CN114591997A

CN114591997A - Expression vector of schizochytrium limacinum, construction method of expression vector and application

Info

Publication number: CN114591997A
Application number: CN202210106434.4A
Authority: CN
Inventors: 李翔宇; 万霞; 刘洋; 刘鹏阳; 李港
Original assignee: Cabio Biotech Wuhan Co Ltd
Current assignee: Cabio Biotech Wuhan Co Ltd
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2022-06-07

Abstract

The invention provides an expression vector of schizochytrium limacinum, a construction method and application of the expression vector, and belongs to the field of genetic engineering. The expression vector comprises: pSc-18S rDNA vector, the combination of promoter and terminator of Schizochytrium limacinum, and the gene sequence of resistance-fluorescence double screening marker; the combination of promoter and terminator includes at least one of the following combinations: a combination of promoter PT and terminator TT, a combination of promoter PA1 and terminator TA, a combination of promoter PA2 and terminator TA, and a combination of promoter PE2 and terminator TE. According to the high-efficiency schizochytrium genome editing and transformant screening technology, a resistance-fluorescence double screening marker is efficiently integrated to a schizochytrium chromosome in a fusion protein expression mode through a gene gun, the testing of a gene editing element and the quick screening of a transformant can be carried out on the basis of the expressed fluorescence intensity, and particularly when an endogenous gene is expressed, the technology has very outstanding advantages.

Description

Expression vector of schizochytrium limacinum, construction method of expression vector and application

Technical Field

The disclosure belongs to the field of genetic engineering, and particularly relates to an expression vector of schizochytrium limacinum, a construction method of the expression vector and application of the expression vector.

Background

Schizochytrium limacinum belongs to heterotrophic eukaryotes of the family thraustochytriaceae, has the capability of rapidly bifurcating and releasing zoospores, and can produce various unsaturated fatty acids, such as docosahexaenoic acid (DHA), by utilizing various carbon sources as substrates to perform high-density fermentation. In recent years it has been found that certain schizochytrium species are also capable of synthesizing carotenoids, such as beta-carotene and astaxanthin (astaxanthhin). The carotenoid can be used as an antioxidant or a colorant in the fields of food, health products, cosmetics and animal breeding, and has considerable application prospect. Since the synthesis mechanism and metabolic regulation mechanism of schizochytrium astaxanthin and carotenoids thereof are not yet revealed, related researches enter a bottleneck period. At the present stage, the carotenoid synthesis of the schizochytrium limacinum is improved mainly by means of random induced mutation, an optimized culture mode and the like.

At present, the electroporation transformation method, the gene gun transformation method and the agrobacterium-mediated transformation method are researched to be applied to the construction of the genetic operation system of the schizochytrium limacinum. For example, electroporation transformation, particle gun method or Agrobacterium transformation have been used to successfully insert the target gene into chromosome by homologous arm integration for gene overexpression, or related gene knockout in schizochytrium chromosome by using CRISPR/Cas 9 system. However, compared with the model strain, the genetic operation system of the schizochytrium still is in an immature stage, the problems of few gene editing elements, complex transformant screening and the like still exist, the gene editing efficiency of the schizochytrium is restricted, and the expansion of the application of the schizochytrium is hindered.

Disclosure of Invention

The embodiment of the disclosure provides an expression vector of schizochytrium limacinum, a construction method and application of the expression vector, and the method can be used for testing gene editing elements and quickly screening transformants. The technical scheme is as follows:

embodiments of the present disclosure provide an expression vector, including: pSc-18S rDNA vector, a combination of promoter and terminator of Schizochytrium limacinum, resistance gene and gene cassette containing fluorescent gene;

the combination of promoter and terminator includes at least one of the following combinations: a combination of promoter PT and terminator TT, a combination of promoter PA1 and terminator TA1, a combination of promoter PA2 and terminator TA2, and a combination of promoter PE2 and terminator TE.

In yet another implementation of the disclosure, the resistance gene is the G418 resistance gene nptii.

In yet another implementation of the present disclosure, the fluorescent gene is an eGFP fluorescent gene or a DsRed fluorescent gene.

In yet another embodiment of the present disclosure, the primer amplification sequences of the promoter PT, the promoter PA1, the promoter PA2, the promoter PE2, the terminator TT, the terminator TA, and the terminator TE (which primers contain partial vector partial sequences for assembly of the genetic elements) are shown as SRQ ID NO: 1-8.

