CN116042688A - Citrus canker high-saturation transposon mutant library and construction method thereof - Google Patents
Citrus canker high-saturation transposon mutant library and construction method thereof Download PDFInfo
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Abstract
The invention relates to a citrus canker germ high-saturation transposon mutant library and a construction method thereof, belonging to the technical field of biological industry. The construction method comprises the following steps: s1: constructing plasmids pLN2-Trans and pLLN 2-Trans; s2: construction of transposon mutant libraries based on plasmids pLN2-Trans and pLLN 2-Trans: (1) preparing a xanthomonas competent cell; (2) Preparation of X.flavus successfully transformed with transposons pLN2-Trans and pLLN 2-Trans; (3) Preparation of mutant pools T1 and T2 transformed with pLN2-Trans and pLLN2-Trans. The invention realizes the efficient amplification of the amplified transposon and the flanking regions thereof, and identifies the essential genes of the citrus canker by high-throughput sequencing.
Description
Technical Field
The invention relates to the technical field of biological industry, in particular to a citrus canker high-saturation transposon mutant library and a construction method thereof.
Background
Citrus canker is a plant disease which seriously threatens the yield and quality of citrus seeds, is a bacterial disease caused by xanthomonas citri subspecies, and causes the citrus fruits and leaves to carry scars when the disease is light; the heavy weight causes fruit drop and leaves of saplings to lead to the reduction of harvest of peasants.
Xanthomonas invades through pores or wounds in the surface of citrus leaves or fruits, and after successful infection and mass reproduction, escapes outwards through eruptive pustules, thus starting the next infection cycle. At present, no perfect method exists for preventing and treating citrus canker, and in the production process, the copper preparation is generally sprayed or disease prevention and control are carried out by cutting off disease trees, but the problems of environment and drug resistance are generated. The invention utilizes transposon mutation and high throughput sequencing technology to identify essential genes of citrus canker pathogens, and is expected to develop a new drug target for disease control.
Disclosure of Invention
Aiming at the problems, the invention provides a citrus canker high-saturation transposon mutant library and a construction method thereof, in particular to a method for constructing the citrus canker high-saturation transposon mutant library and identifying essential genes thereof by using a high-throughput sequencing technology, which is used for researching and preventing bacterial citrus canker caused by citrus subspecies of xanthomonas citri. The invention realizes the efficient amplification of the amplified transposon and the flanking regions thereof, thereby greatly reducing the cost for preparing the sequencing library, and simultaneously, as the inverse PCR amplified fragment can be determined by a PCR program, the flanking sequences are longer and the comparison result is more accurate.
The technical scheme of the invention is as follows:
the construction method of the citrus canker fungus high-saturation transposon mutant library comprises the following steps:
s1: construction of plasmids pLN2-Trans and pLLN 2-Trans:
(1) Constructing pLN2-Trans, and connecting a lactose promoter Plac in a transposase in the pN2-Trans to obtain the pLN2-Trans;
preferably, a primer is used for amplifying a nucleotide sequence 270bp upstream of the LacZ gene in the pBBR1MCS-2 vector to obtain a lactose promoter for driving gene expression; replacing the transposase self-promoter fragment in the known vector pMCS 2-transponsase with a lactose promoter by using a seamless cloning assembly method to obtain a vector pLN2-Trans for constructing a transposon insertion mutant library;
(2) Constructing a pLLN2-Trans, and connecting Plac at the 5' end of the transposase in the pLN2-Trans obtained in the step (1);
preferably, the pLN2-Trans is treated with NdeI and KpnI double cleavage to obtain a linearized vector; amplifying a nucleotide sequence 270bp upstream of the LacZ gene in the pBBR1MCS-2 vector by using a primer; the promoter fragment was ligated into the linearized vector using a seamless cloning assembly method to obtain the vector pLLN2-Trans used to construct the transposon insertion mutant library.
S2: the construction method of the transposon mutant library based on plasmids pLN2-Trans and pLLN2-Trans comprises the following steps:
(1) Preparing a xanthomonas competent cell;
preferably, a xanthomonas broth obtained by single colony culture is prepared, and after centrifugation, it is resuspended using sterile water. Xanthomonas competent cells were obtained by washing the cells multiple times with pre-chilled 10% glycerol.
