阅读说明：本技术 一种微拟球藻遗传转化体系及合成甘油三酯的基因和应用 (Nannochloropsis oculata genetic transformation system, gene for synthesizing triglyceride and application ) 是由 辛一 王勤涛 徐健 于 2020-05-12 设计创作，主要内容包括：本发明属于生物技术领域。本发明涉及一种合成甘油三酯(TAG)的基因、高效、稳定的遗传转化体系及其在提高甘油三酯含量中的应用。合成甘油三酯(TAG)的基因具有SEQ ID NO 1所示的碱基序列；或,2)与序列表中序列1所限定的核酸序列具有95％以上同源性、且编码相同生物学功能蛋白质的DNA序列。以及通用表达载体为：1)具有SEQ ID NO 2所示的碱基序列；或,2)与序列表中序列2所限定的核酸序列具有90％以上同源性、且具有相同功能的DNA序列。并给出优化转化方法,使得本发明所建立的遗传转化体系,对于利用基因工程进行工业微藻性状改良,具有巨大的应用前景。(The invention belongs to the field of biotechnology. The invention relates to a gene for synthesizing Triglyceride (TAG), a high-efficiency and stable genetic transformation system and application thereof in improving the content of triglyceride. The gene for synthesizing Triglyceride (TAG) has a base sequence shown in SEQ ID NO 1; or, 2) DNA sequence which has more than 95 percent of homology with the nucleic acid sequence limited by the sequence 1 in the sequence table and codes the protein with the same biological function. And the universal expression vector is: 1) has a base sequence shown as SEQ ID NO 2; or, 2) DNA sequences which have more than 90 percent of homology with the nucleic acid sequences limited by the sequence 2 in the sequence table and have the same functions. And an optimized transformation method is provided, so that the genetic transformation system established by the invention has a huge application prospect in improving the properties of the industrial microalgae by utilizing genetic engineering.)

1. A gene for synthesizing Triglyceride (TAG), characterized in that:

1) has a base sequence shown as SEQ ID NO 1;

or the like, or, alternatively,

2) DNA sequence which has more than 95% of homology with the nucleic acid sequence limited by the sequence 1 in the sequence table and codes the protein with the same biological function.

2. A protein encoded by the gene of claim 1, wherein: the protein coded by the gene has an amino acid sequence with one of the following conditions:

1) has an amino acid sequence shown as SEQ ID NO 3;

2) an amino acid sequence of a derivative protein produced by substituting, deleting or adding one or more amino acid residues to the amino acid residue sequence of SEQ ID NO 3, the derivative protein having the same biological function as the protein of SEQ ID NO 3.

3. A method for constructing the gene of claim 1, which comprises: the method comprises the following steps:

1) amplifying DGAT gene from cDNA of nannochloropsis oculata;

2) and recovering the amplification product, connecting the amplification product to a sequencing vector, and obtaining the full-length coding sequence of the DGAT gene of the nannochloropsis through sequencing, namely the base sequence shown in SEQ ID NO 1.

4. Use of the gene of claim 1, wherein: use of the Triglyceride (TAG) synthesizing gene for regulating the level of TAG in an organism.

5. A primer for constructing a gene having a Triglyceride (TAG) synthesizing function according to claim 1, wherein: primers 1) and 2):

1)NoDGAT2B-for：

5’GGTACCACATAATGACGCAGGTC 3’；

2)NoDGAT2B-rev：

5’GAATTCTCACTTAATAAGCAGCTTCTTG 3’。

6. a universal nannochloropsis oculata genetic transformation expression vector is characterized in that:

1) comprises a base sequence shown as SEQ ID NO 2;

or the like, or, alternatively,

2) contains DNA sequence with 90% over homology and the same function with the nucleic acid sequence limited by the sequence 2 in the sequence table.

7. The use of the universal nannochloropsis oculata genetic transformation expression vector of claim 6, wherein: the expression vector is applied to the transformation of a nannochloropsis oculata genome sequence and a genetic phenotype.

8. Use of an expression vector according to claim 7, wherein: uniformly mixing nannochloropsis oculata solution, a universal expression vector and salmon sperm DNA in logarithmic phase in an f/2 liquid culture medium, and recovering in a dark place for 48 hours at 25 ℃ and 100 rpm; after recovery, the algae body precipitate is centrifugally collected, is evenly mixed with the corn starch suspension, and is evenly mixed with the suspension containing zeocin and NaHCO₃The cells were cultured on the f/2 agarose plate at 25 ℃ in the dark for 3 days, and then at 25 ℃ at 80. mu. mol m^-2s^-1And (4) performing illumination culture until the clone grows out, so that the nannochloropsis oculata is efficiently and stably transformed.

9. A recombinant vector characterized by: a recombinant vector comprising the DNA sequence of claims 1 and 6.

10. A high-efficiency and stable transformation method of a recombinant vector is characterized in that:

uniformly mixing nannochloropsis oculata solution, recombinant vector and salmon sperm DNA in logarithmic phase in f/2 liquid culture medium, and recovering in dark at 25 deg.C and 100rpm for 48 h; after recovery, the algae body precipitate is centrifugally collected, is evenly mixed with the corn starch suspension, and is evenly mixed with the suspension containing zeocin and NaHCO₃The cells were cultured on the f/2 agarose plate at 25 ℃ in the dark for 3 days, and then at 25 ℃ at 80. mu. mol m^{- 2}s^-1And (4) performing illumination culture until the clone grows out, so that the nannochloropsis oculata is efficiently and stably transformed.