In yet another implementation manner of the present disclosure, there is also provided a method for constructing an expression vector suitable for schizochytrium limacinum, the method comprising:

obtaining a combination of a promoter and a terminator from schizochytrium, the combination of the promoter and the terminator comprising at least one of the following combinations: a combination of promoter PT and terminator TT, a combination of promoter PA1 and terminator TA1, a combination of promoter PA2 and terminator TA2, and a combination of promoter PE2 and terminator TE;

assembling the combination of the promoter and the terminator in pSc-18S rDNA vector to obtain pSc-18S rDNA-P-T vector containing promoter and terminator elements;

assembling a fluorescent gene and a resistance gene into the pSc-18S rDNA-P-T vector to obtain the expression vector.

In yet another implementation form of the present disclosure, the obtaining of the combination of a promoter and a terminator of schizochytrium comprises:

selecting a gene actin, a gene elongation and a gene tubulin from the schizochytrium;

respectively obtaining 2000bp gene sequences of the gene actin, the gene tubulin and the gene ligation as gene element analysis sequences;

analyzing the combination of the promoter and the terminator of the schizochytrium according to the sequence of each gene.

In yet another implementation of the disclosure, the pSc-18S rDNA vector is obtained by:

obtaining a replication element and an ampicillin resistance gene on the pYLXP' vector and an upstream and downstream gene sequence of 18S rDNA;

carrying out double enzyme digestion on the pYLXP' vector;

and (3) assembling the replication element and the ampicillin resistance gene on the pYLXP' vector subjected to double enzyme digestion with the upstream and downstream gene sequences of the 18S rDNA to obtain the pSc-18S rDNA vector.

In yet another implementation of the disclosure, the combination of the promoter and the terminator is assembled in pSc-18S rDNA vector to obtain pSc-18S rDNA-P-T vector containing promoter and terminator elements, comprising:

amplifying the promoter and terminator in said combination of promoter and terminator;

cloning the amplified combination of the promoter and the terminator into the pSc-18S rDNA vector to obtain the pSc-18S rDNA-P-T vector containing promoter and terminator elements.

In yet another implementation of the present disclosure, the resistance gene is the G418 resistance gene nptii, and the fluorescent gene is an eGFP fluorescent gene or a DsRed fluorescent gene.

In still another implementation manner of the disclosure, the application of the expression vector of the schizochytrium limacinum is further provided, and the expression vector is applied to the gene editing of the schizochytrium limacinum.

The technical scheme provided by the embodiment of the disclosure has the following beneficial effects:

when the expression vector of the schizochytrium provided by the embodiment of the disclosure is applied to gene editing of the schizochytrium, the expression vector comprises the resistance gene and the gene cassette containing the fluorescent gene, so that the expression form of the fusion protein can be used as a double-screen marker through the resistance gene and the fluorescent gene during gene editing, so that gene element testing and transformant screening are facilitated, the screening program is simplified, and further, the system of a transformation system is simplified.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present disclosure, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a flow chart of a method for constructing an expression vector for Schizochytrium according to an embodiment of the disclosure;

FIG. 2 is a flow chart of another method for constructing an expression vector for Schizochytrium provided in the embodiments of the present disclosure;

FIG. 3 is a schematic diagram of the assembly of pSc-18S rDNA vector;

FIG. 4 is a schematic diagram of the assembly of pSc-18S rDNA-P-T vector;

FIG. 5 is a schematic diagram of the detection of a promoter and a terminator;

FIG. 6 is a schematic diagram of the assembly of an expression vector;

FIG. 7 is a schematic diagram showing the results of PCR detection of 8 vectors;

FIG. 8 is a schematic diagram of the assembly of pSc-PA2-YREIS-PE 2-eGFP.

Detailed Description

To make the objects, technical solutions and advantages of the present disclosure more apparent, embodiments of the present disclosure will be described in detail with reference to the accompanying drawings.

The embodiment of the present disclosure provides an expression vector of schizochytrium limacinum, which includes: pSc-18S rDNA vector, the combination of promoter and terminator of Schizochytrium limacinum, and resistance-fluorescence double screening marker gene sequence.

