CN109486815B - Artificial chimeric promoter and construction method thereof - Google Patents
Artificial chimeric promoter and construction method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses an artificial chimeric promoter and a construction method thereof; the pTobR and pWIN are combined by a fusion PCR method and an artificial combination element C1 is inserted into the combination to construct a chimeric promoter TC 1W; constructing a chimeric promoter TC1W into a pBI121 binary plant expression vector, and transferring TC1W into tobacco through agrobacterium mediation; GUS tissue staining and GUS protease activity determination are carried out on transgenic tobacco plants, and the expression mode of downstream GUS genes driven by the fusion promoter TC1W is explored. According to the invention, different promoter elements are combined to replace or redesign and construct a new artificial promoter, so that a new promoter with the characteristics of wide induction factors, low background activity, high expression intensity, quick expression starting and the like is constructed, and the expression of a downstream target gene can be better controlled. In the absence of inducing factors, the TC1W promoter exhibits root specificity only.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an artificial chimeric promoter and a construction method thereof.
Background
Currently, the current state of the art commonly used in the industry is such that: at present, two of the most common methods for constructing artificial chimeric promoters are the combination of a core promoter with cis-elements and the bidirectional binding of a unidirectional promoter. In addition, cis-acting elements from different sources are directly connected in series. In general, when genetic improvement is performed on crops, it is often desired that the inserted exogenous gene can be expressed in a specific tissue at a specific development stage or under a specific environmental condition, so that the influence of strong start of the gene on the normal growth of the crops is reduced. Although a large number of cis-acting elements and corresponding transcription factors are widely studied to lay a foundation for constructing artificial promoters, the interaction network of DNA and protein and the theoretical foundation research of signal transduction pathways are not clear, so that the construction of artificial promoters still faces a serious test. The number of cis-elements, the order in which the cis-elements are arranged, and the spacing between them all affect the strength and specificity of the promoter. The tobacco TobRB7 gene promoter has obvious root specificity, and the-299 to-636 bp of the tobacco TobRB7 gene promoter is a key region for realizing the root specificity. The poplar win3.12 gene is related to pest and disease defense, and the promoter has wound inducing property. W1-box (TTGACC) is a class of fungal inducing elements; motifi (ggtacgttggcg) is a type of root-specific cis-element.
In summary, the problems of the prior art are as follows:
(1) at present, no technical means can control the accurate regulation and control of the disease-resistant gene under the condition of the infection of pathogenic bacteria at the root of the plant.
(3) There is no technical means to solve the problem of the effective connection mode of the plant root specific inducible promoter element and the wound inducible specific promoter element.
The difficulty and significance for solving the technical problems are as follows: because the interaction network of DNA and protein and the theoretical basic research of signal transduction path are not clear, the construction of artificial promoter still faces serious test.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an artificial chimeric promoter and a construction method thereof.
The invention is realized by the following steps that the artificial chimeric promoter is shown by transgenic tobacco analysis: GUS histochemical staining shows that the TC1W fusion promoter has promoter activity and can drive a downstream gene to express in a large amount in roots; the TC1W transgenic strain has obvious root specificity, and the GUS activity in the root is obviously higher than that in stems, leaves, flowers and seeds. And the TC1W promoter can drive GUS gene to be specifically expressed in a large amount in roots under the induction of powdery mildew and salicylic acid, and the GUS gene is expressed in a trace amount or is not expressed in stems and leaves.
The invention also aims to provide a construction method of the artificial chimeric promoter, which constructs a new promoter by combining different promoter elements and replacing or redesigning and constructing a new artificial promoter.
Further, the construction method of the artificial chimeric promoter comprises the following steps:
step one, combining pTobR and pWIN by a fusion PCR method and inserting an artificial combination element C1 into the combination to construct a chimeric promoter TC 1W;
step two, constructing the chimeric promoter TC1W into a pBI121 binary plant expression vector, and transferring TC1W into tobacco through agrobacterium-mediated transformation; GUS tissue staining and GUS protease activity determination are carried out on transgenic tobacco plants, and the expression mode of downstream GUS genes driven by the fusion promoter TC1W is explored.