Technical Field

The invention belongs to the field of biotechnology. The invention relates to a gene for synthesizing Triglyceride (TAG), a high-efficiency and stable genetic transformation system and application thereof in improving the content of triglyceride.

Background

Eukaryotic microalgae are regarded as a "cell factory" for the production of sugars, lipids and biologically active substances, due to their characteristics of rapid growth rate, high photosynthetic efficiency, strong environmental suitability, etc. (Hu et al, 2008). In addition, some eukaryotic microalgae such as chlorella and nannochloropsis can be cultured in large scale outdoors, and have great industrialization potential (Wijffels et al, 2010). In recent years, the vigorous development of system biology based on various omics data has opened a new era in microalgae synthetic biology. In the field of microalgae synthetic biology, the design and modification of a specific algal species (strain) depends to a great extent on whether a genetic transformation system of the algal species (strain) is established. Gene transfer and expression have been achieved in more than 30 microalgal species, particularly in Chlamydomonas reinhardtii, a model species of eukaryotic microalgae, where genetic transformation systems have been established in three sets of genomes, nucleus, chloroplast and mitochondria (Kindle et al, 1990; Randol nderson et al, 1993; Boudreau et al, 1997). However, in the process of establishing a microalgae genetic transformation system, two major bottlenecks are needed to be broken through: firstly, the currently established genetic transformation system has the hidden trouble of instability, and in the case of chlamydomonas reinhardtii, the nuclear transformation strain of the system is often lost in the process of passage (kingdle et al, 1990); secondly, in industrial microalgae, an efficient and stable genetic transformation system is not established, and for chlorella as an example, although heterotrophic culture technology is mature, the lack of the genetic transformation system greatly limits the industrialization process of chlorella (Liu et al, 2019).

At present, nannochloropsis has grown up as a model microalgae with industrial development potential, and nannochloropsis has been used for large-scale outdoor culture by Solix, Aurora and Seambitic in Israel; the nannochloropsis oculata genome is small (about 30Mb), the gene structure is compact, and redundant sequences are few, so the nannochloropsis oculata genome is suitable for being used as a chassis organism to create a novel photosynthetic cell factory (Poliner et al, 2018); currently, a variety of genetic manipulation tools have been developed for nannochloropsis, such as homologous recombination (Kilian et al, 2011; Nobusawa et al, 2017), overexpression (Xin et al, 2017), RNA interference (Xin et al, 2017), and CRISPR/Cas 9-based gene editing (Wang et al,2016), which will strongly push the development of nannochloropsis synthetic biology. However, the reported highest transformation efficiency of nannochloropsis oculata is only 2500cfu/μ g DNA (Kilian et al, 2011), which is barely suitable for the targeted modification of target genes, but is not enough to complete the high-throughput construction and screening of various mutant libraries and synthetic biological engineering strains. Therefore, it is urgently needed to establish a set of efficient and stable nannochloropsis oculata genetic transformation system to promote the rapid development of the industrial microalgae synthetic biology.

Triglyceride (TAG) is a main raw material of biodiesel, nannochloropsis oculata can accumulate a large amount of TAG under stress conditions, and compared with terrestrial oil crops, many nannochloropsis oculata strains are regarded as a biodiesel source with huge potential due to the characteristics of strong stress resistance, low environmental requirement and the like (bondiioli et al, 2012). In higher plants and microalgae, there is evidence that the last reaction step in TAG synthesis is the rate-limiting step in this pathway, which is accomplished by diacylglycerol acyltransferase (DGAT for short) (Chen et al, 2012). In higher oil crops, there have been many cases of increasing TAG content by increasing the expression of endogenous DGAT (Roesler et al, 2016). In conclusion, the DGAT is transformed by utilizing the nannochloropsis oculata genetic transformation system, so that the content of the cell TAG is improved, and the obvious necessity and feasibility are provided, but no relevant report proves whether the cell TAG can be realized or not.

Disclosure of Invention

The invention aims to provide a gene for synthesizing Triglyceride (TAG) in nannochloropsis oculata, a high-efficiency and stable genetic transformation system and application thereof in improving the content of triglyceride.

In order to achieve the purpose, the invention adopts the technical scheme that:

a gene that synthesizes a Triglyceride (TAG):

1) has a base sequence shown as SEQ ID NO 1;

or the like, or, alternatively,

2) DNA sequence which has more than 95% of homology with the nucleic acid sequence limited by the sequence 1 in the sequence table and codes the protein with the same biological function.

A protein encoded by a gene that encodes an amino acid sequence having one of the following:

1) has an amino acid sequence shown as SEQ ID NO 3;

2) an amino acid sequence of a derivative protein produced by substituting, deleting or adding one or more amino acid residues to the amino acid residue sequence of SEQ ID NO 3, the derivative protein having the same biological function as the protein of SEQ ID NO 3.

A method for constructing the gene, comprising the steps of:

1) amplifying DGAT gene from cDNA of nannochloropsis oculata;

2) and recovering the amplification product, connecting the amplification product to a sequencing vector, and obtaining the full-length coding sequence of the DGAT gene of the nannochloropsis through sequencing, namely the base sequence shown in SEQ ID NO 1.

The application of the gene and the application of the gene for synthesizing Triglyceride (TAG) in regulating the content of TAG in organisms.

