CN102703498A - Multi-gene plant expression vector and construction method and application thereof - Google Patents

Abstract

The invention discloses a multigene plant expression vector, its construction method and application. The multigene plant expression vector comprises: plant transformation vector p209, cloning vector p6435x and one or more target genes to be transformed. The construction of the multi-gene plant expression vector of the present invention utilizes the characteristic that the original enzyme-cleavage site disappears after the homologous enzyme is joined, so that the two ends of the vector or fragment after enzyme-cleavage are always different sticky segments, which solves the problem of vector endonuclease in the past. The problem of limited sites also avoids self-ligation during vector ligation, no need for phosphorylation of cohesive ends, no intermediate vectors and special engineering strains, and most restriction endonuclease sites are avoided , only need to carry out routine steps such as enzyme digestion, ligation, transformation, etc., which simplifies the operation steps and reduces the difficulty of experiments. The technology is mature and reliable, and the cost is low. It can be completed under ordinary experimental conditions and is easy to promote.

Description

Multigene plant expression vector and its construction method and application

technical field

The present invention relates to a plant expression vector, in particular to a plant expression vector carrying single or multiple exogenous genes and its construction method. The present invention also relates to the application of the plant expression vector in transgenic plants, which belongs to the category of plant expression vectors. Build fields.

Background technique

At present, plant transgenic research mainly focuses on the transformation of a single gene, which has a single function and limited effect. Co-transformation of multiple genes has become a way to solve this problem.

The construction of multigene plant transformation vectors is the basis and important step for the simultaneous transformation of plants with multiple genes. Almost all plant transgenic research begins with the construction of vectors. In the past, the construction of multi-gene expression vectors was limited by restriction enzyme cutting sites, and the number of genes carried by the vectors was small. In some construction processes, dephosphorylation, phosphorylation, sticky end filling and other operations were required. The steps were cumbersome and difficult. Some vectors introduce "gateway technology" or use "homing endonuclease combined with Cre recombination technology". Although the above problems are partially solved, there are also problems such as high cost, special intermediate vectors, and special strains.

Contents of the invention

One of the objects of the present invention is to provide a plant expression vector carrying single or multiple foreign genes;

The second object of the present invention is to provide a method for constructing the plant expression vector;

The third object of the present invention is to apply the plant expression vector to plant transgene.

The above object of the present invention is achieved through the following technical solutions:

A plant expression vector carrying single or multiple exogenous genes, including: plant transformation vector p209, cloning vector p6435x and one or more target genes to be transformed;

Among them, p209 is a Ti plasmid, which is derived from the vector pBI121. A fragment with an Xba I restriction site is introduced between the HindIII and Eco RI restriction sites of the original pBI121 vector; there are Bsp120 I and Xba on the p209 vector I two restriction endonuclease sites; p209 carries the plant selection gene npt II.

The cloning vector p6435x originates from the prokaryotic expression vector pET28a; the vector has a CaMV35S promoter and a NOS termination sequence, and there are two Eam1105 I restriction sites between the two, and the T vector and the target gene PCR product are formed by single restriction digestion Linked together to form a complete open reading frame (ORF) of the target gene, carrying restriction sites Not I and Bsp120I, Spe I and Nhe I restriction sites on both sides of the ORF.

Another object of the present invention is to provide a method for constructing the plant expression vector carrying single or multiple exogenous genes, comprising the following steps:

(1) Construct the plant transformation vector p209 and the cloning vector p6435x respectively;

(2) Digest the cloning vector p6435x with endonuclease Eam1105 I to generate two 3' overhangs containing T to form a T vector, which is connected to the PCR product of the first target gene with a 3' overhang containing A, and the first The amplified fragment of the target gene is complete and forward connected into the cloning vector to construct an open reading frame including CaMV35S promoter + target gene + NOS termination sequence; use Not I and Nhe I double cutting to carry the target gene to express the open reading frame (ORF) cloning vector, recover the first target gene expression open reading frame (ORF) fragment, and reserve;

(3) Use Bsp120 I and Xba I to double-cut p209, and recover the p209 digested fragment; connect the p209 digested fragment with the first target gene expression open reading frame (ORF) fragment recovered in step (2), and the original p209 on p209 The Bsp120I and Xba I sites and the Not I and Nhe I sites at both ends of the open reading frame of the target gene expression disappear, while the Bsp120I and Spe I sites on the newly formed plant expression vector can be digested with the first Two target gene expression open reading frame (ORF) fragments with Not I and Nhe I sites are connected, and so repeated, a plant expression vector carrying multiple target genes is constructed.

