CN113736806A - 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 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 (hereinafter referred to as S1Poe) with high expression of the gene is obtained by means of genetic engineering.

Description

Gene for improving oil synthesis of marine nannochloropsis and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to microalgal protein kinase N_OS1P gene and application thereof.

Background

Due to depletion of fossil fuel reserves and increasing environmental problems, new environmentally friendly fuels have been valued by many countries. The development of the biofuel can effectively reduce the dependence of large-scale industry on non-renewable fossil resources so as to achieve the aims of reducing energy loss and reducing the discharge amount of wastes, bring economic benefits to a certain extent and enhance the international competitiveness of China. Renewable energy and resource potential in China are very large, and the energy supply inevitably occupies an extremely important position in future energy supply. The biological raw materials commonly used for preparing the biofuel comprise microalgae, rape, jatropha curcas and the like, and the microalgae are separated from a plurality of candidate organisms after years of research. Many microalgae can be cultivated in coastal zones, beach and saline-alkali land, and quite a lot of microalgae (such as spirulina and chlorella) are industrially produced. Due to the strong stress resistance of microalgae cells, photosynthesis can be carried out in a plurality of extreme environments, eutrophic water in the environment is used as a carbon source and a nitrogen source to generate a large amount of organic matters through the photosynthesis, wherein the organic matters comprise biological grease, and sunlight and the eutrophic water are used as energy sources and have the characteristics of cleanness and sufficiency.

On the other hand, since microalgae is rich in some unsaturated fatty acids, such as DHA, EPA, etc., which are beneficial to human health, it is a significant matter to increase the lipid content of microalgae cells.

The development of microalgae using genetic engineering techniques has been a hotspot in recent years. The genetic engineering technology is used for improvement, and the cultivation of the microalgae with high oil yield is also a feasible way.

There is a need in the art to develop alternative genes that can increase the lipid accumulation of microalgae, and methods for increasing lipid accumulation of microalgae using genetic engineering techniques.

The Kennedy pathway (Kennedy pathway) is a metabolic pathway for many plant cells to synthesize neutral lipids such as Triacylglycerol (TAG). N is a radical of_OThe S1P gene encodes a protein kinase which has a synergistic effect with many genes in the Kennedy pathway (DGAT I and DGAT II), N_OThe algal strains in which the S1P gene was overexpressed also exhibited increased expression levels of DGAT I and DGAT II, and the oil and fat content in the cells was higher than that in the wild type.

Disclosure of Invention

In view of the above, the present invention provides microalgal protein kinase N_OS1P gene and application thereof. The total lipid content and neutral lipid content of the 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.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a microalgae protein kinase NoS1P gene, wherein the cDNA sequence of the microalgae protein kinase NoS1P gene is as follows:

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

ii) the nucleotide sequence shown as SEQ ID No.1, which is substituted, deleted and/or added with one or more nucleotides and expresses the same functional protein; or

iii) a nucleotide sequence which hybridizes with the sequence shown in SEQ ID No.1 under stringent conditions, which are hybridization at 65 ℃ in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC containing 0.1% SDS solution, and washing the membrane with the solution, and expressing the same functional protein; or

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

Based on the research, the invention also provides a protein coded by the microalgae protein kinase NoS1P gene, which comprises the following components:

(I) an amino acid sequence shown as SEQ ID No. 2; or

(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I); or

(III) and an amino acid sequence having at least 90% homology with the sequence of (I) or (II).

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

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

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

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

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

In some embodiments of the invention, the microalgae comprise one or more of green algae, diatoms, red algae, gold algae, brown algae, and the plants comprise one or more of arabidopsis, wheat, rice, corn, and cotton.

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

In addition, the invention also provides an amplification primer combination of the microalgae protein kinase NoS1P gene, which comprises 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.

In addition, the invention also provides a construction method of the transgenic microalgae, which comprises the following steps:

step 1: extracting total RNA of microalgae, and performing reverse transcription to obtain a first cDNA chain;

step 2: taking a first cDNA 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 step 3: constructing an overexpression vector of the NoS1P gene;

and 4, step 4: the transgenic technology is utilized to transform the overexpression vector with the protein kinase NoS1P gene into a target algae strain, and the algae strain with transgenic stable inheritance is obtained.

The invention provides 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 is shown in SEQ ID NO 1. Functional analysis shows that the total lipid content and neutral lipid content of the 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. The total lipid content of the marine nannochloropsis oceanica strain over-expressing the gene is obviously higher than that of a wild type strain (P < 0.05).