Further, the first step specifically includes:
first round of amplification primers pTOBRC11 and C12pWIN respectively
F-T1:5’GACCATGATTACGCCAAGCTTGATTCGTTCTTTAATATGTC3’
R-TC11:5’CCACGTACCGGTCAACGCCACGTACCTAAGCTTAACTAATATACAAG3’
F-C12W:5’CGTTGACCGGTACGTGGCGTTGACCAAGCTTCCAACATCAATGATTATC3’
R-W1:5’GGACTGACCACCCGGGGATCATTTGTTGAATATGAGTATTAG3’
The PCR reaction procedures were as follows: 0:10 at 98 ℃; 0:15 at 58 ℃; 1:00 at 72 ℃; go to 135 times; 10:00 at 92 ℃;
second round of amplification primers pTOBRC1 and C1pWIN respectively
F-T1:5’GACCATGATTACGCCAAGCTTGATTCGTTCTTTAATATGTC3’
R-TC1:5’CGCCACGTACCGGTCAACGCCACGTACCGGTCAACGCC3’
F-C1W:5’GGCGTTGACCGGTACGTGGCGTTGACCGGTACGTGGCG3’
R-W1:5’GGACTGACCACCCGGGGATCATTTGTTGAATATGAGTATTAG3’
The PCR reaction procedures were as follows: 0:10 at 98 ℃; 0:15 at 58 ℃; 1:00 at 72 ℃; go to 135 times; 10:00 at 92 ℃;
third round of TC1W fragment fusion primer
F-T1:5’GACCATGATTACGCCAAGCTTGATTCGTTCTTTAATATGTC3’
R-W1:5’GGACTGACCACCCGGGGATCATTTGTTGAATATGAGTATTAG3’。
Further, the second step specifically includes:
(1) double digestion pBI121 plasmid vector
pBI121 plasmid vector double restriction system: 10 × buffer (K)2 μ L; hind III 1. mu.L; 1 mu L of BamHI; 6 mu L of DNA; ddH2O10 mu L; adding the reagents in sequence, and placing the mixture on a water bath at 37 ℃ for enzyme digestion2～3h；
(2) EXO III mediated LIC high-efficiency cloning system method for constructing a recombinant expression vector pBI121-TC1W connection system: 25-50ng of carrier; 25-50ng of target fragment; 10 XEXO III buffer 1 u L; ddH2O MμL。
Further, the connection method comprises:
(1) adding into the mixture, mixing, and ice-cooling for 5 min;
(2) adding 20 units of EXO III enzyme into the mixture, mixing, and keeping at 4 deg.C for 60 min;
(3) adding 1 mu L of 0.5M EDTA into the reaction system, blowing, beating and uniformly mixing, and stopping the reaction;
(4) melting at 60 deg.C for 5min, and ice-cooling for 5 min;
(3) the recombinant plasmid pBI121-TC1W is transformed into agrobacterium EHA105 competence by an electrotransformation method;
(4) genetic transformation of tobacco
(5) Relative activity determination of GUS enzyme, GUS protease which is a GUS gene expression product can catalyze p-nitrophenyl-beta-D-glucuronide to hydrolyze into p-nitrophenol, when pH is 7.5, an ion chromophore absorbs light with the wavelength of 400-420nm, and a solution turns yellow; the enzyme reaction is carried out under the condition that the pH value is 7.0, and as the reaction is carried out, the product is generated, gradually alkalized and the color development is enhanced;
(6) treatment of each stress and sampling of each tissue and organ.