The combination of promoter and terminator includes at least one of the following combinations: a combination of promoter PT and terminator TT, a combination of promoter PA1 and terminator TA, a combination of promoter PA2 and terminator TA, and a combination of promoter PE2 and terminator TE.

When the expression vector of the schizochytrium provided by the embodiment of the disclosure is applied to gene editing of the schizochytrium, the expression vector comprises a combination of a promoter and a terminator and a gene sequence of a resistance-fluorescence double-sieve marker, so that the combination of the promoter and the terminator can be used as an expression element of a gene during gene editing, and the expression form of a fusion protein can be used as the double-sieve marker through the resistance gene and the fluorescence gene, so that the gene element test and transformant screening are facilitated, the screening program is simplified, and further, the transformation system is simplified.

Optionally, the resistance gene corresponding to the gene sequence of the resistance-fluorescence double-screening marker is a G418 resistance gene nptII. G418 is an aminoglycoside antibiotic. The Schizochytrium limacinum is completely incapable of growing on a 2mg/mL G418 plate, so that the G418 resistance gene nptII can be used as the resistance gene.

In this example, G418 of 2mg/mL was used as a selection expression vector to form a selection concentration of transformants after transformation by Schizochytrium limacinum.

Optionally, the fluorescent gene corresponding to the gene sequence of the resistance-fluorescent double-screening marker is an eGFP fluorescent gene or a DsRed fluorescent gene.

The DsRed fluorescent gene is a fluorescent gene derived from Corallium shiitanum (Discosoma sp.) and can be excited to emit red fluorescence at 560nm and infrared rays after expression, and the eGFP gene can be excited to emit green fluorescence at 488nm and ultraviolet rays after expression.

Alternatively, the amplification primer sequences for promoter PA1, promoter PT, promoter PE2, promoter PA2, terminator TT, terminator TA, terminator TE are as shown in SRQ ID NO 1-8 or see the following table.

TABLE 1 sequence Listing of amplification primers

The embodiment of the present disclosure provides a method for constructing an expression vector for schizochytrium limacinum, as shown in fig. 1, the method comprises:

s101: obtaining a combination of a promoter and a terminator from schizochytrium, the combination of the promoter and the terminator comprising at least one of the following combinations: a combination of promoter PT and terminator TT, a combination of promoter PA1 and terminator TA, a combination of promoter PA2 and terminator TA, and a combination of promoter PE2 and terminator TE.

S102: the combination of promoter and terminator was assembled on pSc-18S rDNA vector to obtain pSc-18S rDNA-P-T vector containing promoter and terminator elements.

S103: assembling the gene sequence of the resistance-fluorescence double screening marker to pSc-18S rDNA-P-T vector to obtain the expression vector.

When the expression vector of the schizochytrium provided by the embodiment of the disclosure is applied to gene editing of the schizochytrium, because the expression vector comprises pSc-18S rDNA vector sequence, a combination of a promoter and a terminator and a gene sequence of a resistance-fluorescence double screening marker, when the gene editing is carried out, the function of the expression element can be tested by replacing different gene expression elements to express fusion protein of the resistance-fluorescence double screening marker, meanwhile, a target genome can be arranged on the expression vector, and when the endogenous gene is expressed, the resistance gene and the fluorescence gene can be used as the double screening marker, so that transformants can be screened conveniently, the screening program is simplified, and further, a transformation system is simplified.

FIG. 2 is a flow chart of another construction method of an expression vector for Schizochytrium provided in the embodiment of the disclosure, and in combination with FIG. 2, the construction method comprises:

s201: obtaining a combination of a promoter and a terminator from schizochytrium, the combination of the promoter and the terminator comprising at least one of the following combinations: a combination of promoter PT and terminator TT, a combination of promoter PA1 and terminator TA, a combination of promoter PA2 and terminator TA, and a combination of promoter PE2 and terminator TE.

Exemplarily, S201 includes:

2011: selecting gene actin, gene elongation and gene tubulin from Schizochytrium limacinum.

Analyzing the gene transcription levels of the growth logarithmic phase and the stationary phase of the schizochytrium limacinum, and selecting actin with continuously high transcription level, elongation and tubulin with medium transcription level in housekeeping genes as target genes of gene expression elements.

