KR101695465B1 - Method for increasing lipid productivity and growth of microalgae by using an overexpression of bHLH transcription factors - Google Patents

Abstract

The present invention relates to a recombinant vector pNs302 (bHLH2) for overexpressing a gene encoding a basic helical loop helicase transcription factor protein (bHLH2), a transgenic microalga having the bHLH2 (bHLH2) inserted therein, Or osmotic stress medium to increase the lipid productivity and the growth rate of microalgae.

Description

[0001] The present invention relates to a method for increasing the lipid productivity and growth rate of microalgae using overexpression of a gene encoding a basic helical loop helicase transcription factor protein,

The present invention relates to a method for increasing lipid productivity and growth rate of microalgae using overexpression of a gene encoding a basic helical loop helicase transcription factor protein (bHLH2), and more particularly, to a method of increasing basic lipid productivity and growth rate of a basic helical loop helical transcription factor protein (BHLH2), bHLH2 (bHLH2), and the microalgae were cultured in a nitrogen-deficient medium and / or an osmotic stress medium to induce the lipid productivity of the microalgae And a method for increasing the growth rate.

In order to commercialize the micro-algae-based biodiesel production process, it is important to increase the growth rate and lipid content of microalgae. Since it is difficult to meet the economic feasibility for commercialization based on the existing microalgae, it is most important to develop microalgae having characteristics of high quality living.

Chlamydomonas The model strain, such as the reinhardtii , is easy to transform and has a high rate of growth (Canadian Journal of Microbiology 8, 229-239, (1962)), but basically low lipid productivity And thus it has not been evaluated as a suitable strain for the biodiesel process.

On the other hand, the seawater species Nannochloropsis salina is a microalgae (oleaginous microalgse) in lipid production, commercial inde use value is very high microalgae, if the improved strains of N. salina successful, the development of transgenic technology, that can be tailored to the commercial economy The possibility of microalgae increases.

Microalgae belonging to eukaryotes are known to have diverse feedback mechanisms in the metabolic process. To increase lipid productivity in this situation, it is not easy to increase lipid productivity by manipulating several genes in the fatty acid or triglyceride production metabolism (Proceedings of the National Academy of Sciences, 108, 21265 -21269, (2011)).

On the other hand, transcription factors can regulate the expression levels of various genes, overexpressing one transcription factor and overexpressing or suppressing various genes simultaneously. Among them, the gene encoding the basic helical loop helicase transcription factor protein (bHLH2) is known as a transcription factor that either aids growth in the plant system or responds to external stress environment.

On the other hand, as a conventional technique for transforming microalgae, a vector useful for transformation of microalgae and a microorganism capable of over-expressing the transformed microalgae (Korean Patent Publication No. 10-2011-0090565) and FAB2 gene And a method of increasing total fatty acid content and useful fatty acid ratio in microalgae (Korean Patent Laid-Open No. 10-2014-0005001).

The above prior art does not disclose a method for increasing the lipid productivity and the growth rate of microalgae by culturing the transformed microalgae into which the gene encoding the basic helical loop helical transcription factor protein (bHLH2) is inserted , There is a desperate need to develop a method for increasing the lipid productivity and the growth rate of microalgae using the transgenic microalgae and the microalgae.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems occurring in the prior art, and it is an object of the present invention to provide a recombinant vector which overexpresses a gene encoding a basic helical loop helical transcription factor protein (bHLH2).

Another object of the present invention is to provide a transformed microalgae into which the recombinant vector has been introduced.

It is another object of the present invention to provide a method for increasing the lipid productivity of microalgae by culturing the transgenic microalgae under certain conditions.

It is still another object of the present invention to provide a method for increasing the growth rate of microalgae by culturing the transgenic microalgae under certain conditions.

(BHLH2), a tubulin promoter (P _tub ), and a tubulin terminator (T _tub ), and the sequence of the gene encoding the basic helix loop helical transcription factor protein Gt; pNs302 < / RTI > (bHLH2).

The present invention also provides a transgenic microalgae into which the recombinant vector pNs302 (bHLH2) has been introduced and into which a basic helical loop helicase transcription factor (bHLH2) has been inserted.

The present invention also relates to a method for producing a microalgae comprising culturing a transformed microalgae in a nitrogen-restricted medium and / or an osmotic stressed medium to induce overexpression of a gene encoding a basic helical loop helicase transcription factor protein (bHLH2) ≪ / RTI >

In addition, the present invention relates to a method for producing microalgae, comprising culturing the transformed microalgae in a nitrogen-restricted medium and / or an osmotic stressed medium to induce overexpression of a gene encoding a basic helical loop helicase transcription factor protein (bHLH2) / RTI >

In the present invention, growth of an N. salina transformant under various circumstances and an increase in lipid productivity were confirmed through overexpression of a basic helical loop helical factor (bHLH2).