A primer for constructing the gene having a Triglyceride (TAG) synthesis function: primers 1) and 2):

1)NoDGAT2B-for：

5’GGTACCACATAATGACGCAGGTC 3’；

2)NoDGAT2B-rev：

5’GAATTCTCACTTAATAAGCAGCTTCTTG 3’。

a universal nannochloropsis oculata genetic transformation expression vector:

1) comprises a base sequence shown as SEQ ID NO 2;

or the like, or, alternatively,

2) contains DNA sequence with 90% over homology and the same function with the nucleic acid sequence limited by the sequence 2 in the sequence table.

The preferred universal nannochloropsis oculata genetic transformation expression vector is vector pXJ 450.

The application of universal nannochloropsis oculata genetic transformation expression vector in modifying nannochloropsis oculata genome sequence and genetic phenotype.

The method specifically comprises the following steps: uniformly mixing nannochloropsis oculata solution, a universal expression vector and salmon sperm DNA in logarithmic phase in an f/2 liquid culture medium, and recovering in a dark place for 48 hours at 25 ℃ and 100 rpm; after recovery, the algae body precipitate is centrifugally collected, is evenly mixed with the corn starch suspension, and is evenly mixed with the suspension containing zeocin and NaHCO₃The cells were cultured on the f/2 agarose plate at 25 ℃ in the dark for 3 days, and then at 25 ℃ at 80. mu. mol m^-2s^-1And (4) performing illumination culture until the clone grows out, so that the nannochloropsis oculata is efficiently and stably transformed.

A recombinant vector contains DNA sequences shown in SEQ ID NO1 and SEQ ID NO 2.

Further, it is preferable that the recombinant vector is pXJ 419.

A host cell comprising the recombinant vector. The host cell is nannochloropsis.

A high-efficiency and stable transformation method of a recombinant vector comprises the following steps:

uniformly mixing nannochloropsis oculata solution, recombinant vector and salmon sperm DNA in logarithmic phase in f/2 liquid culture medium, and recovering in dark at 25 deg.C and 100rpm for 48 h; after recovery, the algae body precipitate is centrifugally collected, is evenly mixed with the corn starch suspension, and is evenly mixed with the suspension containing zeocin and NaHCO₃The cells were cultured on the f/2 agarose plate at 25 ℃ in the dark for 3 days, and then at 25 ℃ at 80. mu. mol m^-2s^-1And (4) performing illumination culture until the clone grows out, so that the nannochloropsis oculata is efficiently and stably transformed.

The recombinant vector is preferably an expression vector containing SEQ ID NO1 and SEQ ID NO 2.

In conclusion, the gene for synthesizing Triglyceride (TAG) and the transformation method can further improve the content of the triglyceride of nannochloropsis.

The invention has the advantages that:

the transformation efficiency of the transformation method exceeds the highest level reported in marine nannochloropsis, and the obtained vector fragment can stably exist in the marine nannochloropsis without loss. The overexpression of the gene can obviously improve the content of nannochloropsis oculata triglyceride, and the application value of the gene in the aspect of improving the organism production of TAG is also proved. Under the background of relatively complete microalgae omics data, the genetic transformation system established by the invention has a huge application prospect in improving the properties of industrial microalgae by utilizing genetic engineering.

Furthermore, the conversion method can obviously shorten the growth time of the clone by improving the illumination intensity of the plate culture; by using corn starch for embedding, the support and protection of the recovered cells are increased, so that the transformation efficiency is obviously improved.

Drawings

FIG. 1 is a diagram showing the structure of a universal expression vector pXJ450 constructed according to the present invention.

FIG. 2 is a graph showing the comparison of the transformation efficiency between the transformation method constructed according to the present invention and the transformation method before the improvement of resuscitation means.

FIG. 3 is a gene structural diagram of NoDGAT2B used in the present invention.

FIG. 4 is a diagram of the structure of a NoDGAT2B containing recombinant vector pXJ419 constructed in accordance with the present invention.

FIG. 5 shows the results of the electrophoresis detection of the pXJ419 transformant and the control by PCR in the present invention.

Fig. 6 is a graph showing the comparison results of the NoDGAT2B overexpression strain constructed from pXJ450 in the present invention and the control group in terms of the transcription level of NoDGAT 2B.

FIG. 7 is a graph showing the comparison results of the NoDGAT2B overexpression strain constructed by pXJ450 in the present invention and the control group in terms of the TAG content.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The method of the invention realizes the purpose of increasing the content of TAG by constructing an expression vector, optimizing a transformation method and finally over-expressing an endogenous diacylglycerol acyltransferase gene NoDGAT2B in an industrial Nannochloropsis algae strain IMET1 (namely, marine Nannochloropsis oceanica IMET1 which is given by the university of Maryland and can be used for releasing the strain in the research direction of the public). The invention separates the full-length cDNA sequence of the NoDGAT2B gene, and utilizes an optimized nannochloropsis oculata genetic transformation system to carry out overexpression, and experiments prove that the TAG content of nannochloropsis oculata can be obviously improved by overexpressing the gene. The disclosed full-length gene and amino acid sequence are reported for the first time in nannochloropsis, and members of a DGAT gene family are enriched; the overexpression of the gene can obviously improve the TAG synthesis capability of nannochloropsis oculata, and proves the application value of the gene in the aspect of improving the TAG production of organisms by using a genetic engineering means.

The experimental procedures for which specific experimental conditions are not specified in the following examples are generally performed according to conventional conditions, Molecular Cloning (A Laboratory Manual, 3)^rded.) or according to the manufacturer's recommendations.