Wherein, the method for constructing the plant transformation vector p209 comprises: introducing a fragment with an Xba I restriction site between the Hind III and EcoRI restriction sites of the pBI121 vector, to obtain.

p209 is a Ti plasmid derived from vector pBI121. It has the plant selection gene npt II for plant transformation. A fragment with an Xba I restriction site was introduced between the Hind III and EcoR I restriction sites of the original pBI121 vector. There are two restriction endonuclease sites, Bsp120I and XbaI, on the p209 vector. After double digestion with endonucleases Bsp120I and XbaI, two different cohesive ends will be produced, which are identical to the end fragments of the corresponding endonucleases on the cloning vector. Once connected, the spots disappear.

The construction method of the cloning vector p6435x comprises: first cloned a fragment with CaMV 35S promoter, gfp green fluorescent protein gene and NOS termination sequence and introduced a new restriction site from the vector pCAMBIA1302; The fragment with the Eam1105 I site replaced the gfp green fluorescent protein gene to form a new fragment, and then the new fragment was connected to pET28a by enzyme digestion and ligation to obtain the cloning vector p6435x.

The plant expression vector constructed in the present invention can be transformed into competent Agrobacterium by means such as heat shock method. The plants were transformed using the Agrobacterium-mediated method, and the transgenic plants were obtained through kanamycin (Km) resistance selection.

The plant expression vector of the present invention utilizes the characteristics that the same cohesive ends are provided after digestion with the homologous enzymes, and the original restriction sites disappear after being connected with each other to construct a plant expression vector carrying multiple target genes. Generally, this vector can carry foreign fragments less than 30kb to transform plants.

In the present invention, the PCR fragment of the target gene is cloned through A-T, and directly connected into the cloning vector to complete the construction of the open reading frame (ORF) of the target gene, which can be used for further plant transformation vector construction after simple PCR identification. In the process of constructing the multi-gene plant expression vector of the present invention, two different homologous enzyme systems, Not I/Bsp120 I and Spe I/Xba I/Nhe I, are used simultaneously, and the original enzyme cutting site is used after the homologous enzyme is connected. The disappearing feature makes the two ends of the vector or fragment after enzyme digestion always have different sticky segments, which solves the problem of limited endonuclease sites in the past, and also avoids self-ligation during the vector ligation process. Phosphorylation treatment of cohesive ends does not require intermediate vectors and special engineering strains, avoids most restriction endonuclease sites, and only needs conventional steps such as enzyme cutting, ligation, and transformation. The operation steps are simplified and the experiment difficulty is reduced, and the technology is mature and reliable. The method is low in cost, can be completed under ordinary experimental conditions, is easy to popularize, and has great scientific research and practical application value.

Definition of terms involved in the present invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are now described.

The term "host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, pairing, or other methods known in the art. Exogenous polynucleotides may remain as non-integrating vectors such as plasmids or may integrate into the host genome.

The term "polynucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.) . Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the explicitly designated sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which one or more selected (or all) codons are substituted at position 3 with mixed bases and/or deoxyinosine residues (Batzer et al. , Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J.Biol.Chem.260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., MolCell.Probes 8:91 -98(1994)).

The term "exogenous DNA" refers to the DNA sequence being of a source foreign to the particular host cell, or if from the same original source but with modifications or alterations to the original sequence.

The term "transformation": A method of introducing a heterologous DNA sequence into a host cell or organism.

The term "expression": transcription and/or translation of an endogenous or transgene in a plant cell.

The term "coding sequence": a nucleic acid sequence transcribed into RNA.