The over-expression effect of the basic spiral loop helical factor of the present invention as described above can be used industrially because it can reduce time and cost in mass production of biodiesel by increasing lipid productivity in microalgae research .

Figure 1 shows the cleavage map of the bHLH2 over-expression recombinant vector pNs302 (bHLH2).
Fig. 2 shows a cleavage map of the pNs302 vector used for the production of the bHLH2 over-expression recombinant vector pNs302 (bHLH2).
Figure 3 shows the expression level of bHLH transcription factor (bHLH2) in N. gaditana .
Figure 4 shows flag tag western blotting results of transgenic microalgae (NsbHLH2).
FIGS. 5A to 5C show batch screening test results of a candidate transforming microalgae (NsbHLH2) candidate.
FIG. 6 shows results of a well plate screening test for a candidate transformed microalgae (NsbHLH2), wherein (a) is a normal condition, (b) is nitrogen starvation, (c) ) Nitrogen limitation, and (d) osmotic stress conditions.
FIG. 7 shows the results of a normal condition phenotype assay of the transformed microalgae (NsbHLH2) in a steady state, wherein (a) is the growth curve, (b) is the dry cell weight, c) shows the content of fatty acid methyl ester (FAME), and (d) shows the yield of fatty acid methyl ester (FAME).
FIG. 8 shows the results of a nitrogen limitation condition phenotype assay of transformed microalgae (NsbHLH2) under nitrogen restriction conditions, wherein (a) shows the growth curve, (b) shows the dry cell weight, , (c) the content of fatty acid methyl ester (FAME), and (d) the yield of fatty acid methyl ester (FAME).
9 shows the results of osmotic stress condition phenotype assay of osmotic stress condition of transgenic microalgae (NsbHLH2), wherein (a) is the growth curve, (b) is the dry cell weight, , (c) the content of fatty acid methyl ester (FAME), and (d) the yield of fatty acid methyl ester (FAME).
Figure 10 Sh ble The results of Southern blotting on the gene are as follows: V is a vector and W is a wild type.
Fig. 11 shows the results of analysis of the expression amount by the introduced bHLH2 qRT-PCR.
Fig. 12 shows the results of analysis of the expression amount by the entire bHLH2 qRT-PCR.

Microalgae are prokaryotic organisms and have complex mechanisms for lipid synthesis and fertilization processes. Overexpression of one gene in this situation to increase lipid productivity may be negligible by various feedback mechanisms.

Accordingly, in the present invention, overexpression of a transcription factor capable of regulating expression of various genes has been attempted to increase the lipid productivity of microalgae. In the present invention, the present invention focused on bHLH transcription factors.

The present invention relates to a recombinant vector pNs302 (bHLH2) comprising a gene coding for a basic helical loop helicase transcription factor protein (bHLH2), a tubulin promoter (P _tub ) and a tubulin terminator (T _tub ) .

The recombinant vector of the present invention can overexpress the gene (bHLH2) encoding the basic spiral loop helical transcription factor protein.

In the recombinant vector of the present invention, the gene coding for the basic helical loop helicase transcription factor protein (bHLH2) is a plant-derived gene, and in particular, a gene encoding a hormone that protects the jasmonate stress environment in Arabidopsis It is known as a transcription factor that regulates production.

In the recombinant vector of the present invention, the gene (bHLH2) encoding the basic helical loop helicase transcription factor protein may be the nucleotide sequence of SEQ ID NO: 1.

The present invention also relates to a transgenic microalgae into which the recombinant vector has been introduced and into which a basic helical loop helical transcription factor (bHLH2) has been inserted.

In the transgenic microalgae of the present invention, the microalgae may be Nanochioropsis sp.

In the transgenic microalgae of the present invention, the microalgae may be Nanochioropsis salina sp.

In addition, the present invention relates to a method for producing a microalgae, comprising culturing the transformed microalgae in a nitrogen-restricted medium and / or an osmotic stress medium to induce overexpression of a gene encoding a basic helical loop helicase transcription factor protein (bHLH2) To a method for increasing productivity.

