CN113817765A - Agrobacterium homologous recombination system and application thereof - Google Patents
Agrobacterium homologous recombination system and application thereof Download PDFInfo
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Abstract
The invention discloses an agrobacterium homologous recombination system, which consists of a series of expression plasmids of the agrobacterium homologous recombination system, and the expression plasmids are respectively named as pBBR1-kan-Ptet-ETh1h2h3h4_ agroB6, the nucleotide sequence of which is shown in SEQ ID No. 1; pBBR1-kan-Ptet-ETh1h2h3P3 — rhi597, the nucleotide sequence of which is shown in SEQ ID No. 2; pBBR1-kan-PtetET _ rhi145, the nucleotide sequence of which is shown in SEQ ID No. 3; pBBR1-kan-PtetETh _ rhi483, the nucleotide sequence of which is shown in SEQ ID No. 4. The invention also discloses application of the recombination system in mediating homologous recombination of short homologous arms in agrobacterium to genetic modification of genome DNA and application of the recombination system in gene knockout in agrobacterium. The agrobacterium homologous recombination system can greatly promote genome modification of agrobacterium,the genetic modification of the agrobacterium is simple and rapid, and the method has great application prospect in the aspect of constructing agrobacterium strains with high transformation efficiency and host range and in 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 tumefaciens and construction and application thereof, belonging to the field of microbial genetic engineering.
Background
The process of homologous recombination mediated by RecET and Red alpha beta gamma system is collectively called Red/ET homologous recombination engineering. The Red/ET homologous recombination engineering can accurately insert, knock out or replace target genes at any position of an escherichia coli genome, and the like, and can realize the simplification of the escherichia coli genome by being used with systems such as site-specific recombinase (Cre/loxP, Flp/FRT) or CRISPR-Cas and the like. The successful application of Red/ET recombination engineering promotes the development of the whole genetic engineering technology, wherein RecE and RecT homologous recombination proteins are derived from a Rac prophage, and Red alpha, Red beta and Red gamma homologous recombination proteins are derived from a Lambda bacteriophage.
The Red/ET homologous recombination engineering realizes low consumption, high efficiency and accurate genome editing, and 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 the homologous recombination efficiency.
Because the phage recombinase has certain host specificity, the Red/ET system has very low recombination efficiency or no recombination function in bacteria with far relativity, and therefore, a homologous recombination system suitable for different strains needs to be developed. Recombinant systems constructed by using recombinant functional operons of bacteriophages, such as Mycobacterium smegmatis, Pseudomonas syringae, Lactobacillus reuteri, Photorhabdus luminescens, Xenorhabdus nematophilus, Burkholderia strain, Mycobacterium tuberculosis, Bacillus subtilis, and the like have been reported.
Agrobacterium belongs to the family Rhizobiaceae (Rhizobiaceae), and is classified into Agrobacterium tumefaciens (Agrobacterium tumefaciens) capable of infecting plants, Agrobacterium radiobacter (Agrobacterium radiobacter) without infection ability, and Agrobacterium rhizogenes (Rhizobium rhizogenes) that can cause plants to grow hairy roots, according to whether it has infectivity, and the host range and infection symptoms of Agrobacterium. The root cancer and agrobacterium rhizogenes can transfer and integrate a section of DNA (transferred DNA, T-DNA) carried by self plasmid into host plant genome, and the gene in the T-DNA region can be replaced by any DNA sequence, so that the gene transfer and T-DNA transfer integrated gene expression vector has very important significance for gene transfer research, plant gene function research, crop transgenic breeding and other aspects. The transformation efficiency of the agrobacterium is improved, and the host range of the agrobacterium is expanded, so that a plurality of problems to be solved exist, and a set of efficient and simple homologous recombination system is very necessary to be established in the agrobacterium. Through retrieval, no literature report exists on researches on an agrobacterium tumefaciens self homologous recombination system and construction and application thereof.
Disclosure of Invention
Aiming at the defect that an efficient and simple genetic operation system is lacked 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 recombinant 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 mediating homologous recombination of short homologous arms in agrobacterium to genetic modification of genome DNA or gene knockout in agrobacterium.
The agrobacterium homologous recombination system consists of a series of expression plasmids of the agrobacterium homologous recombination system, and comprises a pBBR1 replication origin, a kanamycin resistance gene and tetracyclineInducible promoters and homologous recombination operons derived from Agrobacterium (as shown in FIG. 1); the method is characterized in that: the expression plasmids of the agrobacterium homologous recombination system are respectively named as pBBR1-kan-Ptet-ETh1h2h3h4_ agroB6, the nucleotide sequence of which is shown in SEQ ID No. 1; pBBR1-kan-Ptet-ETh1h2h3P3 — rhi597, the nucleotide sequence of which is shown in SEQ ID No. 2; pBBR1-kan-PtetET _ rhi145, the nucleotide sequence of which is shown in SEQ ID No. 3; pBBR1-kan-Ptet-ETh _ rhi483, the nucleotide sequence of which is shown in SEQ ID No.4 (shown in fig. 2); pBBR1-kan-Ptet-redγ-ETh1h2h3h4_agroB6；pBBR1-kan-Ptet-redγ-ETh1h2h3P3_rhi597；pBBR1-kan-Ptet-redγ-ET_rhi145；pBBR1-kan-Ptet-redγ-ETh_rhi483；pBBR1-kan-Ptet-pluγ-ETh1h2h3h4_agroB6；pBBR1-kan-Ptet-pluγ-ETh1h2h3P3_rhi597；pBBR1-kan-Ptet-pluγ-ET_rhi145；pBBR1-kan-Ptet-plu γ -ETh _ rhi 483; homologous recombinase operon ETh1h2h3h4 related to expression plasmid of agrobacterium homologous recombination systemAGROB6、ETh1h2h3P3RHI597、ETRHI145And EThRHI483Derived from Agrobacterium tumefaciens str. B6, Rhizobium leguminosa Bv. trifolii WSM597, Rhizobium sp. LC145 and Rhizobium sp. Root483D2, respectively.
