CN113817765B - Agrobacterium homologous recombination system and application thereof - Google Patents
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Abstract
The invention discloses an agrobacterium homologous recombination system, which consists of a series of agrobacterium homologous recombination system expression plasmids which are respectively named pBBR1-kan-P tet -ETh1h2h3h4_agrob6, the nucleotide sequence of which is shown in SEQ ID No. 1; pBBR1-kan-P tet -ETh1h2h3p3_ rhi597, the nucleotide sequence of which is shown in SEQ ID No. 2; pBBR1-kan-P tet -et_ rhi145, the nucleotide sequence of which is shown in SEQ ID No. 3; pBBR1-kan-P tet -eth_ rhi483, the nucleotide sequence of which is shown in SEQ ID No. 4. The invention also discloses application of the recombination system in genome DNA genetic modification by agrobacterium-mediated homologous recombination of short homology arms and application of the recombination system in agrobacterium-mediated gene knockout. The agrobacterium homologous recombination system can greatly promote genome modification of agrobacterium, so that genetic modification of the agrobacterium becomes simple and quick, and has great application prospects in the aspects of constructing agrobacterium strains with high transformation efficiency and host range and the field of plant genetic engineering.
Description
Technical Field
The invention relates to a homologous recombination system in gram-negative bacteria and construction and application thereof, in particular to a homologous recombination system of agrobacterium and construction and application thereof, belonging to the field of microbial genetic engineering.
Background
RecET and Red alpha beta gamma system mediated homologous recombination process is called Red/ET homologous recombination engineering. The Red/ET homologous recombination engineering can accurately insert, knock out or replace target genes at any positions of the escherichia coli genome, and can realize the simplification of the escherichia coli genome by being used together with systems such as site-specific recombinase (Cre/loxP, flp/FRT) or CRISPR-Cas. Successful application of Red/ET recombinant engineering has prompted the development of the whole genetic engineering technology, wherein the RecE and RecT homologous recombinant proteins are derived from the Rac prophage, and the Red alpha, red beta and Red gamma homologous recombinant proteins are derived from Lambda phage.
The Red/ET homologous recombination engineering realizes low-consumption, high-efficiency and accurate genome editing, provides an effective means for genetic modification, wherein RecE and Red alpha have 5'-3' exonuclease activity, recT and Red beta are single-stranded DNA annealing proteins, and Red gamma can inhibit the exonuclease activity of RecBCD in escherichia coli and protect exogenous linear DNA molecules from degradation, thereby improving homologous recombination efficiency.
Because phage recombinases have certain host specificity, the Red/ET system has very low recombination efficiency or no recombination function in bacteria with distant relatedness, so that it is necessary to mine homologous recombination systems applicable to different strains themselves. Recombinant systems constructed by using phage recombinant function operons have been reported, such as Mycobacterium smegmatis Mycobacterium smegmatis, pseudomonas syringae Pseudomonas syringae pv.Syringae, lactobacillus reuteri Lactobacillus reuteri, photorhabdus, xenorhabdus, burkholderia Burkholderiales strain, mycobacterium tuberculosis Mycobacterium tuberculosis, bacillus subtilis Bacillus subtilis, and the like.
Agrobacterium (Agrobacterium) belongs to the Rhizobiaceae family (Rhizobiaceae), and is classified into Agrobacterium tumefaciens capable of infecting plants (Agrobacterium tumefaciens), agrobacterium radiobacter without infectivity (Agrobacterium radiobacter), and Agrobacterium rhizogenes that can cause plants to develop hairy roots (Rhizobium rhizogenes) depending on whether it is invasive or not and the host range and infection symptoms of the Agrobacterium. The root cancer and agrobacterium rhizogenes can transfer and integrate a section of DNA (transferred DNA) carried by plasmids of the root cancer and agrobacterium rhizogenes into a host plant genome, and genes in a T-DNA region can be replaced by any DNA sequence, so that the gene has extremely important significance in aspects of gene transfer research, plant gene function research, crop transgenic breeding and the like. There are many urgent problems to be solved in improving the transformation efficiency of agrobacterium and expanding the host range thereof, so it is very necessary to build a set of efficient and simple homologous recombination system in agrobacterium. Through searching, research on the agrobacterium self-homologous recombination system and construction and application thereof is not reported in literature.
Disclosure of Invention
Aiming at the defect of lack of a high-efficiency and simple genetic operating system in agrobacterium, the invention aims to provide a homologous recombination system of agrobacterium and construction and application thereof.
The technical scheme of the invention is as follows: digging agrobacterium phage homologous recombination protein, constructing agrobacterium homologous recombination system expression plasmid, optimizing recombination conditions of the homologous recombination system expression plasmid in agrobacterium tumefaciens, selecting optimal recombination working conditions, and realizing application of the agrobacterium homologous recombination system in genome DNA genetic modification by agrobacterium mediated homologous recombination of short homology arms or gene knockout in agrobacterium.
The agrobacterium homologous recombination system of the invention is composed of a series of agrobacterium homologous recombination system expression plasmids, including pBBR1 replication origin, kanamycin resistance gene, tetracycline inducible promoter and agrobacterium-derived homologous recombination operon (shown in figure 1); the method is characterized in that: the expression plasmids of the agrobacterium homologous recombination system are respectively named pBBR1-kan-P tet -ETh1h2h3h4_agrob6, the nucleotide sequence of which is shown in SEQ ID No. 1; pBBR1-kan-P tet -ETh1h2h3p3_ rhi597, the nucleotide sequence of which is shown in SEQ ID No. 2; pBBR1-kan-P tet -et_ rhi145, the nucleotide sequence of which is shown in SEQ ID No. 3; pBBR1-kan-P tet -eth_ rhi483, the nucleotide sequence of which is shown as SEQ ID No.4 (shown in fig. 2); pBBR1-kan-P tet -redγ-ETh1h2h3h4_agroB6；pBBR1-kan-P tet -redγ-ETh1h2h3P3_rhi597；pBBR1-kan-P tet -redγ-ET_rhi145；pBBR1-kan-P tet -redγ-ETh_rhi483；pBBR1-kan-P tet -pluγ-ETh1h2h3h4_agroB6；pBBR1-kan-P tet -pluγ-ETh1h2h3P3_rhi597；pBBR1-kan-P tet -pluγ-ET_rhi145；pBBR1-kan-P tet -pluγ -eth_ rhi483; homologous recombination enzyme operon ETh1h2h3h4 related to agrobacterium homologous recombination system expression plasmid AGROB6 、ETh1h2h3P3 RHI597 、ET RHI145 And ETh RHI483 Are derived from Agrobacterium Agrobacterium tumefaciens str.B6, rhizobium leguminosarum bv.trifoliWSM 597, rhizobium sp.LC145 and Rhizobium sp.Root483D2, respectively.
The construction method of the agrobacterium homologous recombination system expression plasmid comprises the following steps:
(1)pBBR1-kan-P tet the initial plasmid, ecoRI and NheI double enzyme cutting, gel recovery order linear fragment pBBR1-P tet Kan, about 5kb in length;
(2) The gel obtained in the step (1) is used for recovering the linear fragment pBBR1-P tet The kan is respectively co-transformed with a synthesized recombinase fragment with a vector homology arm, namely, an ETh1h2h3h4_agroB6HA, an ETh1h2h3hP3_ rhi597HA, an ET_ rhi HA and an ETh_ rhi483HA, into E.coli GB05-dir for line recombination;
(3) Performing enzyme digestion verification on the recombinant obtained in the step (2) to obtain a series of expression plasmids of the agrobacterium homologous recombination system, wherein the series of expression plasmids are named pBBR1-kan-P respectively tet -ETh1h2h3h4_agrob6, the nucleotide sequence of which is shown in SEQ ID No. 1; pBBR1-kan-P tet -ETh1h2h3p3_ rhi597, the nucleotide sequence of which is shown in SEQ ID No. 2; pBBR1-kan-P tet -et_ rhi145, the nucleotide sequence of which is shown in SEQ ID No. 3; pBBR1-kan-P tet -eth_ rhi483, the nucleotide sequence of which is shown in SEQ ID No. 4.
The series of expression plasmids pBBR1-kan-P in the above recombination system tet -redγ-ETh1h2h3h4_agroB6、pBBR1-kan-P tet -redγ-ETh1h2h3P3_rhi597、pBBR1-kan-P tet -redγ-ET_rhi145、pBBR1-kan-P tet -redγ-ETh_rhi483、pBBR1-kan-P tet -pluγ-ETh1h2h3h4_agroB6、pBBR1-kan-P tet -pluγ-ETh1h2h3P3_rhi597、pBBR1-kan-P tet -pluγ -ET_ rhi145 or pBBR1-kan-P tet The construction method of the-pluγ -eth_ rhi483 is as follows:
(1) First, pBBR1-kan-P tet -ETh1h2h3h4_agroB6、pBBR1-kan-P tet -ETh1h2h3P3_rhi597、pBBR1-kan-P tet -ET_ rhi145 and pBBR1-kan-P tet -eth_ rhi483 co-transformed with cm-ccdB PCR products with homology arms into e.collgbred-gyrA 462 for wire loop recombination, respectively;
(2) And (3) performing enzyme digestion verification on the recombinant obtained in the step (1), and sequencing plasmids with correct enzyme digestion verification.
(3) Carrying out loop recombination on plasmids with correct enzyme digestion and sequencing in the step (2) and red gamma or plu gamma with homology arms respectively, and carrying out E.coli GB08-red cotransformation to obtain agrobacterium homologous recombination system series expression plasmids; the method comprises the following steps: pBBR1-kan-P tet -redγ-ETh1h2h3h4_agroB6、pBBR1-kan-P tet -redγ-ETh1h2h3P3_rhi597、pBBR1-kan-P tet -redγ-ET_rhi145、pBBR1-kan-P tet -redγ-ETh_rhi483、pBBR1-kan-P tet -pluγ-ETh1h2h3h4_agroB6、pBBR1-kan-P tet -pluγ-ETh1h2h3P3_rhi597、pBBR1-kan-P tet -pluγ -ET_ rhi145 and pBBR1-kan-P tet -pluγ-ETh_rhi483。
The invention relates to application of an agrobacterium homologous recombination system in genome DNA genetic modification by agrobacterium-mediated homologous recombination of short homology arms, wherein the short homology arms refer to homology arms of 80bp-200 bp.
In the above application: the series of expression plasmids of the Agrobacterium homologous recombination system is preferably pBBR1-kan-P tet -pluγ-ET_rhi145、pBBR1-kan-P tet -ETh1h2h3h4_agroB6、pBBR1-kan-P tet -ET_ rhi145 or pBBR1-kan-P tet -eth_ rhi483; the agrobacterium is preferably Agrobacterium tumefaciens C, agrobacterium tumefaciens EHA, 105 or Rhizobium rhizogenesNBRC, 13257; the short homology arm preferably refers to a homology arm of 80bp-100 bp.
In the above application: series expression plasmid pBBR1-kan-P of said agrobacterium homologous recombination system tet Pluγ -ET_ rhi145 in AgrobacteriumOptimal recombination efficiency in um tumefaciens C58, pBBR1-kan-P tet ETh1h2h3h4_agroB6 has optimal recombination efficiency in Agrobacterium tumefaciens EHA105, pBBR1-kan-P tet The-pluγ -eth_ rhi483 has optimal recombination efficiency in Rhizobium rhizogenesNBRC 13257. Wherein:
the expression plasmid pBBR1-kan-P tet The optimal conditions for the promotion of recombination efficiency in Agrobacterium tumefaciens C for pluγ -et_ rhi145 are: initial OD 600 =0.1, transfer 4h at 30 ℃ to OD 600 Inducing at 30 deg.C for 1 hr at 0.6-0.7, and under room temperature conditions, using ddH at room temperature 2 O, preparing competent cells, wherein the amount of exogenous DNA is 1.5 mug, the length of a homology arm is 80bp, the electrotransformation voltage is 1350V/mm, and the recovery time is 2h;
the expression plasmid pBBR1-kan-P tet The optimal conditions for ETh1h2h3h4_agrob6 to increase recombination efficiency in Agrobacterium tumefaciens EHA105 are: initial OD 600 =0.1, transfer 4h at 30 ℃ to OD 600 Inducing at 30 deg.C for 1 hr at 0.6-0.7, preparing competent cells with GH buffer solution of 10% glycerol+2μm HEPES at 4 deg.C, exogenous DNA amount of 1.5 μg, homology arm length of 80bp, electrotransformation voltage 1350V/mm, and recovery time of 2 hr;
the expression plasmid pBBR1-kan-P tet The optimal conditions for the enhancement of recombination efficiency in Rhizobium rhizogenes NBRC13257 for pluγ -eth_ rhi483 are: initial OD 600 =0.1, transfer 6h at 30 ℃ to OD 600 Inducing at 28 deg.c for 0.5-0.4 hr and 10 wt% concentration of SH buffer solution of sucrose+2 mu M HEPES to prepare competent cell at room temperature, with exogenous DNA amount of 4. Mu.g, homology arm length of 80bp, electrotransformation voltage of 1900V/mm and recovery time of 3 hr.
