CN105734077B - Expression vector and application thereof in preparation of transgenic plants - Google Patents

Abstract

The invention discloses an expression vector and application thereof in preparing transgenic plants. The expression vector provided by the invention comprises an expression cassette A and an expression cassette B; the expression cassette A sequentially comprises the following elements: promoter A, selective marker gene and termination sequence A; the expression cassette B sequentially comprises the following elements: promoter B, intron and termination sequence B; in the expression cassette B, a multiple cloning site A is arranged between the promoter and the intron; a multiple cloning site B is arranged between the intron and the termination sequence; the multiple cloning site A and the multiple cloning site B both have more than one enzyme cutting recognition sequence; and the enzyme cutting recognition sequences at other positions in the expression vector are different from the enzyme cutting recognition sequence in the multiple cloning site A and the enzyme cutting recognition sequence in the multiple cloning site B. Experiments prove that the expression vector provided by the invention can be used for preparing a transgenic plant without a selective marker, and has important application value in the aspect of obtaining safe transgenic plants.

Description

Expression vector and application thereof in preparation of transgenic plants

Technical Field

The invention relates to the technical field of biology, in particular to an expression vector and application thereof in preparation of transgenic plants.

Background

Genetic engineering breeding has been considered as an effective way to improve plant yield, quality and stress resistance since the 20 th century. In fact, some new varieties of transgenic plants, such as transgenic soybean, cotton, corn and rape, have been cultivated by genetic engineering breeding techniques and have been used in large areas in production practice. Particularly, in recent 5 years, the development of plant transgenic research is very rapid, and the variety of plants cultivated by transgenic methods is increasing year by year. Meanwhile, due to the importance of many national governments and consumers on the safety of transgenic plants and the inherent safety barrier of transgenic plants, most transgenic plant varieties are difficult to be commercially produced. Among them, the presence of a selection marker gene is one of the greatest risks in the safety of transgenic plants.

Although the widespread use of selectable marker genes has improved the efficiency of obtaining transgenic plants, since selectable marker genes are mostly resistance genes encoding antibiotics or herbicides, the presence and expression of these exogenous selectable genes in the plant genome become redundant as the selection process is completed, and they are passed on to progeny as the transgenic plants are bred, thereby raising many problems regarding the safety of transgenic plants.

The safety problem of transgenic plants is mainly embodied in two aspects of food safety and environmental safety. In terms of food safety, antibiotic genes and their products may have potential toxicity and sensitization to humans or livestock, and once some of the food remains, they may be transmitted into microorganisms, which may increase the number of microorganisms in the digestive tract of humans or animals, thereby endangering the health of humans and livestock. In terms of environmental safety, the selectable marker gene may be transmitted to wild relative species by gene drift, so that weeds acquire corresponding resistance, and the occurrence of super weeds is caused. The elimination of the selective marker gene and other redundant genes can reduce the potential risk of the transgenic plant, and is beneficial to the safety evaluation and popularization of the transgenic plant. Therefore, the cultivation of safe transgenic plants is one of the current research hotspots.

The screening markers involved in obtaining transgenic plants are of two types, one is a bacterial screening marker, such as amp, kan, aadA and the like, and is used for screening constructed vectors and transformed bacteria; another class is plant selection markers, such as nptII, bar, hpt, etc., for selection of transformed plant cells and regenerated plants. The gene gun mediated method not only transfers plant selection markers, but also transfers bacterial selection markers. Although the Agrobacterium-mediated approach may avoid introducing bacterial selection markers into plants, the target gene in the same T-DNA region is tightly linked to the selection marker after transfer into the plant. Therefore, several techniques for obtaining transgenic plants without selection markers have been proposed in turn, including co-transformation, targeted recombination systems, multi-element automatic transformation vector systems, transposon systems, and homologous recombination systems, but only co-transformation is most successful. The particle gun-mediated co-transformation method can remove plant selection markers, but cannot remove bacterial selection markers. The agrobacterium-mediated co-transformation method can avoid transferring bacterial screening markers into plants and screening markers such as nptII, bar, hpt and the like into the plants, and has unique advantages in the aspect of obtaining safe transgenic plants.

