CN111218469B - Double T-DNA vector pBID-RT Enhanced and construction method and application thereof - Google Patents

Double T-DNA vector pBID-RT Enhanced and construction method and application thereof Download PDF

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CN111218469B
CN111218469B CN202010080268.6A CN202010080268A CN111218469B CN 111218469 B CN111218469 B CN 111218469B CN 202010080268 A CN202010080268 A CN 202010080268A CN 111218469 B CN111218469 B CN 111218469B
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刘芳
吴刚
王盼娣
高鸿飞
武玉花
曾新华
闫晓红
罗军玲
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Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
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Abstract

The invention provides a double T-DNA carrier pBID-RT Enhanced and a construction method and application thereof, relating to the technical field of transgenosis; the vector is based on a plasmid of pCAMBIA3300 and comprises two reverse independent T-DNA regions, wherein the nucleotide sequence of the first T-DNA region is shown as SEQ ID NO.1, and the nucleotide sequence of the second T-DNA region is shown as SEQ ID NO. 2. The use of the vector can reduce the frequency of integration of T-DNA into linked sites, T1Generation and F1The unmarked transgenic plants in the generation account for 76.92 percent; the glufosinate-resistant transgenic rape and the glufosinate-resistant and glyphosate-resistant transgenic rape can be obtained by utilizing the vector. The carrier of the invention can be used for successfully and efficiently producing unmarked transgenic cabbage type rape plants and creating two herbicide-resistant varieties.

Description

Double T-DNA vector pBID-RT Enhanced and construction method and application thereof
Technical Field
The invention belongs to the technical field of genetic transformation, and particularly relates to a double T-DNA vector pBID-RT Enhanced and a construction method and application thereof.
Background
Brassica napus (b.napus L.) is one of the most important oil crops in the world, the main plant for the production of protein-rich products. In order to meet the increasing demand, it is necessary to improve existing varieties using genetic engineering methods and create new and superior brassica napus varieties, which provides another approach to create new varieties with improved traits and desirable characteristics that cannot be obtained by traditional breeding methods. Since the successful modification of brassica napus was first reported, agrobacterium-mediated systems have been used to successfully improve brassica napus. Improvements in many important properties, including herbicide, insect and fungal resistance, and improvements in oil and fat content and proteins, have been introduced into brassica napus by transgenic methods. The safety of transgenic brassica napus faces the same problems and debate as other transgenic crops, one of which is the concern of selectable marker genes.
The genetic transformation of a target gene requires the concomitant transformation with other antibiotic or herbicide-resistant selectable marker genes to aid in the selection of positive transgenic plants carrying the target gene. Once the transgenic plant is regenerated and identified, the selectable marker gene is no longer required. Furthermore, transgenic plants carrying marker genes not only affect public acceptance, but also increase the environmental risk of introduction of marker genes into related weeds and non-transgenic crops. These factors complicate the regulatory process of commercialization of transgenic plants. Furthermore, the marker gene limits this multiple gene stacking process for multiple transformations of different genes. Therefore, the production of marker-free transgenic brassica napus varieties will be beneficial to the development and final commercial utilization of brassica napus.
To date, a number of methods have been developed that enable the removal of selectable marker genes, including co-transformation, site-specific recombination, and homologous recombination. Among these methods, co-transformation is a simple, clean technique, and leaves no residual DNA sequences such as inverted repeats and recombination sites in transgenic plants in which marker genes are deleted, thus being widely used. Co-transformation refers to the simultaneous integration of one T-DNA carrying a marker gene and another T-DNA carrying a gene of interest into the genome, and if both genes are integrated at a site that is not linked, they will subsequently recombine and segregate in the progeny. Co-transformation has applications in many species, including soybean, tobacco, corn, sorghum, rice and durum wheat. However, using current co-transformation techniques, a "double T-DNA" vector is constructed by inserting "right and left box" sequences into a binary vector, a T-DNA; GUS is mostly used as a target gene, Bar is used as a marker gene, and finally, a unmarked plant with GUS and Bar removed is obtained, so that the unmarked plant has no application value.
Disclosure of Invention
In view of the above, the invention aims to provide a double T-DNA vector pBID-RT Enhanced and a construction method and application thereof, which can reduce the frequency of T-DNA integration to linked loci, thereby improving the occurrence probability of unmarked transgenic plants and simultaneously obtaining various transgenic materials with practical application values.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a double T-DNA carrier pBID-RT Enhanced, which takes plasmid of pCAMBIA3300 as a basis and comprises two reverse independent T-DNA regions, wherein the nucleotide sequence of the first T-DNA region is shown as SEQ ID NO.1, and the nucleotide sequence of the second T-DNA region is shown as SEQ ID NO. 2; the distance between the first T-DNA region and the second T-DNA region is greater than 2 kb.
Preferably, the first T-DNA region comprises selectable marker genes including the CP4-EPSPS gene and the GOX-V247 gene; the nucleotide sequence of the CP4-EPSPS gene is shown in SEQ ID NO. 3; the nucleotide sequence of the GOX-V247 gene is shown as SEQ ID NO. 4.
Preferably, the second T-DNA region comprises a target gene Bar, and the nucleotide sequence of the target gene Bar is shown as SEQ ID NO. 5.
The invention also provides a construction method of the vector pBID-RT Enhanced, which comprises the following steps: (1) double enzyme digestion is carried out to remove the multiple cloning site MCS of pCAMBIA3300 plasmid, after the cohesive end is filled up, enzyme linkage is carried out to form a ring, thus obtaining pCAMBIA3300 plasmid without MCS;
(2) inserting a new said multiple cloning site MCS between pBR322 bom and pVS1 oriV site of said MCS-free pCAMBIA3300 plasmid;
(3) and inserting a sequence containing a selective marker gene into the new multi-cloning site MCS to obtain a first T-DNA region, and constructing to obtain the vector pBID-RT Enhanced.