(2) Adding the Xanthomonas competent cells obtained in the step (1) into plasmids pLN2-Trans and pLLN2-Trans respectively, mixing, and introducing the plasmids into Xanthomonas by electric shock, wherein the volumes of the plasmids are 90:2; culturing by using NB liquid culture medium added with sucrose with mass fraction of 1%, screening by using NB solid culture medium added with 50 mug/ml kanamycin, and obtaining the transposon pLN2-Trans and the successfully transformed xanthomonas in pLLN2-Trans respectively;
preferably, the xanthomonas competent cells obtained in the step (1) are added to plasmids pLN2-Trans and pLLN2-Trans respectively, and after mixing, the transposon vector is introduced into xanthomonas by 30 electric shocks; culturing by using NB liquid culture medium added with 1% sucrose by mass fraction, standing in a 30 ℃ incubator for incubation of 3 h; subsequently plated on NB solid medium supplemented with 50. Mu.g/ml kanamycin, and incubated upside down at 30℃for 2 d; the transposons pLN2-Trans and pLLN2-Trans were obtained, respectively, and the transformed Xanthomonas was successful.
(3) The xanthomonas successfully transformed by the transposon pLN2-Trans and the xanthomonas successfully transformed by the transposon pLLN2-Trans obtained in the step (2) are scraped respectively, and then the obtained xanthomonas cells are added into NB liquid medium added with 50 mug/ml kanamycin respectively to obtain mutant libraries for transforming pLN2-Trans and pLLN2-Trans respectively, which are named as T1 and T2 respectively. The mutant library was uniformly split and stored at-80℃for cold storage.
The invention also comprises the citrus canker germ high-saturation transposon mutant library obtained by the construction method.
Furthermore, the invention also discloses a sequencing library and a preparation method thereof, and the method comprises the following steps:
amplifying the mutant library with NB liquid medium added with 50 mug/ml kanamycin, extracting Xanthomonas genomic DNA by using a CTAB method, digesting the genomic DNA by using restriction enzymes NcoI and EcoRI, bamHI, sphI, mfeI, mluI, notI, reprecipitating and extracting the DNA by using 100% ethanol, adding T4 ligase for self-ligation, purifying the DNA by using a gel recovery kit, and performing inverse PCR by using restriction enzyme specific primers, wherein the PCR conditions are as follows: 98 ℃ for 3min,98 ℃ for 10s,58 ℃ for 15s,72 ℃ for 2 min,33 cycles, 72 ℃ for 10 min, and 12 ℃ for heat preservation to obtain a vector transposon flanking genomic sequence fragment; and then DNA purification is carried out again by using a gel recovery kit, thus obtaining the sequencing library.
Further, the data processing method of the sequencing library comprises the following steps:
and installing Ubuntu in a window10 system computer to construct a Linux subsystem. The original sequencing data was quality controlled using fastp software, the seqkit software was used to deduplicate sequences, and unidirectional specificity screening was performed with the vector transposon end sequences. The T1 read-out sequence is obtained by screening through the complete matching of the 25 bp O-end sequence or the I-end sequence. After forward and reverse screening, respectively, the reverse sequences are reversed and combined. The O-end sequences were identified using TPP software based on bwa, and the results were mapped to Xanthomonas whole genome (CQ 13 strain chromosome genome and 2 plasmids thereof) to obtain insertion sites. And (3) screening after extracting the data of the insertion sites to delete the situation that the sequenced sequence and the genome sequence are not completely matched, and extracting effective data for subsequent analysis.
Compared with the prior art, the invention has the following advantages:
(1) The T1 transposon adopted by the invention realizes mutation of more than 70 ten thousand random sites on the whole genome, and establishes a high-saturation mutant library with resolution up to 8 bp.
(2) The invention realizes efficient amplification of transposon and amplification of flanking region thereof by combining inverse PCR and various enzyme digestion, thereby greatly reducing the cost for preparing sequencing library, and simultaneously, the flanking sequence is more accurate than the result because the inverse PCR amplified fragment can be determined by PCR program.