In a method for increasing the lipid productivity of the microalgae of the present invention, the nitrogen-restricted medium is selected from the group consisting of 15 g / L of sun salt, 10 mM Tris-HCl, 75 mg / L NaNO ₃ , 30 mg / L NaH ₂ PO ₄ 2H ₂ O, 5 mL / L of trace metal mixture and 2.5 mL / L of vitamin complex, wherein the trace metal mixture contains 4.36 g / L Na ₂ EDTA ₂ H ₂ O, 3.15 g / L FeCl ₃ 6H ₂ O, of 10 mg / L of _{CoCl 2 6H2O, 22 mg / L} ZnSO 4 7H 2 O, 180 mg / L of _{MnCl 2 4H 2 O, 9.8 mg} / L of CuSO ₄ 5H ₂ O and 6.3 mg / L of Na ₂ MoO ₄ 2H ₂ O, and the vitamin complex may comprise 1 mg / L of vitamin B ₁₂ , 1 mg / L of biotin, 200 mg / L of thiamine HCl.

In the method of increasing the lipid productivity of the microalgae of the present invention, the osmotic stress medium comprises 50 g / L of senst salt, 10 mM Tris-HCl, 427.5 mg / L NaNO ₃ , 30 mg / L NaH ₂ PO ₄ 2H ₂ O, 5 mL / L of trace metal mixture and 2.5 mL / L of vitamin complex, wherein the trace metal mixture contains 4.36 g / L Na ₂ EDTA ₂ H ₂ O, 3.15 g / L FeCl ₃ 6H ₂ O, of 10 mg / L of _{CoCl 2 6H2O, 22 mg / L} ZnSO 4 7H 2 O, 180 mg / L of _{MnCl 2 4H 2 O, 9.8 mg} / L of CuSO ₄ 5H ₂ O and 6.3 mg / L of Na ₂ MoO ₄ 2H ₂ O, and the vitamin complex may comprise 1 mg / L of vitamin B ₁₂ , 1 mg / L of biotin, 200 mg / L of thiamine HCl.

In addition, the present invention relates to a method for culturing a transformed microalgae in a nitrogen-restricted medium and / or an osmotic stressed medium to induce overexpression of a gene encoding a basic helical loop helicase transcription factor protein (bHLH2) / RTI >

In the method for increasing the growth rate of the microalgae of the present invention, the nitrogen-restricted medium is selected from the group consisting of 15 g / L of sun salt, 10 mM of Tris-HCl, 75 mg / L of NaNO ₃ , 30 mg / L of NaH ₂ PO ₄ 2H ₂ O, a trace metal mixture of 5 mL / L and a 2.5 mL / L vitamin complex, wherein the trace metal mixture comprises 4.36 g / L Na ₂ EDTA ₂ H ₂ O, 3.15 g / L FeCl ₃ 6H ₂ O, L of CoCl ₂ 6H ₂ O, 22 mg / L of ZnSO ₄ 7H ₂ O, 180 mg / L of MnCl ₂ 4H ₂ O, 9.8 mg / L of CuSO ₄ 5H ₂ O and 6.3 mg / L of Na ₂ MoO ₄ 2H ₂ O, and the vitamin complex may comprise 1 mg / L of vitamin B ₁₂ , 1 mg / L of biotin, 200 mg / L of thiamine HCl.

In the method for increasing the growth rate of the microalgae of the present invention, the osmotic stressed medium comprises 50 g / L of senst salt, 10 mM Tris-HCl, 427.5 mg / L NaNO ₃ , 30 mg / L NaH ₂ PO ₄ 2H ₂ O, a trace metal mixture of 5 mL / L and a 2.5 mL / L vitamin complex, wherein the trace metal mixture comprises 4.36 g / L Na ₂ EDTA ₂ H ₂ O, 3.15 g / L FeCl ₃ 6H ₂ O, L of CoCl ₂ 6H ₂ O, 22 mg / L of ZnSO ₄ 7H ₂ O, 180 mg / L of MnCl ₂ 4H ₂ O, 9.8 mg / L of CuSO ₄ 5H ₂ O and 6.3 mg / L of Na ₂ MoO ₄ 2H ₂ O, and the vitamin complex may comprise 1 mg / L of vitamin B ₁₂ , 1 mg / L of biotin, 200 mg / L of thiamine HCl.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited by the following examples.

<Examples>

1. Materials and Methods

end. Strain and culture conditions

N. salina CCMP 1776 (National Center for Marine Algae and Microbiota) was used as the strain.

The strain can be stably distributed from National Center for Marine Algae and Microbiota (NCMA).

A method of obtaining the strain will be described in detail as follows.

The following figure is the website of "National Center for Marine Algae and Microbiota" (https://ncma.bigelow.org/).

If you select "CCMP1776" on the above website, the following screen appears.

Then, when "VIEW MORE" on the right side of the screen is selected, the following screen is displayed. After confirming the details of the strain on this screen, the strain can be easily obtained.