Preferably, the enzyme used in the double enzyme digestion in step (1) comprises EcoRI and PmeI; filling in the cohesive ends with klenow enzyme; the linear molecules with the cohesive ends aligned are ligated into a loop using ligase.
The invention also provides the vector pBID-RT Enhanced or the application of the vector pBID-RT Enhanced constructed by the construction method in rape genetic transformation.
Preferably, the oilseed rape is transformed using an agrobacterium-mediated genetic transformation method.
The invention provides a double T-DNA carrier pBID-RT Enhanced, wherein the distance between two T-DNAs of the carrier pBID-RT Enhanced is more than 2kb, and the T-DNAs are arranged in an inverted manner. The frequency of integration of the T-DNA into a linkage site can be reduced; in the example of the invention, among the 13T 1 and F1 generations, 10 lines were identified with the presence of marker-free transgenic plants, accounting for 76.92%;
according to the invention, Bar is taken as a target gene, and CP4-EPSPS gene and GOX-V247 gene are taken as marker genes, so that not only can a unmarked plant (glufosinate herbicide resistant transgenic material) containing Bar and with Bar removed CP4-EPSPS be obtained, but also a transgenic plant (glufosinate herbicide resistant transgenic material) with Bar and CP4-EPSPS can be obtained, and a transgenic plant (glyphosate herbicide resistant transgenic material) containing CP4-EPSPS and with Bar removed can be obtained.
The invention adopts an agrobacterium-mediated glyphosate screening method to obtain 76 regenerated brassica napus plants in total, and the result shows that 76 independent T plants0Among the plants, 18 plants are Bar+EPSPS+GOX+39 plants are Bar-EPSPS+GOX +, the cotransformation rate of the vector pBID-RTenhanced is 31.58%; and the Bar gene and the CP4-EPSPS gene are inserted into the same transgene at different copy numbersSince the number of inserted copies of the T-DNA carrying CP4-EPSPS in the genome of the plant is low, a high frequency of progeny segregation marker genes can be obtained because of the large T-DNA fragment.
The vector pBID-RT Enhanced of the double T-DNA can be used for successfully and efficiently producing unmarked transgenic brassica napus plants and creating two herbicide-resistant varieties, can be used for tests, crop improvement and commercial requirements, and has potential for transforming a plurality of plant species in practical application, including dicotyledonous plants and monocotyledonous plants.
Drawings
FIG. 1 is a plasmid structure diagram of vector pBID-RT Enhanced;
FIG. 2 is a diagram showing the results of transgene assay, wherein A is PCR assay Bar gene, B is PCR assay CP4-EPSPS gene, C is PCR assay GOX-V247 gene, and in A, B and C, M represents DL2000 DNA marker; lanes 1-17 represent individual T's obtained by pBID-RT Enhanced transformation0Plant growing; p represents an expression vector pBID-RTenhanced; WT denotes wild-type ZS 6; d is the test strip to detect the EPSPS protein expression in the transgenic cabbage type rape plant, wherein P represents a positive control, and the Bar protein is transformed into MS8 cabbage type rape plant; WT denotes wild-type ZS 6; e is the test strip for detecting Bar protein expression in the transgenic cabbage type rape plants, wherein P represents GT73 transgenic cabbage type rape plants containing EPSPS protein, WT represents wild type ZS 6;
FIG. 3 shows Southern hybridization assay Trans-T0Gene copy number in transgenic brassica napus, wherein: southern hybridization detection of transT0Bar (A, C, E) and EPSPS (B, D, F) gene copy number of transgenic cabbage type rape (A, B, D, F), T is identified from pBID-RT Enhanced double T-DNA system1Generation (C, D, E, F) unmarked plants; lanes 1 to 17 in A and B represent T0The generation plant has Bar+EPSPS+The genotype; C. d, E lanes 1 to 12 of F represent T1The plant has Bar+EPSPS genotype, lanes 13-24 represent T1The plant has Bar+EPSPS+The genotype; and M represents a DNA molecular weight marker, WT represents wild-type ZS6, P represents a positive control;
FIG. 4 shows PCR screening of T1 generation unmarked transgenic plants, wherein A is the Bar gene, B is the EPSPS gene, and C is the GOX gene; in the figure, M represents a DL1000 DNA marker; lanes 1 to 17 represent individual T1Plant growing; p represents a positive control; WT denotes wild-type ZS 6.
Detailed Description
The invention provides a double T-DNA carrier pBID-RT Enhanced, which takes plasmid of pCAMBIA3300 as a basis and comprises two reverse independent T-DNA regions, wherein the nucleotide sequence of the first T-DNA region is shown as SEQ ID NO.1, and the nucleotide sequence of the second T-DNA region is shown as SEQ ID NO. 2; the distance between the first T-DNA region and the second T-DNA region is greater than 2 kb.
In the vector pBID-RT Enhanced, the distance between two T-DNAs is more than 2kb, and the T-DNA containing the CP4-EPSPS marker gene is obviously longer than the other T-DNA, so that the copy number of the T-DNA containing the CP4-EPSPS marker gene integrated into a genome is low, and the T-DNA is integrated into the genome1The T-DNA can be more easily separated and discarded; and the T-DNA is arranged in an inverted manner, so that the frequency of integration of the T-DNA into a linked site can be reduced.