Drawings
FIG. 1 is a graph showing the polar effects of the distribution of transposon insertions in individual genes across the genome of pLN2-Trans and pLLN2-Trans plasmids;
FIG. 2 is a graph showing the polarity effect of the insertion differences of pLN2-Trans and pLLN2-Trans on the respective genes;
FIG. 3 is a graph showing the polarity effect of transposon insertion in an operon;
FIG. 4 is a graph of various clustering studies in essential genomes based on COG functional classes;
FIG. 5 is an enrichment map of COG classification;
FIG. 6 is a KEGG-based essential genome clustering study;
FIG. 7 shows the distribution of essential genes across the genome.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. The embodiments are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
Example 1: citrus canker high-saturation transposon mutant library and construction method thereof
The construction steps are as follows:
s1: construction of plasmids pLN2-Trans and pLLN 2-Trans:
(1) Amplifying a nucleotide sequence 270bp upstream of the LacZ gene in the pBBR1MCS-2 vector by using a primer to obtain a lactose promoter for driving gene expression; replacing the transposase self-promoter fragment in the known vector pMCS 2-transponsase with a lactose promoter by using a seamless cloning assembly method to obtain a vector pLN2-Trans for constructing a transposon insertion mutant library;
(2) Constructing pLLN2-Trans, and carrying out double digestion treatment on the pLN2-Trans by using NdeI and KpnI to obtain a linearization vector; amplifying a nucleotide sequence 270bp upstream of the LacZ gene in the pBBR1MCS-2 vector by using a primer; the promoter fragment was ligated into the linearized vector using a seamless cloning assembly method to obtain the vector pLLN2-Trans used to construct the transposon insertion mutant library.
S2: construction of transposon mutant library based on plasmids pLN2-Trans and pLLN2-Trans
The construction steps are as follows:
(1) Xanthomonas competent cells were prepared, and a Xanthomonas broth obtained by 300 ml single colony culture was prepared, centrifuged, and resuspended in 6 ml sterile water. Washing the cells multiple times with pre-chilled 10% glycerol to obtain xanthomonas competent cells;
(2) Adding the Xanthomonas competent cells obtained in the step (1) into plasmids pLN2-Trans and pLLN2-Trans respectively, mixing, and introducing the transposon vector into Xanthomonas by 30 times of electric shock, wherein the volumes of the two are 90:2; culturing by using NB liquid culture medium added with 1% sucrose by mass fraction, standing in a 30 ℃ incubator for incubation of 3 h; subsequently, the resultant was plated on NB solid medium supplemented with 50. Mu.g/ml kanamycin, and cultured upside down at 30℃for 2 d to obtain the transposons pLN2-Trans and pLLN2-Trans, respectively, for successful transformation of Xanthomonas;
(3) And (3) adding the xanthomonas which is successfully transformed by the transposon pLN2-Trans and the xanthomonas which is successfully transformed by the transposon pLLN2-Trans obtained in the step (2) into NB liquid culture medium added with 50 mu g/ml kanamycin respectively through scraping plates to obtain mutant libraries which are respectively named as T1 and T2 for transforming the pLN2-Trans and the pLLN2-Trans. The mutant library was uniformly split and stored at-80℃for cold storage.
Example 2: tnSeq sequencing library and preparation method thereof
The method comprises the following steps:
amplifying mutant libraries T1 and T2 with NB liquid medium added with 50 μg/ml kanamycin by 100 ml, extracting Xanthomonas genomic DNA by using a CTAB method, digesting the genomic DNA by using restriction enzymes NcoI and EcoRI, bamHI, sphI, mfeI, mluI, notI, reprecipitating and extracting the DNA by using 100% ethanol, adding T4 ligase for self-ligation, and purifying the DNA by using a gel recovery kit (omega); after DNA purification using the gel recovery kit, inverse PCR was performed using restriction enzyme specific primers, the conditions of PCR were: 98 ℃ for 3min,98 ℃ for 10s,58 ℃ for 15s,72 ℃ for 2 min,33 cycles, 72 ℃ for 10 min, and 12 ℃ for heat preservation to obtain a vector transposon flanking genomic sequence fragment; then, DNA purification was performed again using a gel recovery kit (Omega) to obtain a TnSeq sequencing library.