Example 1: construction of universal expression vector for nannochloropsis oculata

Designing two primers, introducing required enzyme cutting sites at two ends of the primers, and performing industrial synthesis by Shanghai:

primer 1)

5’TATGGCATGCGTCGACCCGCGGACGCGTGCTAGCGGCGCCAAGCTTCTGCAGCCCGGGGGATCCCATCATCACCATCACCATCACCATTAAG3’；

Primer 2)

5’TTAAGAATTACCACTACCACTACCACTACTACCCTAGGGGGCCCGACGTCTTCGAACCGCGGCGATCGTGCGCAGGCGCCCAGCTGCGTACGGT 3’。

By utilizing the annealing combination of the two primers, a DNA fragment (both ends respectively contain NdeI and EcoRI cutting sites) with 86bp length and cohesive tail ends is obtained, and the fragment contains 8 effective cutting sites (shown in figure 1) and a label (used for subsequent expression detection) for coding continuous 8 histidines. This fragment was directionally cloned into vector pXJ004(Wang et al,2016) using NdeI and EcoRI enzymatic sites into the vector designated pre-pXJ 450. The fragment of the β -tubulin promoter, cloning sequence and psbA terminator contained in pre-pXJ450 was then subcloned into vector pXJ015(Wang et al,2016) to yield universal expression vector pXJ450 (fig. 1), see sequence listing SEQ ID NO 2.

The universal expression vector pXJ450 inherits the high-efficiency expression characteristics of pXJ004 and pXJ015, and because the two vectors are integrated, and a multiple cloning site and an expression label are introduced, the gene to be expressed is introduced in one step, so that the subsequent construction efficiency is greatly improved.

Example 2:

cloning of the NoDGAT2B Gene

Cloning NoDGAT2B gene and flanking sequence thereof from gDNA and cDNA of nannochloropsis oculata IMET1 by utilizing PCR technology, designing primers used by the method, introducing required enzyme cutting sites at two ends of the primers, and carrying out industrial synthesis by Shanghai, wherein the primers specifically comprise:

1)NoDGAT2B-for：

5’GGTACCACATAATGACGCAGGTC 3’；

2)NoDGAT2B-rev：

5’GAATTCTCACTTAATAAGCAGCTTCTTG 3’。

the PCR instrument used was a MasterCycler from Eppendorf, 50. mu.L of a reaction system comprising 4. mu.L of dNTP (2.5mM each of each, TAKARA), 2. mu.L (10. mu.M) of forward and reverse primers, and 5. mu.L of 10 XBuffer (Mg 2)⁺plus, TAKARA), 0.4. mu.L rTaq enzyme (5U/. mu.L, TAKARA), 1. mu.L wild IMET1cDNA template (50 ng/. mu.L), and 35.6. mu.L ultrapure water. The reaction system is as follows: initial 94 ℃ pre-denaturation for 3min, then 94 ℃ denaturation for 30sec, 55 ℃ annealing for 30sec, 72 ℃ extension for 1-2min, 30 cycles, and finally 72 ℃ reaction for 7 min.

After the reaction, 5. mu.L of the PCR product was mixed with 1. mu.L of 6 × loading buffer (TAKARA), spotted on 1% (w/V) agarose (BIOWEST) gel, electrophoresed at 120V for 25min on an electrophoresis system manufactured by six instruments of Beijing, and then observed and photographed with a UV gel imager of UVP, Bi ℃ hemiHR. The desired fragment was purified and recovered from the PCR product using the Cycle-Pure Kit or Gel Extraction Kit of Omega, the operation of which was completely performed according to the instructions.

The obtained purified fragment was ligated into the pMD18-T vector from TAKARA, and transferred into E.coli competent cell Trans 5. alpha. from whole gold by means of heat shock transformation, and the positive clone was sent to Invitrogen for sequencing to obtain the full-length coding region sequence of the NoDGAT2B gene, see the sequence shown in SEQ ID NO1 of the sequence Listing. Furthermore, by alignment with the genomic sequence, the gene structure of noggat 2B was obtained, which included 5 'flanking fragments, 2 exons, 1 intron and 3' flanking fragments. See fig. 3.

Overexpression of NoDGAT2B in marine nannochloropsis oculata IMET1, and construction of endogenous overexpression vector

The specific method for constructing the vector is as follows: the full-length ORF fragment of noggat 2B was obtained by amplification by the PCR method (same as the PCR conditions of example 2) using the pMD18-T vector of noggat 2B as a template, using the primer combinations 1) and 2) in step (one) of example 2 and the system conditions.

The product of the full-length ORF fragment of noggat 2B amplified by PCR was directionally cloned into the above-mentioned universal expression vector pXJ450 using NdeI and BamHI enzymatic cleavage sites according to the conventional technique and the following primer combination to obtain a recombinant vector pXJ419 (fig. 4).