The term "plant expression vector": one or more DNA vectors used to effect plant transformation; these vectors are often referred to in the art as binary vectors. Binary vectors together with vectors with helper plasmids are the most commonly used for Agrobacterium-mediated transformation. Binary vectors usually include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be expressed in plant cells, heterologous DNA sequences to be transcribed, etc.

Description of drawings

Figure 1 Map of the cloning vector p6435x.

Figure 2 Map of transformation vector p209.

Fig. 3 is a schematic diagram of the process of transforming a multigene plant into a vector of the present invention.

Fig. 4 PCR identification of vector p2096871; M: maker; A: specific amplified fragment of gene BtCryIIIA; B: specific amplified fragment of gene BtCryIA.

Figure 5 The transformed plants (1-8) were detected by PCR; a fragment of the same size as the positive control (CK+) appeared, but the non-transgenic plants (CK-) did not; it was preliminarily proved to be transgenic plants. It is also demonstrated that the plant expression vector of the present invention can be used for plant transformation.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Experimental Materials

1. Plasmids: pUC18 (Dalian TaKaRa Company), pBR322 (Dalian TaKaRa Company), pUCm-T (Shanghai Sangon), pET28a (Novagen Company), pBI121 (Invitrogen Company), pCAMBIA1302 (CambiaLabs)

2. Strains: Escherichia coli JM109 strain (Dalian TaKaRa Company), DH10B strain (Shanghai Sangong)

3. Various reagents and endonucleases: purchased from Dalian TaKaRa Company, new England biolabs (NEB), Shanghai Sangong.

Construction of embodiment 1 cloning vector p6435x

1. Synthesis of vector p1302x

Use 26# primer:

F AGATCTCCACTTCTAAAATGGCTACAGGCATCGTGGTGT (SEQ ID No. 1)

R GGTGACCCCACTTCTAAAATGGATTTTGCCTTCCTGTTTT (SEQ ID No. 2)

Using pUC18 as a template, carry out PCR amplification to obtain a fragment of about 500bp, which is connected into pUCm-T to synthesize vector p26; p26 and pCAMBIA1302 are double digested with BglII and BstEII respectively. The small fragment of p26 and the large fragment of pCAMBIA1302 were recovered and ligated. Synthetic vector p1302x.

2. Synthesis of vector p1302x30

Use 30# primer:

F aagcttagatctCTTGGATCAGATTGTCGT (SEQ ID No. 3)

R ggatccAGAGTCCCCCGTGTTCT (SEQ ID No. 4)

Using pCAMBIA1302 as a template, PCR amplification was performed to obtain a 597bp fragment, which was ligated into pUCm-T to synthesize vector p30. p30 and p1302x were digested with HindIII and BamHI respectively. The small fragment of p30 and the large fragment of p1302x were recovered, and the two were ligated to synthesize the vector p1302x30.

3. Synthesis of vector p35x

Use 4# primer:

F CTTGGATCAGATTGTCGT (SEQ ID No.5)

R TCCCGATCTAGTAACATA (SEQ ID No. 6)

Using pCAMBIA1302 as a template, PCR amplification was performed to obtain a 1622bp fragment, which was ligated into pUCm-T to synthesize vector p4. p4 and p1302x30 were digested with HindIII and BstEII respectively. The small fragment of p1302x30 and the large fragment of p4 were recovered, and the two were connected to synthesize the vector p35x.

4. Synthesis of vector p2858

Use 58# primer:

F ggatcCTACGGCTACACTAGAAGG (SEQ ID No. 7)

R ggtcaccGAAACGCTGGTGAAAGT (SEQ ID No. 8)

Using pBR322 as a template, PCR amplification was performed to obtain a 1122bp fragment, which was connected into pUCm-T to synthesize vector p58. p58 and pET28a were digested with BamHI and BstEII respectively. The small fragment of p58 and the large fragment of pET28a were recovered, and the two were ligated to synthesize vector p2858.

