CN113736806B - Gene for improving oil synthesis of marine nannochloropsis and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a microalgae protein kinase NoS1P gene and application thereof. The invention belongs to the field of metabolic engineering, and particularly relates to a microalgae protein kinase gene and application thereof. The gene is a protein kinase family gene and application thereof. The gene is protein kinase family gene NoS1P, and the sequence thereof is shown in SEQ ID No. 1. Functional analysis shows that the total lipid content and neutral lipid content of nannochloropsis oculata engineering cells over expressing the gene are obviously improved, and the nannochloropsis oculata engineering cells can be used for genetic modification of microalgae, plants, crops and the like, and the engineering microalgae (S1 Poe) with high expression quantity of the gene is obtained by means of genetic engineering.

Description

Gene for improving oil synthesis in marine Nannochloropsis and its use

technical field

The invention relates to the field of biotechnology, in particular to microalgae protein kinase _NOS1P gene and application thereof.

Background technique

Due to the depletion of fossil fuel reserves and the increasingly serious environmental problems, new environmentally friendly fuels have been valued by many countries. The development of biofuels can effectively reduce the dependence of large-scale industries on non-renewable fossil resources, so as to achieve the purpose of reducing energy consumption and waste emissions, and bring economic benefits to a certain extent, and enhance my country's international competitiveness. my country's renewable energy and resources have great potential and will inevitably occupy an extremely important position in the future energy supply. Biological raw materials commonly used to prepare biofuels include microalgae, rapeseed, jatropha, etc. After years of research, microalgae stand out from many candidate organisms. Many microalgae can be cultivated in coastal tidal flats and saline-alkali land, and quite a few microalgae (such as spirulina and chlorella) have been industrialized. Due to the strong stress resistance of microalgae cells, they can perform photosynthesis in many extreme environments. Through photosynthesis, the eutrophic water body in the environment is used as a carbon source and a nitrogen source to produce a large amount of organic matter, including biological oils, and sunlight and nutrient-rich water. As an energy source, nutrient water has the characteristics of cleanliness and adequacy.

On the other hand, because microalgae are rich in some unsaturated fatty acids, such as DHA, EPA, etc., which are beneficial to human health, it is very meaningful to increase the oil content of microalgae cells.

The use of genetic engineering technology to improve microalgae is a hot spot in recent years. It is also a feasible way to use genetic engineering technology to improve and cultivate high-yielding microalgae.

In this field, it is necessary to develop alternative genes capable of increasing the oil accumulation of microalgae, as well as a method for improving the oil accumulation of microalgae by using genetic engineering technology.

The Kennedy pathway is the metabolic pathway for most plant cells to synthesize neutral oils such as triacylglycerol (TAG). The _NOS1P gene encodes a protein kinase, which has a synergistic effect with many genes (DGAT Ⅰ and DGAT Ⅱ) in the Kennedy pathway. The expression levels of DGAT Ⅰ and DGAT Ⅱ also increased in the algal strains after the _NO S1P gene was overexpressed. Its cells also had a higher lipid content than wild-type cells.

Contents of the invention

In view of this, the present invention provides microalgae protein kinase _NOS1P gene and application thereof. The total lipid content and neutral lipid content of Nannochloropsis engineered cells overexpressing the gene are significantly improved, and can be used for genetic modification of microalgae, plants and crops. The present invention obtains a strain of the gene high Expression levels in engineered microalgae.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides microalgae protein kinase NoS1P gene, the cDNA sequence of the microalgae protein kinase NoS1P gene is:

i). A nucleotide sequence as shown in SEQ ID No.1; or

ii). The nucleotide sequence shown in SEQ ID No.1 is substituted, deleted and/or increased by one or more nucleotides and expresses the same functional protein; or

iii). A nucleotide sequence that hybridizes to the sequence shown in SEQ ID No.1 and expresses the same functional protein under stringent conditions, and the stringent conditions are 0.1×SSPE containing 0.1% SDS or 0.1× hybridize at 65°C in SSC solution, and wash the membrane with this solution; or

iv). A nucleotide sequence that has more than 90% identity with the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.

Based on the above research, the present invention also provides the protein encoded by the microalgae protein kinase NoS1P gene, which has:

(I), the amino acid sequence shown in SEQ ID No.2; or

or

(III), an amino acid sequence having at least 90% homology with the sequence described in (I) or (II).