The adopted primers are used for constructing cloning requirements, an NdeI enzyme cutting site and a protective base are added to the 5 'end of a target sequence by a primer introduction method, a BamHI enzyme cutting site is added to the 3' end of the target sequence, and the primer sequences are specifically as follows:

1)NoDGAT2B-oe-for：

5’GGAATTCCATATGATGACGCAGGTCTATGCG 3’；

2)NoDGAT2B-oe-rev:

5’GGAATTCCATATGATGACGCAGGTCTATGCG 3’。

(III) optimization of Nannochloropsis oculata transformation technique

1) Taking the concentration of 1-3 × 10⁷cells/mL Nannochloropsis oculata solution (IMET1 wild) in logarithmic growth phase, centrifuging at 4 deg.C and 4000g for 5min, discarding the supernatant, rinsing with 375mM sorbitol pre-cooled at 4 deg.C for 2 times, and adjusting the cell concentration to 2 × 10 with 375mM sorbitol pre-cooled at 4 deg.C⁸cells/mL。

2) The concentrated algal bodies were divided into 200. mu.l portions, and 3. mu.g of the above-obtained recombinant pXJ419 linearized vector and 1. mu.l of salmon sperm DNA (15. mu.g/mL, pre-denatured at 95 ℃ for 1min) were added to each portion, mixed well and then left to stand on ice for 10 min.

3) The mixture was transferred into a 2mm cuvette and shocked at 2200V (HV), 50. mu.F, 600. omega. immediately after shocking, the algal bodies were transferred into 5mL F/2 liquid medium and then resuscitated in the dark at 25 ℃ and 100rpm for 48 h.

4) Centrifuging at 4000g for 5min, collecting algae, discarding supernatant, adding 1ml corn starch suspension, and blowing and mixing with pipette. The preparation method of the corn starch suspension comprises the following steps: 2g of corn starch was washed with absolute ethanol and pure water alternately 4 times, stored in a 75% ethanol solution, washed 3 times with f/2 liquid medium before use, and then resuspended in 10ml of f/2 liquid medium containing 0.8% PEG8000 (w/v).

5) The mixture was transferred to a medium containing 5. mu.g/mL zeocin and 1.6g/L NaHCO₃The plate was shaken to spread the solution uniformly, incubated at 25 ℃ in the dark for 3 days, and then incubated at 25 ℃ in the dark for 80. mu. mol m^-2s^-1The culture was incubated under light until colonies grew out (about 8-12 days).

The above transformation procedure was compared with the conventional transformation method (Wang et al,2016), in this example, by increasing the light intensity of plate culture (the previously commonly used light intensity was 30. mu. mol m)^-2s^-1) The growth time of the clone can be shortened from 40-50 days to 8-12 days; in addition, in the embodiment, the corn starch is used for embedding, so that the support and the protection of the recovered cells are increased, and the transformation efficiency is increased from 2-3X 10³cfu/ug DNA was elevated to 12-18X 10³cfu/. mu.g DNA (FIG. 2).

(III) electroporation method for introducing vector pXJ419 into Nannochloropsis

1) Taking the concentration of 1-3 × 10⁷cells/mL Nannochloropsis oculata solution (IMET1 wild) in logarithmic growth phase, centrifuging at 4 deg.C and 4000g for 5min, discarding the supernatant, rinsing with 375mM sorbitol pre-cooled at 4 deg.C for 2 times, and adjusting the cell concentration to 2 × 10 with 375mM sorbitol pre-cooled at 4 deg.C⁸cells/mL。

2) The concentrated algal bodies were divided into 200. mu.l aliquots, and 3. mu.g of the above recombinant vector pXJ419 prepared by double digestion with SacI KpnI-HF to form Phsp-Ble-TpsbA-Ptub-NoDGAT2B-His-tag-TpsbA linearized vector (FIG. 4) and 1. mu.l of salmon sperm DNA (15. mu.g/mL, pre-denatured at 95 ℃ for 1min) were added to each aliquot, mixed and then placed on ice for 10 min.

3) The mixture was transferred into a 2mm cuvette and shocked at 2200V (HV), 50. mu.F, 600. omega. immediately after shocking, the algal bodies were transferred into 5mL F/2 liquid medium and then resuscitated in the dark at 25 ℃ and 100rpm for 48 h.

4) Centrifuging at 4000g for 5min, collecting algae, discarding supernatant, adding 1ml corn starch suspension, and blowing and mixing with pipette. The preparation method of the corn starch suspension comprises the following steps: 2g of corn starch was washed with absolute ethanol and pure water alternately 4 times, stored in a 75% ethanol solution, washed 3 times with f/2 liquid medium before use, and then resuspended in 10ml of f/2 liquid medium containing 0.8% PEG8000 (w/v).

5) The mixture was transferred to a medium containing 5. mu.g/mL zeocin and 1.6g/L NaHCO₃The plate was shaken to spread the solution uniformly, incubated at 25 ℃ in the dark for 3 days, and then incubated at 25 ℃ in the dark for 80. mu. mol m^-2s^-1Culturing under illumination until clone grows out to obtain a transformant (Nannochloropsis NoDGAT2B overexpression transformant).

The above transformation procedure was compared with the conventional transformation method (Wang et al,2016), in this example, by increasing the light intensity of plate culture (the previously commonly used light intensity was 30. mu. mol m)^-2s^-1) The growth time of the clone can be shortened from 40-50 days to 8-12 days; in addition, in the embodiment, the corn starch is used for embedding, so that the support and the protection of the recovered cells are increased, and the transformation efficiency is increased from 0.38 multiplied by 10⁴cfu/. mu.g DNA was elevated to 1.46X 10⁴cfu/. mu.g DNA. (IV) analysis of expression level of NoDGAT2B transformed by Nannochloropsis oculata overexpression

And (3) respectively picking the nannochloropsis oculata NoDGAT2B overexpression transformation clone 2 obtained in the step (three) into f/2 culture medium containing 5 mu g/mL zeocin for activation, extracting total DNA of the nannochloropsis oculata NoDGAT2B overexpression transformation clone and carrying out PCR identification on the total DNA, wherein the used primers are as follows:

1)pTub-1for-180322：

5’CTACAAGCCCAACCTTTGCTACACC 3’；

2)NoDGAT2B rev-180419:

5’TCATAAGATTCCCCATAGGCAGCAC 3’；

the PCR instrument used was a MasterCycler from Eppendorf, reaction body50 μ L of the strain, including 4 μ L dNTP (2.5mM each, TAKARA), 2 μ L (10 μ M) of forward and reverse primers, 5 μ L10 XBuffer (Mg 2)⁺plus, TAKARA), 0.4. mu.L rTaq enzyme (5U/. mu.L, TAKARA), 1. mu.L of the gDNA template of the NoDGAT2B overexpressing transformant (50 ng/. mu.L, equal volumes of gDNA of pXJ419 plasmid and wild IMET1 added to positive and negative controls, respectively), and 35.6. mu.L of ultrapure water. The reaction system is as follows: initial 95 ℃ pre-denaturation for 1min, followed by 95 ℃ denaturation for 30sec, 57 ℃ annealing for 30sec, 72 ℃ extension for 1min, 30 cycles, and final 72 ℃ reaction for 5 min. After the reaction, 5. mu.L of the PCR product was mixed with 1. mu.L of 6 × loading buffer (TAKARA), spotted on 1.5% (w/V) agarose (BIOWEST) gel, and subjected to 120V electrophoresis on an electrophoresis system manufactured by Beijing Hei equipment for 25min, followed by observation and photographing using a UV gel imager BioChemiHR of UVP corporation. (FIG. 5)

Specific bands of the transformants OeDgat2b-1, OeDgat2b-2 and the positive control at 600bp are shown, which indicates that pXJ419 has been successfully transferred into nannochloropsis cells. (conclusion is given in connection with the above amplification, and figures and letters are illustrated) the expression amount of each of the above two transformants was analyzed, total RNA was extracted from the transformants by Trizol method, and cDNA was obtained using reverse transcription kit of Takara. Subsequently, CFX96Touch by Bio-Rad was used^TMThe Real-Time PCR Detection System carries out qRT-PCR analysis, and selects housekeeping gene beta-actin as an internal reference. The primers used were as follows:

3)NoDGAT2B-qpcr-for：

5’ATGAAGGCGTGTGGTGGATTGTAAG 3’；

4)NoDGAT2B-qpcr-rev:

5’GTCGAGGTACTGCTTCGCCACG 3’；

5)NoACT-qpcr-for：

5’GACGGCACCAAGGTCAAAAT 3’；

6)NoACT-qpcr-rev:

5’ACGACGTGGAAGAGGAGGAA 3’；

each reaction contained 5. mu.L of iTaq^TM Universal Green Supermix(Bio-Rad),20ng of OeDgat2b-1 and OeDgat2b-2cDNA templates and 280nM primer, in a final reaction system of 10. mu.L. The reaction system is as follows: initial pre-denaturation at 95 ℃ for 30sec, followed by denaturation at 95 ℃ for 5sec, annealing at 60 ℃ for 30sec, 40 cycles, and final fusion at 65-95 ℃.

The result analysis formula is 2^Ct(NoAct)/2^Ct(NoDGATB2). As can be seen from FIG. 5, the mRNA abundance of the transformants was 8-9 times up-regulated compared to the blank vector transformants, and the overexpression effect was good. (V) content analysis of TAG in grease of nannochloropsis oculata NoDGAT2B overexpression transformant

Oil and fat extraction was performed by the chloroform-methanol method of Bligh and Dyer (Bligh and Dyer, 1959). Thin layer chromatography separation of total lipids and TAG acquisition reference Ghosal (Ghosal, 1994). The flow of analysis of TAG by Agilent gas chromatography-quadrupole mass spectrometer (GC-MS) was as follows:

namely, oil extraction, thin layer chromatography separation of total lipid and TAG:

dissolving TAG obtained by total lipid thin-layer chromatography with chloroform-methanol, blowing the lower layer chloroform solution under a nitrogen blowing instrument, adding 1% sulfuric acid-methanol solution (v/v), adding 50uL of n-nonadecanoic acid methanol solution (2.25g/L) as an internal standard, charging nitrogen, sealing with a sealing film, and reacting in a 70 ℃ oven for 60min for methyl esterification. After cooling, the methyl esterification product was extracted with n-hexane and analyzed by GC-MS.

The amount of each fatty acid chain was estimated from the peak area ratio thereof to n-nonadecanoic acid. As can be seen from FIG. 6, the TAG content produced by the over-expressed strain is improved by about 62% compared with that of the blank vector transformant, which proves that nannochloropsis NoDGAT2B has important application value in the aspect of improving the TAG content of organisms.

While specific examples of the invention have been described, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention. It is, therefore, intended that the appended claims cover all such modifications that are within the scope of this present invention.