The agrobacterium homologous recombination system of the invention is applied to gene knockout in agrobacterium, wherein the agrobacterium is agrobacterium tumefaciens or agrobacterium rhizogenes.
In the above application: the agrobacterium tumefaciens is preferably Agrobacterium tumefaciens C58 or Agrobacterium tumefaciens EHA105; the agrobacterium rhizogenes is preferably Rhizobium rhizogenes NBRC 13257.
The gene knockout is preferably performed under the above-described optimized working conditions.
Compared with the prior art, the invention has the remarkable effects that:
the invention discloses an agrobacterium homologous recombination system consisting of a series of agrobacterium homologous recombination system expression plasmids, which is not reported in the current literature. And the present invention expresses plasmid pBBR1-kan-P for recombinant system for the first time tet -ETh1h2h3h4_agroB6、pBBR1-kan-P tet -ETh1h2h3P3_rhi597、pBBR1-kan-P tet -ET_ rhi145 and pBBR1-kan-P tet The recombination efficiency of the recombination system of eth_ rhi483 and its combination with red gamma and plu gamma is optimized and applied in agrobacterium. Experimental results show that Plu gamma ET RHI145 Has the highest recombination efficiency in Agrobacterium tumefaciens C58 and RecETh1h2h3h4 in Agrobacterium tumefaciens EHA105 AGROB6 Has the highest recombination efficiency, plu γReceth in Rhizobium rhizogenes NBRC13257 RHI483 Has the highest recombination efficiency. The invention realizes the gene knockout of the agrobacterium tumefaciens Agrobacterium tumefaciensC and Agrobacterium tumefaciensEHA, and the agrobacterium rhizogenes Rhizobiumrhizogenes NBRC and 13257 by using the constructed recombination system. The agrobacterium recombination system can greatly promote genome modification of agrobacterium, and has great application prospect in the aspects of constructing agrobacterium strains with high transformation efficiency and host range and the field of plant genetic engineering.
Drawings
Fig. 1: construction of homologous recombination operons of Red. Alpha. Beta. Gamma. And RecET operons with Agrobacterium origin. Wherein the proteins encoded by the same color genes function similarly.
Fig. 2: schematic representation of the expression plasmid of the Agrobacterium recombination system.
Fig. 3: optimization of Agrobacterium tumefaciensC electrical transfer conditions.
(A) Agrobacterium tumefaciensC58 growth curve.
(B) Effect of competent cells prepared at different temperature on the electrotransport efficiency of Agrobacterium tumefaciensC with different electrotransport buffers. Error bars, three parallel samples for each experimental group, n=3.
Fig. 4: agrobacterium tumefaciensEHA105 optimization of the electrical transfer conditions.
(A) Agrobacterium tumefaciensEHA 105.
(B) Effect of competent cells prepared at different temperature on Agrobacterium tumefaciensEHA105 electrotransformation efficiency with different electrotransformation buffers. Error bars, three parallel samples for each experimental group, n=3.
Fig. 5: optimization of Rhizobiumrhizogenes NBRC13257 electrical transfer conditions.
(A) Rhizobiumrhizogenes NBRC 13257.
(B) Effect of competent cells prepared at different temperature on the electrotransformation efficiency of Rhizobium rhizogenes NBRC13257 with different electrotransformation buffers.
(C) The effect of different electrical switching voltages on Rhizobium rhizogenes NBRC13257 electrical switching efficiency. Error bars, three parallel samples for each experimental group, n=3.
Fig. 6: comparison of recombination efficiencies in different combinations of recombination systems in Agrobacterium tumefaciensC, agrobacterium tumefaciensEHA, 105 and Rhizobium rhizogenesNBRC, 13257.
(A) In Agrobacterium tumefaciensC, agrobacterium tumefaciensEHA, 105 and Rhizobium rhizogenesNBRC 13257, the tetracycline promoter and recombinase fragment on the recombinant expression plasmid were replaced with the apraR gene, and loop recombination was performed.
(B) Comparison of recombination efficiencies for different combinations of recombination systems in Agrobacterium tumefaciensC.
(C) Agrobacterium tumefaciensEHA105 to recombination efficiency comparison of different combinations of recombination systems.
(D) Comparison of recombination efficiencies for different combinations of recombination systems in Rhizobium rhizogenesNBRC 13257. Error bars, three parallel samples for each experimental group, n=3.
Fig. 7: pBBR1-kan-P tet Optimization of recombination efficiency in Agrobacterium tumefaciensC for pluγ -et_ rhi 145.
(A) Effect of the amount of different exogenous DNA on Agrobacterium tumefaciensC recombination efficiency.
(B) Effects of different homology arm lengths on Agrobacterium tumefaciensC recombination efficiency. Error bars, three parallel samples for each experimental group, n=3.
Fig. 8: pBBR1-kan-P tet Optimization of recombination efficiency of ETh1h2h3h4_agrob6 in Agrobacterium tumefaciens EHA 105.
(A) Effect of the amount of different exogenous DNA on Agrobacterium tumefaciensEHA105 recombination efficiency.
(B) Effects of different homology arm lengths on Agrobacterium tumefaciensEHA105 recombination efficiency. Error bars, three parallel samples for each experimental group, n=3.
Fig. 9: pBBR1-kan-P tet Optimization of recombination efficiency in Rhizobium rhizogenesNBRC 13257 by pluγ -eth_ rhi483.
(A) Effect of the amount of different exogenous DNA on Agrobacterium tumefaciensEHA105 recombination efficiency.
(B) Effects of different homology arm lengths on Agrobacterium tumefaciensEHA105 recombination efficiency.
(C) Recombinant System expression plasmid pBBR1-kan-P at different Induction temperatures and Induction times tet Effect of recombinant efficiency of pluγ -eth_ rhi483. Error bars, three parallel samples for each experimental group, n=3.
Fig. 10: recombinant System expression plasmid pBBR1-kan-P tet Pluγ -ET_ rhi145 at Agrobacterium tumefaciensC, pBBR1-kan-P tet ETh1h2h3h4_agroB6 at Agrobacterium tumefaciens EHA105 and pBBR1-kan-P tet Use of pluγ -eth_ rhi483 for gene knockout in Rhizobium rhizogenesNBRC 13257.
(A) Schematic representation of gene knockout by agrobacterium recombination systems.
(B)pBBR1-kan-P tet Results of colony PCR after knockdown of IS21 transposase family genes istB and istA fusion gene by pluγ -ET_ rhi145 in Agrobacterium tumefaciensC.
(C)pBBR1-kan-P tet Colony PC after the celI gene was knocked out in Agrobacterium tumefaciens EHA105 by ETh1h2h3h4_agroB6Results of R.
(D)pBBR1-kan-P tet Results of colony PCR after knockout of the 3'-5' exonuclease gene in Rhizobium rhizogenesNBRC 13257 by pluγ -eth_ rhi483.
(E)pBBR1-kan-P tet Results of colony PCR after knocking out the endoglucanase gene in Rhizobium rhizogenesNBRC 13257 by pluγ -eth_ rhi483.
Wherein: m is DNA Marker, wt is wild strain. All randomly picked monoclonal were correct.
Detailed Description
The present invention will be described in detail with reference to the following drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are merely for explaining the present invention, and are not limiting in any way, and any simple modification, equivalent variation and modification of the embodiments according to the technical principles of the present invention are within the scope of the technical solutions of the present invention.
General description:
recombinant System expression strains E.coli GB2005, E.coli GB05-dir, E.coli GB08-redE.coli GBred-gyrA462, which are commercially available from GeneBridges, germany, are described in the following examples; agrobacterium tumefaciensC58 and Agrobacterium tumefaciensEHA are derived from the university of northwest agriculture and forestry, rhizobium rhizogenesNBRC and 13257 purchased from the Germany collection of strains (DSMZ).
Plasmid pBBR1-P Rha -GFP-kan、pBBR1-kan-P tet -firefly、pBBR1-P Rha -gba-kan, pBBR1-apra-kan was derived from university of shandong-helmholtz biotechnology institute.
Agrobacterium tumefaciensC58, agrobacterium tumefaciensEHA, 105 and Rhizobium rhizogenes NBRC, 13257 genomic sequences are reported sequences, see related genomic sequences published in NCBI.
Gene sequencing in plasmid construction was done by Huada genes. Gene synthesis was performed by Jin Weizhi. Other non-mentioned plasmids are commercially available conventional plasmids, and the method of electrotransformation into recipient bacteria is conventional.
Other related reagents and consumables are made in China. The experimental methods and reagents in the examples are conventional methods and commercially available reagents in the art unless otherwise specified.
Example 1: construction of series agrobacterium homologous recombination expression plasmid
Proteins homologous to the Rec alpha/beta of the lambda phage of the Escherichia coli or the Rec E/T recombinase of the Rec prophage are searched for potential proteins with recombination functions in Agrobacterium by using the BlastP program of NCBI in all the agrobacteria with complete genome sequencing or bacterial genomes close to the agrobacteria or phage and prophage genomes. Exonuclease-recombinase operons homologous to RecE/T were found in Agrobacterium tumefaciens str.B6, rhizobium leguminosarum bv. Trifolii WSM597, rhizobium sp.LC145 and Rhizobium sp.Root483D2contig_20, respectively, which are ETh1h2h3h 4-agroB 6, ETh1h2h3P3 rhi597, et_ rhi145 and ETh rhi483, respectively. Of these, ETh1h2h3h4_agrob6 is about 3410bp long, encodes six proteins, and there are 4 putative proteins in addition to two proteins homologous to RecE and RecT; ETh1h2h3p3_ rhi597 is 3898bp long, encodes five proteins, 2 putative proteins in addition to proteins homologous to RecE and RecT and Exo-Pol III; et_ rhi145 is 1394bp long, contains proteins homologous to RecE and RecT; eth_ rhi483 was 1951bp long and there was a putative protein in addition to proteins homologous to RecE and RecT, see figure 1.
A series of recombinant expression plasmids of the agrobacterium homologous recombination system are constructed on the basis of a wide-host replicon pBBR1, and the recombinant functional protein is induced to express by using a tetracycline promoter, as shown in figure 2.
Expression plasmid pBBR1-kan-P of homologous recombination system series tet -ETh1h2h3h4_agroB6、pBBR1-kan-P tet -ETh1h2h3P3_rhi597、pBBR1-kan-P tet -ET_ rhi145 and pBBR1-kan-P tet The construction method of the ETh_ rhi483 comprises the following steps:
(1)pBBR1-kan-P tet the initial plasmid, ecoRI and NheI double enzyme cutting, gel recovery order linear fragment pBBR1-P tet Kan, about 5kb in length;
(2) The gel obtained in the step (1) is used for recovering the linear fragment pBBR1-P tet The kan is respectively co-transformed with a synthesized recombinase fragment with a vector homology arm, namely, an ETh1h2h3h4_agroB6HA, an ETh1h2h3hP3_ rhi597HA, an ET_ rhi HA and an ETh_ rhi483HA, into E.coli GB05-dir for line recombination;
(3) Performing enzyme digestion verification on the recombinant obtained in the step (2) to obtain a series of expression plasmids of the agrobacterium homologous recombination system, wherein the series of expression plasmids are named pBBR1-kan-P respectively tet -ETh1h2h3h4_agrob6, the nucleotide sequence of which is shown in SEQ ID No. 1; pBBR1-kan-P tet -ETh1h2h3p3_ rhi597, the nucleotide sequence of which is shown in SEQ ID No. 2; pBBR1-kan-P tet -et_ rhi145, the nucleotide sequence of which is shown in SEQ ID No. 3; pBBR1-kan-P tet -eth_ rhi483, the nucleotide sequence of which is shown in SEQ ID No. 4.