The first T-DNA region of the present invention comprises a selection marker gene, preferably comprising CP4-EPSPS gene (EPSPS gene for short) and GOX-V247 gene (GOX gene for short). The CP4-EPSPS gene is preferably derived from Salmonella typhimurium (Salmonella typhimurium) and can encode 5-enolpyruvate phenyloxalate-3-phosphate lipase, so that the plants generate resistance to herbicide glyphosate; the nucleotide sequence of the CP4-EPSPS gene is preferably shown as SEQ ID NO. 3.
The GOX-V247 gene is preferably derived from human leucobacter canescens (Ochrobactrum anthropi) and is a glyphosate degradation gene, and the nucleotide sequence of the GOX-V247 gene is preferably shown as SEQ ID No. 4. After the CP4-EPSPS gene and the GOX-V247 gene are combined, the glyphosate resistance can be improved while the glyphosate residue is reduced, and the negative influence of the glyphosate residue on sensitive pollination and reproductive development of plants is avoided.
The second T-DNA region of the invention preferably comprises a target gene Bar, and the nucleotide sequence of the target gene Bar is preferably shown as SEQ ID NO. 5.
The invention also provides a construction method of the vector pBID-RT Enhanced, which comprises the following steps: (1) double enzyme digestion is carried out to remove the multiple cloning site MCS of pCAMBIA3300 plasmid, after the cohesive end is filled up, enzyme linkage is carried out to form a ring, thus obtaining pCAMBIA3300 plasmid without MCS;
(2) inserting a new said multiple cloning site MCS between pBR322 bom and pVS1 oriV site of said MCS-free pCAMBIA3300 plasmid;
(3) and inserting a sequence containing a selective marker gene into the new multi-cloning site MCS to obtain a first T-DNA region, and constructing to obtain the vector pBID-RT Enhanced.
The invention removes the multi-cloning site MCS of pCAMBIA3300 plasmid by double enzyme digestion, after filling up the cohesive end, the enzyme links into ring, gets pCAMBIA3300 plasmid without MCS. The enzyme used for the double enzyme digestion of the invention preferably comprises EcoRI and PmeI. The present invention preferably makes use of the klenow enzyme to fill in the sticky ends. In the present invention, the linear molecules after the cohesive ends are filled up are preferably ligated to form a loop by using a ligase. The reaction system and procedure of the enzyme of the present invention are not particularly limited, and conventional systems and procedures in the art may be used.
After obtaining the pCAMBIA3300 plasmid without MCS, the invention inserts a new multi-cloning site MCS between pBR322 bom and pVS1 oriV sites of the pCAMBIA3300 plasmid without MCS. The MCS removed by double enzyme digestion of the multiple enzyme digestion sites in the invention is preferably the same as the MCS of the newly inserted multiple cloning sites.
The invention inserts the sequence containing the selection marker gene into the new multi-cloning site MCS to obtain a first T-DNA region, and constructs and obtains the vector pBID-RT Enhanced. In the present invention, it is preferable that a synthetic sequence including the left border, the enhanced CaMV35S promoter, the CP4-EPSPS gene, the NOS terminator, the enhanced CaMV35S promoter, the GOX-V247 gene, the NOS terminator and the right border is inserted into the new MCS in pCAMBIA3300 to form the first T-DNA region of the sequence shown in SEQ ID NO.1 with a full length of 5776 bp. Since the pCAMBIA3300 plasmid itself contains the Bar gene, and the distance between the second T-DNA region containing the Bar gene and the first T-DNA region is more than 2kb, an Enhanced double T-DNA vector pBID-RT Enhanced is formed.
The invention also provides the vector pBID-RT Enhanced or the application of the vector pBID-RT Enhanced constructed by the construction method in rape genetic transformation. In the present invention, it is preferable to transform rape by using Agrobacterium-mediated genetic transformation method, and the genetic transformation method is not particularly limited, but preferred references include Liu F, Xiong XJ, Wu L, Fu DH, Hayward A, Zeng XH, Cao YL, Wu YH, Li YJ, & Wu G (2014) BraLTP1, a lipid transfer protein gene-infected in ecological tissue displacement, cell promotion and flow reduction in Brassica napus. PLOS (IF 3.534)9(10): e 110272.
The following examples are provided to illustrate the double T-DNA vector pBID-RT Enhanced of the present invention and its construction method and application in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
Construction of pBID-RT Enhanced
For plasmid pCAMBIA3300 containing the Bar gene, the MCS (multiple cloning site) was excised with EcoRI and PmeI enzymes, then the cohesive ends were filled in with klenow enzyme, and the linear molecule was self-ligated into loops with ligase. A new MCS (identical to the excised MCS sequence) was then inserted between pBR322 bom and pVS1 oriV sites. The synthetic sequence comprising the left border, enhanced CaMV35S promoter, CP4-EPSPS gene, NOS terminator, enhanced CaMV35S promoter, GOX-V247 gene, NOS terminator and right border was inserted into the new MCS in pCAMBIA3300, at a full length of 5776bp (SEQ ID NO. 1). The expression vector pBID-RT Enhanced (the plasmid is shown in figure 1) is constructed.