SEQ ID NO1

1041

DNA

Nannochloropsis oceanica IMET1

SEQ ID NO2

6112

DNA

Nannochloropsis oceanica IMET1

SEQ ID NO：3

346

PRT

Nannochloropsis oceanica IMET1

Sequence listing

<110> institute of bioenergy and Process in Qingdao, China academy of sciences

<120> nannochloropsis oculata genetic transformation system, gene for synthesizing triglyceride and application

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1041

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgacgcagg tctatgcgac aggtgctcac aatatgccgg acgaggaccg tctcaaggtg 60

atgaatggac tttccaagcc cttgacggag gccaagcccg gtgatttggg gtttggggat 120

gtggagtcca tgacgttctg tgaagagttt gtagcgatta tgttcttatt gatcattgtc 180

gggagcatgc tttggatacc gattgcagtg ttgggtttcg ccctgtatgt ccgcagcgca 240

atggcgtggg tggtgatgct catcgtgttc ttcaccctga gcctgcaccc agtcccgcgc 300

atccatgata tggttcattc cccattgaat cacttcatat tcaagtactt cagtctaaaa 360

atggcgagtg atgcaccact ggatagtgct gggcgctata tttttgtcgc tccgccacat 420

ggcgtgctgc ctatggggaa tcttatgacg gtgcacgcga tgaaggcgtg tggtggattg 480

gagttccgtg ggctgacgac tgatgtcgcg cttaggctgc ctttgtttcg acactattta 540

ggcgctattg gtactattgc cgcgacgagg cacgtggcga agcagtacct cgacaaagga 600

tggtcaatag gtatatcttc gggcggagtc gcggagattt tcgaagttaa caataaggat 660

gaggtggtgt tgatgaagga gcgaaagggc tttgtgaagc ttgcccttcg cacggggact 720

ccgttggtag cttgttatat ttttgggaat accaagctgt tgtcggcgtg gtatgatgat 780

gggggggtgt tggagggcct gtcgcgttat ttgaaatgtg gtgtattgcc actttggggt 840

cgctttggtt tgccgcttat gcaccgtcat cctgtattgg gcgcgatggc aaagccgatc 900

gtagttccca aggtggaggg agagcccacg caggagatga ttgatgagta ccatagtctc 960

ttctgtcaga cgctggtcga tctatttgat agatacaaga ccttgtatgg ctggccggac 1020

aagaagctgc ttattaagtg a 1041

<210> 2

<211> 6112

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

catcgatgtc tactctcgta aaccctgtcc cactcaatgt agaatacaaa gcaggggctg 60

ctggcacgac tccaagcctc tgcgaagcgt caaattttcg tcttcaggca gagaacgtgc 120

tgtgtgtgtg gctgtggagc ggagagcggc ttcgtcttta aagatgcctt cacccacatg 180

gcgcgtgccc gccctgccga tgcgtcttga taacactacg tgttcagtta cggtgacgat 240

gcatcgtgcc cccgctgtag ccttgtgtca ttttcgttct tctgcgacat ggaaaaggga 300

cagcgaaagc cacagaaaac acaaagaact ttcgacgact cactcaccgc tcccaacctt 360

cacccatacc acagtaacaa aacaatatca caaccaaaga ccctcgaggg cgcgccatgg 420

ccaagctgac cagcgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt 480

tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg 540

tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca 600

ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg 660

tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt 720

gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg 780

agcaggacta accgacgccg acgatatcga attccccggg ccgtatctaa tgttcttcca 840

gttgcattaa acgctcctgc tgttaatgct taagaatatt taattacaca tgagtgattt 900

ttaatcactc atgtgtaata atttttttga tctaatttaa ttaagtaaaa atgaaacggt 960

tagacttttt tatatacttg ctaaattaag tgtaaaaata ctattttata gtattgttgc 1020

tctatccaac ttaactgagc gtctaagtat tatactcaaa ctaacttcat ggataggtaa 1080

aaaagataag cgaacgattt caaatggaat ttaattttac tcaaaaataa agtgaacgcg 1140

gtatctatac attctagtaa attataaaaa ttgatctaat aaagtaagta agtaacaact 1200

agttcatcaa attgatgaac tagttgttac ttatttaaga attataaaac gtaacgttct 1260

ccaggaggat atttgttaac aacataacta tgaatagtgt aacggatgtt acgaccttca 1320

gtttcttgtc tttctagata gaaacctggt tcttcataag gacgttgcgc tataaaagat 1380

aatgcacaac tttcagtccc acgagtatta tcaaaagcat taattttgat atatgttgtt 1440

ggatgtgcag cgcgacattg atctaattca tacataatta cagaagcgtc tttaatatcg 1500

aataaaggca tcccccatag ttcccagtat gtgttacgag gatgtggatc ttccgtccat 1560

tcgatactac aagcccaacc tttgctacac ctaggtgtac actactcctt ttatacgcct 1620

tgacacacaa agcacctagt atgttgtgcg ggaccggcgt atgcgtgggc agagaggcgc 1680

ggtcatggcg tttgtgtgag cgagccgcgc cctccagctt tgtgaatttt tcgtgaaggc 1740

gaaaacgtgc ctatcacact atcccacacg cctacaaaca agccacatag caacaacgcg 1800

agtacactta tgtttcctac gttcgtgccc gcacgaaggg gcgtctgtga aggcgcaggg 1860

ctactctggg ccctccaaat gatgtgcacg cttcccgtct cactttacac acttcctctt 1920

cactccttcc gttcacatca acacacgcac aacttaagca cacatcagtc agcacccctt 1980

caacacactc ctccctccaa ctagtctcaa ccccatatgg catgcgtcga cccgcggacg 2040

cgtgctagcg gcgccaagct tctgcagccc gggggatccc atcatcacca tcaccatcac 2100

cattaaccgt atctaatgtt cttccagttg cattaaacgc tcctgctgtt aatgcttaag 2160

aatatttaat tacacatgag tgatttttaa tcactcatgt gtaataattt ttttgatcta 2220

atttaattaa gtaaaaatga aacggttaga cttttttata tacttgctaa attaagtgta 2280

aaaatactat tttatagtat tgttgctcta tccaacttaa ctgagcgtct aagtattata 2340

ctcaaactaa cttcatggat aggtaaaaaa gataagcgaa cgatttcaaa tggaatttaa 2400

ttttactcaa aaataaagtg aacgcggtat ctatacattc tagtaaatta taaaaattga 2460

tctaataaag taagtaagta acaactagtt catcaaattg atgaactagt tgttacttat 2520

ttaagaatta taaaacgtaa cgttctccag gaggatattt gttaacaaca taactatgaa 2580

tagtgtaacg gatgttacga ccttcagttt cttgtctttc tagatagaaa cctggttctt 2640

cataaggacg ttgcgctata aaagataatg cacaactttc agtcccacga gtattatcaa 2700

aagcattaat tttgatatat gttgttggat gtgcagcgcg acattgatct aattcataca 2760

taattacaga agcgtcttta atatcgaata aaggcatccc ccatagttcc cagtatgtgt 2820

tacgaggatg tggatcttcc gtccattcga tactacaagc ccaacctttg ctacagataa 2880