Example 2: optimization of Agrobacterium tumefaciensC, agrobacterium tumefaciensEHA, 105 and Rhizobiumrhizogenes NBRC13257 electrical transfer conditions
The optimization exploration of Agrobacterium tumefaciensC and Agrobacterium tumefaciensEHA105 electric conversion conditions involves the following two parts:
(1) Determination of Agrobacterium tumefaciensC58 and Agrobacterium tumefaciensEHA105 growth curves
First, the growth curves of Agrobacterium tumefaciensC and Agrobacterium tumefaciensEHA105 need to be measured to determine the optimal length of time to prepare competent cell cultures. Three Agrobacterium tumefaciensC and Agrobacterium tumefaciensEHA monoclonals are picked up respectively, inoculated into 2ml EP tube with 1.3ml LB liquid culture medium with prick hole respectively, shake cultured at 30 deg.C and 950rpm for 24h, 100 μl seed liquid is sucked up and added into 900 μl liquid LB, blown and mixed evenly, and OD is measured 600 . Transfer to 50ml LB, start OD 600 Adjusting to 0.1, culturing at 30deg.C under shaking at 200rpm, sucking 1ml bacterial liquid every 1h, and measuring OD 600 According to the OD at each time point 600 The growth curves of Agrobacterium tumefaciensC and Agrobacterium tumefaciensEHA are plotted and the experimental results are shown in fig. 3A and 4A. Displays Agrobacterium tumefaciensC and Agrobacterium tumefaciensEHA105 is the start of the exponential growth phase from hour 4.
(2) Effects of different electroporation buffers and different temperatures on the electroporation efficiencies of Agrobacterium tumefaciensC and Agrobacterium tumefaciensEHA105
Agrobacterium tumefaciensC58 and Agrobacterium tumefaciensEHA after 4h incubation, each experimental group was incubated with 10% sucrose solution, 10% glycerol solution, SH buffer (10% sucrose+2μM HEPES), GH buffer (10% glycerol+2μMHEPES) and ddH, respectively 2 O, washing 2 times, preparing competent cells at room temperature and 4℃respectively, adding 1. Mu.g of test plasmid pBBR1-P Rha GFP-kan, electrotransformation.
The results of the experiment are shown in FIG. 3B and FIG. 4B, which show that in Agrobacterium tumefaciensC58 ddH is used 2 O is used for preparing competent cells at normal temperature, and the electrotransformation efficiency is highest; in Agrobacterium tumefaciensEHA, GH buffer is used to make competent cells at low temperature, with highest electrotransformation efficiency.
Combining the optimized results of the transformation efficiencies, the invention determines the optimal transformation conditions of Agrobacterium tumefaciensC: initial OD 600 Bacterial liquid=0.1, cultured at 30℃with shaking at 950rpm for 4 hours, and subjected to ddH 2 O is used for preparing competent cells at normal temperature, and the electrotransformation voltage is 1350V/mm. Optimal conversion conditions for Agrobacterium tumefaciensEHA 105: initial OD 600 Bacterial liquid with the concentration of=0.1, the bacterial liquid is subjected to shaking culture for 4 hours at 30 ℃ and 950rpm, and competent cells are prepared from GH buffer at a low temperature, and the electrotransformation voltage is 1350V/mm.
The optimization exploration of Rhizobium rhizogenesNBRC 13257 electric transfer conditions involves the following three parts:
(1) Determination of Rhizobium rhizogenesNBRC 13257 growth curve
The first step requires measurement of the growth curve of Rhizobium rhizogenesNBRC 13257 to determine the optimal length of time to prepare competent cell cultures. Three Rhizobium rhizogenesNBRC 13257 monoclonals were picked, inoculated into 2ml EP tubes with 1.8ml TY liquid medium in the prick holes, cultured overnight at 30℃with shaking at 950rpm, 100. Mu.l seed solution was taken up and added into 900. Mu.l TY liquid, and the mixture was blown and mixed well for OD measurement 600 . Suction fittingThe amount of seed solution was inoculated into 50ml TY liquid medium containing kanamycin to initiate OD 600 =0.1, 30 ℃,200rpm shaking culture, every 2 hours, 1ml bacterial liquid is sucked for OD measurement 600 According to the OD at each time point 600 The growth curve of Rhizobium rhizogenesNBRC and 13257 is plotted, and the experimental results are shown in fig. 5A. Showing when the onset OD 600 When=0.1, the cells entered the logarithmic growth phase after 6h after the adaptation period.
(2) Impact of different electroporation buffers and temperature on the electroporation efficiency of Rhizobium rhizogenesNBRC 13257
After 6h incubation at Rhizobium rhizogenesNBRC 13257, each experimental group was incubated with 10% sucrose solution, 10% glycerol solution, SH buffer (10% sucrose+2. Mu.M HEPES), GH buffer (10% glycerol+2. Mu. MHEPES) and ddH, respectively 2 O, washing 2 times, preparing competent cells at room temperature and 4℃respectively, adding 500ng of test plasmid pBBR1-P Rha GFP-kan,1350V/mm for electrotransformation. The experimental results are shown in FIG. 5B, and the results show that the cells can be washed by SH buffer solution under normal temperature conditions to obtain higher conversion efficiency, and the efficiency is higher than ddH 2 O increases by about 3-fold.
(3) Effects of different transformation voltages on the transformation efficiency of Rhizobium rhizogenesNBRC 13257
To further increase the electrotransformation efficiency of Rhizobium rhizogenesNBRC 13257, applicants used 1350V/mm, 1500V/mm, 1700V/mm, 1900V/mm, and 2100V/mm, respectively, to shock competent cells. The experimental results are shown in FIG. 5C, and the results show that 1900V/mm and 2100V/mm can greatly improve the electrotransformation efficiency. Since high voltages affect cell viability, 1900V/mm was chosen as the optimal electrotransformation voltage for Rhizobium rhizogenes NBRC13257 in this experiment.
Combining the optimized results of the transformation efficiencies, the invention determines the optimal transformation conditions of Rhizobium rhizogenes NBRC 13257: initial OD 600 Bacterial solution of=0.1, shaking culture at 30 ℃,950rpm for 6 hours, SH buffer room temperature preparation competent cells, electrotransformation voltage 1900V/mm.
Example 3: comparison of recombination efficiencies in Agrobacterium tumefaciensC, agrobacterium tumefaciensEHA105 and Rhizobium rhizogenesNBRC 13257 of different combinations of expression plasmids of recombination systems
In the process of digging a recombination system, the applicant connects Red gamma derived from Lambda phage or Plu gamma derived from Pseudomonas in series to an agrobacterium recombination system, adopts a method of reverse screening of Red/ET and ccdB, and constructs recombination system expression plasmids in different combination forms, and the specific names are respectively named as follows: pBBR1-kan-P tet -redγ-ETh1h2h3h4_agroB6、pBBR1-kan-P tet -redγ-ETh1h2h3P3_rhi597、pBBR1-kan-P tet -redγ-ET_rhi145、pBBR1-kan-P tet -redγ-ETh_rhi483、pBBR1-kan-P tet -pluγ-ETh1h2h3h4_agroB6、pBBR1-kan-P tet -pluγ-ETh1h2h3P3_rhi597、pBBR1-kan-P tet -pluγ -ET_ rhi145 or pBBR1-kan-P tet Pluγ -eth_ rhi483. Meanwhile, the experiment adopts pBBR1-P Rha -gba-kan as positive control for recombination experiments.
Recombinant system expression plasmids were electrotransformed into Agrobacterium tumefaciensC, agrobacterium tumefaciensEHA, 105 and Rhizobium rhizogenesNBRC 13257, respectively, using the same electrotransformation method described in the previous examples.
Mu.g of the aprpCR fragment with the 80bp homology arm (obtained by PCR amplification using 80bpHA-apra-5 and 80bpHA-apra-3 as primers and pBBR1-apra-kan as templates) was respectively transferred into Agrobacterium tumefaciensC and Rhizobium rhizogenesNBRC 13257 cells containing the above 13 recombination systems, and the loop recombination replaced the promoter region and the recombinase portion in the recombinase expression plasmid, thereby obtaining the recombinant pBBR1-apra-kan as the basis for comparison of the recombination system efficiency, as shown in FIG. 6A.
80bpHA-apra-5:gcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaACGCTCAGTGGAACGAGGTT
80 bpHA-apra-3. ggagagcctgagcaaaactggcctcagagcattgaagcagcacggttcactgcggtagtataataaaaccggtaagctgagcgtcagccaatcgact (the lower-case letters of the primers are homology arms, capital letters are primers).
The specific procedure for PCR amplification of fragment apraR using primers 80bpHA-apra-5 and 80bpHA-apra-3 was as follows:
PCR reaction procedure
3 of Agrobacterium tumefaciens C and Agrobacterium tumefaciens EHA105 carrying recombinant system expression plasmids were picked up and monoclonal into 2ml EP tubes containing 1.3ml of liquid LB medium, kanamycin was used at a concentration of 10. Mu.g/ml, cultured for 24h at 30℃with shaking at 950rpm, 100. Mu.l of seed solution was taken up and added to 900. Mu.l of liquid LB, and the mixture was blown and mixed well to determine OD 600 . An appropriate amount of seed liquid was aspirated and inoculated into 1.3ml LB liquid medium containing kanamycin (10. Mu.g/ml) to initiate OD 600 The culture was performed for 4h with shaking at 30℃and 950rpm, and anhydrous tetracycline (AHT) was added to a final concentration of 20. Mu.g/mL, and the culture was performed for 1h with shaking at 30℃and 950 rpm. Uniform OD according to minimum in experimental group 600 When competent cells were prepared, agrobacterium tumefaciens EHA 105.105 was prepared using electrotransport buffer GH, agrobacterium tumefaciens C58.58 under low temperature conditions and ddH under normal temperature conditions 2 O was treated, and 1. Mu.g of an apraR PCR fragment (obtained by PCR amplification using 80bpHA-apra-5 and 80bpHA-apra-3 as primers and a recovered gel fragment after cleavage of pBBR1-apra-kan PvuII as a template) with an 80bp homology arm was then electrotransferred into cells. The electrotransformation voltage was 1350V/mm, followed by recovery 2h,Agrobacterium tumefaciens C58 at 30℃with 50. Mu.g/ml of apramycin for resistance screening, agrobacterium tumefaciens EHA105 with 40. Mu.g/ml of apramycin for resistance screening, incubation in a 30℃incubator, and counting the number of colonies, see FIGS. 6B and 6C.
Selecting 3 pieces of Rhizobium rhizogenesNBRC 13257 monoclonal carrying recombinant expression plasmids into 2ml EP tube containing 1.3ml of liquid TY culture medium, culturing for 24h at 30deg.C with shaking at 950rpm, sucking 100 μl of seed solution, adding into 900 μl of liquid LB, blowing, mixing, and measuring OD 600 . Sucking a proper amount ofIs inoculated into 1.3ml LB liquid medium containing kanamycin (30. Mu.g/ml) to make the initial OD 600 The culture was performed at 30℃for 6h with shaking at 950rpm, and anhydrous tetracycline (AHT) was added to a final concentration of 20. Mu.g/mL, and at 30℃for 1h with shaking at 950 rpm. Uniform OD according to minimum in experimental group 600 When competent cells were prepared, they were treated with SH buffer at room temperature, and 1. Mu.g of the apraR PCR fragment (obtained by PCR amplification using 80bpHA-apra-5 and 80bpHA-apra-3 as primers and the recovered gel fragment after cleavage of pBBR1-apra-kan PvuII as a template) with 80bp homology arms was then electrotransferred into the cells. The electrotransformation voltage was 1900V/mm, followed by recovery at 30℃for 2h, resistance screening with 3. Mu.g/ml apramycin, cultivation in a 30℃incubator, and counting the number of colonies, see FIG. 6D.
The experimental results show that:
in Agrobacterium tumefaciens C, the recombination System Receth1h2P3 RHI597 Mediated homologous recombination is superior to the other three recombination systems (Receth 1h2h3h4 AGROB6 、RecET RHI145 And RecETh RHI483 ) The recombination efficiency of the (2) is high; however, after addition of Red gamma, recETh1h2h3h4 AGROB6 The recombination efficiency of (2) is doubled, recETh1h2P3 RHI597 And RecET RHI145 Is unchanged, while RecETh RHI483 But the recombination efficiency of (c) is lowered; after the addition of Plu gamma, receth1h2P3 RHI597 And RecET RHI145 Greatly improves the recombination efficiency of RecETh1h2h3h4 AGROB6 And RecETh RHI483 Almost unchanged. In summary, plu γet RHI145 The highest recombination efficiency was seen in Agrobacterium tumefaciens C, fig. 6B.
In Agrobacterium tumefaciens EHA105, the recombination system RecETh1h2h3h4 AGROB6 、RecETh1h2P3 RHI597 And RecET RHI145 Has higher recombination efficiency, receth RHI483 Has lower recombination efficiency; however, after addition of Red γ or Plu γ, the recombination efficiency of all four recombination systems was greatly reduced. In summary, recath 1h2h3h4 in Agrobacterium tumefaciens EHA105 AGROB6 Has the highest recombination efficiency, as shown in FIG. 6C.
In RhizobiumOf the rhizogenes NBRC13257, only RecETh RHI483 Shows relatively high recombination efficiency, receth1h2P3 RHI597 And RecET RHI145 Has very low recombination efficiency, receth1h2h3h4 AGROB6 There is little recombination function. RecETh after addition of Red gamma or Plu gamma RHI483 The recombination efficiency is slightly improved. In summary, plu gamma RecETh in Rhizobium rhizogenes NBRC13257 RHI483 Has the highest recombination efficiency, as shown in FIG. 6D. .