The data processing method of the TnSeq sequencing library comprises the following steps of:
and installing Ubuntu in a window10 system computer to construct a Linux subsystem. The original sequencing data was quality controlled using fastp software, deduplicated using seqkit software, and screened for unidirectional specificity with the vector transposon end sequences. The T1 read-out sequence is obtained by screening through the complete matching of the 25 bp O-end sequence or the I-end sequence. After forward and reverse screening, respectively, the reverse sequences are reversed and combined. The O-end sequences were identified using TPP software based on bwa, and the results were mapped to Xanthomonas whole genome (CQ 13 strain chromosome genome and 2 plasmids thereof) to obtain insertion sites. And screening the extracted data of the insertion sites to remove the condition of incomplete matching, and extracting effective data for subsequent analysis.
Test example:
1. creation of highly saturated insertion mutant libraries
A single colony of about 400000 mutants was collected by transformation with a pLN2-Trans plasmid, and a transposon mutation library T1 was constructed by example 1; approximately 500000 single colonies of mutants were collected using pLLN2-Trans plasmid transformation, and a transposon mutation library T2 was constructed.
The mutant libraries T1 and T2 obtained in two times are collected and then are respectively cultivated, extracted, digested and stored by inverse PCR. A total of 123958517 sequencing sequences were obtained by high throughput sequencing, mapping to the genome resulted in 737899 independent insertion sites, which data indicated that the abundance of insertions reached high saturation resolution for every 8 bp single insertion.
Identification of essential genes
The essential gene means a gene which is essential for the maintenance of normal life of a living body under certain conditions. Different species have different genome sizes and gene compositions, but these widely differing genomes contain a set of essential genes to maintain critical cellular functions. Most of essential genes are housekeeping genes responsible for vital reactions such as DNA replication, transcription, translation, protein folding and the like in the living body, which reveal the most basic biological mechanism of one species, and the deep penetration of the essential genes is helpful for people to understand the origin of life and the connection between different species in the evolution process.
Tn5 taps in TRANSIT were used to identify the necessity of all genes. The p-value (p-value < 0.05) calculated by Tn5Gaps was screened for 525 essential genes and 181 high fitness genes, wherein the essential genes contained 48 proteins with unknown functions.
Effect of polarity Effect on Adjacent genes within an operon
By comparing the mutant pools T1 and T2, the distribution of transposons over the whole genome was counted, and the ratio of the number of transposons inserted on a single gene to the number of transposons in all genes and the gap between them were calculated, wherein the genesXCQ_RS1890Belonging to an essential gene, and genes upstream of the geneXCQ_RS1885And downstream ofpcaFA non-essential gene; the results are shown in FIGS. 1 and 2, where the trend of T1 and T2 insertion across the whole genome is similar, which is also consistent with the conclusion that the polarity effects described in the literature only affect adjacent genes in a small proportion of cases. In FIG. 2, if the insertion abundance of T1 is greater than T2, the difference is positive; if the insertion abundance of T1 is less than T2, the difference is negative.
The insertion of nonessential genes upstream of the essential genes in the same operon was counted for T1 and T2 and focused on nonessential genes not inserted in the case of T1, 86 nonessential genes were counted in total, and 28 nonessential genes not inserted under the condition of T1 were obtained, 25 of which were expressed as tolerance transposon insertion under the condition of T2, as in one operon shown in FIG. 3, wherein the essential genesXCQ_RS1890Upstream nonessential genes are not transposon inserted in T1, but transposon insertion is allowed in T2, and transposon insertion downstream thereof is not significantly affected. The data show that the outward promoter in pLN2-Trans effectively reduces the influence of the polar effect on non-essential genes around essential genes, and improves the accuracy of essential gene identification.