The present invention also provides an expression cassette containing the microalgae protein kinase NoS1P gene.

The present invention also provides an expression vector containing the microalgae protein kinase NoS1P gene.

The present invention also provides a host containing the microalgal protein kinase NoS1P gene.

The present invention also provides the application of the microalgae protein kinase NoS1P gene in the genetic improvement of microalgae and/or plants.

More importantly, the present invention also provides the application of the microalgae protein kinase NoS1P gene in improving the oil synthesis ability of microalgae and/or plants.

In some embodiments of the present invention, the microalgae include one or more of green algae, diatoms, red algae, golden algae, and brown algae, and the plants include Arabidopsis, wheat, rice, corn, One or more of cotton.

The present invention also provides the application of the microalgae protein kinase NoS1P gene in preparing transgenic plants and/or transgenic microalgae.

In addition, the present invention also provides a combination of amplification primers for the microalgal protein kinase NoS1P gene, including an upstream primer as shown in SEQ ID No.3 and a downstream primer as shown in SEQ ID No.4; or

The upstream primer shown in SEQ ID No.5 and the downstream primer shown in SEQ ID No.6.

In addition, the present invention also provides a method for constructing transgenic microalgae, comprising the following steps:

Step 1: extract the total RNA of microalgae, and reverse transcribe to obtain the first strand of cDNA;

Step 2: Using the first strand of cDNA as a template, the upstream primer SEQ ID No.3 and the downstream primer SEQ ID No.4 as primers, obtain the cDNA sequence of the protein kinase NoS1P gene through PCR amplification reaction;

Step 3: Construct the overexpression vector of NoS1P gene;

Step 4: Using transgenic technology to transform the target algae strain with the overexpression vector carrying the protein kinase NoS1P gene to obtain the algae strain with stable inheritance of the transgene.

The invention provides a microalgae protein kinase gene and its application. The gene is protein kinase family gene and application thereof. The gene is protein kinase family gene NoS1P, and its sequence is shown in SEQ ID NO:1. Functional analysis shows that the total lipid content and neutral lipid content of Nannochloropsis engineering cells overexpressing the gene are significantly increased, which can be used for genetic transformation of microalgae, plants and crops. This patent has obtained a Strain engineered microalgae with high expression of this gene. The total lipid content and neutral lipid content of marine Nannochloropsis strains overexpressing this gene were significantly higher than those of wild-type strains (P<0.05).

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art.

Figure 1 shows the detection of NOS1P transformants by agarose gel electrophoresis in Example 1 of the present invention; the leftmost lane is DNAmaker, and the remaining lanes are transformants; wherein, 33-2 corresponds to No. 2 lane, 33-6 corresponds to No. 6 lane, PC is Positive clone (positive clone). Positive clone is the recipient cell after the exogenous gene is connected to the carrier. The primers are designed according to the vector sequence and the foreign gene sequence. The DNA of the recipient cell is used as a template and can be amplified by PCR. Verify the product, that is, the transformed transformant that has been transformed successfully, and the target band size is 2080bp;

Figure 2 shows the vector map, the transformant can be verified by the upstream primer 5-vcpf and the downstream primer S1Poe33R, and the verified product is the PCR product of the 2080bp fragment in Figure 1; PC is a positive clone, which can be interpreted as only containing the target linear DNA vector can be amplified The resulting fragment, the template of which can be the vector before transformation or the transformed transformant after transformation;

Fig. 3 shows the NOS1P gene relative expression level of the high oil-yielding algae strain S1Poe and the wild-type algae strain inoculation 0 hours in the embodiment of the present invention 3;

Fig. 4 shows the relative expression of NOS1P gene of the high-light-producing algal strain S1Poe and the wild-type algal strain in Example 3 of the present invention for 96 hours;

Fig. 5 shows the relative expression level of NOS1P gene of high oil-yielding algae strain S1Poe and wild-type algae strain in Nitrogen Deficiency culture in Example 3 of the present invention for 96 hours;

Fig. 6 shows the comparison of the oil content of the high-oil-yielding algal strain S1Poe in Example 4 of the present invention with that of the wild-type algal strain in high-light culture for 8 days;

Fig. 7 shows the comparison of the oil content of the high-oil-yielding algae strain S1Poe in Example 4 of the present invention and the wild-type algae strain under nitrogen deficiency for 8 days.