The construction method of the expression plasmid of the agrobacterium homologous recombination system comprises the following steps:
(1)pBBR1-kan-Ptetthe firefly is an initial plasmid, EcoRI and NheI are subjected to double enzyme digestion, and a target linear fragment pBBR1-P is recovered from geltet-kan, about 5kb in length;
(2) recovering the linear fragment pBBR1-P from the gel obtained in the step (1)tet-kan is respectively co-transformed with synthesized recombinase fragments ETh1h2h3h4_ agroB6HA, ETh1h2h3P3_ rhi597HA, ET _ rhi145HA and ETh _ rhi483HA with vector homology arms into E.coli GB05-dir for linear recombination;
(3) enzyme digestion verification is carried out on the recombinants obtained in the step (2), so that expression plasmids of the homologous recombination system series of the agrobacterium are obtained, and the expression plasmids of the series are respectively named as pBBR1-kan-PtetETh1h2h3h4_ agroB6, nucleotide sequence thereof e.gSEQ ID No. 1; pBBR1-kan-Ptet-ETh1h2h3P3 — rhi597, the nucleotide sequence of which is shown in SEQ ID No. 2; pBBR1-kan-PtetET _ rhi145, the nucleotide sequence of which is shown in SEQ ID No. 3; pBBR1-kan-PtetETh _ rhi483, the nucleotide sequence of which is shown in SEQ ID No. 4.
The serial expression plasmid pBBR1-kan-P in the recombination systemtet-redγ-ETh1h2h3h4_agroB6、pBBR1-kan-Ptet-redγ-ETh1h2h3P3_rhi597、pBBR1-kan-Ptet-redγ-ET_rhi145、pBBR1-kan-Ptet-redγ-ETh_rhi483、pBBR1-kan-Ptet-pluγ-ETh1h2h3h4_agroB6、pBBR1-kan-Ptet-pluγ-ETh1h2h3P3_rhi597、pBBR1-kan-Ptet-plu γ -ET _ rhi145 or pBBR1-kan-PtetThe construction method of-plu γ -ETh _ rhi483 is as follows:
(1) first, pBBR1-kan-Ptet-ETh1h2h3h4_agroB6、pBBR1-kan-Ptet-ETh1h2h3P3_rhi597、pBBR1-kan-PtetET _ rhi145 and pBBR1-kan-Ptet-ETh _ rhi483 was co-transformed with cm-ccdB PCR products with homology arms into e.coli gbred-gyrA462 for loop recombination, respectively;
(2) and (3) carrying out enzyme digestion verification on the recombinants obtained in the step (1), and sequencing the plasmids with correct enzyme digestion verification.
(3) Respectively carrying out cotransformation on the plasmid with correct enzyme digestion and sequencing in the step (2) and red gamma or plu gamma with a homologous arm into E.coliGB08-red for loop recombination to obtain an expression plasmid of an agrobacterium homologous recombination system series; the method comprises the following steps: pBBR1-kan-Ptet-redγ-ETh1h2h3h4_agroB6、pBBR1-kan-Ptet-redγ-ETh1h2h3P3_rhi597、pBBR1-kan-Ptet-redγ-ET_rhi145、pBBR1-kan-Ptet-redγ-ETh_rhi483、pBBR1-kan-Ptet-pluγ-ETh1h2h3h4_agroB6、pBBR1-kan-Ptet-pluγ-ETh1h2h3P3_rhi597、pBBR1-kan-Ptet-plu γ -ET _ rhi145 and pBBR1-kan-Ptet-pluγ-ETh_rhi483。
The agrobacterium homologous recombination system is applied to genome DNA genetic modification by mediating homologous recombination of short homologous arms in agrobacterium, wherein the short homologous arms refer to homologous arms of 80bp-200 bp.
In the above application: the preferred expression plasmid series for the homologous recombination system of Agrobacterium is pBBR1-kan-Ptet-pluγ-ET_rhi145、pBBR1-kan-Ptet-ETh1h2h3h4_agroB6、pBBR1-kan-Ptet-ET _ rhi145 or pBBR1-kan-Ptet-ETh _ rhi 483; the Agrobacterium is preferably Agrobacterium tumefaciens C58, Agrobacterium tumefaciens EHA105 or Rhizobium rhizogenes NBRC 13257; the short homology arm preferably refers to 80bp-100bp homology arm.
In the above application: the serial expression plasmid pBBR1-kan-P of the homologous recombination system of agrobacteriumtet-plu γ -ET-rhi 145 having optimal recombination efficiency in Agrobacterium tumefaciens C58, pBBR1-kan-PtetETh1h2h3h4_ agroB6 having optimal recombination efficiency in Agrobacterium tumefaciens EHA105, pBBR1-kan-PtetPlu gamma-ETh _ rhi483 had optimal recombination efficiency in Rhizobium rhizogenes NBRC 13257. Wherein:
the expression plasmid pBBR1-kan-PtetThe optimal conditions for plu γ -ET-rhi 145 to improve recombination efficiency in Agrobacterium tumefaciens C58 were: starting OD600OD was adjusted at 30 ℃ for 4h under 0.16000.6-0.7, inducing at 30 deg.C for 1 hr, and treating with room temperature ddH at room temperature2Preparing competent cells, wherein the amount of exogenous DNA is 1.5 mu g, the length of a homologous arm is 80bp, the electrotransfer voltage is 1350V/mm, and the recovery time is2 h;
the expression plasmid pBBR1-kan-PtetThe optimization conditions for improving the recombination efficiency in the Agrobacterium tumefaciens EHA105 of ETh1h2h3h4_ agroB6 are: starting OD600OD was adjusted at 30 ℃ for 4h under 0.16000.6-0.7, inducing at 30 ℃ for 1h, preparing competent cells by GH buffer solution with a formula of 10% volume ratio of glycerol and 2 MuM HEPES at 4 ℃, wherein the amount of exogenous DNA is 1.5 Mug, the length of a homologous arm is 80bp, the electric conversion voltage is 1350V/mm, and the recovery time is2 h;
the expression plasmid pBBR1-kan-PtetThe optimal conditions for the plu γ -ETh-rhi 483 to improve the recombination efficiency in Rhizobium rhizogenes NBRC13257 are: starting OD600OD was adjusted at 30 ℃ for 6h under 0.16000.4-0.5, inducing at 28 deg.C for 0.5 hr, and using formula of 10Preparing competent cells at room temperature by using SH buffer solution of sucrose +2 mu M HEPES in percentage by mass, wherein the amount of exogenous DNA is 4 mu g, the length of a homologous arm is 80bp, the electric conversion voltage is 1900V/mm, and the recovery time is 3 h.