Reference is made to the procedure of Liu et al for genetic transformation of pBID-RT Enhanced twin T-DNA vectors in oilseed rape (Liu F, Xiong XJ, Wu L, Fu DH, Hayward A, Zeng XH, Cao YL, Wu YH, Li YJ,&wu G (2014) BraLTP1, a lipid transfer protein gene involved in encapsulated in aqueous wall displacement, cell promotion and flow reduction in Brassica napus. PLOS ONE (IF 3.534)9(10) e 110272): the pBID-RT Enhanced plasmid is introduced into Agrobacterium tumefaciens GV3101 by electrotransformation, and LB agar plate is placed at 37 deg.CThe plates were cultured, the plates were supplemented with appropriate concentrations of antibiotics (gentamicin 50mg/L, rifampicin 50mg/L and kanamycin 50mg/L), positive clones were selected and PCR verified. The cabbage type rape is transformed by a single positive agrobacterium colony, and the receptor adopts Zhongshuang No. 6. Soaking seeds of Zhongshuang No. 6 in 75% ethanol for 1min, soaking in 1.5% mercuric chloride for 10-15 min, germinating in the dark for 5-6 d, cutting the yellowed hypocotyl into small segments of 7mm, mixing with 50mL of agrobacterium (the OD value of the agrobacterium is about 0.3) in a liquid DM culture medium (MS +30g/L of sucrose +100 mu M of acetosyringone, pH 5.8), and soaking for 0.5 h. Then transferring the hypocotyls with air-dried surfaces to a co-culture medium (MS +30g/L sucrose +18g/L mannitol +1 mg/L2, 4-D +0.3mg/L kinetin +100 mu M acetosyringone +8.5g agarose, pH value 5.8) for 2D, and then to a selection medium (MS +30g/L sucrose +18g/L mannitol +1 mg/L2, 4-D +0.3mg/L kinetin +20 mg/LAgNO)3+8.5g/L agarose +250mg/L carbenicillin disodium, pH 5.8). After 3 weeks, hypocotyl calli were transferred to regeneration medium (MS +10g/L glucose +0.25g/L xylose +0.6 g/L2- (N-morpholino) ethanesulfonic acid hydrate +2mg/L zeatin +0.1mg/L indole-3-acetic acid +8.5g/L agarose +80mg/L glyphosate +250mg/L carbenicillin disodium, pH 5.8) for 2 weeks. Transferring the hypocotyl into a new regeneration culture medium every 2 weeks for 3-4 regeneration cycles in total, transferring the hypocotyl into a rooting culture medium (MS +10g/L sucrose +10g/L agar, pH 5.8) after seedling formation for rooting (about 3 weeks). Transplanting the transformed rooted plant into a flowerpot for growing.
In the transformation process, as the hypocotyl is sensitive to glyphosate, the glyphosate is added into the culture medium only in the regeneration period, so that the transformed cells are more competitive than the untransformed cells. The regenerated shoots were screened on selection medium containing 80mg/L glyphosate.
Adopting agrobacterium-mediated glyphosate screening method to obtain 76 regenerated cabbage type rape plants. The presence of the Bar, EPSPS and GOX genes in regenerated Brassica napus plants was detected by PCR and the expression of the Bar and EPSPS proteins was detected by a test strip, as shown in Table 1 and FIG. 2, 18 out of 76 independent T0 plants were Bar+EPSPS+GOX+39 plants are Bar-EPSPS+GOX+,pBID-RTThe Enhanced vector cotransformation rate is 31.58%.
TABLE 1 Co-transformation frequency of transformants of the pBID RT Enhanced double T-DNA vector System T0
Figure GDA0002443939430000071
Figure GDA0002443939430000081
Southern hybridization of the double T-DNA system of pBID-RT Enhanced: DNA digested by Hind III is hybridized with Bar probe, the hybridization product is 262bp, and the probe primers are respectively as follows:
Bar-F(SEQ ID NO.6):GAAGGCACGCAACGCCTACGA;
Bar-R(SEQ ID NO.7):TGGCATGACGTGGGTTTCTGG;
the DNA cut by EcoRI is hybridized with an EPSPS probe, the hybridization product is 579bp, and the probe primers are respectively as follows:
EPSPS-F(SEQ ID NO.8):GTGGGGATTGAAGAAGAGTGG;
EPSPS-R(SEQ ID NO.9):CCAAAGACTCCAACGCCAAT。
identification of Co-Integrated T by southern blot0Transgenic plants to detect the integration status and copy number of the Bar and EPSPS genes (FIG. 3). The Blot results showed that the Bar gene was integrated in a single copy in 4 out of 17 transgenic plants and in 3 copies in most plants (A in FIG. 3). However, in 17 Brassica napus plants, there were 9 single copies of the EPSPS gene (B in FIG. 3). The results show that the Bar gene and the EPSPS gene are inserted into the genome of the same transgenic plant at different copy numbers. All T0 positive transgenic plants have normal fertility, and no sterile transgenic plants are found.
Randomly select 8T0 Bar+EPSPS+GOX+Plants, either selfed or crossed with the transformation receptor ZS6, were grown to maturity in the greenhouse. To obtain marker-free transgenic Brassica napus plants (FIG. 4, Table 2), 13T were tested1Is substituted or F1Of breeding linesPlants were analyzed. In the seedling stage, each T1 generation plant is tested for the presence of Bar, EPSPS and GOX genes by a PCR method. 4 transgenic plants were detected in the T1 generation: bar+EPSPS+GOX+、Bar-EPSPS+GOX+、Bar+EPSPS-GOX-And Bar-EPSPS-GOX-. Majority of T1The plant is Bar+EPSPS+GOX+And some are unlabeled bars+EPSPS-GOX-A plant. 13T1Generation and F137 unmarked plants are identified in 10 lines in the generation transgenic lines, the frequency of the unmarked gene lines is 76.92%, and the efficiency of the marker gene separation among different lines is 4.35-52.94%. At T110T of generation unmarked offspring0In the line, the separation efficiency of the marker gene was 21.06% on average.