Detailed ways

The invention discloses the microalgal protein kinase _NO S1P gene and its application, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.

The object of the present invention is to provide protein kinase NoS1P gene and its encoded protein.

Another object of the present invention is to provide the application of protein kinase NoS1P gene in improving microalgae and plant oil synthesis ability.

In order to achieve the purpose of the present invention, the present invention clones and obtains the protein kinase NoS1P gene from Nannochloropsis marine algae, and the cDNA sequence of the NoS1P gene is:

i) the nucleotide sequence shown in SEQ ID No.1; or

ii) A nucleotide sequence in which the nucleotide sequence shown in SEQ ID No.1 is substituted, deleted and/or increased by one or more nucleotides and expresses the same functional protein; or

iii) a nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO:1 and expresses the same functional protein under stringent conditions, and the stringent conditions are 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS solution, hybridize at 65°C, and wash the membrane with this solution; or

iv) A nucleotide sequence having more than 90% homology with the nucleotide sequence of i), ii) or iii) and expressing the same functional protein.

The sequencing results showed that protein kinase NoS1P is a member of the protein kinase family, and the open reading frame of the protein kinase NoS1P gene is 1878 bp in length, encoding a protein consisting of 625 amino acids (SEQ ID No.2). According to the bioinformatics analysis of the protein kinase NoS1P gene and the expression pattern judgment under high light, nitrogen deficiency and other stresses, the gene may be involved in the lipid metabolism regulation pathway of microalgae and so on.

The present invention also provides an expression cassette containing the protein kinase NoS1P gene.

The present invention also provides an overexpression vector containing the NoS1P gene of the protein kinase.

The overexpression vector carrying the target gene can be introduced into plant cells or microalgae cells by conventional biotechnology methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation.

The invention also provides microalgae engineered cells overexpressing the protein kinase NoS1P gene coding region.

The present invention also provides the application of the protein kinase NoS1P gene in improving the oil synthesis ability of microalgae (such as Nannochloropsis, etc.).

The invention also provides the application of the protein kinase NoS1P gene in preparing transgenic microalgae.

The invention also provides a method for constructing the transgenic microalgae.

The present invention further provides fluorescent quantitative PCR detection primers SEQ ID No.5 and SEQ ID No.6 of protein kinase NoS1P gene, and the primer sequences are as follows:

Upstream primer SEQ ID No.5 sequence

NoS1P-QF: 5’-GGCGGTTGCCCCTGTAGA-3’

Downstream primer SEQ ID No.6 sequence

NoS1P-QR: 5'-TCGGGAAATGAGGTCTTGGA-3'.

It can be used to detect the expression of the protein kinase NoS1P gene in the Nannochloropsis engineering cells, and the results show that the transcription level of the NoS1P gene in the engineering cells is higher than that of the wild type.

It can be used to detect the expression of the protein kinase NoS1P gene, and the results show that the expression of the protein kinase NoS1P gene is induced by high light and nitrogen deficiency stress.

It can be used to detect the dry weight, oil content, and neutral lipid content of wild-type algal strains and overexpressed algal strains. The results show that the total lipid content and neutral lipid content of overexpressed marine Nannochloropsis strains are significantly higher than those of wild-type algal strains (P<0.05).

In the microalgal protein kinase _NOS1P gene and its application provided by the present invention, the raw materials and reagents used can be purchased from the market.

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J&Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

Below in conjunction with embodiment, further set forth the present invention:

Embodiment 1: protein kinase NoS1P gene cloning

1. Extract the total RNA of Nannochloropsis sp., and reverse transcribe to obtain the first strand of cDNA.

2. Using the first strand of cDNA as a template, design upstream SEQ ID No. 3 and downstream primer SEQ ID NO: 4 as primers, and obtain the cDNA sequence of NoS1P gene through PCR amplification reaction.

The primer sequences are as follows:

SEQ ID No.3:

5'-AGGATGACGATGACAAGCATGCACTGCCCTAATAAGAGAATG-3',

SEQ ID No.4:

5'-TCCTCCTGGGATCCCCCGGGATCACCTATACTCCCTTTGAAG-3'.

Use 2×Taq PCR MasterMix (Takara) for PCR amplification, the reaction system is: 2 microliters of cDNA template, 1 microliter each of primers F and R (10 μmol/L), 25 microliters of 2×PCR MasterMix, add ddH ₂ O to 50 µl.