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows agarose gel electrophoresis detection of NOS1P transformant in example 1 of the present invention; the leftmost lane is DNA marker, and the remaining lanes are transformants; wherein 33-2 corresponds to lane 2, 33-6 corresponds to lane 6, PC is Positive clone, the Positive clone is receptor cell after the exogenous gene is connected with the vector, a primer is designed according to the vector sequence and the exogenous gene sequence, the DNA of the receptor cell is used as a template, a verification product, namely a successfully transformed transformant, can be amplified by PCR, and the size of a target band is 2080 bp;

FIG. 2 shows a vector map, and transformants can be verified by using the forward primer 5-vcpf and the reverse primer S1Poe33R, and the verified products are the PCR products of the 2080bp fragment in FIG. 1; PC is positive clone, which can be interpreted as a fragment amplified only by a vector containing target linear DNA, and a template of the fragment can be a vector before transformation or a transformant after transformation;

FIG. 3 shows the relative expression levels of NOS1P gene in a high-lipid-producing algal strain S1Poe and a wild-type algal strain inoculated for 0 hour in example 3 of the present invention;

FIG. 4 shows the relative expression amounts of NOS1P gene in oleaginous algal strain S1Poe and wild algal strain cultured in high light for 96 hours in example 3 of the present invention;

FIG. 5 shows the relative expression levels of NOS1P gene in a high-lipid-producing algal strain S1Poe and a wild-type algal strain cultured under nitrogen for 96 hours in example 3 of the present invention;

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

FIG. 7 shows a comparison of the oil content of the high-oil-yielding algal strain S1Poe in example 4 of the present invention in the nitrogen-deficient culture for 8 days with that of the wild-type algal strain.

Detailed Description

The invention discloses a microalgae protein kinase N_OThe S1P gene and its application can be realized by appropriately modifying the process parameters by those skilled in the art with reference to the contents herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention aims to provide a protein kinase NoS1P gene and a protein coded by the same.

The invention also aims to provide application of the protein kinase NoS1P gene in improving the synthesis capacity of microalgae and vegetable oil.

In order to realize the purpose of the invention, the invention clones and obtains protein kinase NoS1P gene from marine nannochloropsis oceanica, the cDNA sequence of the NoS1P gene is as follows:

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

ii) a nucleotide sequence which is shown in SEQ ID No.1 and expresses the same functional protein by replacing, deleting and/or adding one or more nucleotides; or

iii) a nucleotide sequence which hybridizes with the sequence shown in SEQ ID NO.1 under stringent conditions in which hybridization is performed at 65 ℃ in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC containing 0.1% SDS solution and the membrane is washed with the solution and expresses the same functional protein; or

iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.

Sequencing results show that the protein kinase NoS1P is a protein kinase family member, the whole length of an open reading frame of the protein kinase NoS1P gene is 1878bp, and the protein consists of 625 amino acids and is coded (SEQ ID No. 2). Through bioinformatics analysis of a protein kinase NoS1P gene and judgment of an expression mode under stress of high light, nitrogen deficiency and the like, the gene may participate in lipid metabolism regulation and control pathways of microalgae and the like.

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

The 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 using conventional biotechnological methods such as Ti plasmid, plant viral vector, direct DNA transformation, microinjection, electroporation, and the like.

The invention also provides microalgae engineering cells for over-expressing the coding region of the protein kinase NoS1P gene.

The invention also provides application of the protein kinase NoS1P gene in improving the oil and fat synthesis capacity of microalgae (such as nannochloropsis oculata and the like).

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

The invention also provides a construction method of the transgenic microalgae.

The invention further provides primers SEQ ID No.5 and SEQ ID No.6 for the fluorescent quantitative PCR detection of the protein kinase NoS1P gene, wherein 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’。

The kit can be used for detecting the expression condition of protein kinase NoS1P gene in nannochloropsis oculata engineering cells, and the result shows that the transcription level of the NoS1P gene in the engineering cells is higher than that of the wild type.

The method can be used for detecting the expression condition of the protein kinase NoS1P gene, and the result shows that the protein kinase NoS1P gene is induced to express under the stress of high light and insufficient nitrogen.

The method can be used for detecting the dry weight, the oil content and the neutral fat content of wild algae strains and over-expression algae strains, and the result shows that the total fat content and the neutral fat content of the over-expression marine nannochloropsis algae strains are obviously higher than those of the wild algae strains (P < 0.05).

