CN113201555A - Construction method of binary vector containing eGFP marker and hygromycin resistance - Google Patents

Construction method of binary vector containing eGFP marker and hygromycin resistance Download PDF

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CN113201555A
CN113201555A CN202110356294.1A CN202110356294A CN113201555A CN 113201555 A CN113201555 A CN 113201555A CN 202110356294 A CN202110356294 A CN 202110356294A CN 113201555 A CN113201555 A CN 113201555A
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egfp
hygromycin resistance
expression cassette
trpc
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胡懋
曾杨璇
杨正凤
张榕
赵元昊
者园园
黄遵锡
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Yunnan University YNU
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Abstract

The invention discloses a construction method of a binary vector containing an eGFP marker and hygromycin resistance, which constructs a hygromycin resistance binary plasmid based on a vector pCAMBIA3301 and is connected with an expression cassette containing an eGFP marker gene in a seamless cloning manner. Abandons the genome PCR process in the traditional transformant identification, and adopts a more intuitive and efficient identification mode of green fluorescent markers and hygromycin resistance. The experimental cost is reduced, and meanwhile, hazardous reagents such as phenol, chloroform, isoamylol and liquid nitrogen are avoided, so that the safety and reliability are higher.

Description

Construction method of binary vector containing eGFP marker and hygromycin resistance
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a construction method of a binary vector containing an eGFP marker and hygromycin resistance.
Background
Agrobacterium tumefaciens mediated transformation: the cells in the agrobacterium tumefaciens and the agrobacterium rhizogenes respectively contain Ti (Tumour inducing) plasmid and Ri plasmid, and a section of T-DNA (Transf inducing DNA) is arranged on the Ti (Tumour inducing) plasmid and the Ri plasmid, after the agrobacterium tumefaciens enters the cells after being induced by AS (acetosyringone), the T-DNA can be inserted into fungal genomes and can be stably inherited to descendants through meiosis, and the characteristic becomes the theoretical basis of agrobacterium tumefaciens mediated plant and fungus AS representative eukaryotic transgenosis.
A binary vector system (binary system) or a co-integration system is required for agrobacterium transformation, and the basic vector frameworks except an Ori sequence and the like all have a complete Vir gene region, T-DNA and left and right boundaries for specific recognition and cutting of endonuclease. The binary vector is composed of a multifunctional cloning plasmid containing T-DNA and a Ti derived plasmid containing a Vir region, which are both positioned in agrobacterium, wherein the multifunctional cloning plasmid is responsible for replicating and carrying DNA sequences (target genes and marker genes) in the T-DNA boundary in escherichia coli and agrobacterium, and the Ti derived plasmid provides a trans-toxic region function and promotes the transfer of the T-DNA. The co-integration system is mainly composed of Ti plasmid and large intestine shuttle plasmid which simultaneously contain transfer region and virulence region, the Ti plasmid is located in agrobacterium, the shuttle plasmid is located in escherichia coli, and exogenous genes are integrated into the T-DNA region of the Ti plasmid by homologous recombination with the large intestine shuttle plasmid to form co-integration plasmid, so that the co-integration plasmid becomes a part of T-DNA.
Disclosure of Invention
Aiming at the problem of complicated positive identification of genome which is required to be extracted after fungus is transformed by the existing ATM method, the invention provides a preparation method of a binary vector containing eGFP (enhanced Green fluorescent protein) markers and hygromycin resistance, abandons the genome PCR (polymerase chain reaction) process in the traditional transformant identification, adopts a more intuitive and efficient identification mode of green fluorescent markers and hygromycin resistance, and has the characteristics of convenience, low cost, less time consumption, high safety and strong reliability.
In order to achieve the technical purpose, the invention is specifically realized by the following technical scheme:
a construction method of a binary vector containing an eGFP marker and hygromycin resistance is characterized in that a hygromycin resistance binary plasmid is constructed on the basis of a vector pCAMBIA3301 and is connected with an expression cassette containing an eGFP marker gene in a seamless cloning mode.
The specific construction method of the binary vector containing the eGFP marker and the hygromycin resistance comprises the following steps:
1) constructing a hygromycin expression cassette and connecting the hygromycin expression cassette with linearized pCAMBIA3301 plasmid after enzyme digestion by EcoR I and xbal to obtain hygromycin resistance binary plasmid;
2) the gene is obtained by taking pCT74 as a template for amplification, and an expression cassette containing the eGFP marker gene is constructed;
3) seamlessly cloning and connecting the hygromycin resistance binary plasmid in the step 1) with the expression cassette containing the eGFP marker gene in the step 2).
Further, a hygromycin expression cassette gpdA-HygR-trpC is obtained by using a plasmid pAN7-1 as a template and primers shown in SEQ ID No. 1-2.
Further, the expression cassette containing the eGFP marker gene comprises gpdA-eGFP-trpC and glaA-eGFP-trpC.
Furthermore, primers for obtaining each fragment in the expression cassette gpdA-eGFP-trpC are shown as SEQ ID NO. 3-8 in sequence.
Furthermore, primers for obtaining each fragment in the expression cassette glaA-eGFP-trpC are sequentially shown as SEQ ID NO. 9-14.
The invention has the beneficial effects that:
the method of the invention abandons the genome PCR process in the traditional transformant identification and adopts a more intuitive and efficient identification mode of green fluorescent marker and hygromycin resistance. The method does not need to extract a sample genome by phenol-chloroform-isoamyl alcohol and perform PCR positive identification by taking the genome as a template, and can determine a positive transformant only by screening hygromycin resistance of the transformant and then performing fluorescence observation, thereby greatly accelerating the experiment progress, more intuitively and quickly identifying the positive transformant, reducing the experiment cost, avoiding using dangerous reagents such as phenol, chloroform, isoamyl alcohol, liquid nitrogen and the like, and having higher safety and reliability.
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FIG. 1 is an electron micrograph of the hygromycin expression cassette gpdA-HygR-trpC of the present invention;
FIG. 2 is a fluorescent observation of Aspergillus niger transformants under the microscope of the present invention; the single colony photographs of the transformants were gpdA-1, glaA-1 and wild-type-1; gpdA-2 and glaA-2 are observed in the bright field; glaA-3, gpdA-3 and wild type-2 are excited by laser with the wavelength of 510 nm-560 nm in a dark field;
FIG. 3 shows PCR identification of the extracted genome of the transformant in which fluorescence is observed according to the present invention; d1 is gpdA-eGFP, A1 is glaA-eGFP.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 construction of hygromycin resistant binary plasmid
The plasmid pAN7-1 is used as a template, and primers are designed to amplify to obtain a DNA fragment (shown in figure 1) with the fragment size of 4108bp, namely the hygromycin expression cassette gpdA-HygR-trpC.
Wherein, the primer sequences are AGCTATGACCATGATTACGAATTCCCTTGTATCTCTAC and CATGCCTGCAGGTCGACTCTAGATCGAGTGGAGATGTGG.
The reaction system is as follows: PrimeSTAR GXL Premix (2X) 25. mu.L (final concentration 1X), upstream and downstream primers (10-15pmol) 1. mu.L each (final concentration 0.2-0.3. mu.M), template 1. mu.L, and sterilized water 22. mu.L.
PCR amplification conditions: 2min at 98 ℃; 10s at 98 ℃, 15s at 58 ℃, 3min at 68 ℃ and 45s for 35 cycles, and 10min at 68 ℃; the extension time was 1kb/min depending on the size of the fragment.
Purifying and recovering PCR products by using a Gel Extraction Kit, connecting the purified fragments with linearized pCAMBIA3301 plasmid which is cut by EcoR I and xbal enzyme to transform DH5 alpha large intestine competence, and uniformly coating the competent cells on Kan+(final concentration 100. mu.g/mL) resistance plate, selecting single colony to perform colony PCR positive identification, sending the successfully identified positive transformant to the company for correct sequencing, and obtaining the binary plasmid with hygromycin resistance, which is named pCAM-gpd-HygR-trpC. The cleavage system is shown in Table 1.
TABLE 1 double enzyme digestion System
Figure BDA0003003352540000051
Example 2 preparation of the expression cassette gpdA-eGFP-trpC
Using pAN7-1 as a template and primer FgpdA：ACTCGATCTAGAGTCGACCTGCAGGGAATTCCCTTGTATCTC and RgpdA: TGAACAGCTCCTCGCCCTTGCTCACCATGGATCCAGCGCGCTTGGAAAT amplification gave the gpdA promoter. The plot is the restriction site. The reaction system is shown in Table 2.
TABLE 2 gpdA promoter reaction System
Figure BDA0003003352540000052
Using pAN7-1 as a template, the primer FD-trpC: GCATGGACGAGCTGTACAAGTAAGGATCCGAAGCTTGAATC and RD-trpC：AAACGACGGCCAGTGCCAAGCTTCAAGCTCTAGAAAGA amplification gave trpC terminator. The plot is the restriction site. The reaction system is shown in Table 3.
TABLE 3 trpC terminator reaction System
Figure BDA0003003352540000053
Figure BDA0003003352540000061