Functional characteristics of essential genes
Functionally classifying the essential genome of xanthomonas by COG (Cluster of Orthologous Groups) or KEGG, COG (as in fig. 4, the number of genes beside the bar graph) demonstrates that about one third of the essential genes are concentrated in three functional classifications: 15% of the genes are involved in gene translation, ribosome structure and biological synthesis (J), 9.3% of the genes are involved in energy production and conversion (C), 7.6% are involved in biological synthesis of cell walls/membranes (M). Although the enrichment degree of the essential genome of different xanthomonas bacteria is different, the enrichment trend is similar: essential genes are concentrated on basic functions such as translation and energy metabolism. Analysis revealed 62 essential genes with unknown function. While another COG analysis shows the abundance of different functional classifications throughout the genome as shown in fig. 5, a more accurate assessment of functional importance is made by calculating the ratio of a particular class of genes in the essential genome to its total proportion in the genome. Evaluation showed essential gene enrichment cluster classification: gene translation, ribosomal structure and biological synthesis (J), nucleotide transport and metabolism (F), coenzyme transport and metabolism (H), energy production and conversion (C), cell cycle control, cell division and chromosome partitioning (D), intracellular transport, secretion and vesicle transport (U), DNA replication recombination and repair (L), cell membrane biogenesis (M) and post-translational modification, protein turnover and chaperones, and the like (O).
In addition to COG, the KEGG pathway was used to cluster genome functions in more detail, as shown in fig. 6. The figures show that the number of essential genes involved in tRNA-amino acid transport, ribosomes and photosynthesis is significantly higher than the others, while the ratio of essential genes in the genes involved in tRNA-amino acid transport and cell cycle is also much higher than the others (87.5% and 88.3%, respectively).
Distribution and comparison of essential genes across the genome
The distribution of essential genes in citrus canker bacteria on the whole genome is analyzed and mapped, 500000bp bases are used as a moving window, 10000 bases are used as a walking distance, the probability of occurrence of the essential genes in the window is calculated by using the super-geometric distribution, p values are assigned, and the p values on the whole genome are mapped after being processed, as shown in figure 7.
FIG. 7 shows the enrichment of essential genes over the whole genome, the enrichment of essential genes reaching the highest value at about 0.2 (1000000 bases) of the whole genome, and in addition to the concentrated region of the ribosomal structure-encoding protein found here, the enrichment of the chromosomal DNA replication initiation site and the end site of the species Leptospira citri was found to be insufficient, which is different from the other two known bacteria: the essential genes of the bacillus crescent are concentrated in the central and initial terminal regions, and the escherichia coli has higher essential gene enrichment in the region near the initial site.
Claims (9)
1. The construction method of the citrus canker fungus high-saturation transposon mutant library is characterized by comprising the following steps of:
s1: construction of plasmids pLN2-Trans and pLLN 2-Trans:
(1) Constructing pLN2-Trans, and connecting a lactose promoter Plac in a transposase in the pN2-Trans to obtain the pLN2-Trans;
(2) Constructing a pLLN2-Trans, and connecting Plac at the 5' end of the transposase in the pLN2-Trans obtained in the step (1);
s2: the construction method of the transposon mutant library based on plasmids pLN2-Trans and pLLN2-Trans comprises the following steps:
(1) Preparing a xanthomonas competent cell;
(2) Adding the Xanthomonas competent cells obtained in the step (1) into plasmids pLN2-Trans and pLLN2-Trans respectively, mixing, and introducing the plasmids into Xanthomonas by electric shock, wherein the volumes of the plasmids are 90:2; culturing by using NB liquid culture medium added with sucrose with mass fraction of 1%, screening by using NB solid culture medium added with 50 mug/ml kanamycin, and obtaining the transposon pLN2-Trans and the successfully transformed xanthomonas in pLLN2-Trans respectively;
(3) Scraping the xanthomonas successfully transformed by the transposon pLN2-Trans and the xanthomonas successfully transformed by the transposon pLLN2-Trans obtained in the step (2) respectively, and then adding the obtained xanthomonas respectively into NB liquid medium added with 50 mug/ml kanamycin to obtain mutant libraries for transforming pLN2-Trans and pLLN2-Trans respectively, which are named as T1 and T2 respectively; the mutant library was uniformly split and stored at-80℃for cold storage.