Example 4: optimization of recombinant System expression plasmids recombinant working conditions in Agrobacterium tumefaciensC, agrobacterium tumefaciensEHA105 and Rhizobium rhizogenes NBRC13257
In different growth states, the bacterial cells have great difference on the absorption rate of the exogenous DNA, and in order to ensure that the recombination efficiency meets the requirement of genetic modification on the microorganism DNA molecules, the working conditions of a recombination system are necessary to be optimized. Based on the study of the optimal transformation conditions in example 2, the present example optimizes the recombinant working conditions for Agrobacterium tumefaciensC, agrobacterium tumefaciensEHA, 105 and Rhizobium rhizogenes NBRC 13257.
(1) Effect of homology arm Length on recombination efficiency
The Red recombination system consists of three proteins: recE and Red alpha proteins are exonucleases that bind to the ends of double-stranded DNA, degrading the DNA from the 5' end to the 3' end, resulting in 3' end or single-stranded DNA; recT and Red beta are single-stranded DNA binding proteins and mediate complementary single-stranded DNA annealing; red gamma protein binds to RecBCD protein and inhibits the activity of the protein in degrading exogenous DNA. The length of the recognition sequence of RecE and Red alpha proteins with exonuclease activity is within a certain range, and the longer the non-homologous arm is, the better. Therefore, the use of homology arms can be shortened by studying the effect of the length of homology arms on recombination efficiency. The homology arms used in this example were 80bp, 100bp, 150bp, 200bp, respectively.
The experimental results are shown in fig. 7A, 8A and 9A, and the recombination efficiency is improved with the increase of the length of the homology arm. The 80bp homology arm was selected for the next experiment.
(2) Effect of the amount of exogenous DNA on the recombinant efficiency
The cell has a certain threshold value on the intake of the exogenous DNA, and when the capacity of the cell reaches saturation, the redundant DNA can not be absorbed, so that the waste of experimental materials can be greatly reduced by researching the influence of the amount of the added exogenous DNA on the recombination efficiency. After competent cells were prepared, 500ng, 1000ng, 1500ng and 2000ng of apraRPCR product carrying an 80bp homology arm were added to Agrobacterium tumefaciensC, 58 and Agrobacterium tumefaciensEHA, respectively. 1000ng, 2000ng, 3000ng and 4000ng of the apraRPCR product carrying an 80bp homology arm were added to Rhizobium rhizogenes NBRC13257, respectively.
As a result, as shown in FIGS. 7B, 8B and 9B, in Agrobacterium tumefaciensC58, the applicant found that recombination efficiency was low at an amount of 500ng of exogenous DNA, but there was no significant difference in the amount of DNA exceeding 1000 ng; with increasing DNA usage in Agrobacterium tumefaciensEHA105, in the range of 1000ng-2000ng, there was no significant impact on the recombination efficiency; in Rhizobium rhizogenes NBRC13257, the recombination efficiency increases with increasing DNA amount, and the recombination efficiency is highest at 4000ng, but no "saturation effect" occurs at this time, and the amount of DNA used can be increased appropriately in subsequent experiments.
(3) Effect of Induction temperature on Rhizobium rhizogenes NBRC13257 recombination efficiency
The cell growth and the protein expression have the optimal temperature, and the recombinant enzyme expression also has the optimal temperature, so that the optimal expression temperature of the recombinant enzyme can be determined by researching different induction times of the recombinant enzyme, thereby obtaining higher recombination efficiency. In this example, five induction temperatures were selected, 25 ℃,28 ℃,30 ℃, 32 ℃ and 37 ℃ respectively, and three replicates per group. Rhizobium rhizogenes NBRC13257 cells containing pBBR1-kan-Ptet-eth_ rhi483 recombinase plasmid were cultured for 6h (initial OD) 600 =0.1), 20 μl AHT was added to induce 1h at different temperatures, the pcr product was used at 1000ng, the electrotransformation voltage was 1900V/mm, and the OD was uniform before electrotransformation 600 。
The experimental results are shown in FIG. 9C, and the results show that the highest recombination efficiency can be obtained by preparing competent cells after induction at 28 ℃, and the recombinase has no recombination function at 37 ℃. Thus, the optimal induction temperature for the recombination system in Rhizobium rhizogenes NBRC13257 is 28 ℃.
(4) Effect of Induction time on the efficiency of the Rhizobium rhizogenes NBRC13257 recombination
In addition, the induction time can also influence the recombination efficiency, the induction time is respectively selected from 0.5h, 1h, 1.5h and 2h, after 6h of cell culture, 20 μl of AHT is added to induce for different time at 28 ℃, the dosage of PCR product is 1000ng, the voltage of electric transformation is 1900V/mm, and the OD before electric transformation is uniform 600 。
The experimental results are shown in FIG. 9C, and the results show that the competent cells can be prepared to obtain higher recombination efficiency after induction for 0.5h at 28 ℃.
Summary of experimental results:
optimal recombination conditions in Agrobacterium tumefaciensC: initial OD 600 =0.1, transfer at 30 ℃ for 4h (OD 600 About 0.6-0.7), induction at 30℃for 1h, under room temperature conditions, with ddH at room temperature 2 O is used for preparing competent cells, the amount of exogenous DNA is 1.5 mug, the length of a homology arm is 80bp, the electrotransformation voltage is 1350V/mm, and the recovery time is 2h.
Optimal recombination conditions in Agrobacterium tumefaciensEHA 105: initial OD 600 =0.1, transfer at 30 ℃ for 4h (OD 600 About 0.6-0.7), induced at 30℃for 1h, competent cells were prepared with GH buffer (10% glycerol+2. Mu.M HEPES) at 4℃with an amount of exogenous DNA of 1.5. Mu.g, homology arm length of 80bp, electrotransformation voltage 1350V/mm, recovery time of 2h.
The optimal recombination conditions in Rhizobium rhizogenes NBRC13257 are: initial OD 600 =0.1, transfer at 30 ℃ for 6h (OD 600 About 0.4-0.5), induced at 28℃for 0.5h, competent cells were prepared at normal temperature using SH buffer (10% sucrose+2. Mu.M HEPES), the amount of exogenous DNA was 4. Mu.g, the length of homology arm was 80bp, the electrotransformation voltage was 1900V/mm, and recovery time was 3h.
Example 5: application of genetic modification of genome by using novel agrobacterium recombination system
Using Plu gamma ET RHI145 、RecETh1h2h3h4 AGROB6 And Plu gamma RecETh RHI483 The genome was genetically modified in Agrobacterium tumefaciens C, agrobacterium tumefaciens EHA, 105 and Rhizobium rhizogenes NBRC13257, respectively.
The IS21 transposase family gene on the linear chromosome of Agrobacterium tumefaciens C has a full length of 2252bp. The IS21 transposase family gene can be knocked out, so that the genome of the gene can be simplified, the potential risk of transposon pollution to exogenous DNA on Ti plasmid IS eliminated, and the stability of agrobacterium tumefaciens C58 serving as a transgenic vector IS improved. The moderate simplification of genome can optimize the metabolic pathway of the cell, improve the utilization efficiency of the cell to the substrate and the energy, and obviously improve the predictability and the controllability of the physiological performance of the cell.
The gene celI is a member of a transcription regulator MarR/ArsR gene family, is positioned on a Agrobacterium tumefaciensEHA105 linear chromosome, has the total length of 929bp, and is an inhibitor of cellulose synthesis gene celABC, so that the celI is knocked out, the cellulose yield is improved, the attachment of agrobacterium to plants is facilitated, and the parallel transfer efficiency of exogenous DNA is improved.
In Rhizobium rhizogenes NBRC13257, the 3'-5' exonuclease gene is 978bp long, codes 325 amino acids altogether, and the other gene endoglucanase gene is a cellulase gene, belongs to a class of glycoside hydrolase, is a key enzyme for decomposing plant cell walls by bacteria, and is 1050bp long, and codes 349 amino acids.
The target gene is replaced by using the apraR PCR fragment with the homologous arm under the action of recombinase, so as to realize the gene knockout of agrobacterium. The homology arms were 80bp in length, and were loaded at both ends of the apraR gene by primer synthesis combined PCR, see fig. 10A.
C58-IS21-apra-5：
cttcgatgtcaacgccggagcaaattaaggccaggcggcggagtaaaaccaggccactatcggcgcacgcatgagacctcAGCTGAATTACATTCCCAACCG
C58-IS21-apra-3：
ggctggccgctgtcttcggacgcttgggaccagcagatgctgttcttcagccagaatggcttcagggtgagagcggcggtCAACTTAAATGTGAAAGTGGGTC
EHA105-celI-apra-5：
cttagaatatgatataaaccatatttcgtaatatagttgccgagagaatccgcgcctgtcaaccgctaaaatcatacttaAGCTGAATTACATTCCCAACCG
EHA105-celI-apra-3：
ggctttatgacaaacagggctggacaagggcagggcatttaagccagaaattctgatctggatcgataaatctgaagattCAACTTAAATGTGAAAGTGGGTC
13257-exo-apra-5：
ctaggaaaaagcctgcttgatctcctttaggagaaatggcttttgtggagagccatgacatcagtcattacgacgtctgcAGCTGAATTACATTCCCAACCG
13257-exo-apra-3：
tgcaaaagctgcattgtgtgttgcccggtgtttgactgttttctattctaaagcctgatcatcaatcatcgaaagctcgcCAACTTAAATGTGAAAGTGGGTC
13257-glu-apra-5：
ctccatcgtctcacggtctctcatcgtcatgacaactgctgttgccgcgactgccaaggaaaccgtaatcagagaggattAGCTGAATTACATTCCCAACCG
13257-glu-apra-3：
cctcattgctgatcggcataacgacggcgggcattctcgcaaagatcggcaggcgcggatgaagcgctggcgcatcatggCAACTTAAATGTGAAAGTGGGTC (lower case for primer homology arm, upper case for primer).
The specific PCR reaction system and the reaction procedure were the same as in example 3.
Single colonies were picked for colony PCR verification for genetically manipulated mutant strains, see FIGS. 10B-E.
IS21-check-5：CATCACCTGACGCTTGGCAT
IS21-check-3：GGAGAAGTACCTGCCCATCG
celI-check-5：AGCGATAGGCAGAGACGAAG
celI-check-3：GGAGAAGTACCTGCCCATCG
exo-check-5：GGTGAACTTCTCTACGCCAG
exo-check-3：TGGATCAGAGATGATCTGCTC
glu-check-5：TGGTGGTTTGGATCGCTGTC
glu-check-3：TGGATCAGAGATGATCTGCTC
The specific PCR reaction system and the reaction procedure were the same as in example 3.