On the other hand, the medium was used in an improved F ₂ N medium (Kilian et al., 2011). The composition of the medium is as follows.

(One). Normal conditional medium

Steady-state medium is 15 g / L solar salt of (Sigma-Aldrich, USA), 10 mM of Tris-HCl (pH 7.6), 427.5 mg / L of NaNO3, 30 mg / L of _{NaH 2 PO 4 2H 2 O,} 5 mL / L of trace metal mixture 4.36 g / L Na ₂ EDTA ₂ H ₂ O, 3.15 g / L FeCl ₃ 6H ₂ O, 10 mg / L CoCl ₂ 6H ₂ O, 22 mg / L ZnSO ₄ 7H ₂ O L of MnCl ₂ 4H ₂ O, 9.8 mg / L of CuSO ₄ 5H ₂ O, 6.3 mg / L of Na ₂ MoO ₄ 2H ₂ O), and a 2.5 mL / L vitamin mixture (1 mg / L Of vitamin B ₁₂ , 1 mg / L of biotin, 200 mg / L of thiamine HCl) (Guillard Ryther, 1962).

The cultures were maintained at 25 ° C, 120 rpm, 120 photons / m ² / s, and incubated with 2% CO ₂ at 0.5 vvm.

(2). N-limiting medium

Basically, it was the same as the medium in the steady state but the concentration of NaNO ₃ was 75 mg / L and the nitrogen source was decreased by 5 times or more.

(3). Osmotic stress medium

It is basically the same as the badge in a normal state. The concentration of the salt was increased to 50 g / L and cultured.

I. Recombination of vectors

Using the vector pNs000 having the cleavage map of Fig. 2 as the start vector, a recombinant vector pNs302 (bHLH2) having the cleavage map of Fig. 1 was constructed using the Gibson assembly method.

As shown in FIG. 1, bHLH2 was expressed by the TUB promoter which is an endogenous promoter in the overexpression vector, and the expression of the protein was confirmed by western blotting by fusion of a flag tag.

At this time, Sh ble gene, a zeocin resistance gene, was expressed as a selection marker by UEP promoter which is an endogenous promoter.

All. Nannochloropsis saline Transformation

Transformation was performed using the particle bombardment technique. Nannochloropsis salina was inoculated to the steady-state medium with modified F ₂ N at an initial OD of 680 nm as 0.2% based on 0.5% v / v 2% CO ₂ , 120 μE fluorescence and 120 rpm for 1 week, lt; RTI ID = 0.0 &gt; OD680 &lt; / RTI &gt;

A 47 mm cellulosic acetate membrane filter (Sartorius Stedim Biotech, Germany) was placed on an F ₂ N solid medium and 108 cells were placed on it. Plasmids used for transformation were prepared by mixing microcial gold particles (Bio-Rad, USA) in 2.5 M CaCl ₂ , 0.1 M spermidine and 25% glycerol and vigorously stirring for 3 minutes, After washing with 70% ethanol, it was again dissolved in 100% ethanol.

The gene total technique was performed using a low-pressure gene delivery system (GDS-80; Wealtec, USA) under conditions of 1 linearized plasmid per shot at 700 psi helium and 3 cm target distance.

la. Analysis method

(One). Growth analysis

Cell density was measured by hemocytometer and dry cell weight (DCW) was measured after drying for 1 hour at 105 ° C with Whatman GF / C filter paper.

(2). Fatty acid methyl ester (FAME) analysis

10 mg of dried cells were injected with 2 ml of chloroform-methanol (2: 1, vol / vol) mixture and vigorously stirred at room temperature for 10 minutes or more. 1 ml of chloroform (0.5 mg of heptadecinic acid / 1 mL of chloroform) containing an internal standard substance was injected.

1 ml of methanol and 300 μl of sulfuric acid were added and mixed vigorously for 5 minutes. The mixture was reacted at 100 ° C for 10 minutes and then cooled to room temperature. Then, 1 ml of distilled water was added, and the mixture was stirred vigorously for 5 minutes. Then, the mixture was separated by centrifugation, and the organic phase was extracted and subjected to gas chromatography (GC) analysis.

FAME analysis was performed using a gas chromatograph (HP 6890, Agilent, USA) equipped with a flame ionized detector (FID) and HP-INNOWax polyethylene glycol column (HP 19091N-213, Agilent, USA)

The FAME peak was analyzed by increasing the temperature of the column from 50 ° C to 250 ° C at 15 ° C per minute and analyzed using a 37-component FAME standard mix (F.A.M.E.MIX C8-C24, Supelco, USA).