5. Synthesis of vector p285859

Use 59# primer:

F gatatcgggcccagatctCTTCGGTTTCCGTGTT (SEQ ID No.9)

R CTGCAGTGATGCCTCCGTGTAA (SEQ ID No.10)

Using pET28a as a template, PCR amplification was performed to obtain a fragment of about 300 bp, which was ligated into pUCm-T to synthesize vector p59. p59 and p2858 were digested with BglII and EcoRV respectively. The small fragment of p59 and the large fragment of p2858 were recovered, and the two were ligated to synthesize the vector p285859.

6. Synthesis of vector p35N

p285859 and p35x were digested with BglII and PstI respectively. The small fragment of p35x and the large fragment of p285859 were recovered, and the two were ligated to synthesize the vector p35N.

7. Synthesis of cloning vector p6435x

Use 64# primer:

F GGTCACCgggccCTTCGGTTTCCGTGTT (SEQ ID No. 11)

R gatatcGCTAGCactagTGATGCCTCCGTGTAA (SEQ ID No. 12)

Using pET28a as a template, PCR amplification was performed to obtain a fragment of about 300 bp, which was ligated into pUCm-T to synthesize vector p64. p64 and p35N were digested with BstEII and EcoRV respectively. The small fragment of p64 and the large fragment of p35N were recovered, and the two were ligated to synthesize the cloning vector p6435x.

The construction of embodiment 2 transformation vector p209

1. Synthesis of vector p88

Use 88# primer:

F gaattctctagaCTTCGGTTTCCGTGTT (SEQ ID No. 13)

R aagctTGATGCCTCCGTGTAA (SEQ ID No. 14)

Using pET28a as a template for PCR amplification, a fragment with a length of about 300bp was obtained. This fragment was ligated into pUCm-T to synthesize vector p88.

2. Synthesis of plant transformation vector p209

p88 and pBI121 were digested with HindIII and EcoRI respectively. The small fragment of p88 and the large fragment of pBI121 were recovered, and the two were ligated to synthesize the plant transformation vector p209.

Example 3 Construction of Plant Transformation Vectors Carrying BtCryIA and BtCryIIIA Two Insecticidal Toxin Genes

1. Construction of plant transformation vector p2096871

The general PCR reaction system was used, and the insecticidal toxin gene BtCryIA gene and BtCryIIIA gene were used as templates for PCR; the PCR reaction procedure was as follows: 94°C pre-denaturation for 10 minutes, 94°C denaturation for 30 seconds, 48-60°C annealing for 40 seconds, and 72°C extension 1.5min, 20 cycles; 72°C extension for 20min. After the reaction was completed, the presence and length of the amplified product fragments were detected by 1% TAE agarose gel electrophoresis, the gel was cut and suitable fragments were recovered using a gel recovery kit. The primers are listed in Table 1.

Table 1 partial primers

Extract p6435x plasmid, use restriction endonuclease Eam1105I respectively, use 50 μL enzyme digestion reaction system, digest at 37°C for 4 hours, add 10 μL 6×Loadingbuffer at the end of the reaction, check by 1% TAE agarose gel electrophoresis, use gel The corresponding fragments were recovered by the recovery kit and used as T vectors for further research.

The amplified fragment was ligated with the gel-recovered fragment of the vector digestion product, and the reaction system was (10 μL): 2 μL of vector digestion product, 4 μL of PCR product, 1 μL of ddH ₂ O, 1 μL of 50% PEG4000, 1 μL of 10×T ₄ lig buffer, T ₄ 1 μL. 16°C overnight. Transform Escherichia coli JM109 competent medium by the heat shock method (prepared using the competent preparation kit produced by Dalian Takara Company, the method is the same as the instruction manual). Spread on LB solid medium containing 50mg/l Km, and incubate at 37°C for 12h to grow monoclonal colonies.