Sequence list
<110> university of Shandong
<120> Agrobacterium homologous recombination system and application thereof
<141> 2021-09-15
<160> 4
<210> 1
<211> 8496
<212> DNA
<213> artificial sequence
<221> nucleotide sequence of pBBR1-kan-Ptet-ETh1h2h3h 4-agroB 6
<222>（1）…（8496）
<400> 1
atggcggcat acgcgatcat gcgatgcaag aagctggcga aaatgggcaa cgtggcggcc 60
agtctcaagc acgcctaccg cgagcgcgag acgcccaacg ctgacgccag caggacgcca 120
gagaacgagc actgggcggc cagcagcacc gatgaagcga tgggccgact gcgcgagttg 180
ctgccagaga agcggcgcaa ggacgctgtg ttggcggtcg agtacgtcat gacggccagc 240
ccggaatggt ggaagtcggc cagccaagaa cagcaggcgg cgttcttcga gaaggcgcac 300
aagtggctgg cggacaagta cggggcggat cgcatcgtga cggccagcat ccaccgtgac 360
gaaaccagcc cgcacatgac cgcgttcgtg gtgccgctga cgcaggacgg caggctgtcg 420
gccaaggagt tcatcggcaa caaagcgcag atgacccgcg accagaccac gtttgcggcc 480
gctgtggccg atctagggct gcaacggggc atcgagggca gcaaggcacg tcacacgcgc 540
attcaggcgt tctacgaggc cctggagcgg ccaccagtgg gccacgtcac catcagcccg 600
caagcggtcg agccacgcgc ctatgcaccg cagggattgg ccgaaaagct gggaatctca 660
aagcgcgttg agacgccgga agccgtggcc gaccggctga caaaagcggt tcggcagggg 720
tatgagcctg ccctacaggc cgccgcagga gcgcgtgaga tgcgcaagaa ggccgatcaa 780
gcccaagaga cggcccgaga ccttcgggag cgcctgaagc ccgttctgga cgccctgggg 840
ccgttgaatc gggatatgca ggccaaggcc gccgcgatca tcaaggccgt gggcgaaaag 900
ctgctgacgg aacagcggga agtccagcgc cagaaacagg cccagcgcca gcaggaacgc 960
gggcgcgcac atttccccga aaagtgccac ctgggatgaa tgtcagctac tgggctatct 1020
ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc 1080
gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc 1140
cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga 1200
tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga 1260
ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 1320
atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 1380
aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg 1440
acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 1500
acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 1560
tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 1620
ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 1680
agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 1740
atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg 1800
aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 1860
gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 1920
cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 1980
tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 2040
agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc 2100
atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt 2160
ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca 2220
cccccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg 2280
ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt 2340
ttttatgcag atatcaatta atttaagacc cactttcaca tttaagttgt ttttctaatc 2400
cgcatatgat caattcaagg ccgaataaga aggctggctc tgcaccttgg tgatcaaata 2460
attcgatagc ttgtcgtaat aatggcggca tactatcagt agtaggtgtt tccctttctt 2520
ctttagcgac ttgatgctct tgatcttcca atacgcaacc taaagtaaaa tgccccacag 2580
cgctgagtgc atataatgca ttctctagtg aaaaaccttg ttggcataaa aaggctaatt 2640
gattttcgag agtttcatac tgtttttctg taggccgtgt acctaaatgt acttttgctc 2700
catcgcgatg acttagtaaa gcacatctaa aacttttagc gttattacgt aaaaaatctt 2760
gccagctttc cccttctaaa gggcaaaagt gagtatggtg cctatctaac atctcaatgg 2820
ctaaggcgtc gagcaaagcc cgcttatttt ttacatgcca atacaatgta ggctgctcta 2880
cacctagctt ctgggcgagt ttacgggttg ttaaaccttc gattccgacc tcattaagca 2940
gctctaatgc gctgttaatc actttacttt tatctaatct agacatcatt aattcctaat 3000
ttttgttgac actctatcat tgatagagtt attttaccac tccctatcag tgatagagag 3060
aattcatatg gaaaagataa tcgacaaaga ccagtttgag cacatcacca gcccgattga 3120
tgcgatactc aagcgcgtca agccggtgaa gggcgaagtg cagtatgacg gcggcgttat 3180
ctatcagcct ggtgcatacc ggaatgttcc gatcgagcgt tatcaccacg accgcgatct 3240
gttcgacggc ccagctgttt cgaagtcgat catcaaggaa attctgccgg tccacggcgg 3300
cagcccgaag cagttctggg gccggtggaa ctggaacaaa gaccgaattg acccgaaaga 3360
cccgactgag gcgctgatct ttggcaaggc cgcgcactgc ctgcttctgg gcgatgaggt 3420
gtttgaagag aattttatcg ttcgcccggc gacctatcca gacaaggacg ggaaggaaaa 3480
gccatggaac ggcaatgcca catggtgcaa gcaatatctc gatctgcaaa agaaggaagg 3540
gctgatggtc ctgaccgaca agcagatgga aactatcaag cgcatccgcg ccgacgcctc 3600
cacctacccg cttgtgcagc agggcatcct caatggccgc gtagagcgca cactggctgc 3660
gaaagacccg gagacgggta tatggctcaa ggttcgccca gacgcgatgc ctaacgccga 3720
tggcgtcttt gccgacctga aaacgatcgg cagtctggat gaagacttca tgcagcgcca 3780
gatattcgac gcgggctatt acctgcaggc ggcaatgacg cgcatggtct gccggctcct 3840
gaaaatcccg ttcgaaacct tcgtgctcgt ctatgtcctg aacgacgaca ttccagacac 3900
cgcccacgtc gaaatgaacg atcagtcgat ccttctcccc ggcggcgagg aaataccgag 3960
cgagcttgac cacggcgagg cgatgatccg ctgggcgctg cgcacgatcc gcaaatgcct 4020
tgatgacggt cattggcccg gtcgtgagcc gttccaaggc ggcgagcgca agatcacgat 4080
gctcccttac cacaagagca aaatcaccag attcctgaac ggcatcgagg gcgacgttcc 4140
tcctccgtca gatgagcagg aggccgcgta aaggaggcag ctatatgaac cagcttgcaa 4200
ccaacacaga acgtttgccg atggatcagg tcggcatgtc caccggatcg aatggcgcca 4260
agatcgctcc ccagaacctg ggtgaagtcg tgaagtttgc ggaggtcatg tgccgtgccg 4320
atatcgcctt gccgaagcat ctgcgcggca atgccggggc ttgcatggcc gtttcgcttc 4380
aagctcttga ctggcaaatg aacccgtttg cagtggcttc gaagtcctat tcagtcaacg 4440
gcacgatcgc ttacgaagcc cagctcatca tcgctgtgat caatacccgc tccgggatcg 4500
aaggccgtct cgaatactca ttcgaaggcg aaggtggcga tagggtctgc atcgcatccg 4560
gcaagcttga cggcaaagta ttggaagtcc gctcgccaaa gttcaaggac attactccga 4620
agaactcccc tctctggaaa tccgacccgg atcagcagca ctgctattat acaggccgct 4680
cgtggggccg acgccataca cctgaagtca tccttggcgt ctatgaccgt gacgaagtgg 4740
aagagttccg aggccctgac aacgcccgcg acgtgacgcc gaaagcatcg ctatccgcac 4800
gtctcgcaca gtcaaacagc gccacacagc aagccaatga cgagcgcgaa gggttcaccg 4860
catcacctgc cgctgatgag cgctctgacg ctctcacggg cgagatactg cccaacacca 4920
attccgacga tgaaacccct gcctcgtcgt cggataacgc tggcaatacg ccagtagatg 4980
aggccggagc ggaatccccc tccgacgctc cggcctctac cgatccagag cgcgatatcc 5040
tgatccggtt tgccgctgaa atgctgccga tggcagcaac ggcaaagacc gaggtctgga 5100
aggacgtcga gaagggctgg tccgagggcg agatgaaaac tctgtccgaa gccggaaagg 5160
ccaaggcgaa gtccatcagc cagtcgatcc gggcgattgc gctcggtaat gtcagcttgg 5220
agtcggccgc cgagttccac gccgaggttc tggactggaa ggcttctgat ctgggaggcg 5280
tcgatggctg acttaagcta gctgtacagg atccaggagg cagctatatg ccccctttct 5340
ccaccaccaa cacacaagaa tcggatgacc cgaccgagat catcatccgg ctccgcaatt 5400
gcgaaaagca gatcgaccag tcgaccagag agtttcgcag agacgtgaac cgcctcatca 5460
cagggcttct aaccctcggg atcatttcag cctcgtttct gcacttcggc atgcccgcag 5520
atcggaagat ggcgaaggca aatcaggaga tcgatgtcgc atggaaaaga taaggatccg 5580
agctcgtgca cggtaccagg aggcagctat atgttggttc gtcttgtcat catactcatc 5640
gcgcctccgg tcctgatctg gaccttcgta gccgagttct ggggcgacgt gaaaaaagcg 5700
ccttggtacg cgtggaacgc ctgcagccaa gaacttgacg gcattcgccg cgcatggcgc 5760
gcaaaatcta tccgagaaga ggactggaaa tgaggtacct gtacaccgcg gctcgagagg 5820
aggcagctat atggctgacc tgacgaagta cgcagatctc attgagcggc ttgaaaaggt 5880
aacggggcca gatcgtggaa ttgatcgcga tatagcagag catatcgttg ggacgaaata 5940
tcgttcgacg cagcgagggc gcgaatggct tgaagacagc catggcggcg tggagacatg 6000
gacgcgctat cccgtgccgt ataccgcttc catagacgca gccatcgctc tcatcgagaa 6060
gattctaccc ggttcggagc ttgagataac taatctctat ggggtcgcac gcgtcacgct 6120
ccacgatgtg gaaaactctt tccacggctc ggacccttgc aacaggatca atacagcttt 6180
gctgatcgca cttttccgca cccttgaggc gaaggcaggt gcagcatgac tcgagtgcac 6240
gctagcccta ggaggaggca gctatatgaa ccgcttaatc cgccgcgcca tccaccactg 6300
gctcgcttgg aagtcgagac agaagcttgc tcgggaatac aactggcaaa ccgagatcga 6360
cgccgagatc cgacaggcaa agcaatcccg cagcaagact ggacgtgtcc gcgatctgga 6420
acgccgcaag cgggacatga tgacacgcgc gctgggaggg cagaggtgat gacctaggct 6480
taaggagctc ccgcggccca gcccgcctaa tgagcgggct tttttttgaa caaagcttac 6540
cggtttattg actaccggaa gcagtgtgac cgtgtgcttc tcaaatgcct gaggccagtt 6600
tgctcaggct ctccccgtgg aggtaataat tgacgatatg atcatttatt ctgcctccca 6660
gagcctgata aaaacggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag 6720
gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg 6780
aggcggctac agccgatagt ctggaacagc gcacttacgg gttgctgcgc aacccaagtg 6840
ctaccggcgc ggcagcgtga cccgtgtcgg cggctccaac ggctcgccat cgtccagaaa 6900
acacggctca tcgggcatcg gcaggcgctg ctgcccgcgc cgttcccatt cctccgtttc 6960
ggtcaaggct ggcaggtctg gttccatgcc cggaatgccg ggctggctgg gcggctcctc 7020
gccggggccg gtcggtagtt gctgctcgcc cggatacagg gtcgggatgc ggcgcaggtc 7080
gccatgcccc aacagcgatt cgtcctggtc gtcgtgatca accaccacgg cggcactgaa 7140
caccgacagg cgcaactggt cgcggggctg gccccacgcc acgcggtcat tgaccacgta 7200
ggccgacacg gtgccggggc cgttgagctt cacgacggag atccagcgct cggccaccaa 7260
gtccttgact gcgtattgga ccgtccgcaa agaacgtccg atgagcttgg aaagtgtctt 7320
ctggctgacc accacggcgt tctggtggcc catctgcgcc acgaggtgat gcagcagcat 7380
tgccgccgtg ggtttcctcg caataagccc ggcccacgcc tcatgcgctt tgcgttccgt 7440
ttgcacccag tgaccgggct tgttcttggc ttgaatgccg atttctctgg actgcgtggc 7500
catgcttatc tccatgcggt agggtgccgc acggttgcgg caccatgcgc aatcagctgc 7560
aacttttcgg cagcgcgaca acaattatgc gttgcgtaaa agtggcagtc aattacagat 7620
tttctttaac ctacgcaatg agctattgcg gggggtgccg caatgagctg ttgcgtaccc 7680
ccctttttta agttgttgat ttttaagtct ttcgcatttc gccctatatc tagttctttg 7740
gtgcccaaag aagggcaccc ctgcggggtt cccccacgcc ttcggcgcgg ctccccctcc 7800
ggcaaaaagt ggcccctccg gggcttgttg atcgactgcg cggccttcgg ccttgcccaa 7860
ggtggcgctg cccccttgga acccccgcac tcgccgccgt gaggctcggg gggcaggcgg 7920
gcgggcttcg ccttcgactg cccccactcg cataggcttg ggtcgttcca ggcgcgtcaa 7980
ggccaagccg ctgcgcggtc gctgcgcgag ccttgacccg ccttccactt ggtgtccaac 8040