The agrobacterium homologous recombination system disclosed by 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 EHA 105; the Agrobacterium rhizogenes is preferably Rhizobium rhizogenes NBRC 13257.
The knockout is preferably performed under the optimized working conditions described above.
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 expression plasmids of the agrobacterium homologous recombination system, and the recombination system is not reported in the current literature. And the present invention expresses the plasmid pBBR1-kan-P to the recombinant system for the first timetet-ETh1h2h3h4_agroB6、pBBR1-kan-Ptet-ETh1h2h3P3_rhi597、pBBR1-kan-PtetET _ rhi145 and pBBR1-kan-PtetThe recombination efficiency of ETh _ rhi483 and its recombination system combined with red γ and plu γ was optimized and applied in Agrobacterium. The experimental result shows that Plu gamma ETRHI145The highest recombination efficiency was found in Agrobacterium tumefaciens C58, RecETh1h2h3h4 in Agrobacterium tumefaciens EHA105AGROB6Has the highest recombination efficiency, Plu gamma RecETh in Rhizobium rhizogenes NBRC13257RHI483Has the highest recombination efficiency. The invention realizes gene knockout of Agrobacterium tumefaciens C58, Agrobacterium tumefaciens EHA105 and Agrobacterium rhizogenes NBRC13257 by using the constructed recombination system. The agrobacterium recombination system can greatly promote genome modification of agrobacterium, and has great application prospect in the aspect of constructing agrobacterium strains with high transformation efficiency and host range and the field of plant genetic engineering.
Drawings
FIG. 1: structure of Red alpha beta gamma and RecET operons with agrobacterium derived homologous recombinant operons. Wherein, the proteins encoded by the genes with the same color have similar functions.
FIG. 2: schematic representation of expression plasmid of agrobacterium recombination system.
FIG. 3: optimization of the electric conversion conditions of Agrobacterium tumefaciens C58.
(A) Growth curve of Agrobacterium tumefaciens C58.
(B) Effect of competent cells prepared at different electrotransformation buffers and temperatures on the electrotransformation efficiency of Agrobacterium tumefaciens C58. Error bars, three replicates per experimental group, n-3.
FIG. 4: optimization of the electric transfer conditions of the Agrobacterium tumefaciens EHA 105.
(A) Growth curves of Agrobacterium tumefaciens EHA 105.
(B) The effect of different electrotransformation buffers and competent cells prepared at different temperatures on the electrotransformation efficiency of Agrobacterium tumefaciens EHA 105. Error bars, three replicates per experimental group, n-3.
FIG. 5: optimization of transformation conditions of Rhizobium rhizogenes NBRC 13257.
(A) Growth curves of rhizobium rhizogenes NBRC 13257.
(B) Effect of different electrotransfer buffers and competent cells prepared at different temperatures on the electrotransfer efficiency of Rhizobium rhizogenes NBRC 13257.
(C) The effect of different electrical transfer voltages on the electrical transfer efficiency of Rhizobium rhizogenes NBRC 13257. Error bars, three replicates per experimental group, n-3.
FIG. 6: comparison of recombination efficiency in different combinations of Agrobacterium tumefaciens C58, Agrobacterium tumefaciens EHA105 and Rhizobium rhizogenes NBRC 13257.
(A) Schematic representation of the loop recombination by replacing the tetracycline promoter and recombinase fragment on the recombinant system expression plasmid with the aprAR gene in Agrobacterium tumefaciens C58, Agrobacterium tumefaciens EHA105 and Rhizobium rhizogenes NBRC 13257.
(B) Comparison of recombination efficiencies of different combinations of recombination systems in Agrobacterium tumefaciens C58.
(C) Comparison of recombination efficiencies of different combinations of recombination systems in Agrobacterium tumefaciens EHA 105.
(D) Comparison of recombination efficiencies of different combinations of the recombination system in Rhizobium rhizogenes NBRC 13257. Error bars, three replicates per experimental group, n-3.
FIG. 7: pBBR1-kan-PtetOptimization of the recombination efficiency of plu. gamma. -ET-rhi 145 in Agrobacterium tumefaciens C58.
(A) The effect of the amount of different exogenous DNA on the recombination efficiency of Agrobacterium tumefaciens C58.
(B) Effect of different homology arm lengths on the efficiency of Agrobacterium tumefaciens C58 recombination. Error bars, three replicates per experimental group, n-3.
FIG. 8: pBBR1-kan-PtetOptimization of the recombination efficiency of ETh1h2h3h4_ agroB6 in Agrobacterium tumefaciens EHA 105.
(A) The effect of the amount of different exogenous DNA on the efficiency of Agrobacterium tumefaciens EHA105 recombination.
(B) Effect of different homology arm lengths on the efficiency of Agrobacterium tumefaciens EHA105 recombination. Error bars, three replicates per experimental group, n-3.
FIG. 9: pBBR1-kan-PtetOptimization of the recombination efficiency of plu. gamma. -ETh-rhi 483 in Rhizobium rhizogenes NBRC 13257.
(A) The effect of the amount of different exogenous DNA on the efficiency of Agrobacterium tumefaciens EHA105 recombination.
(B) Effect of different homology arm lengths on the efficiency of Agrobacterium tumefaciens EHA105 recombination.
(C) Expression plasmid pBBR1-kan-P for recombinant system with different induction temperatures and induction timestet-effect of plu γ -ETh _ rhi483 recombination efficiency. Error bars, three replicates per experimental group, n-3.
FIG. 10: recombinant System expression plasmid pBBR1-kan-Ptet-plu γ -ET-rhi 145 in Agrobacterium tumefaciens C58, pBBR1-kan-PtetETh1h2h3h4_ agroB6 in Agrobacterium tumefaciens EHA105 and pBBR1-kan-Ptet-plu γ -ETh _ rhi483 for use in Rhizobium rhizogenes NBRC13257 for gene knock-out.
(A) Schematic diagram of agrobacterium recombination system for gene knockout.
(B)pBBR1-kan-PtetResults of colony PCR after knocking out IS21 transposase family gene istB and istA fusion gene in Agrobacterium tumefaciens C58 by plu. gamma. -ET-rhi 145.
(C)pBBR1-kan-PtetResults of colony PCR after knockout of the celI gene in Agrobacterium tumefaciens EHA105 by ETh1h2h3h4_ agroB 6.