(3). Southern blot

Genomic DNA of WT, 3-6, and 3-11 traits was extracted with phenol and purified by 70% ethanol. The WT represents a wild type.

Ten grams of genomic DNA was treated with restriction enzymes SspI and KpnI , separated by electrophoresis and transferred to Amersham Hybond-N + membrane.

For detection, Sh ble The gene was purified by PCR and labeled with DIG-High Prime DNA Labeling and Detection Starter Kit II.

(4). Western blot

1.5X Laemmli sample buffer (62.5 mM Tris-HCl (pH 7.6), 7% sodium dodecyl sulfate (SDS), 25% glycerol, 5% beta-mercaptoethanol and 0.02% Were added to the harvested cells and reacted at 100 ° C for 5 minutes, followed by centrifugation to take the supernatant.

Protein samples were separated by SDS-PAGE (polyacrylamide gel electrophoresis) and transferred to PVDF membrane. The membrane was blocked with 5% skim milk solution in PBS and reacted with DYKDDDDK Tag Antibody (Cell Signaling Technology, USA) for 1 hour and then washed.

Then, the cells were reacted with Horseradish peroxidase (HRP) -conjugated anti-rabbit secondary antibody (Cell Signaling Technology, USA) for 1 hour, washed, and reacted with chemiluminescence (ECL) Respectively.

(5). Real-time polymerase chain reaction (qRT-PCR)

The qRT-PCR was carried out using SsoAdvancedTM Universal SYBRGreen Supermix (Bio-Rad, USA). The primers used are shown in Table 1 below.

The expression level of bHLH2 was normalized by actin and transgene bHLH2 was found to be the amount of bHLH2 expression in the transgene and endogenous and transgenic bHLH2 was found to be the total expression level of bHLH2 there was.

2. Results

end. bHLH  Transcription factor ( bHLH2 ) Expression level

There are three bHLH transcription factors in N. gaditana . Among them, the expression level of bHLH2 is increased in the exponential phase as shown in FIG.

Thus, bHLH2 can be regarded as a transcription factor that can support growth under stress conditions and various conditions, thus overexpressing the endogenous bHLH2 transcription factor of N. salina to increase growth under various stress conditions, .

I. Flag tag Western Blotting

Western blotting was performed using a flag tag antibody to confirm over-expression of the introduced bHLH2 after transformation with the pNs302 (bHLH2) vector, and the expected bands were detected in the vicinity of 65 kD First, phenotype screening was carried out by selective screening, and the results are shown in Fig.

All. NsbHLH2  Screening test of candidates

(Fig. 5A to Fig. 5C), the well plate screening result (Fig. 6) and the transformant 3-6 were found to show high lipid productivity for the candidates selected primarily by the flag tag, The transformant 3-11 showed a good growth pattern under various stress conditions, and a detailed phenotype assay was carried out with the two transformants.

la. By each condition NsbHLH2  Phenotype of transformant Assay

(One). Steady state

FIG. 7 shows the phenotypic assay results of the transformed microalgae (NsbHLH2) in a steady state, and the phenotypic assay results for WT and 3-6, 3-11 transformants were summarized in a steady state.

7, when WT and NsbHLH2 3-6 and 3-11 were cultured in a steady state, when the growth curve according to cell density was analyzed, it was found that 3-6, 3-11 transformants versus WT had better growth .

Analysis of the dry cell weight (DCW) showed that the DCW of 3-6 and 3-11 relative to WT increased by 36 and 48%, respectively, on the 8th day of culture. However, it was confirmed that the DCW difference became very small as the culture became 12 days.

In the case of the FAME content, the transformant was increased by about 5% or more compared to WT on the 8th day of culture and increased by 10% or more on the 12th day.

On the other hand, when the FAME yield was calculated considering the DCW and FAME contents, 43% of 3-6 and 60% of 3-11 were increased on the 8th day of culturing, 3 to 11%.

Thus, in the case of bHLH2 overexpressed transformants in the steady state, the growth rapidly occurs in the early stage and in the early stage, which may lead to an increase in FAME yield. However, when the culture reached the stationary phase, the difference in the DCW due to growth was decreased, and the FAME content and yield were increased, but the rate of increase was lower than that at the initial stage of culture.

(2). Nitrogen restriction condition

FIG. 8 shows the result of a nitrogen limitation condition phenotype assay of the transformed microalgae (NsbHLH2) under nitrogen restriction conditions. The phenotypic assay for WT and 3-6, 3-11 transcripts The results are summarized.