TABLE 2T1Generation strain EPSPS and Bar genotyping detection
Figure GDA0002443939430000091
Figure GDA0002443939430000101
Selection of 12 unmarked Brassica napus Bar+EPSPS-GOX-Southern hybridization analysis was performed (C and D in FIG. 3). Since EPSPS and GOX are closely linked sites in the same T-DNA, identification of EPSPS represents both genes. All 12T1 Bar+EPSPS-GOX-All showed 1-2 bars, no EPSPS bands, and showed four different band patterns, representing four independent T0A transgenic line. Corresponding four T0Lines all show an EPSPS band ( lines 2, 6, 8 and 15 of B in FIG. 3), representing four T0The EPSPS gene is present in a single copy in the strain.
By using the scheme of the inventionObtaining Bar+EPSPS-GOX-Bar is obtained in addition to the plants (glufosinate-resistant)+EPSPS+GOX+Transgenic brassica napus (glufosinate and glyphosate resistant) is resistant to both herbicides. Therefore, 12 bars were selected+EPSPS+GOX+T of genotype1Or F1Progeny, verified by Southern hybridization with untransformed wild-type ZS6 as control (E and F in fig. 3). 12T1Or F1 Bar+EPSPS+GOX+Progeny showed 1-4 Bar bands and 1-2 EPSPS bands, while all progeny showed 6 different banding patterns, representing 6 independent transgenic T0Lines (E and F in FIG. 3).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> institute of oil crop of academy of agricultural sciences of China
<120> double T-DNA vector pBID-RT Enhanced and construction method and application thereof
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<170> SIPOSequenceListing 1.0
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atcccgggcg cgccagggag ctccccgatc tagtaacata gatgacaccg cgcgcgataa 180
tttatcctag tttgcgcgct atattttgtt ttctatcgcg tattaaatgt ataattgcgg 240
gactctaatc ataaaaaccc atctcataaa taacgtcatg cattacatgt taattattac 300
atgcttaacg taattcaaca gaaattatat gataatcatc gcaagaccgg caacaggatt 360
caatcttaag aaactttatt gccaaatgtt tgaacgatcg gatctgaggc tgcagttact 420
agttccggat ctttaggatg caggaccagt ttgcttggac ttaccaatac cgaaacggtt 480
tggtgcgaat ggagagatgt cgatagaggt cttctcacct gcgaggagct cagaaacgag 540
ggttgcggtc attggagcac cagtcatacc gaggtgaccg tgaccgaaag cgtagataac 600
gtctggagta cgggtagcac gaccaatcac tggaagggaa tccgggatgc ttggacggaa 660
acccatccac ttggagtaac gttcttcaga actggcagga gcgagagctg gaagcaactt 720
acgagcgcga gtgtagagaa cgtgagcacg cttccagtta ggagcagcag tgagaccagc 780
gaactcaacg gttccagcaa cacgaagacc catctccata ggagtagcga tgaactttcc 840
agaagcatcg gtagttggaa tacgtggagc agcttctggg ttggcgatca cgatgtggta 900
tccacgttcg gtatccaatg ggatgtcatc accaagggag ttagcaagag acttggagtg 960
tgcaccagct gcaacaacag ctgcatcaac agcaagaaca ccgttggtgg tggtgatacc 1020
cttgagagca cgaccttcag tctcgaatcc gataacacga gcagacacga actctccacc 1080
gttagcgatg aaacgacgaa acaagagagt cacgagacct tgtgggttga tggtgtgacc 1140
gttctcttcg ataaggattc ccttggtaaa ggcgtgagac aagttaggat cgaaatcacg 1200
caatgcatca gcgctgagga tttgagtacg aacaccgttg agacgacgaa gttcccaacc 1260
tccacggtcc ctggcgaagt ctgcttctcc acggtacacg gtaaggtgac cttcgtgacg 1320
gataaggtgg ctagcatcag cctcctcagc caaggacttg atcaaaggca cagtggactt 1380
gatgaggtta cggagtgcct tagcttgctc cttcaccttg tttggtcttc cagcaagcaa 1440
gaaacgaatc aaccaaggca tgatggttgg aaagtagccg aaacggatgg acaatggacc 1500
cattgggtca agaagccact ttggaacgct agtcaagttt cctggcatgg acattggaac 1560
aacggaggaa ccgttgaagc aaccagcgtt accgaaagag gcaccttcac ctggtgggtt 1620
tggatcaatc aaggtaacct tgaatccacg acgttgaagc atcaaagcag tgcaaacacc 1680
aacgattcca gctccagcga taccaacctt cttgtggttc tcagccatgg cctgcatgca 1740
gttgacgcga ccaccggaat cggtaaggtc aggaaggtaa gagagagtct caaacttctt 1800
ctttccaatc ggaggccaca cctgcatgca gttaactctt ccgccgttgc ttgtgatgga 1860
agtaatgtcg ttgttagcct tgcgggtggc tgggaaggca gcggaggact taagtccgtt 1920
gaaaggagcg accatagtgg cctgagccgg agaggcaacc atagtagcgg aagagagcat 1980
agaggaagcc atggtggatc tgaggctgca gttactagtt ccggatctga gatagatttg 2040
tagagagaga ctggtgattt cagcgtgtcc tctccaaatg aaatgaactt ccttatatag 2100
aggaaggtct tgcgaaggat agtgggattg tgcgtcatcc cttacgtcag tggagatatc 2160