(D)pBBR1-kan-PtetResults of colony PCR after knocking out the 3 '-5' exouclase gene in Rhizobium rhizogenes NBRC13257 by-plu. gamma. -ETh-rhi 483.
(E)pBBR1-kan-PtetResults of colony PCR after knocking out endoglucanase gene in Rhizobium rhizogenes NBRC13257 by-plu. gamma. -ETh-rhi 483.
Wherein: m is DNA Marker, and Wt is wild strain. All randomly picked monoclonals were correct.
Detailed Description
The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.
General description:
recombinant system expression strains E.coli GB2005, E.coli GB05-dir and E.coli GB08-redE.coli GBred-gyrA462 related to the following examples are all purchased from GeneBridges, Germany; the Agrobacterium tumefaciens C58 and Agrobacterium tumefaciens EHA105 were obtained from the university of agricultural and forestry, Rhizobium rhizogenes NBRC13257, and purchased from German culture Collection (DSMZ).
Plasmid pBBR1-PRha-GFP-kan、pBBR1-kan-Ptet-firefly、pBBR1-PRha-gba-kan, pBBR1-apra-kan were from university of Shandong-Helmholtz institute of biotechnology.
The genomic sequences of Agrobacterium tumefaciens C58, Agrobacterium tumefaciens EHA105 and Rhizobium rhizogenes NBRC13257 are reported sequences, see for details the relevant genomic sequences published in NCBI.
Gene sequencing in plasmid construction was accomplished by Huada Gene. Gene synthesis was performed by Jinzhi corporation. The plasmids are all conventional plasmids which are sold on the market, and the method for electrotransformation into recipient bacteria is a conventional method.
Other related reagents and consumables are all made in China. Unless otherwise specified, the experimental methods and reagents in the examples are those conventionally used in the art and those commercially available.
Example 1: construction of homologous recombination expression plasmid of series agrobacterium
The NCBI BlastP program is used for searching proteins which are homologous with Red alpha/beta of Escherichia coli lambda phage or RecE/T recombinase of Rec prophage in all agrobacterium subjected to genome sequencing or bacterial genomes close to the genome or phage and prophage genomes of the agrobacterium or the phage and the prophage genomes, and searching potential proteins with recombination function in the agrobacterium. The operons of exonuclease-recombinase homologous to RecE/T are found in Agrobacterium tumefaciens str. B6, Rhizobium leguminosarum bv. Trifolii WSM597, Rhizobium sp. LC145 and Rhizobium sp. Root483D2 contig-20, respectively, ETh1h2h3h4_ agroB6, ETh1h2h3P3_ rhi597, ET _ rhi145 and ETh _ rhi 483. Among them, ETh1h2h3h4_ agroB6 is about 3410bp long, encodes six proteins, in addition to two proteins homologous to RecE and RecT, 4 putative proteins; ETh1h2h3P3 — rhi597 is 3898bp long and encodes five proteins, 2 putative proteins in addition to proteins homologous to RecE and RecT, and Exo-Pol III; ET-rhi 145 is 1394bp long and contains proteins homologous to RecE and RecT; ETh-rhi 483 was 1951bp long, with a putative protein present in addition to proteins homologous to RecE and RecT, as shown in FIG. 1.
The agrobacterium homologous recombination system is a series of recombination system expression plasmids which are constructed on the basis of a wide-host replicon pBBR1, and the recombination functional protein is induced and expressed by using a tetracycline promoter, which is shown in figure 2.
Homologous recombination system series expression plasmid pBBR1-kan-Ptet-ETh1h2h3h4_agroB6、pBBR1-kan-Ptet-ETh1h2h3P3_rhi597、pBBR1-kan-PtetET _ rhi145 and pBBR1-kan-Ptet-ETh _ rhi483, comprising the following steps:
(1)pBBR1-kan-Ptetthe firefly is an initial plasmid, EcoRI and NheI are subjected to double enzyme digestion, and a target linear fragment pBBR1-P is recovered from geltet-kan, about 5kb in length;
(2) recovering the linear fragment pBBR1-P from the gel obtained in the step (1)tet-kan is respectively co-transformed with synthesized recombinase fragments ETh1h2h3h4_ agroB6HA, ETh1h2h3P3_ rhi597HA, ET _ rhi145HA and ETh _ rhi483HA with vector homology arms into E.coli GB05-dir for linear recombination;
(3) enzyme digestion verification is carried out on the recombinants obtained in the step (2), so that expression plasmids of the homologous recombination system series of the agrobacterium are obtained, and the expression plasmids of the series are respectively named as pBBR1-kan-Ptet-ETh1h2h3h4_ agroB6, the nucleotide sequence of which is shown in SEQ ID No. 1; pBBR1-kan-Ptet-ETh1h2h3P3 — rhi597, the nucleotide sequence of which is shown in SEQ ID No. 2; pBBR1-kan-PtetET _ rhi145, the nucleotide sequence of which is shown in SEQ ID No. 3; pBBR1-kan-PtetETh _ rhi483, the nucleotide sequence of which is shown in SEQ ID No. 4.
Example 2: optimization of the Electrical transfer conditions of Agrobacterium tumefaciens C58, Agrobacterium tumefaciens EHA105 and Rhizobium rhizogenes NBRC13257
The optimized exploration of the electric transfer conditions of the Agrobacterium tumefaciens C58 and the Agrobacterium tumefaciens EHA105 involves the following two parts:
(1) determination of growth curves for Agrobacterium tumefaciens C58 and Agrobacterium tumefaciens EHA105
First, the growth curves of Agrobacterium tumefaciens C58 and Agrobacterium tumefaciens EHA105 need to be determined to determine the optimal length of time for the preparation of competent cell cultures. Respectively pick threeInoculating Agrobacterium tumefaciens C58 and Agrobacterium tumefaciens EHA105 monoclonals, respectively, into a perforated 2ml EP tube containing 1.3ml LB liquid culture medium, performing shake culture at 30 deg.C and 950rpm for 24h, sucking 100 μ l seed liquid, adding into 900 μ l liquid LB, blowing, mixing, and measuring OD600. Transfer to 50ml LB separately, starting OD600Adjusting to 0.1 deg.C, culturing at 30 deg.C and 200rpm under shaking, and sucking 1ml of bacterial liquid every 1 hr for OD measurement600According to OD of each time point600Growth curves of Agrobacterium tumefaciens C58 and Agrobacterium tumefaciens EHA105 were plotted and the experimental results are shown in FIGS. 3A and 4A. It was shown that both Agrobacterium tumefaciens C58 and Agrobacterium tumefaciens EHA105 entered the exponential growth phase starting from hour 4.