The PCR amplification program was: denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 90 seconds, extension at 72°C for 2 minutes, a total of 35 cycles; and finally extension at 72°C for 10 minutes.

3. The amplified products were detected by 1% agarose gel electrophoresis and sequenced.

4. Cut out the target fragment, recover and purify it by gel, and clone it into the pMD18-T vector. After colony PCR detection, send it to Shanghai Sangon Biotechnology Co., Ltd. for sequencing verification. The correct plasmid after sequencing verification is named NoS1P-18T. The cDNA sequence of NoS1P gene (SEQ ID No.1) is 1878 bp, encoding the protein NoS1P (SEQ ID No.2) consisting of 625 amino acids.

Example 2: Preparation of Nannochloropsis engineering cells overexpressing the NoS1P gene

1. Bacterial strains and their cultivation: Nannochloropsis grows in F2 solid medium and F2 liquid medium.

2. The resistance gene tag method was used to construct the overexpression vector of Nannochloropsis, using the bleomycin resistance tag. According to molecular biology techniques familiar to those skilled in the art, the resistance tag is amplified, and the fragment containing the screening marker is cut with an appropriate restriction endonuclease, and Nannochloropsis is transformed.

3. Electrotransform Nannochloropsis cells, and introduce the fragment containing the resistance tag into the cells. That is, 0.5 micrograms of linearized plasmid and 6 million algal cells were placed in a 0.2 cm electroporation cell (Bio-Rad) with 2.2 kV pulses (Bio-Rad electroporation instrument, 50 μF).

4. Through antibiotic screening and according to the following protein kinase NoS1P gene-specific primers, transformant cells containing protein kinase NoS1P gene overexpression exogenous vectors were identified.

5'-TTTAACTAGGATACTGCCGGGTG-3' (as shown in SEQID No.7)

5'-TTACGGCTGAAGTCCACATCCT-3' (as shown in SEQ ID No. 8).

Example 3: Identification of NoS1P gene transcription level in engineered cells

1. Use a centrifuge tube to take a certain amount of S1Poe algal strain sample into a 1.5 ml centrifuge tube, and use the Adelaide Plant RNA Extraction Kit to extract S1Poe RNA.

2. Take 500 microliters of CLB lysate to a 1.5 ml centrifuge tube (if crystals are precipitated in the lysate, just put it in a water bath at 65°C for a period of time), add 25 microliters of 5% β-mercaptoethanol to the lysate, and the above solution Preheat at 65°C after mixing.

3. Bake the glass beads for grinding in an oven at 200°C for 3 hours, and let them cool at room temperature. Take an appropriate amount of dried glass beads and 100 microliters of cooled lysate in the sample, and vibrate with a bead mill for 1-2 minutes. Add the remaining 425 μl of lysate to the sample and shake for 15 seconds on a shaker. Water bath at 65°C for 5 minutes, invert and mix once every minute.

4. The above product was placed in a centrifuge at 13000 r/min for 10 minutes. Collect 500 microliters of supernatant in a new centrifuge tube, add 0.5 times the volume of absolute ethanol, and mix by pipetting.

5. Transfer all the above solutions to the genome cleanup column (note that each time the solution should be less than 700 microliters, and add in several times after centrifugation), centrifuge at 13000 r/min for 2 minutes, and pour off the filtrate.

6. Put the genome cleanup column back into a new centrifuge tube, add 500 microliters of RLT plus lysate, centrifuge at 13000r/min for 30 seconds. Collect the filtrate, add 0.5 volume of absolute ethanol and mix well.

7. Add the above mixture into a new adsorption column, centrifuge at 13000 r/min for 2 minutes. After discarding the filtrate, add 700 microliters of deproteinized solution to the adsorption column, place at room temperature for 1 minute, centrifuge at 13000 r/min for 2 minutes, and pour off the filtrate.

8. Add 500 microliters of rinse solution to the adsorption column in the above step, centrifuge at 13000 r/min for 2 minutes, and discard the filtrate. Repeat this step once, and centrifuge the adsorption column discarding the filtrate once at 13000 r/min for 2 minutes.

9. Take out the adsorption column, put it in an enzyme-free centrifuge tube, add 30-50 microliters of enzyme-free water, place the solution at room temperature for 1 minute, 13000 r/min, 1 minute. After verification by gel electrophoresis, store at -20°C for later use.