The (3) further includes:
(1) adding 5 mu L of recombinant plasmid pBI 121-prooters into the competence of the agrobacterium EHA105, slightly blowing and stirring uniformly, and carrying out ice bath for 30 min;
(2) adding the mixture into an electric shock cup, selecting Agr mode electric shock, adding 800 μ LYEP liquid culture medium, performing shake culture at 28 deg.C and 200rpm for 3-5h, centrifuging at 8000rpm for 30s, discarding supernatant, and coating YEP plate;
(3) and (3) carrying out inverted culture in an incubator at 28 ℃ for 48 h.
The invention also aims to provide application of the artificial chimeric promoter constructed by the construction method of the artificial chimeric promoter in artificial regulation of gene expression in plants.
Another objective of the invention is to provide a method for constructing the artificial chimeric promoter, which combines different promoter elements, replaces or redesigns a new artificial promoter, and constructs a new promoter.
The invention has the advantages and positive effects that: by combining different promoter elements and replacing or redesigning and constructing a new artificial promoter, the new promoter with the characteristics of wide induction factors, low background activity, high expression intensity, quick expression starting and the like can be constructed, and the expression of a downstream target gene can be better controlled. The result shows that the TC1W promoter fragment shows pathogenicity inducibility, wound inducibility and root specificity, and the TC1W promoter only shows root specificity under the condition of no induction factor.
Drawings
FIG. 1 is a flow chart of a method for constructing an artificial chimeric promoter according to an embodiment of the present invention.
FIG. 2 is a flow chart of a method for constructing an artificial chimeric promoter according to an embodiment of the present invention.
FIG. 3 is a schematic diagram of the activity assay of TC1W in promoting GUS gene expression under different treatments provided in the examples of the present invention;
in the figure: a, no treatment is carried out; b, powdery mildew; c, treating single injury; and D, treating the wound for multiple times.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention constructs a new artificial promoter by combining different promoter elements and replacing or redesigning the promoter elements.
As shown in fig. 1, the method for constructing an artificial chimeric promoter provided by the embodiment of the present invention includes the following steps:
s101: combining and splicing pTobR and pWIN into a TRWI (pTobR-pWIN) promoter by a fusion PCR method, and inserting an artificial combination element C1 into the spliced and chimeric TRWI promoter to construct another chimeric promoter TC1W (pTobR-C1-pWIN);
s102: constructing a chimeric promoter TC1W into a pBI121 binary plant expression vector, and transferring TC1W into tobacco through agrobacterium mediation; GUS tissue staining and GUS protease activity determination are carried out on transgenic tobacco plants, and the expression mode of downstream GUS genes driven by the fusion promoter TC1W is explored.
The construction method of the artificial chimeric promoter provided by the embodiment of the invention comprises the following steps:
step one, combining and splicing pTobR and pWIN into a TRWI promoter by a fusion PCR method, and inserting an artificial combination element C1 into the spliced and chimeric TRWI promoter to construct another chimeric promoter TC 1W;
first round of amplification primers pTOBRC11 and C12pWIN respectively
F-T1:5’GACCATGATTACGCCAAGCTTGATTCGTTCTTTAATATGTC3’
R-TC11:
5’CCACGTACCGGTCAACGCCACGTACCTAAGCTTAACTAATATACAAG3’
F-C12W:5’CGTTGACCGGTACGTGGCGTTGACCAAGCTTCCAACATCAATGATTATC3’
R-W1:
5’GGACTGACCACCCGGGGATCATTTGTTGAATATGAGTATTAG3’
PCR reaction system of pTOBRC11 fragment
PCR reaction system of C12pWIN fragment
The PCR reaction procedures were as follows:
second round of amplification primers pTOBRC1 and C1pWIN respectively
F-T1:
5’GACCATGATTACGCCAAGCTTGATTCGTTCTTTAATATGTC3’
