD mutants (High-Cell-sensitivity abbreviations, representing the High Cell Density mutants screened) in serial subculture. After approximately 30 cycles of passaging, only a few bands remain in RFLP, and mutants containing these fragments are considered as candidate mutants for HCD.

In order to detect the change condition of the algal strain in the passage process, RFLP analysis of the mutant strain with the specificity of the Ble sequence is carried out. The specific operation is as follows: collecting the cell samples of the primary culture cells, passage 10, 20 and 30, cracking by CTAB buffer solution, and extracting genome DNA by phenol-chloroform method. The DNA was digested with restriction enzyme NcoI, and the RFLP pattern was examined. Detection probes were synthesized from the template plasmid pSP124S using a PCR DIG Probe Synthesis Kit (Roche, PCR DIG Probe Synthesis Kit, cat # 11636090910) and the primers were synthesized as follows:

Ble_E2_F：5'-TGGCCAAGCTGACCAGCGCCGTTCC-3'；

Ble_E3_R：5'-CCTCCGACCACTCGGCGTACAGC-3'，

the sequence of the finally obtained probe is shown as SEQ ID No.2, and the probe is coupled with an alkaline phosphatase anti-digoxin antibody.

Southern hybridization was performed according to DIG-labeled membrane hybridization. The specific steps are as follows, separating the genomic DNA after electrophoresis, transferring the DNA fragments to a nylon membrane, hybridizing the DNA fragments with the synthesized DIG-labeled probe, and detecting. alpha-DIG-AP is diluted according to the ratio of 1: 15000 in the detection reaction, and for optimizing the detection step, the eluent components are 0.1M maleic acid, 3M NaCl, 0.3 percent Tween20 and the pH value is 8.0.

In 3 repeated passages of example 2, 1, 3 and 3 dominant fragments were found in experiments a, B and C, respectively (fig. 3). A dominant fragment STN _ a1 present in experiment a was present in both experimental groups B and C. The fragment STN _ B3 present in experiment B was also present in C (STN _ C2). Thus, after serial passages, enrichment yielded at least 4 possible HCD mutants.

Example 4

The mutant strains obtained after 30 passages of example 2 are screened by solid plate single algae colonies, 5-6 single algae colonies are picked from each plate and RFLP verification analysis is carried out again. The results showed that all 5 mutants in experiment A showed a fragment similar to STN _ A1. In experiment B, isolated 2, 4, 6 were similar to STN _ B1 fragment, isolated 3 and 5 fragments were similar to STN _ B3, and isolated 1 was similar to STN _ B2 fragment. In experiment C, all isolated fragments were similar to STN _ C1, and no isolates containing the same size as the STN _ C2 and STN _ C3 fragments were found (FIG. 4).

To verify that the fragments observed in the isolates were indeed identical to those enriched after 30 passages, the genome of each isolate was subjected to an RFLP analysis after double digestion with NcoI and KpnI. The size of the double digested fragment was identical to that of the enriched strain after 30 passages (FIG. 5). The results show that the isolate is the dominant species among all the inserted mutants. Thus, mutants containing fragments of STN _ B1 (isolates STN _ A1_ i1-i 5; STN _ B1_ i2, i4 and i 6; STN _ C1_ i1 and i3-i6), STN _ B2 (isolates STN _ B2_ i1) and STN _ B3 (isolates STN _ B3_ i3 and i5) were designated as HCD1, HCD2 and HCD3 mutants, respectively. Therefore, the invention adopts a continuous passage way to enrich and obtain the mutant strain with the above 3 high cell growth density, and the continuous passage way can separate the mutant strain which grows rapidly or densely.

Example 5

To analyze the growth rate and maximum cell density of HCD mutants, the mutants were exposed to continuous light at an intensity of 50. mu. mol m^-2s^-1Growth curves (FIG. 6), growth rates and biomass were determined. The results show that the growth rates of HCD1 and HCD2 are higher than that of wild type (p-value)<0.001) (fig. 7). HCD1, HCD2 and HCD3 grown in HS medium had a maximum cell density (maximum dry cell weight, CDW) 22-40% higher than wild type; the maximum cell density of the HCD mutant was 50-100% higher than the wild type when the medium was grown in 2-fold HS medium (fig. 8). Meanwhile, chlorophyll content (fig. 9) and cell morphology showed that no significant difference was seen in HCD mutant compared to wild type. In conclusion, the HCD mutant strains already have a growth density and biomass which are superior to the wild type Chlamydomonas reinhardtii.

Claims (3)

1. A screening method of microalgae mutants with high cell growth density is characterized by comprising the following steps:

(1) converting the resistance gene into microalgae, obtaining single colonies of the mutant microalgae transformed with the resistance gene sequence through solid plate resistance screening, and collecting each single colony to obtain a microalgae mutant library;

(2) sampling each single algal colony in the microalgae mutant library, mixing, culturing in a liquid culture medium, and continuously carrying out generation;

(3) detecting by an RFLP analysis method of sequence specificity of a plurality of alternative resistance genes every time, finishing screening when the detection result shows that only one microalgae mutant remains, and obtaining the microalgae mutant with high cell growth density; when the detection result shows that 2-5 microalgae mutants remain, performing single-algal colony separation by using a solid culture medium to obtain the high-cell-growth-density microalgae mutant,

the inoculation density is OD750 of 0.1-0.2 during passage, the number of passages is not less than 30,

in the step (3), the RFLP analysis method of the sequence specificity of the resistance gene is used for detecting 5-15 times per passage,

the microalgae is chlamydomonas reinhardtii,

the resistance gene is aadA gene, ble gene, aphVIII gene or als gene,

in RFLP analysis, restriction with restriction enzymes is performed, followed by Southern detection, in which DIG-labeled probes for resistance genes are hybridized.

2. The screening method according to claim 1, wherein the resistance gene is a ble gene.

3. The screening method of claim 1, wherein the transformation of microalgae with the resistance gene is by electrotransformation.

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