(2) Effect of different electrotransformation buffers and different temperatures on the electrotransformation efficiency of Agrobacterium tumefaciens C58 and Agrobacterium tumefaciens EHA105
After 4h of incubation of Agrobacterium tumefaciens C58 and Agrobacterium tumefaciens EHA105, each experimental group was treated with 10% sucrose solution, 10% glycerol solution, SH buffer (10% sucrose + 2. mu.M HEPES), GH buffer (10% glycerol + 2. mu.M HEPES) and ddH, respectively2O, washing 2 times, preparing competent cells at room temperature and 4 ℃ respectively, and adding 1. mu.g of test plasmid pBBR1-PRhaGFP-kan, for electrotransformation.
The results of the experiment are shown in FIGS. 3B and 4B, and the results show that ddH was used in Agrobacterium tumefaciens C582Preparing competent cells at normal temperature, wherein the electrotransformation efficiency is highest; in Agrobacterium tumefaciens EHA105, competent cells were prepared at low temperature using GH buffer, and the electrotransformation efficiency was the highest.
By integrating the optimization results of the transformation efficiency, the invention determines the optimal transformation conditions of Agrobacterium tumefaciens C58: starting OD6000.1 medium, cultured at 30 ℃ for 4h with shaking at 950rpm, and then treated with ddH2And O preparing competent cells at normal temperature, wherein the electrotransfer voltage is 1350V/mm. Optimal transformation conditions for Agrobacterium tumefaciens EHA 105: starting OD6000.1 of the strain solution, shaking and culturing at 30 ℃ and 950rpm for 4h, preparing competent cells by GH buffer solution at low temperature, and the electrotransfer voltage is 1350V/mm.
The optimization exploration of the Rhizobium rhizogenes NBRC13257 electrotransformation condition involves the following three parts:
(1) determination of the growth Curve of Rhizobium rhizogenes NBRC13257
The first step requires the determination of the growth curve of Rhizobium rhizogenes NBRC13257 to determine the optimal length of time for preparing competent cell cultures. Selecting three Rhizobium rhizogenesis NBRC13257 monoclonals, inoculating to a 2ml EP tube containing 1.8ml TY liquid culture medium, culturing at 30 deg.C and 950rpm overnight under shaking, adding 100 μ l seed liquid into 900 μ l liquid TY, blowing and mixing, and measuring OD600. The appropriate amount of seed solution was aspirated and inoculated into 50ml TY broth containing kanamycin to obtain the starting OD600Shaking culture at 30 deg.C and 200rpm for 2h at 0.1 deg.C, and sucking 1ml of bacterial liquid to measure OD600According to OD of each time point600The growth curve of Rhizobium rhizogenes NBRC13257 was plotted, and the experimental results are shown in FIG. 5A. Shows when the starting OD is600When the cell count is 0.1, the cells are in the logarithmic growth phase after the acclimation phase and after 6 hours.
(2) Effect of different electrotransfer buffers and temperatures on the electrotransfer efficiency of Rhizobium rhizogenes NBRC13257
After 6h of incubation in Rhizobium rhizogenes NBRC13257, each experimental group was treated with 10% sucrose solution, 10% glycerol solution, SH buffer (10% sucrose + 2. mu.M HEPES), GH buffer (10% glycerol + 2. mu.M HEPES) and ddH2O, washing 2 times, preparing competent cells at room temperature and 4 ℃ respectively, and adding 500ng of test plasmid pBBR1-PRhaGFP-kan, 1350V/mm. The results are shown in FIG. 5B, and show that higher transformation efficiency can be obtained by washing cells with SH buffer solution at room temperature, compared with ddH2The increase in O was about 3-fold.
(3) Effect of different electric conversion voltages on Rhizobium rhizogenes NBRC13257 electric conversion efficiency
To further improve the electrotransformation efficiency of Rhizobium rhizogenes NBRC13257, the Applicant shocked the competent cells with 1350V/mm, 1500V/mm, 1700V/mm, 1900V/mm and 2100V/mm, respectively. The results of the experiment are shown in FIG. 5C, and the results show that 1900V/mm and 2100V/mm can greatly improve the electrical conversion efficiency. Since high voltage affects the viability of the cells, 1900V/mm was chosen as the optimal voltage for transformation of Rhizobium rhizogenes NBRC13257 in this experiment.
By combining the optimization results of the transformation efficiency, the invention determines the optimal transformation conditions of Rhizobium rhizogenes NBRC 13257: starting OD600The cells were cultured in 0.1 g of the bacterial solution at 30 ℃ for 6 hours with shaking at 950rpm, and then in SH buffer at room temperature, and the voltage for electroporation was 1900V/mm.
Example 3: recombination System comparison of the efficiency of recombination of expression plasmids in Agrobacterium tumefaciens C58, Agrobacterium tumefaciens EHA105 and Rhizobium rhizogenes NBRC13257
In the process of recombinant system mining, the applicant connects Red gamma derived from Lambda phage or Plu gamma derived from Pseudomonas in series to an agrobacterium recombinant system, and constructs recombinant system expression plasmids with different combinations by adopting a method of reverse screening of Red/ET and ccdB, wherein the specific names are respectively named as follows: pBBR1-kan-Ptet-redγ-ETh1h2h3h4_agroB6、pBBR1-kan-Ptet-redγ-ETh1h2h3P3_rhi597、pBBR1-kan-Ptet-redγ-ET_rhi145、pBBR1-kan-Ptet-redγ-ETh_rhi483、pBBR1-kan-Ptet-pluγ-ETh1h2h3h4_agroB6、pBBR1-kan-Ptet-pluγ-ETh1h2h3P3_rhi597、pBBR1-kan-Ptet-plu γ -ET _ rhi145 or pBBR1-kan-Ptet-plu γ -ETh _ rhi 483. Meanwhile, pBBR1-P was used in this experimentRhaGba-kan as a positive control for recombination experiments.