R-TC1:5’CGCCACGTACCGGTCAACGCCACGTACCGGTCAACGCC3’
F-C1W:5’GGCGTTGACCGGTACGTGGCGTTGACCGGTACGTGGCG3’
R-W1:
5’GGACTGACCACCCGGGGATCATTTGTTGAATATGAGTATTAG3’
PCR reaction system of pTOBRC1 fragment
PCR reaction system of C1pWIN fragment
The PCR reaction procedures were as follows:
third round of TC1W fragment fusion primer
F-T1:5’GACCATGATTACGCCAAGCTTGATTCGTTCTTTAATATGTC3’
R-W1:
5’GGACTGACCACCCGGGGATCATTTGTTGAATATGAGTATTAG3’
TC1W fragment fusion PCR reaction system
The PCR reaction program is:
step two, constructing the chimeric promoter TC1W into a pBI121 binary plant expression vector, and transferring TC1W into tobacco through agrobacterium-mediated transformation; GUS tissue staining and GUS protease activity determination are carried out on transgenic tobacco plants, and the expression mode of downstream GUS genes driven by the fusion promoter TC1W is explored. In order to identify whether each fusion fragment has the capability of promoting the expression of downstream genes and what expression driving mode, each promoter fragment is connected into a plant binary expression vector pBI121 to replace the original CaMV 35S constitutive promoter, and a new recombinant expression vector pBI121-TC1W is constructed.
(1) Double digestion pBI121 plasmid vector
pBI121 plasmid vector double restriction system:
and sequentially adding the reagents, and placing the mixture on a water bath at 37 ℃ for enzyme digestion for 2-3 h.
(2) EXO III mediated LIC high-efficiency clone system method for constructing recombinant expression vector pBI121-TC1W
A connection system:
the connection method comprises the following steps:
(1) adding into the above system, mixing, and ice-cooling for 5 min;
(2) adding 20 units of EXO III enzyme into the mixture, mixing, and keeping at 4 deg.C for 60 min;
(3) adding 1 mu L of 0.5M EDTA (pH 8.0) into the reaction system, blowing, stirring and mixing uniformly, and stopping the reaction;
(4) melting at 60 deg.C for 5min, and ice-cooling for 5 min;
(3) electrotransformation method for transferring recombinant plasmid pBI121-TC1W into agrobacterium EHA105 competence
a.5 mu L of recombinant plasmid pBI 121-prooters is added into the competence of the agrobacterium EHA105, and the mixture is lightly blown and uniformly mixed and ice-bathed for 30 min;
b. adding the mixture into an electric shocking cup, selecting an "Agr" mode (voltage 2.2KV time 4.9ms) electric shock, adding 800 μ L YEP (antibiotic-free) liquid culture medium, shaking and culturing at 28 deg.C and 200rpm for 3-5h, centrifuging at 8000rpm for 30s, discarding the supernatant, and coating YEP plate (Rif +, Kan +);
c.28 ℃ in an incubator for 48 h.
(4) Genetic transformation of tobacco
a preculture
Cutting leaves of sterile tobacco seedling, cutting off leaf edge, cutting into size of 0.5 × 0.5cm, inoculating on MS differentiation culture medium at 25 deg.C, dark culturing for 2 days, and illuminating at 2000 ×.
b regeneration by transformation
And (3) placing the pre-cultured individual plant into the prepared agrobacterium engineering bacterial liquid to be soaked for about 10min, and slightly shaking the culture bottle every 2-3min to ensure that the leaves can be fully contacted with the bacterial liquid. Transferring the explant to sterile filter paper, sucking off residual bacterial liquid, putting the back of the leaf downwards on a co-culture medium, and performing dark culture at 25 ℃ for 2 days. Taking out the co-cultured leaf disc, transferring to selective medium, pressing the edge into the medium with the back facing downwards, culturing at 25 deg.C with photoperiod of 16/8, and performing resistance screening. The selective culture medium is replaced once a week, and the leaf disc materials polluted by the agrobacterium are cleaned in time.
c rooting culture
When the differentiated bud grows to about 2-3cm, cutting off the bud and inserting the bud into a rooting culture medium. The adventitious roots can grow out in about one week. After 2-3 weeks, hardening the well-grown transformed seedlings, transplanting the seedlings in a flowerpot or carrying out asexual propagation and expanded culture.