The invention provides a microalgae protein kinase N_OThe S1P gene and its application, the raw material and reagent are all available in market.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.

The invention is further illustrated by the following examples:

example 1: cloning of protein kinase NoS1P Gene

1. Extracting total RNA of nannochloropsis oculata, and reverse transcribing to obtain the first cDNA chain.

2. Designing an upstream SEQ ID No.3 and a downstream primer SEQ ID NO: 4 as a primer, and obtaining the cDNA sequence of the NoS1P gene by PCR amplification reaction.

The primer sequences are as follows:

SEQ ID No.3：

5’-AGGATGACGATGACAAGCATGCACTGCCCTAATAAGAGAATG-3’，

SEQ ID No.4：

5’-TCCTCCTGGGATCCCCCGGGATCACCTATACTCCCTTTGAAG-3’。

PCR amplification was performed using 2 XTaq PCR MasterMix (Takara) in the following reaction scheme: 2. mu.l cDNA template, 1. mu.l each of primer F, R (10. mu. mol/L), 2 XPCR MasterMix 25. mu.l, ddH₂O to 50 microliters.

The PCR amplification procedure was: denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 90 seconds, and extension at 72 ℃ for 2 minutes for 35 cycles; final extension at 72 ℃ for 10 min.

3. And detecting the amplification product by 1% agarose gel electrophoresis, and sequencing.

4. Cutting off a target fragment, recovering and purifying the gel, cloning the gel to a pMD18-T vector, detecting the gel by colony PCR, sending the gel to Shanghai Biotechnology Limited company for sequencing verification, and naming the plasmid with correct sequencing verification as NoS 1P-18T. The cDNA sequence (SEQ ID No.1) of the NoS1P gene is 1878bp, and encodes a protein NoS1P (SEQ ID No.2) consisting of 625 amino acids.

Example 2: preparation of nannochloropsis oculata engineering cell of over-expression NoS1P gene

1. Bacterial strain and culture: nannochloropsis was grown in F2 solid medium and F2 liquid medium.

2. The resistance gene label method is used for constructing an overexpression vector of nannochloropsis, and a bleomycin resistance label is adopted. According to molecular biology techniques familiar to those skilled in the art, the resistance tag is amplified, and the fragment containing the selection marker is cleaved with an appropriate restriction enzyme to transform Nannochloropsis.

3. And (3) electrically transforming the nannochloropsis cell, and introducing the fragment containing the resistance label into the cell. That is, 0.5. mu.g of linearized plasmid and 600 ten thousand algal cells were placed in a 0.2 cm electrical transformation cell (Bio-Rad), 2.2kV pulse (Bio-Rad electrical transformer, 50. mu.F).

4. Transformant cells containing the protein kinase NoS1P gene over-expression exogenous vector were identified by antibiotic screening and based on primers specific to the protein kinase NoS1P gene described below.

5'-TTTAACTAGGATACTGCCGGGTG-3' (shown as SEQ ID No. 7)

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

Example 3: engineering cell NoS1P gene transcription level identification

1. A certain amount of S1Poe algal strain sample was taken in a 1.5 ml centrifuge tube by a centrifuge tube, and S1Poe RNA was extracted using the Elder plant RNA extraction kit.

2. 500 microliters of CLB lysate was taken into a 1.5 milliliter centrifuge tube (if the lysate had crystals precipitated, the tube was placed in a 65 ℃ water bath for a while), 25 microliters of 5% beta-mercaptoethanol was added to the lysate, and the solution was pre-heated at 65 ℃ after being mixed uniformly.

3. The glass beads for grinding are baked in an oven at 200 ℃ for 3 hours and cooled at room temperature. A proper amount of air-dried glass beads and 100 microliters of air-cooled lysate are taken to be placed in a sample, and a bead mill oscillates for 1-2 minutes. The remaining 425 microliters of lysate were added to the sample and the shaker shaken for 15 seconds. The mixture was mixed by inversion every minute in a water bath at 65 ℃ for 5 minutes.

4. The product is put in a centrifuge at 13000r/min for 10 minutes. Collecting 500 microliters of supernatant in a new centrifuge tube, adding 0.5-fold volume of absolute ethyl alcohol, and blowing and uniformly mixing by a pipette gun.