10. Use the TaKaRa PrimeScript ^TM RT reagent Kit with gDNA Eraser kit to prepare a solution according to the following system to remove genomic DNA, and perform it on ice.

Table 1

Mix the solution evenly. After the PCR instrument is preheated, react at 42°C for 2 minutes and keep warm at 4°C.

11. Use the TaKaRa PrimeScript ^TM RT reagent Kit with gDNA Eraser kit to reverse the RNA to cDNA, prepare the reaction system according to the following table, and proceed on ice.

Table 2

Mix well, and after the PCR machine is preheated, set the reverse transcription conditions to 37°C for 15 minutes; 85°C for 5 seconds; 4°C.

12. Fluorescence quantitative detection of S1P gene expression level.

table 3

The fluorescence quantification operation is:

step1: Pre-denaturation, 95°C, 30 s, 20°C/s, 1cycle.

step2: PCR reaction, 95°C, 5 s, 20°C/s; 55°C, 30 s, 20°C/s; 72°C, 30 s, 20°C/s, 40 cycles.

Step3: Draw the melting curve.

95°C, 0s, 20°C/s

65°C, 15s, 20°C/s

95°C, 0s, 0.1°C/s

As shown in Figures 3-5 and Table 4, the relative expression level of NoS1P in S1Poe was significantly higher than that of the wild-type marine Nannochloropsis strain.

Table 4. Relative expression of S1P gene in WT, 33-2, 33-6 cultured under high light (200 μmol/m ² /s ^-1 ) and nitrogen deficiency

Among them, 33-2 corresponds to No. 2 swimming lane in Fig. 1, and 33-6 corresponds to No. 6 swimming lane in Fig. 1 .

Both 33-2 and 33-6 are SIP overexpressed transformants, and both are Nannochloropsis screened in the experiment process of the present invention.

Example 4: Production of Oil by Nannochloropsis Engineering Cells Overexpressing the Protein Kinase NoS1P Gene

Utilize the Nannochloropsis engineering cell S1Poe of overexpressing the NoS1P gene obtained in Example 1 for oil production, the specific method is as follows:

Activated Nannochloropsis wild-type and S1Poe strains were inoculated in photobioreactors (PBR; diameter 33 mm, height 60 cm) containing freshly modified F2 medium.

Culture PBR under the culture conditions set in Table 5, high light (200 μmol/m ² /s ^-1 ), and nitrogen deficiencies, that is, the improved F2 medium without adding sodium nitrate as a nitrogen source.

Improved F2 medium, 35 g/L sea salt self-made seawater, 1.21 g Tris, 1000 mg/L NaNO ₃ , 67 mg/L NaH ₂ PO ₄ ·H ₂ O, 0.0196 mg/L CuSO ₄ ·5H ₂ O, 0.0126 mg/L NaMoO ₄ 2H ₂ O, 0.044 mg/L ZnSO ₄ 7H ₂ O, 0.01092 mg/L CoCl ₂ 6H ₂ O, 0.36 mg/L MnCl ₂ 4H ₂ O, 3.65 mg/L FeCl ₃ 6H ₂ O, 4.37 mg/L Na ₂ EDTA·2H ₂ O, 0.005 mg/L cobalamin, 0.005 mg/L biotin, 0.1 mg/L thiamine hydrochloride

Cells were harvested eight days later to determine dry weight and total lipid content.

The results showed that under the nitrogen deficiency treatment, compared with the wild type, the oil content of Nannochloropsis strain S1Poe was increased (Fig. 6~7).

Table 5. Lipid content and TAG content of WT, 33-2, 33-6 under high light (200 μmol/m ² /s ^-1 ), nitrogen deficiency, high light and nitrogen deficiency culture

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Hainan University

<120> Gene for improving oil synthesis in marine Nannochloropsis and its use

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aacggggaga gggtatcgat atcgacgggc ctgtcgagta ggtcgtttgt gtctgcggcg 1260

gaggcggttg cccctgtaga catgcgttgg gaagcggaca attcgtcgtc aatgccgact 1320

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Ser Ser Ser Thr Ser Ala Ser Ser Ser Thr Ala Val Ser Asp Lys Pro Pro

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Arg Pro Pro Ile Pro Ile Thr Val Thr Asp Val Asn Ser Thr Thr Asp