(5) GUS enzyme relative activity assay
GUS protease which is a GUS gene expression product can catalyze P-nitrophenyl-beta-D-glucuronide (PNPG) to hydrolyze P-nitrophenol (P-nitrophenol), and when the pH value is 7.5, the ion chromophore absorbs light with the wavelength of 400-420nm, and the solution turns yellow. The enzyme reaction was carried out at pH 7.0, and as the reaction proceeded, the product was produced, gradually alkalinized, and the color developed increased. Therefore, the GUS enzyme activity can be detected by spectrophotometry.
a extraction of GUS protease from different tissues and organs of each transgenic line under different stress treatment
And (3) placing the transgenic tobacco of each promoter fragment obtained by rapid propagation and the wild tobacco which is subjected to subculture under proper conditions, and selecting the tobaccos with consistent growth vigor to divide into six groups when the tobaccos grow to 7-8 leaves normally for different stress treatments.
(6) Method for treating stress and sampling and determining tissue and organ
Pathogenic bacteria (powdery mildew) infestation: powdery mildew is prepared into bacterial liquid through pre-culture, and each tobacco plant is treated in a mode of injecting stem sections through an injector. When powdery mildew symptoms appear (the plants are withered and frost spots appear on the leaves), sampling and extracting protein, and determining GUS.
Salicylic acid treatment: salicylic acid is easily soluble in ethanol and insoluble in water, so it is first solubilized with a small amount of ethanol and then diluted with distilled water to a final concentration of 50mM (pH 6.8) and treated by smearing the leaves. And (3) coating a proper amount of the prepared solution on a fifth leaf blade from the stem tip, covering the fifth leaf blade with a preservative film for 12 hours, sampling, extracting protein, and determining GUS.
The result shows that the TC1W promoter fragment shows pathogenicity inducibility, wound inducibility and root specificity, and the TC1W promoter only shows root specificity under the condition of no induction factor.
The application of the principles of the present invention will now be described in further detail with reference to the accompanying drawings.
The promoter of the tobacco gene TobRB7 has obvious root specificity, the promoter-299 bp-636 bp is a key region for realizing the root specificity, and the promoter is transferred into tobacco, so that the promoter is confirmed to have the root specificity. win3.12 is a member of gene family related to pest and disease defense in poplar genome, and when win3, 12 promoter is transferred into tobacco, the transgenic tobacco shows obvious wound induction characteristic. In the experiment, the ptobR and the pWIN are combined and spliced by a fusion PCR method, an artificial combination element- "C1" (shown in figure 2) is inserted into the two promoters to construct a chimeric promoter TC1W (ptobR-C1-Pwin) (shown in figures 1 and 2), the tobacco is transformed after the chimeric promoter is inserted into pBI121, and the transgenic positive tobacco is subjected to GUS tissue staining and GUS protease activity determination to identify the functions of the promoters.
TABLE 1 promoter fragment fusion PCR primers
Fusion of fragment WC1T also required three rounds of PCR. In the first round, populus tomentosa genomic DNA and tobacco genomic DNA are respectively used as templates, and specific primers F-W1/R-WC11 and F-C12T/R-T1 (shown in table 1) are used to obtain a fragment C12pTOBR of-299 to-634 bp in promoter partial sequences pWINC11 and TobRB7 of Win3.12 promoters. The second round respectively takes recovered fragments pWINC11 and C12pTOBR as templates, F-W1/R-WC1 and F-C1T/R-T1 as primers to respectively carry out PCR amplification to obtain partial fusion fragments pWINC1 and C1pTOBR with 'C1' elements, the PCR product mixture in the second round is directly taken as a template solution to carry out third round of amplification, F-W1/R-T1 is taken as a specific primer, and the pWINC1 and the C1pTOBR are fused into a chimeric fragment WC1T through the same sequence 'C1' carried by the respective fragments.