2012: respectively obtaining the gene sequences of 2000bp upstream and downstream of the gene actin, the gene tubulin and the gene elongation to obtain the sequences for analyzing the promoter and the terminator elements.

The sequences of 2000bp upstream and downstream of the gene actin, the gene tubulin and the gene elongation are obtained by analyzing the genome sequence of the gene with high transcription level expression.

2013: based on the sequence of the promoter and terminator element analysis, the combination of the promoter and terminator of the schizochytrium limacinum is obtained by analysis.

Promoter preliminary prediction is carried out on the upstream of the gene sequence through TSSP software, and the promoter sequence is obtained by combining with specific analysis. The terminator of the gene sequence is preliminarily predicted by RibEx software, and the terminator is obtained according to whether the gene sequence can form a hairpin structure or not.

S202: pSc-18S rDNA vector was constructed.

Exemplarily, S202 includes:

2021: obtaining the replication element and ampicillin resistance gene on the pYLXP' vector and the gene sequence of the 18S rDNA upstream and downstream.

Both the pYLXP' vector and the 18S rDNA gene sequence were from the laboratory of the group of microbial subjects applied to the institute of oil crops, academy of agricultural sciences, China.

The middle of the 18S rDNA gene sequence is designed with AvrII, XhoI, NheI, SalI and other enzyme cutting sites and 2 homodromous loxp sites, which is convenient for the subsequent assembly of promoter and terminator and the iterative integration of gene.

2022: carrying out double enzyme digestion on the pYLXP' vector.

pYLXP' was double-digested with SspI enzyme and SalI enzyme, and the whole reaction system was shown in Table 2, and reacted at 37 ℃ for 1 hour.

TABLE 2 SspI and SalI double digestion pYLXP' reaction System

Composition (component)	V(μL)
		SspI	1
SalI	1
		Cut Smart (enzyme digestion buffer)	3
ddH₂O (double distilled water)	15
		plasmid (vector, pYLXP' vector)	10

In this example, after the double digestion reaction was completed, the target band of 3674bp was recovered by agarose gel electrophoresis.

2023: and assembling the replicon and the ampicillin resistance gene after the pYLXP' double enzyme digestion with the upstream and downstream gene sequences of the 18S rDNA to obtain the pSc-18S rDNA vector.

The specific assembly system is shown in Table 3, wherein the reaction conditions of the assembly system are 50 ℃ and the reaction time is 60 min.

TABLE 3 pSc-18S rDNA vector Assembly System

FIG. 3 is a schematic diagram of the assembly of pSc-18S rDNA vector, and referring to FIG. 3, it can be seen that the whole vector sequence is assembled by three sequences, i.e., two sequences are obtained by gene synthesis, and iii is a replication element and an ampicillin resistance gene obtained by double digestion of pYLXP' vector by SspI and SalI. In the recombination process, SalI in the third step is destroyed and BamHI restriction enzyme cutting sites are introduced.

S203: the combination of promoter and terminator was assembled into pSc-18S rDNA vector to obtain pSc-18S rDNA-P-T vector containing promoter and terminator elements.

Exemplarily, S203 includes:

2031: the promoter and terminator in the combination of promoter and terminator were subjected to PCR amplification (polymerase chain reaction).

Illustratively, the predicted Schizochytrium limacinum promoters PT (tubulin) and terminator TT (tubulin), promoter PA1(actin) and terminator TA (actin), promoter PA2(actin), terminator TA (actin), promoter PE2 (electrophoresis), and terminator TE (electrophoresis) were amplified and cloned into pSc-18S rDNA vectors, respectively.

Promoter and terminator amplification was performed according to the amplification system of table 4 below.

TABLE 4 reaction System for promoter and terminator amplification

PCR amplification	V(μL)
		F-primer (Forward primer)	2
R-primer (reverse primer)	2
		genomic DNA (promoter or terminator to be amplified)	5
2x Clonexpress Mix (buffer)	25
		H₂O	16

In this example, the PCR detection primers are shown in Table 5 below.

TABLE 5 PCR detection primers

2032: cloning the combination of the amplified promoter and terminator into pSc-18S rDNA vector to obtain pSc-18S rDNA-P-T vector containing the promoter and the terminator.