In the aspect of obtaining safe transgenic plants by utilizing an agrobacterium-mediated co-transformation method, the construction of a double T-DNA vector containing a target gene is very tedious work. Although it has been reported that transgenic tobacco, soybean and the like without selection marker genes are obtained by constructing double T-DNA vectors, the double T-DNA vectors are not universal because of lack of regulatory sequences and multiple cloning sites, and the double T-DNA vectors need to be constructed from the beginning through a plurality of intermediate vectors for every gene transformation, which wastes time and labor and is difficult to succeed. Therefore, a binary expression vector containing double T-DNA segments and multiple cloning sites, as well as a regulatory sequence and a suitable screening marker is developed, the full-length cDNA or gDNA of a target gene can be conveniently and quickly inserted, and the method has important significance for obtaining safe transgenic plants. In addition, generally, when the function of a gene is verified by using transgenes, two ways of over-expressing the gene and silencing the gene are mainly adopted, so that the construction of a screening marker-free safe transgenic vector which can perform over-expression and silencing (RNA interference) is also particularly important.

Disclosure of Invention

The technical problem to be solved by the invention is how to obtain safe transgenic plants.

In order to solve the above technical problems, the present invention provides an expression vector.

The expression vector provided by the invention comprises an expression cassette A and an expression cassette B; the expression cassette A sequentially comprises the following elements: promoter A, selective marker gene and termination sequence A; the expression cassette B sequentially comprises the following elements: promoter B, intron and termination sequence B; in the expression cassette B, a multiple cloning site A is arranged between the promoter B and the intron; a multiple cloning site B is arranged between the intron and the termination sequence; the multiple cloning site A and the multiple cloning site B both have more than one enzyme cutting recognition sequence; and the enzyme cutting recognition sequences at other positions in the expression vector are different from the enzyme cutting recognition sequence in the multiple cloning site A and the enzyme cutting recognition sequence in the multiple cloning site B.

The promoter A can be a 35S promoter or a UBI promoter.

The promoter B can be a 35S promoter or a UBI promoter.

The terminator sequence A can be a NOS terminator or ployA.

The terminator sequence B can be a NOS terminator or ployA.

The nucleotide sequence of the 35S promoter can be a reverse complementary sequence of 2213 th to 3039 th sites from the 5' tail end of the sequence 1 in the sequence table.

The nucleotide sequence of the UBI promoter can be a reverse complementary sequence from 10950 to 12942 th positions from the 5' end of the sequence 1 in the sequence table.

The nucleotide sequence of the NOS terminator can be a reverse complementary sequence from 10121 to 10395 th from the 5' end of the sequence 1 in the sequence table.

The nucleotide sequence of ployA can be a reverse complementary sequence from 1479 th site to 1647 th site of the 5' end of the sequence 1 in the sequence table.

The selectable marker gene may be a bar gene.

The nucleotide sequence of the bar gene can be a complementary sequence of 1648 th to 2212 th positions from the 5' end of the sequence 1 in the sequence table.

The multiple cloning site A can have the following restriction enzyme cutting recognition sequences: PstI, BamHI, SmaI, KpnI, SalI, Nhe I and XholI.

The multiple cloning site B can have the following restriction enzyme cutting recognition sequences: SpeI and SacI.

The nucleotide sequence of the multiple cloning site A can be a reverse complementary sequence from 10894 th to 10949 th positions of the 5' tail end of the sequence 1 in the sequence table.

The nucleotide sequence of the multiple cloning site B can be a reverse complementary sequence from 10396 th to 10404 th positions of the 5' tail end of a sequence 1 in a sequence table.

The expression vector may include the expression cassette a and the expression cassette b in order from the 5 'end to the 3' end.

The nucleotide sequence of the expression vector can be specifically shown as a sequence 1 in a sequence table.

The application of any expression vector in the preparation of transgenic plants also belongs to the protection scope of the invention.

The application of any one of the expression vectors in preparing transgenic plants can be specifically realized by introducing a target gene into a receptor plant through any one of the expression vectors to obtain the transgenic plant; the transgenic plant has an increased expression of the target gene compared to the recipient plant. In one embodiment of the present invention, the target gene is specifically GUS gene.