The recombinant system expression plasmids were respectively transformed into Agrobacterium tumefaciens C58, Agrobacterium tumefaciens EHA105 and Rhizobium rhizogenes NBRC13257 by the same transformation method as described in the above examples.
Mu.g of aprRPCR fragment with 80bp homology arm (obtained by PCR amplification with 80bpHA-apra-5 and 80bpHA-apra-3 as primers and pBBR1-apra-kan as template) was respectively transferred into Agrobacterium tumefaciens C58 and Rhizobium rhizogenes NBRC13257 cells containing the above 13 recombination systems by loop recombination to replace the promoter region and recombinase portion in the recombinase expression plasmid, and finally the recombinant pBBR1-apra-kan was obtained as the basis for the efficiency comparison of the recombination systems, as shown in FIG. 6A.
80bpHA-apra-5:gcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaACGCTCAGTGGAACGAGGTT
80bpHA-apra-3: ggagagcctgagcaaactggcctcaggcatttgagaagcacacggtcacactgcttccggtagtcaataaaccggtaagcTGAGCGTCAGCCAATCGACT (lower case in the primer is the homology arm, upper case is the primer).
The specific procedure for PCR amplification of fragment aprAR with primers 80bpHA-apra-5 and 80bpHA-apra-3 was as follows:
PCR reaction procedure
Selecting 3 Agrobacterium tumefaciens C58 and Agrobacterium tumefaciens EHA105 carrying recombinant system expression plasmids, monoclonally culturing into 2ml EP tube containing 1.3ml liquid LB culture medium, using kanamycin at 10 μ g/ml, shaking at 30 deg.C and 950rpm for 24h, sucking 100 μ l seed liquid, adding into 900 μ l liquid LB, blowing, mixing, measuring OD600. An appropriate amount of the seed liquid was aspirated and inoculated into 1.3ml of LB liquid medium containing kanamycin (10. mu.g/ml) to initiate OD600After shaking culture at 950rpm at 30 ℃ for 4h and 0.1, Anhydrotetracycline (AHT) was added to a final concentration of 20. mu.g/mL, and shaking culture at 950rpm for 1h and 30 ℃. Uniform OD according to minimum in experimental group600In the preparation of competent cells, Agrobacterium tumefaciens EHA105 used an electric transfer buffer GH under a low temperature condition, and Agrobacterium tumefaciens C58 used ddH under a normal temperature condition2O treatment, then 1. mu.g of aprAR PCR fragment with 80bp homology arm (obtained by PCR amplification with 80bpHA-apra-5 and 80bpHA-apra-3 as primers and pBBR1-apra-kan PvuII gel recovery fragment after enzyme digestion as template). The voltage for electric transformation was 1350V/mm, and then recovered at 30 ℃ for 2 hours, Agrobacterium tumefaciens C58 was resistance-screened with 50. mu.g/ml apramycin, Agrobacterium tumefaciens EHA105 was resistance-screened with 40. mu.g/ml apramycin, cultured in 30 ℃ incubator, and then the number of colonies was counted, see FIGS. 6B and 6C.
Selecting 3 Rhizogene NBRC13257 monoclonals carrying recombinant system expression plasmid into 2ml EP tube containing 1.3ml liquid TY culture medium, using concentration of kanamycin is 30 mug/ml, shaking culture is carried out at 30 ℃, 950rpm is carried out for 24h, 100 mul seed liquid is sucked and added into 900 mul liquid LB, evenly blowing and beating are carried out, OD is measured600. An appropriate amount of the seed liquid was aspirated and inoculated into 1.3ml of LB liquid medium containing kanamycin (30. mu.g/ml) to initiate OD600After shaking culture at 950rpm at 30 ℃ for 6h and 0.1, Anhydrotetracycline (AHT) was added to a final concentration of 20. mu.g/mL, and shaking culture at 950rpm for 1h and 30 ℃. Uniform OD according to minimum in experimental group600When preparing competent cells, SH buffer solution is used for treatment at normal temperature, and then 1 mu g of aprAR PCR fragment (obtained by PCR amplification by taking 80bpHA-apra-5 and 80bpHA-apra-3 as primers and taking a gel recovery fragment obtained after enzyme digestion of pBBR1-apra-kan PvuII as a template) with 80bp homology arms is electrotransferred into cells. The voltage of the electric transfer is 1900V/mm, then the electric transfer is recovered for 2h at 30 ℃, resistance screening is carried out on the apramycin with the concentration of 3 mu g/ml, the apramycin is cultured in an incubator with the temperature of 30 ℃, and then the number of bacterial colonies is counted, and the diagram is shown in figure 6D.
The experimental results show that:
in Agrobacterium tumefaciens C58, recombination System RecETh1h2P3RHI597The mediated homologous recombination is more than other three recombination systems (RecETh1h2h3h 4)AGROB6、RecETRHI145And RecEThRHI483) The recombination efficiency is high; however, after addition of Red γ, RecETh1h2h3h4AGROB6The recombination efficiency of (1) is improved by one time, RecETh1h2P3RHI597And RecETRHI145The recombination efficiency of (A) is unchanged, while RecEThRHI483The recombination efficiency of (2) is reduced; RecETh1h2P3 after addition of Plu γRHI597And RecETRHI145The recombination efficiency of (1) RecETh (1 h2h3h 4)AGROB6And RecEThRHI483Hardly changed. General assemblyOf Plu gamma ETRHI145The highest recombination efficiency was found in Agrobacterium tumefaciens C58, shown in FIG. 6B.
In Agrobacterium tumefaciens EHA105, recombination System RecETh1h2h3h4AGROB6、RecETh1h2P3RHI597And RecETRHI145Has high recombination efficiency, RecEThRHI483Has lower recombination efficiency; however, after adding Red gamma or Plu gamma, the recombination efficiency of the four recombination systems is greatly reduced. In summary, RecETh1h2h3h4 in Agrobacterium tumefaciens EHA105AGROB6With the highest recombination efficiency, see fig. 6C.
In Rhizobium rhizogenes NBRC13257, only RecEThRHI483Shows relatively high recombination efficiency, RecETh1h2P3RHI597And RecETRHI145The recombination efficiency of (1) is very low, RecETh1h2h3h4AGROB6There is little recombination function. RecETh after addition of Red γ or Plu γRHI483The recombination efficiency is slightly improved. In summary, Plu γ RecETh in Rhizobium rhizogenes NBRC13257RHI483With the highest recombination efficiency, see fig. 6D. .