Using pCT74 as a template and a primer FD-gfp: TTTCCAAGCGCGCTGGATCCATGGTGAGCAAGGGCGAGGAGCTGT and RD-gfp: GATTCAAGCTTCGGATCCTTACTTGTACAGCTCGTCCATGCCGTG amplified to obtain eGFP marker gene. The reaction system is shown in Table 4.
TABLE 4 eGFP marker Gene reaction System
Figure BDA0003003352540000062
The reaction procedure of the above systems is shown in Table 5.
TABLE 5 PCR procedure for the Components of the eGFP expression cassette
Figure BDA0003003352540000063
EXAMPLE 3 preparation of the expression cassette glaA-eGFP-trpC
Taking the genome of the hexosaminidase as a template and using a primer FglaA：CACTCGATCTAGAGTCGACCTGCAGGCCATGGCTGAGGTGTAAT and RglaA: CCTCGCCCTTGCTCACCATGTCGACAGCGCGCTTGGAAATCACATTTG the gla promoter and the glucoamylase signal peptide are obtained by amplification, the drawn part is the restriction enzyme site. The reaction system is shown in Table 6.
TABLE 6 reaction system for glaA promoter
Figure BDA0003003352540000064
Figure BDA0003003352540000071
Using pCT74 as a template and a primer FA-gfp: CAAATGTGATTTCCAAGCGCGCTGTCGACATGGTGAGCAAGGGCGAGG and RA-gfp: ATTCAAGCTTCGTCGACTTACTTGTACAGCTCGTCCAT amplified to obtain eGFP marker gene. The reaction system is shown in Table 7.
TABLE 7 eGFP marker Gene reaction System
Figure BDA0003003352540000072
Using pAN7-1 as a template, the primer FA-trpC: ATGGACGAGCTGTACAAGTAAGTCGACGAAGCTTGAAT and RA-trpC：AAACGACGGCCAGTGCCAAGCTTCAAGCTCTAGAAAGA amplification gave trpC terminator. The plot is the restriction site. The reaction system is shown in Table 8.
TABLE 8 trpC terminator reaction System
Figure BDA0003003352540000073
The PCR procedure for each component of the above eGFP expression cassette is as shown in Table 5.
Example 4 construction of binary vector containing eGFP marker and hygromycin resistance
Using TreliefTMThe SoSoSoSoSoo Cloning Kit Ver.2 seamless Cloning Kit performs homologous recombination on the expression cassette fragments prepared in Experimental example 2 and example 3 and the linearized binary plasmid fragment in example 1, and the reaction system is shown in Table 9.
TABLE 9 recombinant ligation System for eGFP Gene expression plasmids
Figure BDA0003003352540000074
Figure BDA0003003352540000081
Preparing a connecting system according to the above system, placing the connecting system in a PCR instrument, connecting for 0.5h at 50 ℃, transferring the connecting product to ice after the reaction is finished, standing for 3min, and transforming DH5 alpha large intestine competent cells to obtain a transforming daughter board.
EXAMPLE 5 Positive identification of transformants with recombinant plasmids
Single colonies were randomly picked from the transformation plates and cultured in 500. mu.L of LB (50mg/L Kan) -containing liquid medium at 37 ℃ for 3-4h at 200rpm/min in a constant temperature shaker. After the culture medium is turbid, the bacterial liquid is used as a template, and an expression cassette primer F is usedgpdA(5'-ACTCGATCTAGAGTCGACCTGCAGGGAATTCCCTTGTATCTC-3') and RD-trpC(5′-AAACGACGGCCAGTGCCAAGCTTCAAGCTCTAGAAAGA-3′)、FglaA(5'-CACTCGATCTAGAGTCGACCTGCAGGCCATGGCTGAGGTGTAAT-3') and RA-trpC(5′-AAACGACGGCCAGTGCCAAGCTTCAAGCTCTAGAAAGA-3′)、FT-eGFP(5'-GTTGCGTGCCTTCCAGGGGCCCGATCTAGTAACATAG-3') and RT-eGFP(5′-CCCGGGTACCGAGCTCGAATTCCTATTCCTTTGCCCT-3') were subjected to PCR analysis of bacterial suspension as shown in Table 10, and the procedure was the same as in examples 2 and 3. And extracting the recombinant plasmid of the positive strain for enzyme digestion verification, wherein each enzyme digestion system is shown in Table 11. The identification result is positive and the correct strain is sent to Kunming Shuozhi Biotech limited for sequencing.
TABLE 10 Positive screener validation PCR reaction System (50. mu.L)
Figure BDA0003003352540000091
TABLE 11 double restriction enzyme validation System
Figure BDA0003003352540000092
The Aspergillus niger transformants are subjected to fluorescence observation under a body type microscope, and as a result, as shown in FIG. 2, the GpdA-eGFP-2 and glaA-eGFP-2 Aspergillus niger mycelia can be obviously observed, the sporangium excites green fluorescence in a fluorescence field, and the wild type has no fluorescence phenomenon, so that the expression cassettes of the GpdA-eGFP-trpC and glaA-eGFP-trpC are integrated into the genome of the Aspergillus niger and stably exist and express, and therefore, the three vectors can be used for screening the fungal transformants, directly observing fluorescence, and not extracting and identifying the genome.
The PCR identification is carried out on the transformant extracted genome observed to be fluorescent, the positive rate reaches 100%, and as shown in figure 3, the result shows that the binary vector containing the GFP marker gene is practical and usable, the experimental steps are greatly reduced, the efficiency is improved, the use of toxic reagents is avoided, and the accuracy is high.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
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Claims (5)