5. The whole solution was transferred to a genome removal column (note less than 700. mu.l of solution each time, and added in portions after centrifugation), and centrifuged at 13000r/min for 2 minutes, and the filtrate was decanted off.

6. The genomic DNA clean-up column was replaced with a new centrifuge tube, 500. mu.l of RLT plus lysate was added thereto, 13000r/min, and centrifugation was carried out for 30 seconds. Collecting the filtrate, adding 0.5 volume times of anhydrous ethanol, and mixing.

7. The mixture was added to a new adsorption column, 13000r/min, and centrifuged for 2 min. Removing the filtrate, adding 700 microliters of deproteinized solution into the adsorption column, standing at room temperature for 1 minute, 13000r/min, centrifuging for 2 minutes, and pouring off the filtrate.

8. Adding 500 microliters of rinsing liquid into the adsorption column in the above step, carrying out 13000r/min, carrying out centrifugation for 2 minutes, and discarding the filtrate. This procedure was repeated once, and the column 13000r/min from which the filtrate was discarded was centrifuged once for 2 minutes.

9. Taking out the adsorption column, placing in an enzyme-free centrifuge tube, adding 30-50 microliters of enzyme-free water, standing the solution at room temperature for hydrolysis for 1 minute, 13000r/min, and 1 minute. After gel electrophoresis verification, the gel is stored at-20 ℃ for later use.

10. Using TaKaRa PrimeScript^TMThe RT reagent Kit with gDNA Eraser Kit was prepared as follows to prepare a solution to remove genomic DNA and was performed on ice.

TABLE 1

The solution is mixed evenly, preheated by a PCR instrument, reacted for 2 minutes at 42 ℃, and kept warm at 4 ℃.

11. Using TaKaRaPrimeScript^TMThe RT reagent kit Kitwith gDNA Eraser kit inverts RNA into cDNA, prepares a reaction system according to the following table and carries out the reaction on ice.

TABLE 2

Mixing, preheating by a PCR instrument, setting reverse transcription conditions at 37 ℃ for 15 minutes; 5 seconds at 85 ℃; 4 ℃ is prepared.

12. And (3) carrying out fluorescence quantitative detection on the expression level of the S1P gene.

TABLE 3

The fluorescent quantitative operation comprises the following steps:

step 1: pre-denaturation, 95 ℃, 30s, 20 ℃/s, 1 cycle.

step 2: PCR reaction at 95 deg.c for 5 sec at 20 deg.c/s; 55 ℃, 30s, 20 ℃/s; 72 ℃, 30s, 20 ℃/s, 40 cycles.

Step 3: and drawing a dissolution curve.

95℃，0s，20℃/s

65℃，15s，20℃/s

95℃，0s，0.1℃/s

The detection results are shown in fig. 2-4 and table 4, and the relative expression quantity of the NoS1P of the S1Poe is obviously higher than that of the wild marine nannochloropsis strain.

TABLE 4 high gloss (200. mu. mol/m)²/s^-1) Relative expression level of S1P gene of WT, 33-2, 33-6 in nitrogen deficient culture

Among them, 33-2 corresponds to lane 2 in FIG. 1, and 33-6 corresponds to lane 6 in FIG. 1.

33-2 and 33-6 are transformants with SIP overexpression, and are all nannochloropsis selected in the experimental process of the invention.

Example 4: production of grease by nannochloropsis oculata engineering cell over expressing protein kinase NoS1P gene

The nannochloropsis oculata engineering cell S1Poe with the over-expressed NoS1P gene obtained in example 1 is used for oil production, and the specific method is as follows:

the activated nannochloropsis wild type and S1Poe strains were inoculated separately into photobioreactors (PBR; diameter 33mm, height 60cm) containing fresh modified F2 medium.

PBR were cultured under the culture conditions set forth in Table 5, with highlight (200. mu. mol/m)²/s^-1) The nitrogen deficiency means that sodium nitrate was not added as a nitrogen source to the modified F2 medium.

Modified F2 culture medium, self-made seawater of 35g/L sea salt, 1.21g Tris, 1000mg/LNaNO₃，67mg/L NaH₂PO₄·H₂O，0.0196mg/L CuSO₄·5H₂O，0.0126mg/LNaMoO₄·2H₂O，0.044mg/L ZnSO₄·7H₂O，0.01092mg/L CoCl₂·6H₂O，0.36mg/L MnCl₂·4H₂O，3.65mg/L FeCl₃·6H₂O，4.37mg/L Na₂EDTA·2H₂O, 0.005mg/L cobalamin, 0.005mg/L biotin, 0.1mg/L thiamine hydrochloride

Eight days later, cells were harvested and assayed for dry weight and total lipid content.