Illustratively, assembly of the amplified combination of promoter and terminator into pSc-18S rDNA vector was performed by the following reaction system.

TABLE 6 combination of promoter and terminator Assembly into pSc-18S rDNA vector

FIG. 4 is a schematic diagram of the assembly of pSc-18S rDNA-P-T vector, and in conjunction with FIG. 4, it can be seen that pSc-18S rDNA-P-T vector is based on pSc-18S rDNA vector, and the corresponding promoter and terminator are assembled between NheI and SalI cleavage sites of 18S rDNA vector.

In this example, the promoters PA1(749bp), PA2(1530bp), PT (603bp), PE2(1229bp), and the terminators TE (140bp), TA (154bp), and TT (150bp) were amplified, respectively.

The obtained promoter (PA1, PT) and terminator (TA and TT) are assembled into the pSc-18S rDNA vector digested by NheI and SalI respectively, the detection result is shown in figure 5, the vector sizes of the

sequence numbers

9, 10, 15 and 16 are correct, and then the sequence is sequenced to detect the correctness.

Then on the basis of pSc-18S rDNA-PA1-TA vector, PA2, TA, PE2 and TE are respectively replaced to the vector, so that pSc-18S rDNA-PA2-TA vector and pSc-18S rDNA-PE2-TE vector can be obtained.

S204: and assembling the gene sequence of the resistance-fluorescence double-screening marker into an pSc-18S rDNA-P-T vector containing a promoter and a terminator to obtain an expression vector.

The resistance gene is G418 resistance gene nptII, and the fluorescent gene is eGFP or DsRed gene.

The Schizochytrium limacinum cannot grow at all on a 2mg/mL G418 plate, so that the G418 resistance gene nptII can be used as a resistance gene of an expression vector.

G418 resistance gene nptII is selected to be respectively expressed with eGFP and DsRed fluorescent genes in a fusion protein form, and a gene sequence corresponding to the fusion protein is directly synthesized after codon optimization.

In this example, the PT promoter, PA1 promoter, PA2 promoter, and PE2 promoter were used to initiate the transcription and expression of the gene composed of nptII plus eGFP and DsRed.

The whole recombination system is shown in Table 7, the whole recombination reaction system is reacted at 50 ℃ for 1 hour, and the specific assembly process is shown in FIG. 6.

TABLE 7 fluorescent genes and resistance genes assembled into pSc-18S rDNA-P-T vectors.

FIG. 6 is a schematic diagram showing the assembly of the expression vector, and it can be seen from FIG. 6 that the G418 resistance gene nptII, the fluorescent genes eGFP and DsRed are assembled between the NheI and SalI cleavage sites of the 18S rDNA vector.

Finally, the synthesized cassettes of the fluorescent gene and the resistance gene were ligated to pSc-18S rDNA containing a promoter and a terminator, to construct pSc-PA1-DsRed, pSc-PA1-eGFP, pSc-PT-DsRed, pSc-PT-eGFP, pSc-PA2-DsRed, pSc-PA2-eGFP, pSc-PE2-DsRed, pSc-PE2-eGFP, and a total of 8 vectors, whose PCR-detected results are shown in FIG. 7, wherein pSc-PA1-DsRed (12), pSc-PA1-eGFP (15), pSc-PT-DsRed (21), pSc-sRed-eGFP (37), pSc-PA2-DsRed (1), pSc-PA2-eGFP (23), pSc-PE2-DsRed (34), and pSc-2-eGFP (46) in the correct size, and further sequencing to verify correctness, DH5 alpha containing correct expression vector is cultured in AMP resistant medium at 37 ℃ overnight and plasmid is extracted, and then the plasmid is transferred to Schizochytrium by gene gun or electric transformation for promoter function verification.

The predicted promoter and terminator were verified for promoter function by expression of fusion proteins by constructing 8 vectors of pSc-PT-DsRed, pSc-PA1-DsRed, pSc-PA2-DsRed, pSc-PE2-DsRed and pSc-PT-eGFP, pSc-PA1-eGFP, pSc-PA2-eGFP, pSc-PE 2-eGFP. Both PA1 and PA2 and PE2 successfully initiated fluorescent gene expression, except that the PT promoter did not successfully initiate fluorescent gene expression.

The transformation efficiency of the specific gene gun is shown in Table 8, and the transformation efficiency of the schizochytrium through the gene gun is between 60% and 100%.