TABLE 2 TC1W sequences obtained by PCR fusion
As can be seen from FIG. 3A, in the absence of any treatment in the normal growth state, the genes have certain expression in each tissue and organ of each transgenic line, the tissue expression difference is not obvious, but the expression in the root is higher as a whole, which indicates that the TC1W fusion promoter has promoter activity and can drive a large amount of expression of downstream genes in the root. The TC1W transgenic line has root specificity, and GUS activity in roots is remarkably higher than that in stems, leaves and flowers (as shown in FIG. 3B). Powdery mildew is a common fungal disease in tobacco, and in the experiment, powdery mildew can strongly induce the expression of gus gene in TC1W transgenic line tobacco, and the expression is limited in roots (as shown in FIG. 3C); to some extent, salicylic acid also has the same induction pattern, and as shown in FIG. 3D, TC 1W-transfected tobacco showed strong root specificity and 3-5 times higher GUS gene expression level in roots in the treatment group with high salicylic acid concentration (50mM, pH 6.8).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> university of Chongqing teacher
<120> an artificial chimeric promoter and a construction method thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 335
<212> DNA
<213> tobacco (Nicotiana tabacum)
<400> 1
ttagattgct ctacggtata tcatatgatt ataaataaaa aaaattagca aaagaatata 60
atttattaaa tattttacac cataccaaac acaagcgcat tatatataat cttaattatc 120
attatcacca gcatcaacat tataatgatt cccctatgcg ttggaacgtc attatagtta 180
ttctaaacaa gaaagaaatt tgttcttgac atcagacatc tagtattata actctagtgg 240
agcttacctt ttcttttcct tctttttttc ttcttaaaaa attatcactt tttaaatctt 300
gtatattagt taagcttagg gacgtggcgt tgacc 335
<210> 2
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggtacgtggc gttgaccggt acgtggcgtt gaccggtacg tggcgttgac c 51
<210> 3
<211> 536
<212> DNA
<213> Populus trichocarpa (Populus trichocarpa)
<400> 3
tattgaagca aaccaaccta gattagtgat taatcaagcc tctcaaagtg tctagtgcaa 60
agaaaggacc agattttttt ttagaatact cggacatgta atctcatttt aaatgactcc 120
tttgggcttg acaacttgaa tttgattttg aagatttaat tagtagtttt aattgaagtt 180
attaatagag tagactattt gtgtgcgttt tgttcaattt tatacatggt gtttattttc 240
aagttgtcaa gttcaccaat tattacacga taaatgaata caatgatagt cgtttcaaaa 300
aagtatagtt tatttattta aatttaaggt atttaaaatt ttaagttcat tgatcagatc 360
aattttaagc acgattaaaa cacttttgtg ggataaatga aggaggcaag aggcaa 416
Claims (4)
1. A construction method of an artificial chimeric promoter is characterized by comprising the following steps:
step one, combining pTobR and pWIN by a fusion PCR method and inserting an artificial combination element C1 into the combination to construct a chimeric promoter TC 1W;
step two, constructing the chimeric promoter TC1W into a pBI121 binary plant expression vector, and transferring TC1W into tobacco through agrobacterium-mediated transformation; GUS tissue staining and GUS protease activity determination are carried out on transgenic tobacco plants, and the expression mode of downstream GUS genes driven by the fusion promoter TC1W is explored;
the first step specifically comprises:
first round of amplification primers pTOBRC11 and C12pWIN respectively
F-T1: 5’GACCATGATTACGCCAAGCTTGATTCGTTCTTTAATATGTC3’