2. The construction method according to claim 1, wherein the S1 step (1) uses a primer to amplify a nucleotide sequence 270bp upstream of the LacZ gene in the pBBR1MCS-2 vector to obtain a lactose promoter driving gene expression; the transposase self-promoter fragment in the known vector pMCS 2-transponsase was replaced with the lactose promoter using a seamless cloning assembly method to give the vector pLN2-Trans for constructing a library of transposon insertion mutants.
3. The construction method according to claim 1, wherein the step (2) of S1 is performed by treating pLN2-Trans with NdeI and KpnI double cleavage to obtain a linearized vector; amplifying a nucleotide sequence 270bp upstream of the LacZ gene in the pBBR1MCS-2 vector by using a primer; the promoter fragment was ligated into the linearized vector using a seamless cloning assembly method to obtain the vector pLLN2-Trans used to construct the transposon insertion mutant library.
4. The construction method according to claim 1, wherein the xanthomonas fluid obtained by single colony culture is prepared in step (1) of S2, and is resuspended by using sterile water after centrifugation; xanthomonas competent cells were obtained by washing the cells multiple times with pre-chilled 10% glycerol.
5. The construction method according to claim 1, wherein in the step (2) of S2, the competent cells of xanthomonas obtained in the step (1) of S2 are added to the plasmids pLN2-Trans and pLN2-Trans, respectively, and then the transposon vector is introduced into xanthomonas by 30 electric shocks; culturing by using NB liquid culture medium added with 1% sucrose by mass fraction, standing in a 30 ℃ incubator for incubation of 3 h; subsequently plated on NB solid medium supplemented with 50. Mu.g/ml kanamycin, and incubated upside down at 30℃for 2 d; the transposons pLN2-Trans and pLLN2-Trans were obtained, respectively, and the transformed Xanthomonas was successful.
6. The library of highly saturated transposon mutants of citrus canker pathogenic bacteria obtained by the construction method according to any one of claims 1 to 5.
7. A sequencing library prepared using the library of highly saturated transposon mutants of Leptosphaeria citri according to claim 6.
8. The method of preparing a sequencing library of claim 7, wherein the steps of the method of preparing are as follows:
amplifying and culturing the citrus canker high-saturation transposon mutant library by using NB liquid culture medium added with 50 mug/ml kanamycin, extracting xanthomonas genome DNA by using a CTAB method, digesting and treating the genome DNA by using restriction enzymes NcoI and EcoRI, bamHI, sphI, mfeI, mluI, notI, reprecipitating and extracting the DNA by using 100% ethanol, adding T4 ligase for self-ligation, and purifying the DNA by using a glue recovery kit; and then carrying out inverse PCR by using an endonuclease specific primer to obtain a genome sequence fragment flanking the carrier transposon, wherein the PCR conditions are as follows: 3min at 98 ℃,10 s at 98 ℃, 15s at 58 ℃, 2 min at 72 ℃,33 cycles, 10 min at 72 ℃ and 12 ℃ for heat preservation; and then DNA purification is carried out again by using a gel recovery kit, thus obtaining the sequencing library.