The application of any expression vector in the preparation of transgenic plants can be specifically that the function of a target gene in a receptor plant is lost through any expression vector to obtain the transgenic plants; the transgenic plant has reduced expression of the target gene compared to the recipient plant; the "loss of function of a gene of interest in a recipient plant" is achieved by RNA interference.

In order to solve the above problems, the present invention also provides a method for producing a transgenic plant.

The method for preparing the transgenic plant provided by the invention can be specifically a method I, and comprises the following steps: inserting a target gene into the enzyme digestion recognition sequence in the expression cassette B in any one of the expression vectors, then transforming a receptor plant, and screening by virtue of a selection marker gene to obtain T₀Plant generation; will be the T₀Selfing the generation plants to obtain T containing the target gene and not containing the selectable marker gene₁And (4) generating plants, namely transgenic plants without the selection marker.

The method for preparing the transgenic plant provided by the invention can be specifically a second method, and comprises the following steps: taking any one of the expression vectors as a starting vector, inserting a specific segment in a target gene into a multiple cloning site A, and inserting a reverse complementary sequence of the specific segment in the target gene into a multiple cloning site B to obtain a target vector; transforming the target vector into a receptor plant, and screening by virtue of a selective marker gene to obtain T₀Plant generation; will be the T₀Selfing the generation plants to obtain T which does not contain the target gene and does not contain the selectable marker gene₁And (4) generating plants, namely transgenic plants without the selection marker.

In the above method, the selectable marker gene may specifically be a bar gene.

In the above method, the recipient plant may be any one of the following a1) to a 8):

a1) a dicotyledonous plant;

a2) a monocot plant;

a3) rice;

a4) corn;

a5) barley;

a6) rye;

a7) wheat;

a8) wheat variety Fielder.

Experiments prove that GUS gene (target gene) is inserted into the expression vector provided by the invention, and then the wheat variety Fielder is transformedScreening Bar-assisted gene to obtain GUS gene and Bar gene-transferred T₀Plant generation; will be T₀Selfing the generation plant to obtain T containing the GUS gene and not containing the Bar gene₁Transgenic wheat plants, i.e., transgenic plants without selectable marker, were obtained at a rate of 3.23%. Therefore, the expression vector provided by the invention has important application value in the aspect of obtaining safe transgenic plants.

Drawings

FIG. 1 is T₀Bar test paper for testing transgenic wheat plants.

FIG. 2 is T₀GUS staining of transgenic wheat plants.

FIG. 3 is T₀Southern hybridization of transgenic wheat plants.

FIG. 4 is T₁And (3) carrying out molecular detection on the transgenic wheat plants.

FIG. 5 is T₁Southern hybridization of transgenic wheat plants.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

Agrobacterium tumefaciens C58C1 is a product of Biovector NTCC China plasmid vector strain cell gene collection center (website address ishttp://biovector.chemdrug.com/(ii) a Telephone: 18901268599). The MS basic culture medium (containing no vitamin) is a product of Beijing Ximeijie science and technology Limited, and the product number is M524. The MS basic culture medium (containing vitamins) is a product of Beijing Western Meijie science and technology Limited, and the product number is M519.

The illumination is alternately cultured as lightCulturing and dark culturing are alternated, and the conditions are as follows: 28 ℃; 14 hours of light culture/10 hours of dark culture; the illumination intensity of the illumination culture is 90 muE/m²/s。

The vector pPTN290 is described in the following documents: howe a1, Sato S, Dweikat I, Fromm M, clement t.rapid and reproducible Agrobacterium-mediated transformation of plant Cell rep.2006 aug; 25(8): 784-91.

Dyeing liquid: 38mL of NaH₂PO₄Aqueous solution (concentration 0.2 mol. L)^-1)、62mL Na₂HPO₄Aqueous solution (concentration 0.2 mol. L)^-1)、1mL K₃[Fe(CN)₆]Aqueous solution (concentration 0.1 mol. L)^-1)、1mL K₄[Fe(CN)₆]Aqueous solution (concentration 0.1 mol. L)^-1)、4mL Na₂EDTA aqueous solution (concentration 0.5 mol. L)^-1) 200mg of X-Gluc, 50mL of methanol and 120. mu.l of Triton-X were dissolved in distilled water, and the volume was adjusted to 250mL with distilled water.