The pSc-PE 2-DsRed-transformed Schizochytrium limacinum can be excited to emit strong fluorescence under infrared light, while the pSc-PE 2-eGFP-transformed Schizochytrium limacinum can be excited to emit green fluorescence under ultraviolet light, while the wild-type (WT) Schizochytrium limacinum is not excited to emit fluorescence.

TABLE 8 promoter validation and transformation efficiency statistics

In this example, the strength of the promoter was also verified by experiments.

The promoter is verified by respectively starting the predicted promoter and terminator with the fusion protein consisting of eGFP or DsRed and nptII, and the strength of the promoter is judged according to the expressed fluorescence intensity. Wild Type (WT) Schizochytrium limacinum has no visible light under various signal channels when the exposure time is 400 msec. pSc-PE2-DsRed fluorescent protein expressed by Schizochytrium limacinum is very strong, when the exposure time is 40msec, the red fluorescent protein is equivalent to the fluorescence expressed by pSc-PA2-DsRed with the exposure time of 400msec, which indicates that the PE2 promoter is stronger than the PA2 promoter. When pSc-PA2-eGFP and pSc-PA1-eGFP respectively express the green fluorescent protein, the fluorescence intensity of the green fluorescent protein initiated by PA2 is stronger than that of the green fluorescent protein initiated by PA1 when the exposure time is 400 msec. In total, 4 pairs of promoters were verified, and since it was predicted that the PT promoter did not successfully initiate gene expression, the strength of the promoters was compared: PE2> PA2> PA 1.

In addition, in this example, expression vectors of genes related to the MVA pathway (mevalonate pathway) of schizochytrium were constructed by the above method.

Firstly, 4 main enzymes in the MVA path of the schizochytrium, namely CrtE, HMGR, HMGS and IDI are synthesized after codon optimization to obtain corresponding target genes.

Then, the target gene was cloned into pSc-PA2-TA vector digested with XbaI and SphI by Gibson Assembly, respectively.

Next, the target gene containing the promoter and terminator regions was digested by HpaI and SnaBI in a double digestion, and ligated to the AvrII single digested pSc-PE2-eGFP vector again in the Gibson Assembly manner, yielding pSc-PA2-hmgs-PE2-eGFP, pSc-PA2-hmgr-PE2-eGFP, pSc-PA2-crtE-PE2-eGFP, pSc-PA2-idi-PE 2-eGFP.

In addition, the 4 enzymes and the crtIBY gene were linked by linker and ligated to pSc-PA1-TA vector in the same manner, which was named pSc-PA1-YREIS-PE 2-eGFP. The whole recombination system is shown as (Table 3.6), the whole recombination reaction system is at 50 ℃ for 60min, and the specific assembly process is shown as figure 8.

Alternatively, the above expression vector can be used in gene editing of schizochytrium limacinum, for example, in synthesis of high value-added products such as carotenoids.

The invention establishes an efficient schizochytrium genome editing and transformant screening technology, and integrates a resistance-fluorescence double screening marker into a schizochytrium chromosome in a form of fusion protein through a gene gun in an efficient manner.

When the system is used for expressing the endogenous gene of the schizochytrium limacinum, the advantages are very outstanding, and the reasons are as follows: by using the resistance-fluorescence double screening marker, the positive transformant can grow on a resistance plate and can observe fluorescence. When designing a vector, an endogenous gene and a double-screening marker gene are in one expression vector, and the expression of the endogenous gene is started first, and then the expression of the resistance-fluorescence double gene is started. Theoretically, the resistance-fluorescent gene arranged later, if expressed, has the previous endogenous gene already expressed. Therefore, when the endogenous gene is overexpressed, only transformants which can grow and fluoresce on the resistant plate need to be picked up, and no additional transformant verification work needs to be carried out, so that the time and the cost are remarkably saved.