Example 4: recombinant System expression plasmids optimization of recombinant working conditions in Agrobacterium tumefaciens C58, Agrobacterium tumefaciens EHA105 and Rhizobium rhizogenes NBRC13257
The absorption rate of the bacteria to the exogenous DNA is greatly different under different growth states, and in order to ensure that the recombination efficiency meets the requirement of genetic modification on microbial DNA molecules, the working condition of a recombination system needs to be optimized. Based on the study of the optimal transformation conditions in example 2, this example performed the optimization of the recombinant working conditions for Agrobacterium tumefaciens C58, Agrobacterium tumefaciens EHA105 and Rhizobium rhizogenes NBRC 13257.
(1) Effect of homology arm length on recombination efficiency
The Red recombination system consists of three proteins: the RecE and Red α proteins are exonucleases, bind to the ends of double-stranded DNA, and degrade the DNA from the 5' end to the 3' end, producing a 3' end or single-stranded DNA; RecT and Red β are single-stranded DNA binding proteins and mediate annealing of complementary single-stranded DNA; the Red gamma protein is combined with RecBCD protein to inhibit the activity of degrading exogenous DNA. The length of recognition sequences of RecE and Red alpha proteins having exonuclease activity is in a certain range, and it is not the longer the homology arm, the better. Therefore, the use of homology arms can be shortened by studying the effect of the length of the homology arms on recombination efficiency. The lengths of the homology arms used in this example were 80bp, 100bp, 150bp, and 200bp, respectively.
The results of the experiments are shown in FIG. 7A, FIG. 8A and FIG. 9A, and the recombination efficiency increases with the increase of the length of the homology arm. The 80bp homology arm was selected for the next step of the experiment.
(2) Effect of the amount of exogenous DNA on the efficiency of recombination
The uptake of exogenous DNA by cells has a certain threshold, and when the capacity of the cells reaches saturation, redundant DNA cannot be absorbed, so that the waste of experimental materials can be greatly reduced by researching the influence of the addition of exogenous DNA on the recombination efficiency. After preparation of competent cells, 500ng, 1000ng, 1500ng and 2000ng of apraRPCR product carrying an 80bp homology arm were added to Agrobacterium tumefaciens C58 and Agrobacterium tumefaciens EHA105, respectively. 1000ng, 2000ng, 3000ng and 4000ng of aprRPCR products carrying 80bp homology arms were added to Rhizobium rhizogenes NBRC13257, respectively.
As a result, referring to FIGS. 7B, 8B and 9B, in Agrobacterium tumefaciens C58, the applicants found that the recombination efficiency was low when the amount of the foreign DNA was 500ng, but there was no significant difference when the amount of the DNA was more than 1000 ng; in the Agrobacterium tumefaciens EHA105, the recombination efficiency is not significantly influenced in the range of 1000ng to 2000ng with the increase of the DNA dosage; in Rhizobium rhizogenes NBRC13257, the recombination efficiency is increased correspondingly with the increase of the DNA amount, the recombination efficiency is the highest when the DNA amount is 4000ng, but the saturation effect is not generated at this time, and the use amount of the DNA can be increased properly in subsequent experiments.
(3) Effect of Induction temperature on the efficiency of Rhizobium rhizogenes NBRC13257 recombination
The optimal temperature for growth of bacteria and expression of protein, and the optimal temperature for expression of recombinant enzymeTherefore, the optimal expression temperature of the recombinase can be determined by studying different induction times of the recombinase, so that higher recombination efficiency can be obtained. In this example, five induction temperatures were selected, 25 ℃, 28 ℃, 30 ℃, 32 ℃ and 37 ℃ respectively, and each set was repeated three times. Rhizobium rhizogenes NBRC13257 cells containing pBBR1-kan-Ptet-ETh _ rhi483 recombinase plasmid were cultured for 6h (starting OD6000.1), adding 20 μ l AHT, inducing at different temperatures for 1h, respectively, using 1000ng PCR product, using 1900V/mm voltage for electric conversion, and obtaining uniform OD before electric conversion600。
The results are shown in FIG. 9C, which shows that the highest recombination efficiency can be obtained by preparing competent cells after induction at 28 ℃ and that the recombinase has no recombination function at 37 ℃. Therefore, the optimal induction temperature of the recombinant system in Rhizobium rhizogenes NBRC13257 is 28 ℃.
(4) Effect of Induction time on the efficiency of 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, 20 mul AHT is added to induce at 28 ℃ for different time after the cell is cultured for 6h, the dosage of PCR products is 1000ng, the voltage of electric conversion is 1900V/mm, and the uniform OD before the electric conversion is performed600。
The results are shown in FIG. 9C and show that higher recombination efficiency can be obtained by preparing competent cells after 0.5h of induction at 28 ℃.
Summary of the experimental results:
optimal recombination conditions in Agrobacterium tumefaciens C58: starting OD6000.1, 4h (OD) at 30 ℃600About 0.6-0.7)), induced at 30 ℃ for 1h, at room temperature with room temperature ddH2Preparing competent cells, wherein the amount of exogenous DNA is 1.5 mu g, the length of a homologous arm is 80bp, the electrotransfer voltage is 1350V/mm, and the recovery time is2 h.
Optimal recombination conditions in Agrobacterium tumefaciens EHA 105: starting OD6000.1, 4h (OD) at 30 ℃600About 0.6-0.7), inducing at 30 deg.C for 1h, preparing competent cells with GH buffer (10% glycerol +2 μ M HEPES) at 4 deg.C, exogenous DNA amount of 1.5 μ g, homology arm length of 80bp, and electric transition voltage of 1350 ℃V/mm, recovery time 2 h.
The optimal recombination conditions in Rhizobium rhizogenes NBRC13257 are: starting OD6000.1, 6h (OD) at 30 ℃600Approximately 0.4-0.5), induction at 28 ℃ for 0.5h, preparation of competent cells at room temperature using SH buffer (10% sucrose + 2. mu.M HEPES) with an amount of exogenous DNA of 4. mu.g, a length of homology arm of 80bp, an electric transition voltage of 1900V/mm, and a recovery time of 3 h.