9. The method for processing data of a sequencing library obtained by the preparation method according to claim 8, wherein the method for processing data comprises the following steps:
installing Ubuntu in a Window10 system computer to construct a Linux subsystem; performing quality control on the original sequencing data by using fastp software; the seqkit software is used for removing repeated sequences, and unidirectional specificity screening is carried out by using the terminal sequences of the transposons of the vector; the T1 readout sequence is obtained through screening by completely matching the 25 bp O-end sequence or the I-end sequence; respectively carrying out forward and reverse screening, reversing reverse sequences, and merging; identifying an O-end sequence by using TPP software based on bwa, and mapping the result onto a whole genome of Xanthomonas to obtain an insertion site; and (3) screening after extracting the data of the insertion sites to delete the situation that the sequenced sequence and the genome sequence are not completely matched, and extracting effective data for subsequent analysis.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002000916A2 (en) * | 2000-06-28 | 2002-01-03 | California Institute Of Technology | Methods for identifying an essential gene in a prokaryotic microorganism |
| CN101260399A (en) * | 2008-03-13 | 2008-09-10 | 邓子牛 | Orange canker resistant pthA-nls gene and its construction method and application |
| WO2011109031A1 (en) * | 2010-03-05 | 2011-09-09 | Synthetic Genomics, Inc. | Methods for cloning and manipulating genomes |
| GB201322084D0 (en) * | 2013-12-13 | 2014-01-29 | Discuva Ltd | Method for identifying genes involved in antibiotic resistance and sensitivity in gram-negative bacteria |
| CN106086070A (en) * | 2016-06-07 | 2016-11-09 | 中山大学 | A kind of ProtoRAG Transposon System and application thereof |
| WO2016180379A1 (en) * | 2015-05-14 | 2016-11-17 | Universidad De Los Andes | Novel transposon that promotes the functional expression of genes in episomal dna, and method for increasing dna transcription in the functional analysis of metagenomic libraries |
| CN106754606A (en) * | 2017-01-11 | 2017-05-31 | 中国农业科学院柑桔研究所 | The screening technique of the unmarked deletion mutant of citrus processing hfq genes |
| CN106916774A (en) * | 2015-12-25 | 2017-07-04 | 沈阳中化农药化工研发有限公司 | A kind of reporting system for being applied to the type excretory system of plant pathogenetic bacteria III and its application |
| CN110283843A (en) * | 2019-07-04 | 2019-09-27 | 西南大学 | A method of mediate CsWRKY22 fixed point editor to improve citrus bacterial canker disease resistance based on CRISPRCas9 |
| CN114317584A (en) * | 2021-11-23 | 2022-04-12 | 海南医学院 | Construction system of novel transposon mutant library, novel transposon mutant library and application |
-
2023
- 2023-03-06 CN CN202310200163.3A patent/CN116042688B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002000916A2 (en) * | 2000-06-28 | 2002-01-03 | California Institute Of Technology | Methods for identifying an essential gene in a prokaryotic microorganism |
| CN101260399A (en) * | 2008-03-13 | 2008-09-10 | 邓子牛 | Orange canker resistant pthA-nls gene and its construction method and application |
| WO2011109031A1 (en) * | 2010-03-05 | 2011-09-09 | Synthetic Genomics, Inc. | Methods for cloning and manipulating genomes |
| GB201322084D0 (en) * | 2013-12-13 | 2014-01-29 | Discuva Ltd | Method for identifying genes involved in antibiotic resistance and sensitivity in gram-negative bacteria |
| WO2016180379A1 (en) * | 2015-05-14 | 2016-11-17 | Universidad De Los Andes | Novel transposon that promotes the functional expression of genes in episomal dna, and method for increasing dna transcription in the functional analysis of metagenomic libraries |
| CN106916774A (en) * | 2015-12-25 | 2017-07-04 | 沈阳中化农药化工研发有限公司 | A kind of reporting system for being applied to the type excretory system of plant pathogenetic bacteria III and its application |
| CN106086070A (en) * | 2016-06-07 | 2016-11-09 | 中山大学 | A kind of ProtoRAG Transposon System and application thereof |
| CN106754606A (en) * | 2017-01-11 | 2017-05-31 | 中国农业科学院柑桔研究所 | The screening technique of the unmarked deletion mutant of citrus processing hfq genes |
| CN110283843A (en) * | 2019-07-04 | 2019-09-27 | 西南大学 | A method of mediate CsWRKY22 fixed point editor to improve citrus bacterial canker disease resistance based on CRISPRCas9 |
| CN114317584A (en) * | 2021-11-23 | 2022-04-12 | 海南医学院 | Construction system of novel transposon mutant library, novel transposon mutant library and application |
Non-Patent Citations (2)
| Title |
|---|
| KEREN LASKER: "Selective sequestration of signalling proteins in__a membraneless organelle reinforces the spatial__regulation of asymmetry in Caulobacter crescentus", 《NATURE MICROBIOLOGY 》, pages 1 - 26 * |
| 黄坤炫;韩铖潇;谭棋仁;蔡璐璐;马文秀;邹丽芳;陈功友;: "大豆斑疹病菌tal基因Tn5插入突变体的构建", 上海交通大学学报(农业科学版), no. 02 * |
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