The N limitation condition is a condition in which the culture is performed at a temperature about 5 times lower than the NaNO ₃ concentration in the steady state. When the growth curve according to the cell density at the nitrogen restriction condition is analyzed, And it was confirmed that the growth rapidly occurred in the early stage.

In addition, when DCW was analyzed, it was confirmed that on the eighth day of culture, both transformants were increased by 20% or more compared to WT.

On the other hand, in the case of FAME content, the content of 3-6 was increased by 8% compared with that of WT, and the content of FAME was slightly decreased in 3-11. From the analysis of FAME yield, it was confirmed that the increase of DCW increased the ratio of WT to 3-6 to 33.2% and that of WT to 3-11 to 18.1%.

Analysis of the overall phenotype showed that bHLH2 overexpressed transformants grew relatively well, similar to the normal state, resulting in increased FAME y yield.

Under the stress condition of nitrogen limitation, it is considered that the growth of transformant compared to WT is maximized more than the normal state.

(3). Osmotic stress conditions

FIG. 9 shows phenotypic assay results of transgenic microalgae (NsbHLH2) under osmotic stress conditions. The phenotypic assay results for WT and 3-6, 3-11 transformants are summarized.

The osmotic stress condition is that the concentration of sea salt in osmotic stress condition is increased up to 50 g / L compared with that of sea salt in normal condition of 15 g / L.

In FIG. 9, when the cell growth curve (a) was analyzed under osmotic stress conditions, there was no significant difference between the wild type (WT) and the transformant. However, as shown in the DCW analysis results (b), bHLH2 over- High DCW was observed on day 8 of culturing, and the difference was decreased on day 12 of culture. On the other hand, in case of 3-11, DCW decreased on day 12 of culture.

In the case of FAME content (c), 3-6 was slightly decreased to less than 10% compared to WT. On the other hand, the FAME yield (d) d tended to increase by more than 30% on day 8 and 28% on WT and 3-6 on day 8, And in the case of 3-11, it decreased by 7.4%.

Under osmotic stress conditions, bHLH2 overexpressing transformants showed a positive effect on initial growth and a slight tendency in the latter half of culture.

(4). Sintering

Thus, bHLH2 overexpressing transgenic aids the early growth of the bHLH2 transcription factor, and it helps the early growth in comparison with the wild type (WT) under stress conditions, which also affects the FAME yield .

hemp. Sh ble Southern for genes Blotting

FIG. 10 shows results of Southern blotting in order to confirm whether the genes introduced into the 3-6 transformants and the 3-11 transformants are well inserted into the genomic DNA.

10, each of Sh ble It can be seen that one copy was introduced into the gene.

bar. qRT - PCR  Expression level analysis

Analysis of bHLH2 expression level and total bHLH2 expression level introduced under each stress condition were analyzed by qRT-PCR. The results are shown in FIGS. 11 and 12.

11 shows the result of analysis of the expression amount by the introduced bHLH2 qRT-PCR. Referring to FIG. 11, a primer for measuring the expression amount of the transgene of the transgene was prepared and subjected to qRT-PCR analysis. As a result, (WT), but it is expressed in 3-6 and 3-11.

FIG. 12 shows the results of analysis of the expression amount by the entire bHLH2 qRT-PCR. Referring to FIG. 12, the transformant 3-11 expresses a relatively large amount and shows normal conditions, nitrogen restriction conditions, And the osmotic stress condition, the expression level decreases under the stress condition.

In the case of the transformant 3-6, too, the expression amount itself is small, so that it is difficult to make accurate comparison, but the expression level is the lowest in osmotic stress conditions. A similar trend can be found by analyzing the results of qRT-PCR comparing total bHLH2 expression.

In the case of WT, 3-6 and 3-11, bHLH2 expression was the highest in normal condition, followed by nitrogen restriction, and finally bHLH2 expression in osmotic stress condition.

Compared to the wild type (WT), 3-6 was slightly increased in each condition, and 3-11 was clearly higher than that of wild type (WT).

Considering that the early growth rate of phenotype assay is 3-11 higher than 3-6, it is considered that the increase in the expression level of bHLH2 in 3-11 is due to the increase in the initial growth rate under various stress conditions.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. It will be understood that the present invention can be changed.