1. A method for constructing a binary vector containing an eGFP marker and hygromycin resistance is characterized by comprising the following steps of:
1) constructing a hygromycin expression cassette and connecting the hygromycin expression cassette with linearized pCAMBIA3301 plasmid after enzyme digestion by EcoR I and xbal to obtain hygromycin resistance binary plasmid;
2) the gene is obtained by taking pCT74 as a template for amplification, and an expression cassette containing the eGFP marker gene is constructed;
3) seamlessly cloning and connecting the hygromycin resistance binary plasmid in the step 1) with the expression cassette containing the eGFP marker gene in the step 2).
2. The method for constructing the binary vector containing the eGFP marker and the hygromycin resistance according to claim 1, wherein the hygromycin expression cassette gpdA-HygR-trpC is obtained by using a primer shown in SEQ ID NO. 1-2 and a plasmid pAN7-1 as a template.
3. The method of claim 1, wherein the expression cassette containing the eGFP marker gene comprises gpdA-eGFP-trpC and glaA-eGFP-trpC.
4. The method for constructing the binary vector containing the eGFP marker and the hygromycin resistance of claim 3, wherein the primers for obtaining the fragments in the expression cassette gpdA-eGFP-trpC are shown as SEQ ID NO. 3-8 in sequence.
5. The method for constructing the binary vector containing the eGFP marker and the hygromycin resistance according to claim 3, wherein the primers for obtaining the fragments in the expression cassette glaA-eGFP-trpC are shown as SEQ ID NO. 9-14 in sequence.
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CN113667688A (en) * 2021-09-21 2021-11-19 江苏医药职业学院 Trichoderma longibrachiatum plasmid vector and construction method and application thereof

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