Wheat variety Fielder is described in the following literature: liupan wheat TaERF5 gene diversity and functional analysis [ D ]. Beijing: the wheat variety Fielder is hereinafter referred to as Fielder, 2015, the academy of agricultural sciences.

The media involved in the following examples are as follows:

callus induction culture medium I (SD for short)₂Culture medium): 4.33g of MS minimal medium (containing no vitamin) and Vb₁Dissolving 1mg, asparagine 150mg, sucrose 30g, 2, 4-D2 mg and plant gel 2.4g in 1L distilled water, adjusting pH to 5.8, and sterilizing at 121 deg.C for 15 min.

Coculture medium (wcci medium for short): 0.443g of MS minimal medium (containing vitamins), 40g of maltose and MgCl₂0.75g, MES 1.95g, glutamine 0.5g, hydrolyzed casein 0.1g, glucose 10g, vitamin C100 mg, Picloram 2.2mg and Acetosyringone (AS) 39mg were dissolved in 1L of distilled water, pH was adjusted to 5.4, and sterilized at 121 ℃ for 15 min.

Recovering the culture medium: SD containing 250mg/L carbenicillin₂And (4) a culture medium.

Screening a culture medium: 4.43g of MS minimal medium (containing vitamins), 30g of sucrose, 150mg of aspartyl, 2, 4-D2 mg, 5mg of Glufosinate, 250mg of carbenicillin and 2.4g of plant gel are dissolved in 1L of distilled water, the pH is adjusted to 5.8, and the mixture is sterilized at 121 ℃ for 15 min.

Differentiation medium: 4.43g of MS minimal medium (containing vitamins), 30g of sucrose, 0.2mg of 2,4-D, 0.1mg of vitamin C, 5mg of Glufosinate, 250mg of carbenicillin and 2.4g of plant gel are dissolved in 1L of distilled water, the pH is adjusted to 5.8, and the mixture is sterilized at 121 ℃ for 15 min.

Strong seedling culture medium: 4.43g of MS minimal medium (containing vitamins), 20g of sucrose, 0.2mg of IAA, 0.5mg of MET, 5mg of Glufosinate, 100mg of carbenicillin and 2.4g of plant gel were dissolved in 1L of distilled water, the pH was adjusted to 5.8, and sterilization was carried out at 121 ℃ for 15 min.

Example 1 obtaining of vector pWMB122

The nucleotide sequence of the artificially synthesized plasmid pWMB122 and the (circular) plasmid pWMB122 is shown as the sequence 1 in the sequence table. Plasmid pWMB122 has two expression cassettes, designated expression cassette a and expression cassette b, respectively.

The reverse complementary sequence of the expression cassette A is shown as 1479 th to 3039 th from the 5 'end of the sequence 1 in the sequence table, wherein the 2213 rd to 3039 th from the 5' end of the sequence 1 in the sequence table is the reverse complementary sequence of a 35S promoter, the 1648 th to 2212 th positions are the reverse complementary sequence of a coding gene of a bar protein, and the 1479 th to 1647 th positions are the reverse complementary sequence of ployA (for terminating transcription).

The reverse complement of expression cassette B is shown as sequence No. 1 from position 10121 to 12942 from the 5' end, wherein sequence No. 1 from position 10950 to 12942 is the reverse complement of the UBI promoter, sequence No. 10894 to 10949 is the reverse complement of the multiple cloning site A (multiple cloning site A includes restriction enzymes Pst I, BamH I, Sma I, Kpn I, Sal I, Nhe I and Xhol I, etc.), sequence No. 10405 to 10893 is the reverse complement of an intron derived from rice, sequence No. 10396 to 10404 is the reverse complement of multiple cloning site B (multiple cloning site B includes the reverse complements of restriction enzymes SpeI and Sac I), and sequence No. 10121 to 10395 is the reverse complement of the NOS terminator.

The plasmid pWMB122 is the obtained vector pWMB 122.

Example 2 genetic transformation and T of wheat₀Detection of transgenic wheat plants

Construction of recombinant plasmid pWMB123

1. Artificially synthesizing a primer GusF 110-3: 5' -AAAGGATCCATGACCACCAGTGCAAG-3' (underlined is the recognition sequence for restriction endonucleases BamHI) and GusR 110-3:

5'-AAAGAGCTCTCTCACACGTGATGGTGTGGTG-3' (recognition sequence for SacI is underlined).