atttaagttg acctaaaatt tgttcgtttg ttaaatccgg taagtacgaa aatgcacctt 2940

gtgttaatct cactcttcaa atactccttt agtatgttaa actgattgat attgttttta 3000

tacgattact gtttagttgc agtttcaatg taatcagcag tatctgttga tgtatagttg 3060

aaagcaatat ctttccaaag atctaaagca gaacgtaatg gcgcacaact ttctgccgcc 3120

gcacgtaaaa tttttggtcc ttcgttaatg taatctttac cttcatattt agctaaaagc 3180

actgactcca tagctacgcg gttagcagtc gcaccagaag cgataccatc agggtgacca 3240

attgagctga gctccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc 3300

gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 3360

catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 3420

cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg 3480

cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc 3540

cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga 3600

gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg 3660

atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag 3720

cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga 3780

acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg 3840

tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg 3900

cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 3960

tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 4020

gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 4080

cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 4140

atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 4200

agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 4260

gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 4320

ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 4380

cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 4440

ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 4500

atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 4560

gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 4620

tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 4680

gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 4740

gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 4800

tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 4860

ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 4920

tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 4980

ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 5040

ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 5100

tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 5160

ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 5220

tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 5280

tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 5340

actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 5400

cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 5460

gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 5520

tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 5580

ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 5640

ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 5700

cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 5760

cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 5820

gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 5880

attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 5940

ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 6000

gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 6060

attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctgggt ac 6112

<210> 3

<211> 346

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Thr Gln Val Tyr Ala Thr Gly Ala His Asn Met Pro Asp Glu Asp

1 5 10 15

Arg Leu Lys Val Met Asn Gly Leu Ser Lys Pro Leu Thr Glu Ala Lys

20 25 30

Pro Gly Asp Leu Gly Phe Gly Asp Val Glu Ser Met Thr Phe Cys Glu

35 40 45

Glu Phe Val Ala Ile Met Phe Leu Leu Ile Ile Val Gly Ser Met Leu

50 55 60

Trp Ile Pro Ile Ala Val Leu Gly Phe Ala Leu Tyr Val Arg Ser Ala

65 70 75 80

Met Ala Trp Val Val Met Leu Ile Val Phe Phe Thr Leu Ser Leu His

85 90 95

Pro Val Pro Arg Ile His Asp Met Val His Ser Pro Leu Asn His Phe

100 105 110

Ile Phe Lys Tyr Phe Ser Leu Lys Met Ala Ser Asp Ala Pro Leu Asp

115 120 125

Ser Ala Gly Arg Tyr Ile Phe Val Ala Pro Pro His Gly Val Leu Pro

130 135 140

Met Gly Asn Leu Met Thr Val His Ala Met Lys Ala Cys Gly Gly Leu

145 150 155 160

Glu Phe Arg Gly Leu Thr Thr Asp Val Ala Leu Arg Leu Pro Leu Phe

165 170 175

Arg His Tyr Leu Gly Ala Ile Gly Thr Ile Ala Ala Thr Arg His Val

180 185 190

Ala Lys Gln Tyr Leu Asp Lys Gly Trp Ser Ile Gly Ile Ser Ser Gly

195 200 205

Gly Val Ala Glu Ile Phe Glu Val Asn Asn Lys Asp Glu Val Val Leu

210 215 220

Met Lys Glu Arg Lys Gly Phe Val Lys Leu Ala Leu Arg Thr Gly Thr

225 230 235 240

Pro Leu Val Ala Cys Tyr Ile Phe Gly Asn Thr Lys Leu Leu Ser Ala

245 250 255

Trp Tyr Asp Asp Gly Gly Val Leu Glu Gly Leu Ser Arg Tyr Leu Lys

260 265 270

Cys Gly Val Leu Pro Leu Trp Gly Arg Phe Gly Leu Pro Leu Met His

275 280 285

Arg His Pro Val Leu Gly Ala Met Ala Lys Pro Ile Val Val Pro Lys

290 295 300

Val Glu Gly Glu Pro Thr Gln Glu Met Ile Asp Glu Tyr His Ser Leu

305 310 315 320

Phe Cys Gln Thr Leu Val Asp Leu Phe Asp Arg Tyr Lys Thr Leu Tyr

325 330 335

Gly Trp Pro Asp Lys Lys Leu Leu Ile Lys

340 345