cggcaagcga agcgcgcagg ccgcaggccg gaggcttttc cccagagaaa attaaaaaaa 8100
ttgatggggc aaggccgcag gccgcgcagt tggagccggt gggtatgtgg tcgaaggctg 8160
ggtagccggt gggcaatccc tgtggtcaag ctcgtgggca ggcgcagcct gtccatcagc 8220
ttgtccagca gggttgtcca cgggccgagc gaagcgagcc agccggtggc cgctcgcggc 8280
catcgtccac atatccacgg gctggcaagg gagcgcagcg accgcgcagg gcgaagcccg 8340
gagagcaagc ccgtagggcg ccgcagccgc cgtaggcggt cacgactttg cgaagcaaag 8400
tctagtgagt atactcaagc attgagtggc ccgccggagg caccgccttg cgctgccccc 8460
gtcgagccgg ttggacacca aaagggaggg gcaggc 8496
<210> 2
<211> 8978
<212> DNA
<213> artificial sequence
<221> nucleotide sequence of pBBR1-kan-Ptet-ETh1h2h3P3_ rhi597
<222>（1）…（8978）
<400> 2
atggcggcat acgcgatcat gcgatgcaag aagctggcga aaatgggcaa cgtggcggcc 60
agtctcaagc acgcctaccg cgagcgcgag acgcccaacg ctgacgccag caggacgcca 120
gagaacgagc actgggcggc cagcagcacc gatgaagcga tgggccgact gcgcgagttg 180
ctgccagaga agcggcgcaa ggacgctgtg ttggcggtcg agtacgtcat gacggccagc 240
ccggaatggt ggaagtcggc cagccaagaa cagcaggcgg cgttcttcga gaaggcgcac 300
aagtggctgg cggacaagta cggggcggat cgcatcgtga cggccagcat ccaccgtgac 360
gaaaccagcc cgcacatgac cgcgttcgtg gtgccgctga cgcaggacgg caggctgtcg 420
gccaaggagt tcatcggcaa caaagcgcag atgacccgcg accagaccac gtttgcggcc 480
gctgtggccg atctagggct gcaacggggc atcgagggca gcaaggcacg tcacacgcgc 540
attcaggcgt tctacgaggc cctggagcgg ccaccagtgg gccacgtcac catcagcccg 600
caagcggtcg agccacgcgc ctatgcaccg cagggattgg ccgaaaagct gggaatctca 660
aagcgcgttg agacgccgga agccgtggcc gaccggctga caaaagcggt tcggcagggg 720
tatgagcctg ccctacaggc cgccgcagga gcgcgtgaga tgcgcaagaa ggccgatcaa 780
gcccaagaga cggcccgaga ccttcgggag cgcctgaagc ccgttctgga cgccctgggg 840
ccgttgaatc gggatatgca ggccaaggcc gccgcgatca tcaaggccgt gggcgaaaag 900
ctgctgacgg aacagcggga agtccagcgc cagaaacagg cccagcgcca gcaggaacgc 960
gggcgcgcac atttccccga aaagtgccac ctgggatgaa tgtcagctac tgggctatct 1020
ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc 1080
gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc 1140
cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga 1200
tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga 1260
ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 1320
atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 1380
aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg 1440
acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 1500
acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 1560
tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 1620
ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 1680
agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 1740
atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg 1800
aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 1860
gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 1920
cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 1980
tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 2040
agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc 2100
atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt 2160
ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca 2220
cccccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg 2280
ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt 2340
ttttatgcag atatcaatta atttaagacc cactttcaca tttaagttgt ttttctaatc 2400
cgcatatgat caattcaagg ccgaataaga aggctggctc tgcaccttgg tgatcaaata 2460
attcgatagc ttgtcgtaat aatggcggca tactatcagt agtaggtgtt tccctttctt 2520
ctttagcgac ttgatgctct tgatcttcca atacgcaacc taaagtaaaa tgccccacag 2580
cgctgagtgc atataatgca ttctctagtg aaaaaccttg ttggcataaa aaggctaatt 2640
gattttcgag agtttcatac tgtttttctg taggccgtgt acctaaatgt acttttgctc 2700
catcgcgatg acttagtaaa gcacatctaa aacttttagc gttattacgt aaaaaatctt 2760
gccagctttc cccttctaaa gggcaaaagt gagtatggtg cctatctaac atctcaatgg 2820
ctaaggcgtc gagcaaagcc cgcttatttt ttacatgcca atacaatgta ggctgctcta 2880
cacctagctt ctgggcgagt ttacgggttg ttaaaccttc gattccgacc tcattaagca 2940
gctctaatgc gctgttaatc actttacttt tatctaatct agacatcatt aattcctaat 3000
ttttgttgac actctatcat tgatagagtt attttaccac tccctatcag tgatagagag 3060
aattcatatg tcggttgaat gcatcctgcc tggcgatcgc gccgactggc ttgaactccg 3120
ccgcggcttc gtaaccgctt ccgtcgccgg cgcgctccta cagtgccacc cctatacgac 3180
ggcctatcag ctctgggcgc tcaagtccgg ccgcctcgaa gagagcaccg aagaaaacga 3240
agcgatgcgg cgcggccgcc tgcttgagcc ggtcgctgta cagatgctcc gcgaggagcg 3300
tccgacatgg acgatcgatt acagggcgga taacgccttt tacctcgatc gcggtctgaa 3360
gctcggcgct acgccggact ccttcgcata tcggccggac ataagcggtc gcggcatcgt 3420
gcagttcaag acctcgtctg aggaagcttt tcgcgacaac tggatcgatc cggaatcccg 3480
cgaagttgag gttccgctct ggatcgccgt ccaggcgatc gtcgaggcaa agctgaccgg 3540
ggccgcctgg gcggcggtgg cggttcttgt tgtcggtcgc ggcatcaaga tggaagtcat 3600
cgacatccct ctccatgccc aagtatggga ggcgcttctc ggcgccgctt ccgaattctg 3660
gcgcatcact caggtcggag agcatccgcc tgtggactgg gatcgggacg gctcgaccgt 3720
tctcgacgtc aaccgctgga ccgaggcaaa gcggatcgac ctcacggccg acgagactgt 3780
cgacctgttt gccagcaatc tcgaggacac gcgcaccaag cggcgggagc tgcagaagcg 3840
cgaggacgtg ctccgcgcgc agatccttta tgcgctcggc tcggctgagg tcgcgaccac 3900
ccggcgcttt gagatcctcg ctccaaccgt cgtgcgcgcc gacggcgccg cacaacgcgc 3960
catccgcatc aaactaaagg atcaaagcaa tggacgcttc tgaaggaggc agctatatga 4020
acgacgtagc tatttcgaac gatccgaacg tccagacgct tcgggaccaa cttgagaaga 4080
ggcttggcag tttcgctgag gccctcccgt cccacatcac gccggagcag ttcaaggcaa 4140
ccctgatccg agccgctatg ggcgatgcca acctactgct cgccgatcgg gtttccttct 4200
tcgaagccgc cctcgccgcc gcaattgacg ggctgatgcc cgacaaaaag gaaggcgcga 4260
tggtggtcta caacaccaag atcaaagaga acggcaagga tatctggatc aagaaggtcc 4320
agtggatgcc gatgatccgt ggcatcttca gtaaggtcta caacaccggc ctggtgaaaa 4380
gcgccacagt cggcatcgtc tacggcggcg accagttccg ctcgtggacg gatgatgacg 4440
gcgagcacct ttttcacgag gaggcagaag agcaggacag gaaagtcatc cgccgcattt 4500
atgcgcaggt ggtaatgaaa gccggtggct gcttcgtcga cacgatgcga tccacagata 4560
tcgagaaggt tcgacagtcc tctaaaaaca aggacagcgg accgtgggcc gactggtggg 4620
aggagatggc gtttaagacc gtcttccggc acctctccaa gcgtttgccc ttctcgcgcg 4680
agatcactcc gctgctcgat cgagacaact tcctctacga cctcgcggct caagcgcgcg 4740
acatcacacc ggccggtgca cggccgcgtg gcataggcaa ccgcctggac gccctcgtcg 4800
gcatcaccga ccagtccgga tcctcgatgc cgatggaaaa gcttggggaa aagcaggggg 4860
agaaggaacc ggcgcagcgg aagaactccg agcgcaagcc ggccgagcag cgccagcagc 4920
gccgcgacaa ccagcggagc gatgaccggc gcaaccaaga caaccgcgac gacggtggtg 4980
acggggatca gtccgaagac gcggcagcag aggaagccgc cgcatatcgc gccggccggg 5040
aagcccgcgc caaggagatg agccgcaagg ccattcccgc cgaattcaag aaggacgagg 5100
cgctgacgac agcctggctc gaaggcttcg atgaggaggg caactgagct agcactagtc 5160
ctaggaggag gcagctatat ggatgacttc aaaccgggcg acaaaatgcc ggatagcgtg 5220
ccggtctcaa tcggagcccg ttcgcgccta accgtcgccg cctgcattcg caaatggtcc 5280
gccggcgctg cgctcaccga gcgcgagcgc gacctcgtcg acgaagccat cttccgcaac 5340
cgacttcaaa tgatcgaggc cgtcttcgag gaccgggaga ctgccgatgt cggttgacct 5400
aggcgcgcct taattaagga ggcagctata tggacgcttc tgacgtggcc ggcatcggcc 5460
acaacatgcc cccgctcgcc gaccggctgg cgattgacca caaggctctt ctcgacaagg 5520
tccagtcgct tgccgatcgg gcgaacgcgg cgaaagccgt cgccgacgag aagggcctca 5580
acggcgacga cgatatcctg ccgctgatcg agatcggcaa ggatgccagc aagctcgtga 5640
aggaaatcgg ggacacacgt ctcgccacca cgaagccgct gcgcgacgac atcgaaacca 5700
tcaacggctt tttcaacgtg gcggggaccc gggcggatcg catcaaaggt gctttcgccg 5760
aaaaggttgg ggaatacgac cgggagaagc gcgcgcggga agcacgcgac gcagcggagc 5820
gcgctcggat cgcccaggag gaagccgccg ccaagctcga ggaggcacag aacgcagagc 5880
attccgttct aggcgacgtc gtcatgaacg aggctgctgt gctggaagaa gccgcacaga 5940
aagctgctcg cgaggccgtc aaggctggta ccagccccac tcgcactgaa gctgggaccg 6000
tcagcaccag cggccgttgg accgctgaag tgatcgatag cgacaagatc ccgctcgagc 6060
agctccgccc attcatcaaa cttgctgatt tcgagaagtt ttgccgcgcc tacgcgcagg 6120
caaaccagga ccgcaagccc ctgcccggcg tccggatctt ccgcgactcc aagacctcct 6180
tccgctgatt aattaactag tgagctcagg aggcagctat atggatatcg cccctctctc 6240
tgttttccgc ctttgcgata ccgagacgac aggctttccg ccgcgcgctg agatgtgcga 6300
aatcggttgg gtcgacttcg ttctctatcc agatggctgg aagatcgaag gtgaacacca 6360
gagcaggttc gtcaatccag gccacccgat cccggcaaaa tgcaccgaaa tccacggcat 6420
caccgatgac atggttatcg acggtatgta cccgaatgaa gcccgcgcat ttctctcacg 6480
cggagctgcg gttctcggcg cccacaatgc agcgttcgac aagcagtttg tgcgctcggc 6540
actgccatgg atctgcaccc tcgaatgcgc tcgctatgtc tggccacagg cgccgaacca 6600
caagaacgag acgttgaagg actttcttgg cattacggtc gatggtgatg ctcatcgtgc 6660
cggctacgac gcggccgtgt ccgcgggcat cttcctgcaa ctgatcaagc atttgacgtt 6720
cgaagagatg cttgctctct ccgacccggg cgctgtcccg cttaagatgc cctttggcgt 6780
ccataagggg agccggttca ctgaaattcc cgactcctac ctgaagttca ttgtcggcag 6840
cgacatgcgc aagggcgtca agaccgccgc tcagaatgag atcaaccgcc ggaccgccac 6900
gaggccgtcg gtgccggcac ttcgcccccg ctcgagttcc tgggatcggg gtttctgaga 6960
gctcgctagc ggcgcgcccc cagcccgcct aatgagcggg cttttttttg aacaaagctt 7020
accggtttat tgactaccgg aagcagtgtg accgtgtgct tctcaaatgc ctgaggccag 7080
tttgctcagg ctctccccgt ggaggtaata attgacgata tgatcattta ttctgcctcc 7140
cagagcctga taaaaacggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg 7200
aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg 7260
cgaggcggct acagccgata gtctggaaca gcgcacttac gggttgctgc gcaacccaag 7320
tgctaccggc gcggcagcgt gacccgtgtc ggcggctcca acggctcgcc atcgtccaga 7380
aaacacggct catcgggcat cggcaggcgc tgctgcccgc gccgttccca ttcctccgtt 7440
tcggtcaagg ctggcaggtc tggttccatg cccggaatgc cgggctggct gggcggctcc 7500
tcgccggggc cggtcggtag ttgctgctcg cccggataca gggtcgggat gcggcgcagg 7560
tcgccatgcc ccaacagcga ttcgtcctgg tcgtcgtgat caaccaccac ggcggcactg 7620
aacaccgaca ggcgcaactg gtcgcggggc tggccccacg ccacgcggtc attgaccacg 7680