2. And (3) carrying out PCR amplification by using the vector pPTN290 as a template and the GusF110-3 and the GusR110-3 synthesized in the step (1) as primers to obtain a double-stranded DNA molecule of about 2084 bp.

3. After the step 2 is finished, the double-stranded DNA molecules obtained in the step 2 are subjected to double digestion by using restriction endonucleases BamH I and Sac I, and digestion fragments are recovered.

4. The plasmid pWMB122 was digested both with BamHI and SacI by restriction endonucleases and the vector backbone of about 13kb was recovered.

5. And (4) connecting the enzyme digestion fragment recovered in the step (3) with the vector skeleton obtained in the step (4) to obtain a recombinant plasmid pWMB 123.

According to the sequencing result, the structure of the recombinant plasmid pWMB123 is described as follows: the fragment between the BamH I and Sac I recognition sequences of vector pWMB122 (into which the vector pWMB122 is cut into a large fragment and a small fragment by restriction endonucleases BamH I and Sac I, the DNA being the small fragment) is replaced by a DNA molecule represented by sequence 2 in the sequence listing. The recombinant plasmid pWMB123 contains an expression cassette for promoting GUS gene expression by UBI promoter.

II, obtaining recombinant agrobacterium

The recombinant plasmid pWMB123 is introduced into agrobacterium tumefaciens C58C1 to obtain recombinant agrobacterium tumefaciens which is named C58C1/pWMB 123.

The plasmid pWMB122 was introduced into Agrobacterium tumefaciens C58C1 to obtain a recombinant Agrobacterium, designated C58C1/pWMB 122.

Genetic transformation of wheat

1. Single clone C58C1/pWMB123 was inoculated into 20mL of a strain containing 50mg/L kanamycin andculturing Fuping 50mg/L YEB liquid culture medium with shaking at 28 deg.C and 220rpm for 12-16 h, inoculating into YEB liquid culture medium containing acetosyringone 100 μ M at 2-4% (volume percentage), and culturing at 28 deg.C and 220rpm until OD₆₀₀The value reaches 0.6-0.8, the mixture is centrifuged at 3500rpm for 10min at 4 ℃, and the thalli are collected.

2. Suspending the thallus collected in the step 1 by using a WCCI culture medium to obtain OD₆₀₀The value is about 0.7 of the agrobacterium infection liquid.

3. Taking young embryo of Fielder, and placing in SD₂Culturing in culture medium at 28 deg.c for 4 days to obtain wheat embryo explant.

4. Soaking wheat embryo explants in agrobacterium infection solution, infecting at room temperature for 30min, placing on a co-culture medium paved with a layer of sterilized filter paper, and performing dark culture at 25 ℃ for 2 days.

5. After completing step 4, the callus was placed in a recovery medium and cultured in the dark at 25 ℃ for 5 days.

6. And (5) after the step 5 is completed, placing the callus in a screening culture medium, and performing dark culture at 25 ℃ for 14-21 days to obtain the embryogenic callus.

7. And (6) after the step 6 is finished, taking the embryonic callus, placing the embryonic callus in a differentiation culture medium, and alternately culturing for 21-28 days at the temperature of 25 ℃ under illumination to obtain the resistant seedlings.

8. And (4) after the step (7) is finished, placing the resistant seedlings in a strong seedling culture medium, and alternately culturing under illumination at 25 ℃ to obtain resistant plants. When the resistant plants grow to 6-10 cm, cleaning agar and transferring the agar to a flowerpot to obtain 235T₀To simulate transgenic wheat plants.

According to the steps, C58C1/pWMB123 is replaced by C58C1/pWMB122, and other steps are not changed, so that an empty vector plant is obtained.

Four, T₀Detection of transgenic-pseudowheat plants

1. Bar immune test paper identification

(1) Get T₀The leaves of the pseudotransgenic wheat plants (0.1 g) were placed in a centrifuge tube (2 mL volume) and ground. Then adding a protein extract EB2 buffer solution added by a QuickStix Kit (product of Envirologix, USA)500 μ L, mix well.