acatcaatcc acttgctttg aagacgtggt tggaacgtct tctttttcca cgatgctcct 2220
cgtgggtggg ggtccatctt tgggaccact gtcggcagag gcatcttgaa cgatagcctt 2280
tcctttatcg caatgatggc atttgtaggt gccaccttcc ttttctactg tccttttgat 2340
gaagtgacag atagctgggc aatggaatcc gaggaggttt cccgatatta ccctttgttg 2400
aaaagtctca atagcccttt ggtcttctga gactgtatct ttgatattct tggagtagac 2460
gagagtgtcg tgctccacca tgttatcaca tcaatccact tgctttgaag acgtggttgg 2520
aacgtcttct ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc 2580
ggcagaggca tcttgaacga tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc 2640
accttccttt tctactgtcc ttttgatgaa gtgacagata gctgggcaat ggaatccgag 2700
gaggtttccc gatattaccc tttgttgaaa agtctcaata gccctttggt cttctgagac 2760
tgtatctttg atattcttgg agtagacgag agtgtcgtgc tccaccatgt tggcaagctg 2820
ctctagccaa tggatctgag gctgcagtta ctagttccgg atctcccgat ctagtaacat 2880
agatgacacc gcgcgcgata atttatccta gtttgcgcgc tatattttgt tttctatcgc 2940
gtattaaatg tataattgcg ggactctaat cataaaaacc catctcataa ataacgtcat 3000
gcattacatg ttaattatta catgcttaac gtaattcaac agaaattata tgataatcat 3060
cgcaagaccg gcaacaggat tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc 3120
ggatctgagg ctgcagttac tagttccgga tcttcaagca gccttagtgt cggagagttc 3180
gatcttagct ccaagaccag ccatcaaatc catgaactct gggaagctag tagcgatcat 3240
agtagcatca tcaacagtaa cagggttttc agaaacgaga cccataacga ggaagctcat 3300
agcgatacgg tgatcgaggt gggtagcgac agctgctcca gaagcgttac cgagaccctt 3360
accgtcagga cgaccacgca cgacgagaga agtctcacct tcatcgcaat caacaccgtt 3420
gagcttgaga ccgtttgcga cagcagaaag acggtcgctt tccttaacac ggagttcttc 3480
caaaccgttc ataacggtag caccttcagc gaatgcagct gcaacagcga gaattggata 3540
ctcgtcgatc atagaaggag cacggtcttc tggaacagta acacccttca aagtagaaga 3600
acgaacacgc aagtcagcca cgtcttctcc accagcaaga cgtgggttga tcacttcgat 3660
gtcggcaccc atttcctgca gagtcaagat gagaccagta cgggttgggt tcatcaaaac 3720
gttaaggatg gtgacgtcgg aacctggaac aagcaaggca gcaaccaatg ggaaagcagt 3780
agaggatgga tcacctggaa catcaatcac ttgaccggtg agcttaccac gaccttcaag 3840
acggatggta cgcacaccgt cagcatcagt ctcaacggta aggttagcac caaaaccttg 3900
aagcatcttt tcagtgtggt cacgagtcat gattggctcg ataacagtgg tgatacctgg 3960
ggtgttgaga ccagcaagca gaacagcgga cttcacttga gcggaagcca taggtaccct 4020
gtaggtgatt ggcgttggag tctttggtcc acgcaaggta actggaagac gatcaccgtc 4080
ttcagacttc acctgcacac ccatttcgcg aagtgggttc aacacacgac ccattggacg 4140
cttagtgaga gaagcgtcac caatgaaagt gctatcgaaa tcgtaaacac caacaagacc 4200
catagtcaaa cggcaaccag ttgcagcgtt accgaaatcg agaggagcct caggagcaag 4260
gagtccaccg ttaccaacac catcaatgat ccaagtatca ccttccttac ggattctggc 4320
acccatagct tgcatagcct taccagtgtt gataacatct tcaccttcca aaagaccggt 4380
gatacgagtt tcaccgctag cgagacctcc aaacatgaag gacctgtggg agatagactt 4440
gtcacctgga atacggacgg ttccagaaag accagaggac ttacgagcag ttgctggacg 4500
gctgcttgca ccgtgaagca tgcacgccgt ggaaacagaa gacatgacct taagaggacg 4560
aagctcagag ccaattaacg tcatcccact cttcttcaat ccccacgacg acgaaatcgg 4620
ataagctcgt ggatgctgct gcgtcttcag agaaaccgat aagggagatt tgcgttgact 4680
ggatttcgag agattggaga taagagatgg gttctgcaca ccattgcaga ttctgctaac 4740
ttgcgccatg gtggatctga gatagatttg tagagagaga ctggtgattt cagcgtgtcc 4800
tctccaaatg aaatgaactt ccttatatag aggaaggtct tgcgaaggat agtgggattg 4860
tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg aagacgtggt 4920
tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt tgggaccact 4980
gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc atttgtaggt 5040
gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc aatggaatcc 5100
gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt ggtcttctga 5160
gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca tgttatcaca 5220
tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga tgctcctcgt 5280
gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaacga tagcctttcc 5340
tttatcgcaa tgatggcatt tgtaggtgcc accttccttt tctactgtcc ttttgatgaa 5400
gtgacagata gctgggcaat ggaatccgag gaggtttccc gatattaccc tttgttgaaa 5460
agtctcaata gccctttggt cttctgagac tgtatctttg atattcttgg agtagacgag 5520
agtgtcgtgc tccaccatgt tggcaagctg ctctagccaa ttctagaggg ggatccggaa 5580