The results show that the fat content of the nannochloropsis strain S1Poe is improved compared with the wild type under the nitrogen deficiency treatment (figures 5-6).

TABLE 5 high gloss (200. mu. mol/m)²/s^-1) Oil content and TAG content of WT, 33-2, 33-6 under nitrogen deficiency, highlight and nitrogen deficiency culture

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of Hainan

<120> gene for improving oil synthesis of marine nannochloropsis and application thereof

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acagacgtaa attcgacgac agatacgtat tctttttcct ttcctaaagt gttgaacaag 180

gaggaggacg aggaggggga ggacgaggag gacgacgaag atactgtggc tacagcgctc 240

agcaatgcgt gcatcaccga aagtgcgaag gaaaaggaag caatgaagga tgtcccgcag 300

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caagagagca tccacggcat catcacccgc ctggcctgcg atgacggggt ctctgtccgc 420

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ttcccattca cctcccacct accaaccatc accgacaacg taaacagcat aacggctgaa 540

ggcggaggtg acggtcccga agccatggcg gacgccctgt acgacctcct tcatcttgaa 600

tggcgctcct ccctccctcc ctctccttcc tcctcccctt cgatccagac aagcaaagtc 660

gctgtgctca ttgccgatgc tccccctcac ggattgggag agcgtgaaga cggttttcca 720

aatggctgcc ctctccatca tgaccctctc gctttgactc gacagctcgc agcttgcggc 780

attactctct atactgtggg gtgtgaaccc gccatttcca cctatgagaa ttgtaaggat 840

atattgatct ttatggcgga ggcaacggga gggcaggcct tggtgctgga gagtgcgagt 900

ctgttggctt ctgttatttt agcgggggcg caggaggagg tggggctgac acgactggag 960

agggaggtga aggaggtgat gagggaggca gggagggagg caggggaaga ggaggatgaa 1020

gatgtgtggg tggcgagggt taaggaggcg ttgaggagga aagggactag aacaaggcag 1080

ttaaagacaa acatgaggag gatgacgtca gttcaaactg aggtgattgc gtcatgctct 1140

tcccttgagg aggcgaagag gaggttgggg agcgagagag cgcgggaggt agaggaggaa 1200

aacggggaga gggtatcgat atcgacgggc ctgtcgagta ggtcgtttgt gtctgcggcg 1260

gaggcggttg cccctgtaga catgcgttgg gaagcggaca attcgtcgtc aatgccgact 1320

tcaggtgtag gatgggaagg agagagggag ggaggaaagg gcggaggacg aggttgcgga 1380

cgactcatgg tccaagacct catttcccga cggggaggcg gtggtattat ttccgctcct 1440

actgttcgtg ctgctcctac tcatgctgct gctaccacta cgactactgt tggtgcagcc 1500

acccctactt ctactgctgc tactgagata ccgtccgcag aagccacgcc gaaaagaggt 1560

ggtcttcctc ggagcaggga gaaggagctt tcggcctccc acactgaagc gaccgataca 1620

tttccacgga ctgtggttcc tactccatgc tcctcttccg tctcctcctc ctcctccccc 1680

ttcctcccgg cccccctctt ctcccccacg catacgtact cgcttacggc atgtgccacg 1740

gttggtgctg gaggaggagg aggaagggct ggtgggcggg agggagggag ggccgaggtg 1800

gcggaagagg agatttcgat cgatcaaatt cgacggttgg tccgcaaagc ccagagcctt 1860

caaagggagt ataggtga 1878

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Met Gln Ala Phe Val Thr Ser Ile Phe Cys Arg Thr Lys Ser Asp Ile

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Ser Ser Ser Thr Ser Ala 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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Thr Tyr Ser Phe Ser Phe Pro Lys Val Leu Asn Lys Glu Glu Asp Glu

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Glu Gly Glu Asp Glu Glu Asp Asp Glu Asp Thr Val Ala Thr Ala Leu

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Ser Asn Ala Cys Ile Thr Glu Ser Ala Lys Glu Lys Glu Ala Met Lys

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Asp Val Pro Gln Leu Asp Leu Val Phe Val Val Asp Ser Thr Ser Ser