<110> Advanced Biomass R & D Center <120> ethod for increasing lipid productivity and growth of microalgae          by using an overexpression of bHLH transcription factors <130> 10114 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 1818 <212> DNA <213> Arabidopsis <400> 1 atgcgtagaa tgcatccatg ttgctcacgg ccccctcctc gcccgccctt gactcgcctc 60 tttccctgcc agtacgggtc tacctccatc cctcagggtc ctcccaccca tgcccacact 120 gcgccccctc ttcagccaac atcagcctac ccgcgctggc tctatgaagg cggagcgggg 180 acaggcgctg cgcccagcct tccatatcca cagcaacatc aaccaccatc gcagccaccg 240 ccaccgccgc agcagactgg cggaagttgg atcccggggg ccgggggagg agagctggta 300 aatggaggga ggagaatagg agaggcacgg ggggggggag aaggagggga aggaggaaca 360 ggaggaaccg cgcggccgca gacgttgggg tactgggggg ctggcgtgcc cagcgcgggc 420 tacagtggtg gagggctcgg gggaggggga ggggcggcga gcaaagggag cgagagcgac 480 tcctctgacg agggcagcac agcccgtcct ggcgggggca aggggcggaa gctgccgtcg 540 aacggccggg actcttcgga gctggagaag cgcctgaaga agaatgcccg ggagcaaaag 600 cgcgcgaaca agatcaacga ccagatcgcc atcatgaaga cgatcctgga gtctgtgggg 660 ctccgcccgc ctcccaccaa gtcctccatc ctcgccgagg gcatccagct tatccgcagc 720 ctgctcgcgc agatcggccc cgagggcaag gcgccaggca ccgagcccag ccaggcggag 780 ccctactggc ttctgtttcg gaagtcggcc acgcccctgg ccatgtgccg gccggacggg 840 cacgggcgct ttctggactg caatacccgc ttctgcgagg tcttcgggtg cacgcccgag 900 gagctgcaga aggagacgcc cctctccttc acccgcgagt acgatgcgga gaacacccag 960 cgggtcctgg ccctggtgag tcagcccggg ctcttggacg agagctctcc cctggaggtg 1020 gtgctcacca acaagcacca gcagcgcttc ctcgttcgcg tctcctccct cctggacgac 1080 taccagaagc cgcgcttcct cctcttcacg cccaaggcca tgctccgggg cgtgaacgga 1140 caggaaccct cctctgcccc gccctcctct ttcgcctctg ccccgcccct cccttcgcag 1200 cccgcccccc accccgcagt ctcggctggc accgtggccc cgcccctcat gccctcccag 1260 tcctccacca tgccttcctc cgcctcctcc tcctcctcct cgctgggcgc catgctgccc 1320 attcccatca gtcaagctcc cgacccactc ttgccgcaga tctccctcct atccccgcag 1380 gcaccgccca ctcgcgagcc tcgagccccc cacatccaca atcagctcca tcaccagcac 1440 taccagcaac atctacacca catcctccag cagcataacc caggatcgcc gcaagacgcg 1500 ccgccacagc agcagcggca gcagcaagcc tttcctgccg ccctgcagga ggcatcctca 1560 tcctctcccc aggcgggacg cgtggccgag aacgatcaga agccttcccc tcccccctct 1620 tcccttcaac cttcgtttcc ctcctccttc tacaacggag gccgcgtcgg tggtggaccg 1680 agcaacagcc attgctcgcc ccgctcctcg cccaacaact cgtcaaggga gccagtcttg 1740 ggcgttgtct cctctttgac cacaggtcct ccgccaacac taaagccgag cggcaagagt 1800 caagaggcgg ctgtttga 1818 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RT-actin fwd <400> 2 gtgtttccct ccatcgtg 18 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RT-actin rev <400> 3 ccagttcgtc acaataccg 19 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RT-TbHLH2 fwd <400> 4 ttctgtctcc ctcacacg 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RT-TbHLH2 rev <400> 5 gtagacccgt actggcag 18 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RT-EbHLH2 fwd <400> 6 gaacaagatc aacgaccaga t 21 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> RT-EbHLH2 rev <400> 7 cgaggatgga ggacttgg 18

Claims (12)