ctagtaactg cagcctcaga tctcggtgaa ttctgtagat atccatcaca ctggcggccg 5640
cagctcgagc atgtcgactc tcgtgggccc aattcgccct atagtgagtc gtattacaat 5700
tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgaatt tgtttacacc 5760
acaatatatc ctgcca 5776
<210> 2
<211> 1896
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 60
gacgttttta atgtactgaa ttaacgccga attaattcgg gggatctgga ttttagtact 120
ggattttggt tttaggaatt agaaatttta ttgatagaag tattttacaa atacaaatac 180
atactaaggg tttcttatat gctcaacaca tgagcgaaac cctataggaa ccctaattcc 240
cttatctggg aactactcac acattattat ggagaaactc gagggtcatc agatctcggt 300
gacgggcagg accggacggg gcggtaccgg caggctgaag tccagctgcc agaaacccac 360
gtcatgccag ttcccgtgct tgaagccggc cgcccgcagc atgccgcggg gggcatatcc 420
gagcgcctcg tgcatgcgca cgctcgggtc gttgggcagc ccgatgacag cgaccacgct 480
cttgaagccc tgtgcctcca gggacttcag caggtgggtg tagagcgtgg agcccagtcc 540
cgtccgctgg tggcgggggg agacgtacac ggtcgactcg gccgtccagt cgtaggcgtt 600
gcgtgccttc cagggacccg cgtaggcgat gccggcgacc tcgccgtcca cctcggcgac 660
gagccaggga tagcgctccc gcagacggac gaggtcgtcc gtccactcct gcggttcctg 720
cggctcggta cggaagttga ccgtgcttgt ctcgatgtag tggttgacga tggtgcagac 780
cgccggcatg tccgcctcgg tggcacggcg gatgtcggcc gggcgtcgtt ctgggctcat 840
atctcattgc cccccgggat ctgcgaaagc tcgagagaga tagatttgta gagagagact 900
ggtgatttca gcgtgtcctc tccaaatgaa atgaacttcc ttatatagag gaaggtcttg 960
cgaaggatag tgggattgtg cgtcatccct tacgtcagtg gagatatcac atcaatccac 1020
ttgctttgaa gacgtggttg gaacgtcttc tttttccacg atgctcctcg tgggtggggg 1080
tccatctttg ggaccactgt cggcagaggc atcttgaacg atagcctttc ctttatcgca 1140
atgatggcat ttgtaggtgc caccttcctt ttctactgtc cttttgatga agtgacagat 1200
agctgggcaa tggaatccga ggaggtttcc cgatattacc ctttgttgaa aagtctcaat 1260
agccctttgg tcttctgaga ctgtatcttt gatattcttg gagtagacga gagtgtcgtg 1320
ctccaccatg ttatcacatc aatccacttg ctttgaagac gtggttggaa cgtcttcttt 1380
ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg cagaggcatc 1440
ttgaacgata gcctttcctt tatcgcaatg atggcatttg taggtgccac cttccttttc 1500
tactgtcctt ttgatgaagt gacagatagc tgggcaatgg aatccgagga ggtttcccga 1560
tattaccctt tgttgaaaag tctcaatagc cctttggtct tctgagactg tatctttgat 1620
attcttggag tagacgagag tgtcgtgctc caccatgttg gcaagctgct ctagccaaac 1680
ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 1740
ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgct agagcagctt 1800
gagcttggat cagattgtcg tttcccgcct tcagtttaaa ctatcagtgt ttgacaggat 1860
atattggcgg gtaaacctaa gagaaaagag cgttta 1896
<210> 3
<211> 1596
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tcaagcagcc ttagtgtcgg agagttcgat cttagctcca agaccagcca tcaaatccat 60
gaactctggg aagctagtag cgatcatagt agcatcatca acagtaacag ggttttcaga 120
aacgagaccc ataacgagga agctcatagc gatacggtga tcgaggtggg tagcgacagc 180
tgctccagaa gcgttaccga gacccttacc gtcaggacga ccacgcacga cgagagaagt 240
ctcaccttca tcgcaatcaa caccgttgag cttgagaccg tttgcgacag cagaaagacg 300
gtcgctttcc ttaacacgga gttcttccaa accgttcata acggtagcac cttcagcgaa 360
tgcagctgca acagcgagaa ttggatactc gtcgatcata gaaggagcac ggtcttctgg 420
aacagtaaca cccttcaaag tagaagaacg aacacgcaag tcagccacgt cttctccacc 480
agcaagacgt gggttgatca cttcgatgtc ggcacccatt tcctgcagag tcaagatgag 540
accagtacgg gttgggttca tcaaaacgtt aaggatggtg acgtcggaac ctggaacaag 600
caaggcagca accaatggga aagcagtaga ggatggatca cctggaacat caatcacttg 660
accggtgagc ttaccacgac cttcaagacg gatggtacgc acaccgtcag catcagtctc 720
aacggtaagg ttagcaccaa aaccttgaag catcttttca gtgtggtcac gagtcatgat 780
tggctcgata acagtggtga tacctggggt gttgagacca gcaagcagaa cagcggactt 840
cacttgagcg gaagccatag gtaccctgta ggtgattggc gttggagtct ttggtccacg 900
caaggtaact ggaagacgat caccgtcttc agacttcacc tgcacaccca tttcgcgaag 960
tgggttcaac acacgaccca ttggacgctt agtgagagaa gcgtcaccaa tgaaagtgct 1020
atcgaaatcg taaacaccaa caagacccat agtcaaacgg caaccagttg cagcgttacc 1080
gaaatcgaga ggagcctcag gagcaaggag tccaccgtta ccaacaccat caatgatcca 1140
agtatcacct tccttacgga ttctggcacc catagcttgc atagccttac cagtgttgat 1200
aacatcttca ccttccaaaa gaccggtgat acgagtttca ccgctagcga gacctccaaa 1260