taggccgaca cggtgccggg gccgttgagc ttcacgacgg agatccagcg ctcggccacc 7740
aagtccttga ctgcgtattg gaccgtccgc aaagaacgtc cgatgagctt ggaaagtgtc 7800
ttctggctga ccaccacggc gttctggtgg cccatctgcg ccacgaggtg atgcagcagc 7860
attgccgccg tgggtttcct cgcaataagc ccggcccacg cctcatgcgc tttgcgttcc 7920
gtttgcaccc agtgaccggg cttgttcttg gcttgaatgc cgatttctct ggactgcgtg 7980
gccatgctta tctccatgcg gtagggtgcc gcacggttgc ggcaccatgc gcaatcagct 8040
gcaacttttc ggcagcgcga caacaattat gcgttgcgta aaagtggcag tcaattacag 8100
attttcttta acctacgcaa tgagctattg cggggggtgc cgcaatgagc tgttgcgtac 8160
cccccttttt taagttgttg atttttaagt ctttcgcatt tcgccctata tctagttctt 8220
tggtgcccaa agaagggcac ccctgcgggg ttcccccacg ccttcggcgc ggctccccct 8280
ccggcaaaaa gtggcccctc cggggcttgt tgatcgactg cgcggccttc ggccttgccc 8340
aaggtggcgc tgcccccttg gaacccccgc actcgccgcc gtgaggctcg gggggcaggc 8400
gggcgggctt cgccttcgac tgcccccact cgcataggct tgggtcgttc caggcgcgtc 8460
aaggccaagc cgctgcgcgg tcgctgcgcg agccttgacc cgccttccac ttggtgtcca 8520
accggcaagc gaagcgcgca ggccgcaggc cggaggcttt tccccagaga aaattaaaaa 8580
aattgatggg gcaaggccgc aggccgcgca gttggagccg gtgggtatgt ggtcgaaggc 8640
tgggtagccg gtgggcaatc cctgtggtca agctcgtggg caggcgcagc ctgtccatca 8700
gcttgtccag cagggttgtc cacgggccga gcgaagcgag ccagccggtg gccgctcgcg 8760
gccatcgtcc acatatccac gggctggcaa gggagcgcag cgaccgcgca gggcgaagcc 8820
cggagagcaa gcccgtaggg cgccgcagcc gccgtaggcg gtcacgactt tgcgaagcaa 8880
agtctagtga gtatactcaa gcattgagtg gcccgccgga ggcaccgcct tgcgctgccc 8940
ccgtcgagcc ggttggacac caaaagggag gggcaggc 8978
<210> 3
<211> 6473
<212> DNA
<213> artificial sequence
<221> nucleotide sequence of pBBR1-kan-Ptet-ET_ rhi145
<222>（1）…（6473）
<400> 3
atggcggcat acgcgatcat gcgatgcaag aagctggcga aaatgggcaa cgtggcggcc 60
agtctcaagc acgcctaccg cgagcgcgag acgcccaacg ctgacgccag caggacgcca 120
gagaacgagc actgggcggc cagcagcacc gatgaagcga tgggccgact gcgcgagttg 180
ctgccagaga agcggcgcaa ggacgctgtg ttggcggtcg agtacgtcat gacggccagc 240
ccggaatggt ggaagtcggc cagccaagaa cagcaggcgg cgttcttcga gaaggcgcac 300
aagtggctgg cggacaagta cggggcggat cgcatcgtga cggccagcat ccaccgtgac 360
gaaaccagcc cgcacatgac cgcgttcgtg gtgccgctga cgcaggacgg caggctgtcg 420
gccaaggagt tcatcggcaa caaagcgcag atgacccgcg accagaccac gtttgcggcc 480
gctgtggccg atctagggct gcaacggggc atcgagggca gcaaggcacg tcacacgcgc 540
attcaggcgt tctacgaggc cctggagcgg ccaccagtgg gccacgtcac catcagcccg 600
caagcggtcg agccacgcgc ctatgcaccg cagggattgg ccgaaaagct gggaatctca 660
aagcgcgttg agacgccgga agccgtggcc gaccggctga caaaagcggt tcggcagggg 720
tatgagcctg ccctacaggc cgccgcagga gcgcgtgaga tgcgcaagaa ggccgatcaa 780
gcccaagaga cggcccgaga ccttcgggag cgcctgaagc ccgttctgga cgccctgggg 840
ccgttgaatc gggatatgca ggccaaggcc gccgcgatca tcaaggccgt gggcgaaaag 900
ctgctgacgg aacagcggga agtccagcgc cagaaacagg cccagcgcca gcaggaacgc 960
gggcgcgcac atttccccga aaagtgccac ctgggatgaa tgtcagctac tgggctatct 1020
ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc 1080
gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc 1140
cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga 1200
tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga 1260
ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 1320
atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 1380
aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg 1440
acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 1500
acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 1560
tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 1620
ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 1680
agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 1740
atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg 1800
aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 1860
gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 1920
cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 1980
tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 2040
agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc 2100
atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt 2160
ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca 2220
cccccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg 2280
ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt 2340
ttttatgcag atatcaatta atttaagacc cactttcaca tttaagttgt ttttctaatc 2400
cgcatatgat caattcaagg ccgaataaga aggctggctc tgcaccttgg tgatcaaata 2460
attcgatagc ttgtcgtaat aatggcggca tactatcagt agtaggtgtt tccctttctt 2520
ctttagcgac ttgatgctct tgatcttcca atacgcaacc taaagtaaaa tgccccacag 2580
cgctgagtgc atataatgca ttctctagtg aaaaaccttg ttggcataaa aaggctaatt 2640
gattttcgag agtttcatac tgtttttctg taggccgtgt acctaaatgt acttttgctc 2700
catcgcgatg acttagtaaa gcacatctaa aacttttagc gttattacgt aaaaaatctt 2760
gccagctttc cccttctaaa gggcaaaagt gagtatggtg cctatctaac atctcaatgg 2820
ctaaggcgtc gagcaaagcc cgcttatttt ttacatgcca atacaatgta ggctgctcta 2880
cacctagctt ctgggcgagt ttacgggttg ttaaaccttc gattccgacc tcattaagca 2940
gctctaatgc gctgttaatc actttacttt tatctaatct agacatcatt aattcctaat 3000
ttttgttgac actctatcat tgatagagtt attttaccac tccctatcag tgatagagag 3060
aattcatatg gcaaacgcag ttgaaatcca gcgtccgggc gagcagacgg cagtggcgcc 3120
ggcggcagct atgacgccta tggaaatgct cgaccgcgct gtttcgcaag gcgcctcggt 3180
cgaaacgctg tccaaactga tggacctgca ggagcgctgg gaaaagaacc aggcacgcaa 3240
ggcgtttgct gcggcaatgt cagcagtcaa ggctgaactg ccgcggatcg tcaaaacccg 3300
caaggtagac ttcaccagcg ccaagggccg gaccaactac cagtacgaag acctcgcagg 3360
gatcatggat caagtgggcc cggtgctgtc ccgccatggt ctttctgtcc gatatcgcac 3420
ggttgccgag ccgaaccagc cgatatctgt cacctgcatc atcgagcatt ccgacggcca 3480
ccacgaggag aacaccctga tggccggccg cgacgacagc ggcaacaaga acagcattca 3540
gcagatcggt tccactgtca cgtacctcca gcgctacacg ctcaaggccg cgctcggtct 3600
ggcggcagct gctgacgatg atggctcgaa ggccgacgat accggcacga tcaccgaagc 3660
cgagcgcgaa atcatcctca gcatgatcga tgagacggaa tccgacatcg agaaattctg 3720
cgcggctctg caaatcgaga gcgttgccac gatgccggcc gccaagttcc gccgagctgt 3780
cggcatgctc gaagccaaga aaaagaaggt ggccgcaaat ggatgagatc attcagggaa 3840
gcgcggaatg gcatgctctc cgctgcggga aggtcacggc ctctcgcgtg gcggacgtta 3900
tcacccgcac caagacagga tggggcgcat cccgggccaa ctatgccgcc gagctgatcg 3960
cggagcgcct gacgggtgtt gcggccgaag gcttcaccaa cgccgcgatg cagtggggca 4020
ccgatcagga gccgaacgcg cgcatggctt acgagttcat gcacgacgtc acagtcgagc 4080
agatcgcttt cgtgattcat ccgtccatcc cggatgccgg cgcgtctcct gacggccttg 4140
tgggcgaaag cgggctggtg gagatcaagt gcccgaatac cgcaacccac atcgacacgc 4200
tgatcaagca ggagataccg gccaagtacg tcacgcagat gatgtggcag atggcctgca 4260
ccggccggca atggtgcgat ttcgtctcct acgaccctcg cctgccggag agcatgcagt 4320
tgttcgtcaa gcgcattgag cggaacgacg acatgatcac cgatcttgag gatgccgtcc 4380
ggttgttcct cgatacggag gtcgtttcga aggtggacgc tcttcggagc atctacgagc 4440
aggaggccgc ataaaggagg gtgcactgct agccccagcc cgcctaatga gcgggctttt 4500
ttttgaacaa agcttaccgg tttattgact accggaagca gtgtgaccgt gtgcttctca 4560
aatgcctgag gccagtttgc tcaggctctc cccgtggagg taataattga cgatatgatc 4620
atttattctg cctcccagag cctgataaaa acggtgaatc cgttagcgag gtgccgccgg 4680
cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga 4740
caaggtatag ggcggcgagg cggctacagc cgatagtctg gaacagcgca cttacgggtt 4800
gctgcgcaac ccaagtgcta ccggcgcggc agcgtgaccc gtgtcggcgg ctccaacggc 4860
tcgccatcgt ccagaaaaca cggctcatcg ggcatcggca ggcgctgctg cccgcgccgt 4920
tcccattcct ccgtttcggt caaggctggc aggtctggtt ccatgcccgg aatgccgggc 4980
tggctgggcg gctcctcgcc ggggccggtc ggtagttgct gctcgcccgg atacagggtc 5040
gggatgcggc gcaggtcgcc atgccccaac agcgattcgt cctggtcgtc gtgatcaacc 5100
accacggcgg cactgaacac cgacaggcgc aactggtcgc ggggctggcc ccacgccacg 5160
cggtcattga ccacgtaggc cgacacggtg ccggggccgt tgagcttcac gacggagatc 5220
cagcgctcgg ccaccaagtc cttgactgcg tattggaccg tccgcaaaga acgtccgatg 5280
agcttggaaa gtgtcttctg gctgaccacc acggcgttct ggtggcccat ctgcgccacg 5340
aggtgatgca gcagcattgc cgccgtgggt ttcctcgcaa taagcccggc ccacgcctca 5400
tgcgctttgc gttccgtttg cacccagtga ccgggcttgt tcttggcttg aatgccgatt 5460
tctctggact gcgtggccat gcttatctcc atgcggtagg gtgccgcacg gttgcggcac 5520
catgcgcaat cagctgcaac ttttcggcag cgcgacaaca attatgcgtt gcgtaaaagt 5580
ggcagtcaat tacagatttt ctttaaccta cgcaatgagc tattgcgggg ggtgccgcaa 5640
tgagctgttg cgtacccccc ttttttaagt tgttgatttt taagtctttc gcatttcgcc 5700
ctatatctag ttctttggtg cccaaagaag ggcacccctg cggggttccc ccacgccttc 5760
ggcgcggctc cccctccggc aaaaagtggc ccctccgggg cttgttgatc gactgcgcgg 5820
ccttcggcct tgcccaaggt ggcgctgccc ccttggaacc cccgcactcg ccgccgtgag 5880
gctcgggggg caggcgggcg ggcttcgcct tcgactgccc ccactcgcat aggcttgggt 5940
cgttccaggc gcgtcaaggc caagccgctg cgcggtcgct gcgcgagcct tgacccgcct 6000
tccacttggt gtccaaccgg caagcgaagc gcgcaggccg caggccggag gcttttcccc 6060
agagaaaatt aaaaaaattg atggggcaag gccgcaggcc gcgcagttgg agccggtggg 6120
tatgtggtcg aaggctgggt agccggtggg caatccctgt ggtcaagctc gtgggcaggc 6180
gcagcctgtc catcagcttg tccagcaggg ttgtccacgg gccgagcgaa gcgagccagc 6240
cggtggccgc tcgcggccat cgtccacata tccacgggct ggcaagggag cgcagcgacc 6300
gcgcagggcg aagcccggag agcaagcccg tagggcgccg cagccgccgt aggcggtcac 6360
gactttgcga agcaaagtct agtgagtata ctcaagcatt gagtggcccg ccggaggcac 6420
cgccttgcgc tgcccccgtc gagccggttg gacaccaaaa gggaggggca ggc 6473
<210> 4
<211> 7029
<212> DNA
<213> artificial sequence
<221> nucleotide sequence of pBBR1-kan-Ptet-eth_ rhi483
<222>（1）…（7029）
<400> 4
atggcggcat acgcgatcat gcgatgcaag aagctggcga aaatgggcaa cgtggcggcc 60
agtctcaagc acgcctaccg cgagcgcgag acgcccaacg ctgacgccag caggacgcca 120
gagaacgagc actgggcggc cagcagcacc gatgaagcga tgggccgact gcgcgagttg 180
ctgccagaga agcggcgcaa ggacgctgtg ttggcggtcg agtacgtcat gacggccagc 240
ccggaatggt ggaagtcggc cagccaagaa cagcaggcgg cgttcttcga gaaggcgcac 300