The 35# primer designed according to the terminal sequence of the CaMV35S promoter is the forward primer, which forms a pair of detection primers with the anti-primer of the fragment to be tested. Pick a single colony for colony PCR identification. If the length of the amplified fragment is consistent with the length of the target gene, then It was considered that the amplified fragment was complete and forward-connected into the cloning vector, and an open reading frame (ORF) including the CaMV 35S promoter + target gene + NOS termination sequence was constructed. The colonies that passed the inspection were shaken, and the bacterial solution was sent to Shanghai Sangon for sequencing for further inspection. After sequencing, the sequence-unchanged vectors were used for further experiments. In the experiment, the cloning vector carrying BtCryIA was called p6468, and the cloning vector carrying BtCryIIIA was called p6471.

When constructing the multigene transformation vector containing BtCryIA and BtCryIIIA, first, p209 was double-cut with Bsp120I and XbaI, and p6468 was double-cut with NotI and NheI. After recovering the p209 restriction fragment and the ORF fragment of the target gene BtCryIA, connect the two, the original Bsp120I and XbaI sites on p209 and the NotI and NheI sites at both ends of the ORF of the target gene BtCryIA disappear, and the newly synthesized plants are transformed The Bsp120I and SpeI sites on the vector p20968.

Once again, p20968 was double-cut with Bsp120I and SpeI, and p6471 was double-cut with NotI and NheI. After recovering the p20968 restriction fragment and the ORF fragment of the target gene BtCryIIIA, connect the two, the original Bsp120I and XbaI sites on p20968 and the NotI and NheI sites at both ends of the ORF of the target gene BtCryIIIA disappeared again, and the newly synthesized plants were transformed into The Bsp120I and SpeI sites on the vector p2096871. p2096871 is the required plant transformation vector carrying two insecticidal toxin genes, BtCryIA and BtCryIIIA.

2. Identification of plant transformation vector p2096871

p2096871 was identified by PCR technique. Using the carrier to be detected as a template, use the specific primers used to amplify BtCryIA and BtCryIIIA for PCR respectively. The PCR reaction procedure is as follows: 94°C pre-denaturation for 10 minutes, 94°C denaturation for 30s, 48-60°C annealing for 40s, 72°C Extend for 1.5 min, 20 cycles; extend for 5 min at 72°C. After the reaction was completed, the presence and length of the amplified product fragments were detected by 1% TAE agarose gel electrophoresis. If the length of the amplified fragment is consistent with the theoretical length of the target gene, it can be judged that the BtCryIA and BtCryIIIA genes are connected into the vector, and the vector synthesis is successful (Figure 4).

3. Expression of 2 exogenous genes in transgenic tobacco

The expression of two exogenous genes in transgenic tobacco was detected by enzyme-linked immunosorbent immunoassay (ELISA) kit. After transforming the BtCryIA and BtCryIIIA genes, the transgenic tobacco will express two toxic proteins.

Detection method:

Sample preparation: 5 transgenic tobacco plants were selected as samples, and non-transgenic tobacco was used as control. Take 100mg of fresh transgenic tobacco leaves, add 1ml of PBST extraction buffer, thoroughly grind on ice to form a homogenate, centrifuge at 12000rpm at 4°C for 5min, and transfer the supernatant to a new Eppendorf centrifuge for later use.

Determination of total protein content:

(1) Drawing of the standard curve Take bovine serum albumin solutions of different concentrations and add water to make up to 1ml. Accurately draw 0.1ml of each bamboo solution prepared, add 5ml Coomassie Brilliant Blue G-250 reagent, mix repeatedly several times, let stand for 2min, measure the absorbance value at 595nm on a spectrophotometer, and draw a standard curve.

(2) Determination of protein concentration in the sample extract solution Draw 0.1ml of the sample extract solution, add 5ml Coomassie Brilliant Blue G-250 reagent (set 2 repetitions), fully mix, stand for 2min and then compare the color at 595nm, record the absorbance value, And check the protein content through the standard curve.

ELISA detection of Btl toxin protein (BtCryIA):

(1) preparation

1) Dilute 10g of PBST with eubacteria water to 1×PBST.

2) Prepare the reaction box and seal it.

3) Dissolve the positive control provided in the kit with 2mL 1XPBST.

4) Aliquot the positive control, 120g1 per tube, and store at -20°C.