R-TC11: 5’CCACGTACCGGTCAACGCCACGTACCTAAGCTTAACTAATATACAAG3’
F-C12W :5’CGTTGACCGGTACGTGGCGTTGACCAAGCTTCCAACATCAATGATTATC3’
R-W1: 5’GGACTGACCACCCGGGGATCATTTGTTGAATATGAGTATTAG3’
The PCR reaction procedures were as follows: 0:10 at 98 ℃; 0:15 at 58 ℃; 1:00 at 72 ℃; go to 135 times; 10:00 at 92 ℃;
second round of amplification primers pTOBRC1 and C1pWIN respectively
F-T1: 5’GACCATGATTACGCCAAGCTTGATTCGTTCTTTAATATGTC3’
R-TC1: 5’CGCCACGTACCGGTCAACGCCACGTACCGGTCAACGCC3’
F-C1W: 5’GGCGTTGACCGGTACGTGGCGTTGACCGGTACGTGGCG3’
R-W1: 5’GGACTGACCACCCGGGGATCATTTGTTGAATATGAGTATTAG3’
The PCR reaction procedures were as follows: 0:10 at 98 ℃; 0:15 at 58 ℃; 1:00 at 72 ℃; go to 135 times; 10:00 at 92 ℃;
third round of TC1W fragment fusion primer
F-T1:5’GACCATGATTACGCCAAGCTTGATTCGTTCTTTAATATGTC3’
R-W1:5’GGACTGACCACCCGGGGATCATTTGTTGAATATGAGTATTAG3’。
2. The method for constructing an artificial chimeric promoter according to claim 1, wherein the second step specifically comprises:
(1) double digestion pBI121 plasmid vector
pBI121 plasmid vector double restriction system: 10 × buffer K2 μ L;Hind III1 μL；BamH I1 μL；DNA 6 μL；dd H2o10 mu L; sequentially adding reagents, and placing the mixture on a water bath at 37 ℃ for enzyme digestion for 2-3 h;
(2) EXO III mediated LIC high-efficiency cloning system method for constructing a recombinant expression vector pBI121-TC1W connection system: 25-50ng of carrier; 25-50ng of target fragment; 10 XEXO III buffer 1 u L; dd H2O M μL。
3. The method for constructing an artificial chimeric promoter according to claim 2, wherein the ligation method comprises:
(1) adding into the mixture, mixing, and ice-cooling for 5 min;
(2) adding 20 units of EXO III enzyme into the mixture, mixing, and standing at 4 deg.C for 60 min;
(3) adding 1 mu L of 0.5M EDTA into the reaction system, blowing, beating and uniformly mixing, and stopping the reaction;
(4) melting at 60 deg.C for 5min, and ice-cooling for 5 min;
(3) the recombinant plasmid pBI121-TC1W is transformed into agrobacterium EHA105 competence by an electrotransformation method;
(4) genetic transformation of tobacco
(5) Relative activity determination of GUS enzyme, GUS protease which is an expression product of GUS gene can catalyze p-nitrophenyl-beta-D-glucuronide to hydrolyze into p-nitrophenol, when pH =7.5, the ion chromophore absorbs light with the wavelength of 400-420nm, and the solution turns yellow; the enzyme reaction is carried out under the condition of pH =7.0, and as the reaction is carried out, the product is generated, gradually alkalized and the color development is enhanced;
(6) treatment of each stress and sampling of each tissue and organ.
4. The method for constructing an artificial chimeric promoter according to claim 3, wherein the step (3) further comprises:
(1) adding 5 mu L of recombinant plasmid pBI 121-prooters into the competence of the agrobacterium EHA105, slightly blowing and stirring uniformly, and carrying out ice bath for 30 min;
(2) adding the mixture into an electric shock cup, selecting an Agr mode electric shock, adding 800 mu L YEP liquid culture medium, carrying out shaking culture at 28 ℃, 200rpm for 3-5h, centrifuging at 8000rpm for 30s, discarding the supernatant, and coating a YEP plate;
(3) and (5) carrying out inverted culture in an incubator at 28 ℃ for 48 h.
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