(2) Sample detection

And (3) returning the Bar immune test paper to room temperature, vertically inserting the test paper into the centrifugal tube completing the step (1), submerging to the depth of about 0.5cm, taking out after 1min, and flatly reading the test result.

(3) Result judgment

The detection line and the control line can generally appear within 30s, and the detection standard is as follows: only one mauve quality control line appears on the test strip as a negative result; two purple-red strips appear on the detection strip, one is a purple-red detection line, and the other is a purple-red quality control line, which is a positive result.

All that can give two purple-red bands₀Identifying the pseudotransgenic wheat plant as a bar transgenic wheat plant; all that can give a purple-red band₀The pseudotransgenic wheat plant is identified as a non-bar transgenic wheat plant. The results of the experiment are shown in FIG. 1.

2. GUS staining detection

(1) Get T₀Leaves of a pseudotransgenic wheat plant are cut into small sections with the length of about 0.5cm and placed in a culture dish.

(2) And (4) adding a staining solution into the culture dish for GUS staining.

(3) And (5) judging a result: if the leaves appear blue, the result is positive; if the leaves do not appear blue, a negative result is obtained.

All-blade presenting blue T₀The transgenic wheat plant is identified as a GUS gene-transferred wheat plant; t with blue color not appearing on the leaf₀And (4) the transgenic wheat plant is identified as a non-transgenic GUS gene wheat plant. The results of the experiment are shown in FIG. 2.

3. Southern hybridization

Extraction of T₀The genome DNA of the transgenic wheat plant is simulated, and Southern hybridization is carried out by respectively taking the reverse complementary sequence of 675 th to 1670 th sites of the sequence 2 in the sequence table as a probe sequence and the probe sequence shown in 1654 th to 2204 th sites of the sequence 1 in the sequence table.

The reverse complementary sequence of 675 th to 1670 th positions of the sequence 2 in the sequence table isThe Southern hybridization results of the probe sequences are shown in A of FIG. 3 (lanes 1-11 are T)₀A pseudotransgenic wheat plant is replaced, a lane P is a recombinant plasmid pWMB123, and a lane M is a DNA Marker). The results of Southern hybridization with the probe sequence shown at positions 1654 to 2204 of sequence 1 in the sequence listing are shown in B in FIG. 3 (lanes 1-11 are T₀A pseudotransgenic wheat plant is replaced, a lane P is a recombinant plasmid pWMB123, and a lane M is a DNA Marker).

If the reverse complementary sequence of 675 th to 1670 th of the sequence 2 in the sequence table is used as a probe sequence for Southern hybridization and the probe sequence of 1654 th to 2204 th of the sequence 1 in the sequence table is used as a probe sequence for Southern hybridization, the T₀The pseudotransgenic wheat plant is identified as a wheat plant with GUS gene and Bar gene.

Obtaining of safe GUS transgenic wheat plant

And D, culturing the GUS gene and Bar gene transferred wheat plant obtained in the step four in a greenhouse until the wheat plant is mature, selfing, and harvesting seeds. Each wheat plant transformed with GUS gene and Bar gene is taken as a T₁Generation line (i.e. T)₁Generation transgenic wheat lines). Randomly select 14T₁Transgenic wheat lines (numbered L1-L14) were subjected to detection molecular detection and Southern hybridization.

1. Molecular detection

Respectively using T in heading stage₁Genome DNA of leaves of transgenic wheat is taken as a template, and a primer F1: 5'-CAAGGAAATCCGCAACCATATC-3' and primer R1: 5'-TCAAACGTCCGAATCTTCTCCC-3' PCR amplification is carried out to the A by the primer for identifying GUS gene; with primer F2: 5'-ACCATCGTCAACCACTACATCG-3' and primer R2: 5'-GCTGCCAGAAACCCACGTCATG-3' PCR amplification of B was performed to identify the bar gene.

The electrophoretogram of PCR amplification using primers for A is shown in FIG. 4A, and the electrophoretogram of PCR amplification using primers for B is shown in FIG. 4B (in FIG. 4, lanes 1-14 are L1-L14, lane P is recombinant plasmid pWMB123, lane CK is genomic DNA of Fielder leaf, and lane M is DNA Marker). The results showed that lanes 6, 7, 8 and 9 with primer pair A gave a band of approximately 995bp in size (A in FIG. 4)Arrow head) and a band of about 429bp in size could not be obtained with primer pair B (arrow head B in FIG. 4). Thus, L6, L7, L8 and L9 are T containing only the GUS gene₁A safe transgenic wheat plant.