5) Design the test sample arrangement diagram, record the serial number A-H, 1-120

(2) Ellsa reaction

1) Add 100μl enzyme conjugate to each well.

2) According to the design diagram, add the positive control and the prepared samples to the corresponding wells, 100 μL per well, and set 2 replicates.

3) Transfer the ELISA plate to a box covered with damp filter paper, and place it at room temperature for 2 hours or in a refrigerator at 4°C overnight.

4) After the heat preservation is over, wash the plate. Shake off the samples on the ELISA plate, fill each well with 1×PBST, let it sit for 3 minutes (in the box), then quickly shake off the liquid in the well, and pat dry on the filter paper. Be careful not to contaminate each well, especially do not shake In the blank control. Repeat 6-7 times (at room temperature).

5) Add enzyme substrate. Add 100 μl of TMB enzyme substrate to each well, place the microtiter plate in a box covered with damp filter paper, let it stand at room temperature, and wait for the color change.

6) Detection. A color change occurs in about 5-15 minutes. If blue appears in the well, it is a positive result, and if colorless or light blue appears, it is a negative result. Measure the absorbance at 450nm on a microplate reader (OD450)

7) Make a standard curve. The concentration of BtCryIA in the sample was detected, and the expression amount per gram of leaf was calculated. ELISA detection of Bt3 toxin protein (BtCryIIIA)

(1) preparation

1) Dilute a bag of 20×PBST wash buffer into 1×PBST wash buffer with 1L of distilled water.

2) Prepare 100mL MEB sample extraction buffer

Nonfat dried milk 0.4g

Tween 20 0.5g

Add 1×PBST to 100mL, stir slowly at room temperature until completely dissolved.

3) with ECM 25mL

Nonfat dried milk 0.1g

Add 1×PBST to 25mL, stir slowly at room temperature until completely dissolved.

4) with PNP buffer

Prepare 5ml of PNP solution for each PNP tablet with a concentration of 1mg/mL. , prepared 15 minutes before the end of incubation.

(2) El.lsa reaction

1) Distribution of samples According to the sample loading diagram, add 10 μl of the sample to be tested, control and standard samples in appropriate wells, and set 2 replicates.

2) Incubating the plate Put the plate into a humid box and incubate at room temperature for 1 hour.

3) Prepare enzyme conjugate 10 minutes before the end of incubation, prepare enzyme conjugate, 100μl 1×ECM buffer per well. Calculate the amount of enzyme conjugate according to the number of holes used in the test and the dilution method on the bottle and the amount of I1X ECM buffer. Use new, sterile pipettes to prevent contamination.

4) Wash the plate After the first incubation is completed, wash the plate, fill each well with 1×PBST, quickly turn over and pour the liquid in the well, do not contaminate each other between the wells, and repeat 4-8 times. Each well was then filled with 1×PBST, and left to stand at room temperature for 3 minutes in a humid box. Remove the plate from the box, quickly empty the liquid in the well, and tap on the folded filter paper to remove the residual liquid in the well.

5) Add enzyme conjugate and add 100μl enzyme conjugate to each well.

6) Incubate the plate Put the plate into a humid box and incubate at room temperature for 1 hour.

7) Prepare PNP solution

Prepare 5ml of PNP solution for each PNP tablet, the concentration is 1mg/mL, which is enough for 5 strips with 8 holes. 15 minutes before the end of the incubation, take 5ml of 1×PNP buffer at room temperature for each tablet to be used, add the PNP tablet to the buffer, and mix well. Note: Do not touch the tablet or expose the PNP solution to strong light. Light or pollution will cause the background of the negative control to darken.

8) Wash the plate Repeatedly wash the plate 4-8 times with 1×PBST. Check the holes for air bubbles, wash the plate repeatedly, and clap the plate to remove air bubbles.

9) Add PNP solution Add 100 μl PNP solution to each well.

10) Incubate the plate Put the plate into a humid box and incubate for 1 h.

11) The test result is detected by a microplate reader at 405nm, the wells with color are positive, and the wells without obvious color change are negative.