In the recombinant vector pNs302 (bHLH2), which contains a gene coding for a basic helical loop helicase transcription factor protein (bHLH2), a tubulin promoter (P _tub ) and a tubulin terminator (T _tub )
The recombinant vector pNs302 (bHLH2), wherein the gene (bHLH2) encoding the basic helical loop helicase transcription factor protein consists of the nucleotide sequence of SEQ ID NO: 1. 2. The recombinant vector pNs302 (bHLH2) according to claim 1, wherein said vector overexpresses a gene encoding a basic helical loop helicase transcription factor protein (bHLH2). delete A transgenic microalgae into which the recombinant vector of claim 1 has been introduced and into which a basic helical loop helical transcription factor (bHLH2) has been inserted. The transgenic microalgae of claim 4, wherein the microalgae are Nanochioropsis sp. The transformed microalgae according to claim 5, wherein the microalgae is Nanochioropsis salina sp. The transformed microalgae of any one of claims 4 to 6 is cultured in at least one culture medium selected from the group consisting of nitrogen-restricted medium and osmotic stress medium to produce a gene encoding a basic helical loop helicase transcription factor protein (bHLH2) Wherein the over-expression of the microalgae is induced by over-expression of the microalgae. The method of claim 7, wherein the nitrogen-limiting medium is 15 g / L solar salt in, 10 mM of Tris-HCl, 75 mg / L of NaNO _3, 30 mg / L of _{NaH 2 PO 4 2H 2 O,} 5 mL / L Of a trace metal mixture and 2.5 mL / L of a vitamin complex,
The trace metal mixture contained 4.36 g / L Na ₂ EDTA ₂ H ₂ O, 3.15 g / L FeCl ₃ 6H ₂ O, 10 mg / L CoCl ₂ 6H ₂ O, 22 mg / L ZnSO ₄ 7H ₂ O, 180 mg / MnCl ₂ 4H ₂ O, 9.8 mg / L CuSO ₄ 5H ₂ O and 6.3 mg / L Na ₂ MoO ₄ 2H ₂ O,
Wherein said vitamin complex comprises 1 mg / L of vitamin B ₁₂ , 1 mg / L of biotin, 200 mg / L of thiamine HCl. The osmotic stress medium according to claim 7, wherein the osmotic stressed medium comprises 50 g / L of senst salt, 10 mM Tris-HCl, 427.5 mg / L NaNO ₃ , 30 mg / L NaH ₂ PO ₄ 2H ₂ O, 5 mL / L Of a trace metal mixture and 2.5 mL / L of a vitamin complex,
The trace metal mixture contained 4.36 g / L Na ₂ EDTA ₂ H ₂ O, 3.15 g / L FeCl ₃ 6H ₂ O, 10 mg / L CoCl ₂ 6H ₂ O, 22 mg / L ZnSO ₄ 7H ₂ O, 180 mg / MnCl ₂ 4H ₂ O, 9.8 mg / L CuSO ₄ 5H ₂ O and 6.3 mg / L Na ₂ MoO ₄ 2H ₂ O,
Wherein said vitamin complex comprises 1 mg / L of vitamin B ₁₂ , 1 mg / L of biotin, 200 mg / L of thiamine HCl. The transformed microalgae of any one of claims 4 to 6 is cultured in at least one culture medium selected from the group consisting of nitrogen-restricted medium and osmotic stress medium to produce a gene encoding a basic helical loop helicase transcription factor protein (bHLH2) Of the microalgae in the microalgae. 11. The method of claim 10, wherein the nitrogen-limiting medium is 15 g / L solar salt in, 10 mM of Tris-HCl, 75 mg / L of NaNO _3, 30 mg / L of _{NaH 2 PO 4 2H 2 O,} 5 mL / L Of a trace metal mixture and 2.5 mL / L of a vitamin complex,
The trace metal mixture contained 4.36 g / L Na ₂ EDTA ₂ H ₂ O, 3.15 g / L FeCl ₃ 6H ₂ O, 10 mg / L CoCl ₂ 6H ₂ O, 22 mg / L ZnSO ₄ 7H ₂ O, 180 mg / MnCl ₂ 4H ₂ O, 9.8 mg / L CuSO ₄ 5H ₂ O and 6.3 mg / L Na ₂ MoO ₄ 2H ₂ O,
Wherein said vitamin complex comprises 1 mg / L of vitamin B ₁₂ , 1 mg / L of biotin, 200 mg / L of thiamine HCl. The osmotic stress medium according to claim 10, wherein the osmotic stressed medium comprises 50 g / L of senst salt, 10 mM Tris-HCl, 427.5 mg / L NaNO ₃ , 30 mg / L NaH ₂ PO ₄ 2H ₂ O, 5 mL / L Of a trace metal mixture and 2.5 mL / L of a vitamin complex,
The trace metal mixture contained 4.36 g / L Na ₂ EDTA ₂ H ₂ O, 3.15 g / L FeCl ₃ 6H ₂ O, 10 mg / L CoCl ₂ 6H ₂ O, 22 mg / L ZnSO ₄ 7H ₂ O, 180 mg / MnCl ₂ 4H ₂ O, 9.8 mg / L CuSO ₄ 5H ₂ O and 6.3 mg / L Na ₂ MoO ₄ 2H ₂ O,
Wherein said vitamin complex comprises 1 mg / L of vitamin B ₁₂ , 1 mg / L of biotin, 200 mg / L of thiamine HCl.

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