catgaaggac ctgtgggaga tagacttgtc acctggaata cggacggttc cagaaagacc 1320
agaggactta cgagcagttg ctggacggct gcttgcaccg tgaagcatgc acgccgtgga 1380
aacagaagac atgaccttaa gaggacgaag ctcagagcca attaacgtca tcccactctt 1440
cttcaatccc cacgacgacg aaatcggata agctcgtgga tgctgctgcg tcttcagaga 1500
aaccgataag ggagatttgc gttgactgga tttcgagaga ttggagataa gagatgggtt 1560
ctgcacacca ttgcagattc tgctaacttg cgccat 1596
<210> 4
<211> 1560
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttaggatgca ggaccagttt gcttggactt accaataccg aaacggtttg gtgcgaatgg 60
agagatgtcg atagaggtct tctcacctgc gaggagctca gaaacgaggg ttgcggtcat 120
tggagcacca gtcataccga ggtgaccgtg accgaaagcg tagataacgt ctggagtacg 180
ggtagcacga ccaatcactg gaagggaatc cgggatgctt ggacggaaac ccatccactt 240
ggagtaacgt tcttcagaac tggcaggagc gagagctgga agcaacttac gagcgcgagt 300
gtagagaacg tgagcacgct tccagttagg agcagcagtg agaccagcga actcaacggt 360
tccagcaaca cgaagaccca tctccatagg agtagcgatg aactttccag aagcatcggt 420
agttggaata cgtggagcag cttctgggtt ggcgatcacg atgtggtatc cacgttcggt 480
atccaatggg atgtcatcac caagggagtt agcaagagac ttggagtgtg caccagctgc 540
aacaacagct gcatcaacag caagaacacc gttggtggtg gtgataccct tgagagcacg 600
accttcagtc tcgaatccga taacacgagc agacacgaac tctccaccgt tagcgatgaa 660
acgacgaaac aagagagtca cgagaccttg tgggttgatg gtgtgaccgt tctcttcgat 720
aaggattccc ttggtaaagg cgtgagacaa gttaggatcg aaatcacgca atgcatcagc 780
gctgaggatt tgagtacgaa caccgttgag acgacgaagt tcccaacctc cacggtccct 840
ggcgaagtct gcttctccac ggtacacggt aaggtgacct tcgtgacgga taaggtggct 900
agcatcagcc tcctcagcca aggacttgat caaaggcaca gtggacttga tgaggttacg 960
gagtgcctta gcttgctcct tcaccttgtt tggtcttcca gcaagcaaga aacgaatcaa 1020
ccaaggcatg atggttggaa agtagccgaa acggatggac aatggaccca ttgggtcaag 1080
aagccacttt ggaacgctag tcaagtttcc tggcatggac attggaacaa cggaggaacc 1140
gttgaagcaa ccagcgttac cgaaagaggc accttcacct ggtgggtttg gatcaatcaa 1200
ggtaaccttg aatccacgac gttgaagcat caaagcagtg caaacaccaa cgattccagc 1260
tccagcgata ccaaccttct tgtggttctc agccatggcc tgcatgcagt tgacgcgacc 1320
accggaatcg gtaaggtcag gaaggtaaga gagagtctca aacttcttct ttccaatcgg 1380
aggccacacc tgcatgcagt taactcttcc gccgttgctt gtgatggaag taatgtcgtt 1440
gttagccttg cgggtggctg ggaaggcagc ggaggactta agtccgttga aaggagcgac 1500
catagtggcc tgagccggag aggcaaccat agtagcggaa gagagcatag aggaagccat 1560
<210> 5
<211> 552
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tcagatctcg gtgacgggca ggaccggacg gggcggtacc ggcaggctga agtccagctg 60
ccagaaaccc acgtcatgcc agttcccgtg cttgaagccg gccgcccgca gcatgccgcg 120
gggggcatat ccgagcgcct cgtgcatgcg cacgctcggg tcgttgggca gcccgatgac 180
agcgaccacg ctcttgaagc cctgtgcctc cagggacttc agcaggtggg tgtagagcgt 240
ggagcccagt cccgtccgct ggtggcgggg ggagacgtac acggtcgact cggccgtcca 300
gtcgtaggcg ttgcgtgcct tccagggacc cgcgtaggcg atgccggcga cctcgccgtc 360
cacctcggcg acgagccagg gatagcgctc ccgcagacgg acgaggtcgt ccgtccactc 420
ctgcggttcc tgcggctcgg tacggaagtt gaccgtgctt gtctcgatgt agtggttgac 480
gatggtgcag accgccggca tgtccgcctc ggtggcacgg cggatgtcgg ccgggcgtcg 540
ttctgggctc at 552
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gaaggcacgc aacgcctacg a 21
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tggcatgacg tgggtttctg g 21
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gtggggattg aagaagagtg g 21
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ccaaagactc caacgccaat 20

Claims (2)

1. The application of the double T-DNA carrier pBID-RT Enhanced in the cabbage rape genetic transformation is characterized in that the carrier pBID-RT Enhanced takes a plasmid of pCAMBIA3300 as a base and comprises two reverse independent T-DNA regions, the nucleotide sequence of the first T-DNA region is shown as SEQ ID NO.1, and the nucleotide sequence of the second T-DNA region is shown as SEQ ID NO. 2; the distance between the first T-DNA region and the second T-DNA region is greater than 2 kb.
2. The use according to claim 1, wherein the method of agrobacterium-mediated genetic transformation is used to transform oilseed rape.
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