100 105 110

Met Asp Ala Tyr Ile Arg Ala Ala Gln Glu Ser Ile His Gly Ile Ile

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Thr Arg Leu Ala Cys Asp Asp Gly Val Ser Val Arg Phe Ala Leu Ile

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Ser Tyr Arg Asp His Ala Pro Gln Asp Ser Ser Tyr Val Thr Arg Val

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Phe Pro Phe Thr Ser His Leu Pro Thr Ile Thr Asp Asn Val Asn Ser

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Ile Thr Ala Glu Gly Gly Gly Asp Gly Pro Glu Ala Met Ala Asp Ala

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Leu Tyr Asp Leu Leu His Leu Glu Trp Arg Ser Ser Leu Pro Pro Ser

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Pro Ser Ser Ser Pro Ser Ile Gln Thr Ser Lys Val Ala Val Leu Ile

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Arg Glu Gly Gly Lys Gly Gly Gly Arg Gly Cys Gly Arg Leu Met Val

450 455 460

Gln Asp Leu Ile Ser Arg Arg Gly Gly Gly Gly Ile Ile Ser Ala Pro

465 470 475 480

Thr Val Arg Ala Ala Pro Thr His Ala Ala Ala Thr Thr Thr Thr Thr

485 490 495

Val Gly Ala Ala Thr Pro Thr Ser Thr Ala Ala Thr Glu Ile Pro Ser

500 505 510

Ala Glu Ala Thr Pro Lys Arg Gly Gly Leu Pro Arg Ser Arg Glu Lys

515 520 525

Glu Leu Ser Ala Ser His Thr Glu Ala Thr Asp Thr Phe Pro Arg Thr

530 535 540

Val Val Pro Thr Pro Cys Ser Ser Ser Val Ser Ser Ser Ser Ser Pro

545 550 555 560

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

565 570 575

Ala Cys Ala Thr Val Gly Ala Gly Gly Gly Gly Gly Arg Ala Gly Gly

580 585 590

Arg Glu Gly Gly Arg Ala Glu Val Ala Glu Glu Glu Ile Ser Ile Asp

595 600 605

Gln Ile Arg Arg Leu Val Arg Lys Ala Gln Ser Leu Gln Arg Glu Tyr

610 615 620

Arg

625

<210> 3

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aggatgacga tgacaagcat gcactgccct aataagagaa tg 42

<210> 4

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tcctcctggg 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 (11)

1. The microalgae protein kinase NoS1P gene is characterized in that the cDNA sequence of the microalgae protein kinase NoS1P gene is as follows:

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

ii) the nucleotide sequence shown as SEQ ID No.1, which is substituted, deleted and/or added with one or more nucleotides and expresses the same functional protein; or

iii) a nucleotide sequence which hybridizes with the sequence shown in SEQ ID No.1 under stringent conditions, which are hybridization at 65 ℃ in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC containing 0.1% SDS solution, and washing the membrane with the solution, and expressing the same functional protein; or

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

2. The protein encoded by the microalgal protein kinase NoS1P gene of claim 1, having:

(I) an amino acid sequence shown as SEQ ID No. 2; or

(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I); or

(III) and an amino acid sequence having at least 90% homology with the sequence of (I) or (II).

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. A host comprising the microalgal protein kinase NoS1P gene of claim 1.

6. Use of the microalgal protein kinase NoS1P gene of claim 1 for genetic modification of microalgae and/or plants.

7. The microalgae protein kinase NoS1P gene as claimed in claim 1, for improving the oil and fat synthesis capability of microalgae and/or plants.

8. The use of claim 6 and/or claim 7, wherein the microalgae comprise one or more of green algae, diatoms, red algae, golden algae, brown algae, and the plants comprise one or more of Arabidopsis, wheat, rice, corn, and cotton.

9. Use of the microalgal protein kinase NoS1P gene of claim 1 in the preparation of transgenic plants and/or transgenic microalgae.

10. The microalgae protein kinase NoS1P gene amplification primer combination as claimed in claim 1, which comprises 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.

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

step 1: extracting total RNA of microalgae, and performing reverse transcription to obtain a first cDNA chain;

step 2: taking a first cDNA 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 step 3: constructing an overexpression vector of the NoS1P gene;

and 4, step 4: the transgenic technology is utilized to transform the overexpression vector with the protein kinase NoS1P gene into a target algae strain, and the algae strain with transgenic stable inheritance is obtained.

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