Example 5: application of genetic modification on genome by using novel agrobacterium tumefaciens recombination system
Using Plu gamma ETRHI145、RecETh1h2h3h4AGROB6And Plu gamma RecEThRHI483The genome is genetically modified in Agrobacterium tumefaciens C58, Agrobacterium tumefaciens EHA105 and Rhizobium rhizogenes NBRC13257, respectively.
The IS21 transposase family gene on the linear chromosome of Agrobacterium tumefaciens C58 has a total length of 2252 bp. Knocking out the IS21 transposase family gene not only can simplify the genome, but also eliminates the potential risk of transposon pollution to exogenous DNA on Ti plasmid, and improves the stability of Agrobacterium tumefaciens C58 as a transgenic vector. The moderate simplification of the genome can optimize the metabolic pathway of the cell, improve the utilization efficiency of the cell to substrate and energy, and obviously improve the predictability and controllability of the cell physiological performance.
The gene celI is a member of a transcription regulator MarR/ArsR gene family, is positioned on an Agrobacterium tumefaciens EHA105 linear chromosome, has the total length of 929bp, and the product is an inhibitor of cellulose synthesis gene celABC, so that the celI is knocked out, the yield of cellulose 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' exouclase gene is 978bp long and codes 325 amino acids in total, and the other endoglucanase gene is a cellulase gene, belongs to a class of glycoside hydrolase and is a key enzyme for bacteria to decompose plant cell walls, is 1050bp long and codes 349 amino acids.
And replacing the target gene by using an aprAR PCR fragment with a homology arm under the action of recombinase to knock out the gene of the agrobacterium. The length of the homology arm is 80bp, and the homology arm is loaded at both ends of the apraR gene by primer synthesis combined with PCR, as shown in 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 letters in the primers are homology arms and upper case letters are primers).
The specific PCR reaction system and procedure were the same as in example 3.
Single colonies were picked for colony PCR validation for the 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 procedure were the same as in example 3.
Sequence listing
<110> Shandong university
<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-ETh1h2h3h4_ agroB6
<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 (6)
1. An agrobacterium homologous recombination system, which consists of a series of expression plasmids of the agrobacterium homologous recombination system and comprises a pBBR1 replication origin, a kanamycin resistance gene, a tetracycline inducible promoter and an agrobacterium-derived homologous recombination operon; the method is characterized in that:
the expression plasmids of the agrobacterium homologous recombination system are respectively named as pBBR1-kan-Ptet-ETh1h2h3h4_ agroB6, the nucleotide sequence of which is shown in SEQ ID No. 1; pBBR1-kan-Ptet-ETh1h2h3P3 — rhi597, the nucleotide sequence of which is shown in SEQ ID No. 2; pBBR1-kan-PtetET _ rhi145, the nucleotide sequence of which is shown in SEQ ID No. 3; pBBR1-kan-Ptet-ETh _ rhi483, the nucleotide sequence of which is shown in SEQ ID No. 4; wherein the related homologous recombinase operon ETh1h2h3h4AGROB6、ETh1h2h3P3RHI597、ETRHI145And EThRHI483Derived from Agrobacterium tumefaciens str. B6, Rhizobium leguminosa Bv. trifolii WSM597, Rhizobium sp. LC145 and Rhizobium sp. Root483D2, respectively.
2. The use of the agrobacterium homologous recombination system of claim 1 for mediating homologous recombination of short homology arms in agrobacterium for genetic modification of genomic DNA, wherein the short homology arms are homology arms of 80bp to 200 bp.
3. Use according to claim 2, characterized in that:
the serial expression plasmid of the agrobacterium homologous recombination system is pBBR1-kan-Ptet-pluγ-ET_rhi145、pBBR1-kan-Ptet-ETh1h2h3h4_agroB6、pBBR1-kan-Ptet-ET _ rhi145 or pBBR1-kan-Ptet-ETh_rhi483；
The Agrobacterium is Agrobacterium tumefaciens C58, Agrobacterium tumefaciens EHA105 or Rhizobium rhizogenes NBRC 13257;
the short homology arm refers to 80bp-100 bp.
4. Use according to claim 2 or 3, characterized in that:
the expression plasmid pBBR1-kan-Ptet-plu γ -ET _ rhi145 in AgrobaThe optimal conditions for improving the recombination efficiency in the cteerium tumefaciens C58 are as follows: starting OD600OD was adjusted at 30 ℃ for 4h under 0.16000.6-0.7, inducing at 30 deg.C for 1 hr, and treating with room temperature ddH at room temperature2Preparing competent cells, wherein the amount of exogenous DNA is 1.5 mu g, the length of a homologous arm is 80bp, the electrotransfer voltage is 1350V/mm, and the recovery time is2 h;
the expression plasmid pBBR1-kan-PtetThe optimization conditions for improving the recombination efficiency in the Agrobacterium tumefaciens EHA105 of ETh1h2h3h4_ agroB6 are: starting OD600OD was adjusted at 30 ℃ for 4h under 0.16000.6-0.7, inducing at 30 ℃ for 1h, preparing competent cells by GH buffer solution with a formula of 10% volume ratio of glycerol and 2 MuM HEPES at 4 ℃, wherein the amount of exogenous DNA is 1.5 Mug, the length of a homologous arm is 80bp, the electric conversion voltage is 1350V/mm, and the recovery time is2 h;
the expression plasmid pBBR1-kan-PtetThe optimal conditions for the plu γ -ETh-rhi 483 to improve the recombination efficiency in Rhizobium rhizogenes NBRC13257 are: starting OD600OD was adjusted at 30 ℃ for 6h under 0.16000.4-0.5, inducing at 28 deg.C for 0.5h, preparing competent cells at room temperature with SH buffer solution containing 10% sucrose +2 μ M HEPES by using exogenous DNA 4 μ g, homologous arm length 80bp, electric conversion voltage 1900V/mm, and recovery time 3 h.
5. The use of the agrobacterium homologous recombination system of claim 1 for gene knockout in agrobacterium, wherein the agrobacterium is agrobacterium tumefaciens or agrobacterium rhizogenes.
6. Use according to claim 5, characterized in that:
the Agrobacterium tumefaciens is Agrobacterium tumefaciens C58 or Agrobacterium tumefaciens EHA 105; the Agrobacterium rhizogenes is Rhizobium rhizogenes NBRC 13257.
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