aagtggctgg cggacaagta cggggcggat cgcatcgtga cggccagcat ccaccgtgac 360
gaaaccagcc cgcacatgac cgcgttcgtg gtgccgctga cgcaggacgg caggctgtcg 420
gccaaggagt tcatcggcaa caaagcgcag atgacccgcg accagaccac gtttgcggcc 480
gctgtggccg atctagggct gcaacggggc atcgagggca gcaaggcacg tcacacgcgc 540
attcaggcgt tctacgaggc cctggagcgg ccaccagtgg gccacgtcac catcagcccg 600
caagcggtcg agccacgcgc ctatgcaccg cagggattgg ccgaaaagct gggaatctca 660
aagcgcgttg agacgccgga agccgtggcc gaccggctga caaaagcggt tcggcagggg 720
tatgagcctg ccctacaggc cgccgcagga gcgcgtgaga tgcgcaagaa ggccgatcaa 780
gcccaagaga cggcccgaga ccttcgggag cgcctgaagc ccgttctgga cgccctgggg 840
ccgttgaatc gggatatgca ggccaaggcc gccgcgatca tcaaggccgt gggcgaaaag 900
ctgctgacgg aacagcggga agtccagcgc cagaaacagg cccagcgcca gcaggaacgc 960
gggcgcgcac atttccccga aaagtgccac ctgggatgaa tgtcagctac tgggctatct 1020
ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc 1080
gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc 1140
cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga 1200
tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga 1260
ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 1320
atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 1380
aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg 1440
acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 1500
acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 1560
tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 1620
ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 1680
agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 1740
atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg 1800
aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 1860
gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 1920
cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 1980
tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 2040
agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc 2100
atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt 2160
ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca 2220
cccccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg 2280
ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt 2340
ttttatgcag atatcaatta atttaagacc cactttcaca tttaagttgt ttttctaatc 2400
cgcatatgat caattcaagg ccgaataaga aggctggctc tgcaccttgg tgatcaaata 2460
attcgatagc ttgtcgtaat aatggcggca tactatcagt agtaggtgtt tccctttctt 2520
ctttagcgac ttgatgctct tgatcttcca atacgcaacc taaagtaaaa tgccccacag 2580
cgctgagtgc atataatgca ttctctagtg aaaaaccttg ttggcataaa aaggctaatt 2640
gattttcgag agtttcatac tgtttttctg taggccgtgt acctaaatgt acttttgctc 2700
catcgcgatg acttagtaaa gcacatctaa aacttttagc gttattacgt aaaaaatctt 2760
gccagctttc cccttctaaa gggcaaaagt gagtatggtg cctatctaac atctcaatgg 2820
ctaaggcgtc gagcaaagcc cgcttatttt ttacatgcca atacaatgta ggctgctcta 2880
cacctagctt ctgggcgagt ttacgggttg ttaaaccttc gattccgacc tcattaagca 2940
gctctaatgc gctgttaatc actttacttt tatctaatct agacatcatt aattcctaat 3000
ttttgttgac actctatcat tgatagagtt attttaccac tccctatcag tgatagagag 3060
aattcatatg ggcgcagtag cacgtcacga cgaacagagc gttattccca tttccgacgc 3120
gccaatggtg gcgatgatcg aacgcatcgt tatggacccg tccattccga tcgatcgcct 3180
tgagcaaatg atggcgatga aagagcgcat ggaagaccgc gctcgaacga cggcgaagga 3240
agaccgggac tttgaagccc gcaaggcata cttcgcagct atgtcggctt gccagaagca 3300
gttgccggtt gtcgtcaagt cccaacgcaa cagccacacc aattcgaact atgccgacct 3360
ggctgcgatc gaggcacagg ccatgccgat catccatgac cacggctttg gcgtctcgtt 3420
ccagccggac ggatacaacg acaacggcga gctgcgcatc ctttgggaga tttcccacaa 3480
tgaaggatac gtgcgcaacg gcgtcggcga aatccccgta gatggggccg ggaccggcgg 3540
caaggtcaac aagaccggaa ctcaggcttt cggaagcact gccacctacg gccgccggta 3600
cctgctctgc atgctgttca acatcagcac aggtgatgat cgggacggca acaaggctcc 3660
tgacaccggc ggcccgatct ctgatgagca gaccattgcc cttcgcgaaa agatcgaggc 3720
cgtcgaggct gatatcgagc gcttctgcag ccggtggaaa atcgaagctc tgaaagacct 3780
ccccaagggc caattcaagg acgccatggt ttcgcttgac cggttcggcg agcagaaaaa 3840
acagcgcaat gcggaggatc gcaacaatgg ataatatcgt tcagggctca cccgaatggc 3900
tggccatgcg ggctggcaag gtaaccgcct cgcgtgtcgc cgatgtcatc gccaagacca 3960
agaccggcgt gtcggcatcc cgagccaagt atgctggcga gcttatcgcc gagcggttga 4020
ccggacaacc ggcggaacgc ttcaccaacg gcgctatggc atggggaacc gaaaaggaac 4080
ccgatgcccg caaggcctat gagttctatc gggatactga tgttgcggag gtcgccttcg 4140
ttcctcaccc gacgatcgcc gacagcggcg catcgcctga cggcttggtg gacgtcgaag 4200
gtctcttgga gattaagtgc cccgaaacgc acacccacat cgaaacgctg ctcaacaagg 4260
ccgtgccgtc aaagtacgtc acccagatga tgtggcagat ggcatgcacg gggcggaaat 4320
ggtgcgattt cgtatccttc gaccctcgcc tgcccgaatc catgcagttc ttttgccagc 4380
gcgttcatcg cgatgaagcc gtgatcgccg agcttgagcg cgaagtggtc gtgtttatca 4440
acgaggtgcg cggcaaggtc gccgagcttc gccgcctcta tgaaccggcc gaagcggatc 4500
ctgcagcttc gatgctgatg gctggctgaa ggagggtgca ctatgggcac cgtcaaccgc 4560
atcgtagcaa acgaactcga caaggatatc ctcatccggc tgatccgggc gaggaagccc 4620
ccgttcacga caacgatgac ggatggcaag caccggacga acccgcagaa caaactgcag 4680
cggaaatgga tgaccgagat cgccgaccag ctcggcgatc ggacagccga ggaagtgcgc 4740
ggcgaatgca agctcatgct tggcgtaccg atcctgcgcg ccgagaacga ggcattctgc 4800
aaggcctatg acgaacacgt caagccgctg tcctacgagc agaagctggc cttcatgatg 4860
atgccgcttg acttcccagt tacccggcta atgaccacgg cgcagagcaa gcaatacctg 4920
gatgcgatcc accggcatta ctcggcgcag ggtatctatc tcaccaatcc cgaagatcgc 4980
ggtcgcgccg agatggcgag ggcgtcatga aggagggtgc actgctagcc ccagcccgcc 5040
taatgagcgg gctttttttt gaacaaagct taccggttta ttgactaccg gaagcagtgt 5100
gaccgtgtgc ttctcaaatg cctgaggcca gtttgctcag gctctccccg tggaggtaat 5160
aattgacgat atgatcattt attctgcctc ccagagcctg ataaaaacgg tgaatccgtt 5220
agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc 5280
aacgcgggga ggcagacaag gtatagggcg gcgaggcggc tacagccgat agtctggaac 5340
agcgcactta cgggttgctg cgcaacccaa gtgctaccgg cgcggcagcg tgacccgtgt 5400
cggcggctcc aacggctcgc catcgtccag aaaacacggc tcatcgggca tcggcaggcg 5460
ctgctgcccg cgccgttccc attcctccgt ttcggtcaag gctggcaggt ctggttccat 5520
gcccggaatg ccgggctggc tgggcggctc ctcgccgggg ccggtcggta gttgctgctc 5580
gcccggatac agggtcggga tgcggcgcag gtcgccatgc cccaacagcg attcgtcctg 5640
gtcgtcgtga tcaaccacca cggcggcact gaacaccgac aggcgcaact ggtcgcgggg 5700
ctggccccac gccacgcggt cattgaccac gtaggccgac acggtgccgg ggccgttgag 5760
cttcacgacg gagatccagc gctcggccac caagtccttg actgcgtatt ggaccgtccg 5820
caaagaacgt ccgatgagct tggaaagtgt cttctggctg accaccacgg cgttctggtg 5880
gcccatctgc gccacgaggt gatgcagcag cattgccgcc gtgggtttcc tcgcaataag 5940
cccggcccac gcctcatgcg ctttgcgttc cgtttgcacc cagtgaccgg gcttgttctt 6000
ggcttgaatg ccgatttctc tggactgcgt ggccatgctt atctccatgc ggtagggtgc 6060
cgcacggttg cggcaccatg cgcaatcagc tgcaactttt cggcagcgcg acaacaatta 6120
tgcgttgcgt aaaagtggca gtcaattaca gattttcttt aacctacgca atgagctatt 6180
gcggggggtg ccgcaatgag ctgttgcgta cccccctttt ttaagttgtt gatttttaag 6240
tctttcgcat ttcgccctat atctagttct ttggtgccca aagaagggca cccctgcggg 6300
gttcccccac gccttcggcg cggctccccc tccggcaaaa agtggcccct ccggggcttg 6360
ttgatcgact gcgcggcctt cggccttgcc caaggtggcg ctgccccctt ggaacccccg 6420
cactcgccgc cgtgaggctc ggggggcagg cgggcgggct tcgccttcga ctgcccccac 6480
tcgcataggc ttgggtcgtt ccaggcgcgt caaggccaag ccgctgcgcg gtcgctgcgc 6540
gagccttgac ccgccttcca cttggtgtcc aaccggcaag cgaagcgcgc aggccgcagg 6600
ccggaggctt ttccccagag aaaattaaaa aaattgatgg ggcaaggccg caggccgcgc 6660
agttggagcc ggtgggtatg tggtcgaagg ctgggtagcc ggtgggcaat ccctgtggtc 6720
aagctcgtgg gcaggcgcag cctgtccatc agcttgtcca gcagggttgt ccacgggccg 6780
agcgaagcga gccagccggt ggccgctcgc ggccatcgtc cacatatcca cgggctggca 6840
agggagcgca gcgaccgcgc agggcgaagc ccggagagca agcccgtagg gcgccgcagc 6900
cgccgtaggc ggtcacgact ttgcgaagca aagtctagtg agtatactca agcattgagt 6960
ggcccgccgg aggcaccgcc ttgcgctgcc cccgtcgagc cggttggaca ccaaaaggga 7020
ggggcaggc 7029
Claims (1)
1. Agrobacterium homologous recombination system expression plasmid pBBR1-kan-P tet Use of pluγ -eth_ rhi483 for gene knockout in agrobacterium rhizogenes (Rhizobium rhizogenes) NBRC 13257; wherein said pBBR1-kan-P tet The nucleotide sequence of the-eth_ rhi483 is shown as SEQ ID No.4, the gene knockout is that a 3'-5' exonuclease gene or an endoglucanase gene is replaced by using an apr PCR fragment with a homology arm under the action of recombinase to realize the 3'-5' exonuclease gene or the endoglucanase gene knockout of agrobacterium, wherein the length of the homology arm is 80bp, and the homology arm is loaded at two ends of the apr gene through primer synthesis and combination PCR; the primer is as follows, lower case letters of the primer are homologous arms, and upper case letters of the primer are primers;
13257-exo-apra-5：
ctaggaaaaagcctgcttgatctcctttaggagaaatggcttttgtggagagccatgacatcagtcattacgacgtctgcAGCTGAATTA CATTCCCAACCG
13257-exo-apra-3：
tgcaaaagctgcattgtgtgttgcccggtgtttgactgttttctattctaaagcctgatcatcaatcatcgaaagctcgcCAACTTAAATGT GAAAGTGGGTC
13257-glu-apra-5：
ctccatcgtctcacggtctctcatcgtcatgacaactgctgttgccgcgactgccaaggaaaccgtaatcagagaggattAGCTGAATTA CATTCCCAACCG
13257-glu-apra-3：
cctcattgctgatcggcataacgacggcgggcattctcgcaaagatcggcaggcgcggatgaagcgctggcgcatcatggCAACTTAA ATGTGAAAGTGGGTC
expression plasmid pBBR1-kan-P of agrobacterium homologous recombination system tet The recombination conditions for eth_ rhi483 are: initial OD 600 =0.1, transfer 6h at 30 ℃ to OD 600 Inducing at 28 deg.c for 0.5-0.4 hr and 10 wt% concentration of SH buffer solution with sucrose+2 micron MHEPES to prepare competent cell at room temperature, exogenous DNA amount of 4 μg, homology arm length of 80bp, electrotransformation voltage of 1900V/mm and recovery time of 3 hr.
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