In general, when endogenous or exogenous genes are transformed into a host, even if transformants that can grow are selected on a resistant plate, a certain proportion of false positive strains exist, so that the selected transformants need to be further tested. At the time of expressing an endogenous gene, since the host already contains the gene, it is only possible to amplify a resistance marker on the vector or amplify the integration position of the gene to judge whether the gene of interest is integrated into the genome, thereby identifying a false-positive transformant. In addition, there may be cases where the gene sequence is integrated into the genome, but the gene is not necessarily expressed, in positive transformants. If the expressed foreign gene is the foreign gene, judging whether the foreign gene is transcribed or expressed through qRT-PCR or Western Blot; if an endogenous gene is expressed, since the host already contains the gene, whether the gene is successfully expressed can be judged only based on the amount of the protein or the transcription of the gene in the host by the above-mentioned method. However, some genes are relatively weak in expression, and when endogenous genes are converted, resistance genes are carried to increase burden on host bacteria, multiple uncontrollable factors exist in the processes of strain culture, experiment operation and the like, the change of transcription or expression quantity is possibly not obvious, whether the genes are successfully expressed or not cannot be judged, and time and labor are wasted in one set of process. Compared with the prior art, the method provided by the invention has the advantage that the time and labor cost for screening the positive transformants are remarkably saved.

The above description is intended to be exemplary only and not to limit the present disclosure, and any modification, equivalent replacement, or improvement made without departing from the spirit and scope of the present disclosure is to be considered as the same as the present disclosure.

Claims

1. An expression vector for schizochytrium, comprising: pSc-18S rDNA vector, the combination of promoter and terminator of Schizochytrium limacinum, and resistance-fluorescence double screening marked gene sequence;

2. The expression vector of claim 1, wherein the resistance gene corresponding to the gene sequence of the resistance-fluorescence double screening marker is the G418 resistance gene nptII.

3. The expression vector of claim 1, wherein the fluorescent gene corresponding to the gene sequence of the resistance-fluorescent double-screening marker is an eGFP fluorescent gene or a DsRed fluorescent gene.

4. The expression vector of claim 1, wherein the amplified sequences of the promoter PT, the promoter PA1, the promoter PA2 and the promoter PE2, the terminator TT, the terminator TA and the terminator TE are shown in SRQ ID NO 1-8.

5. A method for constructing an expression vector suitable for Schizochytrium, which is used for constructing the expression vector of any one of claims 1-4, the method comprising:

obtaining a combination of a promoter and a terminator from schizochytrium, the combination of the promoter and the terminator comprising at least one of the following combinations: a combination of promoter PT and terminator TT, a combination of promoter PA1 and terminator TA, a combination of promoter PA2 and terminator TA, and a combination of promoter PE2 and terminator TE;

assembling the combination of the promoter and the terminator on pSc-18S rDNA vector to obtain pSc-18S rDNA-P-T vector containing promoter and terminator elements;

assembling the gene sequence of the resistance-fluorescence double screening marker to the pSc-18S rDNA-P-T vector to obtain the expression vector.

6. The construction method according to claim 5, wherein the obtaining of the combination of the promoter and the terminator of the Schizochytrium limacinum comprises:

respectively obtaining 2000bp gene sequences of the gene actin, the gene tubulin and the gene ligation, and obtaining sequences for analyzing promoter and terminator elements;

analyzing the combination of the promoter and the terminator of the schizochytrium according to the analyzed sequence of the promoter and the terminator elements.

7. The method of construction according to claim 5, wherein the pSc-18S rDNA vector is obtained by:

carrying out double enzyme digestion on the pYLXP' vector;

and assembling the replicon and the ampicillin resistance gene after the pYLXP' double enzyme digestion with the upstream and downstream gene sequences of the 18S rDNA to obtain the pSc-18S rDNA vector.

8. The method of constructing a recombinant vector according to claim 7, wherein the combination of the promoter and the terminator is assembled into pSc-18S rDNA vector to obtain pSc-18S rDNA-P-T vector containing promoter and terminator elements, comprising:

cloning the amplified combination of the promoter and the terminator onto the pSc-18S rDNA vector to obtain the pSc-18S rDNA-P-T vector containing the promoter and the terminator.

9. The construction method according to claim 5, wherein the resistance gene corresponding to the gene sequence of the resistance-fluorescence double-screening marker is G418 resistance gene nptII, and the fluorescent gene corresponding to the fusion protein is eGFP fluorescent gene or DsRed fluorescent gene.

10. Use of an expression vector of Schizochytrium, wherein the expression vector is the expression vector of any one of claims 1-4, in gene editing of the Schizochytrium.