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Ala Asp Ala Pro Pro His Gly Leu Gly Glu Arg Glu Asp Gly Phe Pro

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Asn Gly Cys Pro Leu His His Asp Pro Leu Ala Leu Thr Arg Gln Leu

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Ala Ala Cys Gly Ile Thr Leu Tyr Thr Val Gly Cys Glu Pro Ala Ile

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Ser Thr Tyr Glu Asn Cys Lys Asp Ile Leu Ile Phe Met Ala Glu Ala

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Thr Gly Gly Gln Ala Leu Val Leu Glu Ser Ala Ser Leu Leu Ala Ser

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Val Ile Leu Ala Gly Ala Gln Glu Glu Val Gly Leu Thr Arg Leu Glu

305 310 315 320

Arg Glu Val Lys Glu Val Met Arg Glu Ala Gly Arg Glu Ala Gly Glu

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Glu Glu Asp Glu Asp Val Trp Val Ala Arg Val Lys Glu Ala Leu Arg

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Arg Lys Gly Thr Arg Thr Arg Gln Leu Lys Thr Asn Met Arg Arg Met

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Thr Ser Val Gln Thr Glu Val Ile Ala Ser Cys Ser Ser Leu Glu Glu

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Ala Lys Arg Arg Leu Gly Ser Glu Arg Ala Arg Glu Val Glu Glu Glu Glu

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Asn Gly Glu Arg Val Ser Ile Ser Thr Gly Leu Ser Ser Arg Ser Phe

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Val Ser Ala Ala Glu Ala Val Ala Pro Val Asp Met Arg Trp Glu Ala

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Asp Asn Ser Ser Ser Met Pro Thr Ser Gly Val Gly Trp Glu Gly Glu

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Phe Leu Pro Ala Pro Leu Phe Ser Pro Thr His Thr Tyr Ser Leu Thr

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Arg

625

<210> 3

<211> 42

<212>DNA

<213> Artificial Sequence

<400> 3

aggatgacga tgacaagcat gcactgccct aataagagaa tg 42

<210> 4

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 4

tccctcctggg atcccccggg atcacctata ctccctttga ag 42

<210> 5

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggcggttgcc cctgtaga 18

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tcgggaaatg aggtcttgga 20

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tttaactagg atactgccgg gtg 23

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ttacggctga agtccacatc ct 22

Claims (10)

1. The microalgae protein kinase NoS1P gene is characterized in that the cDNA sequence of the microalgae protein kinase NoS1P gene is a nucleotide sequence shown as SEQ ID No. 1.

2. The protein encoded by the microalgal protein kinase NoS1P gene of claim 1, having the amino acid sequence shown in SEQ ID No. 2.

3. An expression cassette comprising the microalgal protein kinase NoS1P gene of claim 1.

4. An expression vector comprising the microalgal protein kinase NoS1P gene of claim 1.

5. Nannochloropsis containing the microalga protein kinase NoS1P gene of claim 1.

6. Use of a microalgal protein kinase NoS1P gene of claim 1 in the genetic improvement of microalgae; the microalgae is nannochloropsis.

7. The application of the microalgae protein kinase NoS1P gene in improving the oil and fat synthesis capability of microalgae according to claim 1; the microalgae is nannochloropsis.

8. The use of the microalgal protein kinase NoS1P gene of claim 1 in the preparation of transgenic microalgae; the microalgae is nannochloropsis.

9. The microalgae protein kinase NoS1P gene amplification primer combination as claimed in claim 1, which is characterized by comprising an upstream primer shown as SEQ ID No.3 and a downstream primer shown as SEQ ID No. 4; or

An upstream primer shown as SEQ ID No.5 and a downstream primer shown as SEQ ID No. 6.

10. The construction method of the transgenic microalgae is characterized by comprising the following steps:

step1: extracting total RNA of microalgae, and performing reverse transcription to obtain a first cDNA chain;

step2: taking a cDNA first chain as a template, and taking an upstream primer SEQ ID No.3 and a downstream primer SEQ ID No.4 as primers, and obtaining a cDNA sequence of a protein kinase NoS1P gene through PCR amplification reaction;

and step3: constructing an overexpression vector of the NoS1P gene;

and 4, step 4: transforming an overexpression vector with a protein kinase NoS1P gene into a target algae strain by using a transgenic technology to obtain a transgenic stably inherited algae strain;

the microalgae is nannochloropsis.

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