Toxic protein test results

The test results are shown in Table 2. In the five transgenic tobacco plants, two kinds of toxic proteins were produced, and non-toxic proteins were synthesized in non-transgenic tobacco plants. It shows that the plant transformation vector p2096871 with BtCryIA and BtCryIIIA constructed by this vector system can carry exogenous genes to transform plants and express in transgenic plants.

Table 2 Ratio of transgenic plants and control poisoning protein to total protein

Claims (10)

1. carry the plant expression vector of single or a plurality of foreign genes, it is characterized in that, comprising: plant conversion carrier p209, cloning vector p6435x and one or more goal gene to be transformed.

2. according to the described plant expression vector of claim 1; It is characterized in that: said plant conversion carrier p209 is a Ti-plasmids; Be derived from carrier pBI121, between the Hind of original pBI121 carrier III and two restriction enzyme sites of EcoR I, introduce a fragment that has Xba I restriction enzyme site; Two restriction endonuclease sites of Bsp120 I and Xba I are arranged on p 209 carriers; P209 has foliage filter screening gene npt II;

Said cloning vector p6435x originates from prokaryotic expression carrier pET28a; The p6435x carrier has CaMV 35S promoter and NOS terminator sequence, and 2 Eam1105 I restriction enzyme sites are arranged between the two; The p6435x carrier carries Not I and Bsp120 I, Spe I and Nhe I restriction enzyme site.

3. according to claim 1 or 2 described plant expression vectors, it is characterized in that: described goal gene is that desinsection toxoprotein gene BtCryIA is or/and desinsection toxoprotein gene BtCryIIIA.

4. one kind makes up claim 1 or 2 said methods of carrying the plant expression vector of single or a plurality of foreign genes, may further comprise the steps:

(1) makes up plant conversion carrier p209 and cloning vector p6435x respectively;

(2) produce 23 ' overhangs that contain T with restriction endonuclease Eam1105 I enzyme cutting clone carrier p6435x; Form the T carrier; Link to each other with the 1st target gene PCR product that has the 3 ' overhang that contains A; Complete and forward is connected in the cloning vector with the 1st goal gene amplified fragments, is built into the ORFs that comprises CaMV35S promotor+goal gene+NOS terminator sequence; Utilize Not I and Nhe I pair to cut the cloning vector that carries the destination gene expression ORFs, reclaim the 1st destination gene expression ORFs fragment, subsequent use;

(3) utilize the two p209 that cut of Bsp120 I and Xba I, reclaim the p209 endonuclease bamhi; The p209 endonuclease bamhi is connected with the 1st the destination gene expression ORFs fragment that step (2) reclaims; The Bsp120 I that has on the new synthetic plant expression vector cuts through enzyme with Spe I site and has Not I and link to each other with the destination gene expression ORFs fragment in NheI site with the 2nd; So constantly repeat, construct the plant expression vector that carries a plurality of goal gene.

5. according to the described method of claim 4, it is characterized in that the construction process of described plant conversion carrier p209 comprises: between the Hind of pBI121 carrier III and two restriction enzyme sites of EcoR I, introduce a fragment that has Xba I restriction enzyme site, promptly get.

6. according to the described method of claim 4; It is characterized in that the construction process of said cloning vector p6435x comprises: cloned the fragment that has CaMV 35S promoter, gfp green fluorescence protein gene and NOS terminator sequence and introduced new restriction enzyme site from carrier pCAMBIA1302 earlier; The fragment that all has Eam1105 I site with two ends replaces the gfp green fluorescence protein gene, forms a new segment, links new segment and obtains cloning vector p6435x on the pET28a.

7. the host cell that contains claim 1 or 2 described plant expression vectors.

8. according to the described host cell of claim 7, it is characterized in that: described host cell is a vegetable cell.

9. claim 1 or 2 described plant expression vectors are transformed into the application in vegetable cell or the tissue with foreign gene.

10. according to the described application of claim 9, it is characterized in that: described foreign gene comprises desinsection toxoprotein gene BtCryIA or/and desinsection toxoprotein gene BtCryIIIA.

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