2. Southern hybridization

Further validation was performed using Southern hybridization.

Extraction of T at heading stage₁The genome DNA of the leaf of the transgenic wheat is substituted,

southern hybridization was performed using the reverse complementary sequence of the 675 th to 1670 th positions of the sequence 2 in the sequence listing as a probe sequence and the probe sequence of the 1654 th to 2204 th positions of the sequence 1 in the sequence listing, respectively.

The Southern hybridization results using the reverse complement of the sequence No. 675 to 1670 of the sequence No. 2 in the sequence Listing as the probe sequence are shown in A in FIG. 5 (lanes 1-11 are L3 to L13, lane P is the recombinant plasmid pWMB123, and lane M is the DNA Marker). The Southern hybridization results using the probe sequences as shown at positions 1654 to 2204 of the sequence 1 in the sequence listing are shown in B in FIG. 5 (lanes 1-11 are L3 to L13, lane P is the recombinant plasmid pWMB123, and lane M is the DNA Marker). As a result, it was revealed that lanes 4-7 could be subjected to Southern hybridization with the reverse complementary sequence of positions 675 to 1670 of the sequence No. 2 in the sequence Listing as the probe sequence and could not be subjected to Southern hybridization with the probe sequences of positions 1654 to 2204 of the sequence No. 1 in the sequence Listing, indicating that L6, L7, L8 and L9 are T containing only GUS gene₁A safe transgenic wheat plant.

For 12T₁The segregation conditions of GUS gene and Bar gene in the generation transgenic wheat strain are counted, the statistical result is shown in Table 1, and the result shows that T only containing GUS gene is obtained₁The rate of transgenic wheat plants was 3.23%.

TABLE 1.T₁Segregation proportion statistics of generation wheat transgenic line

Claims (6)

1. An expression vector comprising an expression cassette a and an expression cassette b;

the expression cassette A sequentially comprises the following elements: promoter A, selective marker gene and termination sequence A;

the expression cassette B sequentially comprises the following elements: promoter B, intron and termination sequence B;

in the expression cassette B, a multiple cloning site A is arranged between the promoter B and the intron; a multiple cloning site B is arranged between the intron and the termination sequence;

the multiple cloning site A and the multiple cloning site B both have more than one enzyme cutting recognition sequence;

enzyme cutting recognition sequences at other positions in the expression vector are different from the enzyme cutting recognition sequence in the multiple cloning site A and the enzyme cutting recognition sequence in the multiple cloning site B;

the nucleotide sequence of the expression vector is shown as a sequence 1 in a sequence table.

2. Use of the expression vector of claim 1 for the preparation of a transgenic plant.

3. A method of making a transgenic plant comprising the steps of: inserting a target gene into the enzyme digestion recognition sequence in the expression cassette B in the expression vector of claim 1, then transforming a receptor plant, and screening by virtue of a selection marker gene to obtain T₀Plant generation; will be the T₀Selfing the generation plants to obtain T containing the target gene and not containing the selectable marker gene₁And (4) generating plants, namely transgenic plants without the selection marker.

4. A method of making a transgenic plant comprising the steps of: using the expression vector of claim 1 as a starting vector, inserting a specific segment in a target gene into the multiple cloning site A, and inserting a reverse complementary sequence of the specific segment in the target gene into the multiple cloning site B to obtain a target vector; transforming the target vector into a receptor plant, and screening by virtue of a selective marker gene to obtain T₀Plant generation; will be the T₀Selfing the generation plants to obtain T which does not contain the target gene and does not contain the selectable marker gene₁And (4) generating plants, namely transgenic plants without the selection marker.

5. The method of claim 3 or 4, wherein: the recipient plant is a1) or a2) as follows:

a1) a dicotyledonous plant;

a2) a monocotyledonous plant.

6. The method of claim 3 or 4, wherein: the recipient plant is any one of the following a3) -a 8):

a3) rice;

a4) corn;

a5) barley;

a6) rye;

a7) wheat;

a8) wheat variety Fielder.

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