CN116042688B - Highly saturated transposon mutant library and construction method of X. citri - Google Patents

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

Highly saturated transposon mutant library and construction method of X. citri

technical field

The invention relates to the technical field of biological industry, in particular to a highly saturated transposon mutant library of X. citri and a construction method thereof.

Background technique

Citrus canker is a plant disease that seriously threatens the yield and quality of citrus species. It is a bacterial disease caused by Xanthomonas citri subsp. If it is serious, it will cause the fruit to drop and the young trees to lose their leaves, resulting in a reduction in farmers' income.

Xanthomonas invades through stomata or wounds on the surface of citrus leaves or fruits. After successful infection and massive reproduction, it escapes outwards through eruptive pustules, thus starting the next round of infection cycle. At present, there is no perfect method for the prevention and treatment of citrus canker. In production, the disease is generally prevented and controlled by spraying copper preparations or cutting down diseased trees, but environmental and drug resistance problems arise. The invention uses transposon mutation combined with high-throughput sequencing technology to identify essential genes of X. citri, which is expected to discover new drug targets for disease prevention and control.

Contents of the invention

In view of the above problems, the present invention provides a highly saturated transposon mutant library of X. citri and a construction method thereof, specifically by constructing a highly saturated transposon mutant library of X. citri and using high-throughput sequencing technology to identify its essential genes , for the research and control of bacterial citrus canker caused by Xanthomonas citri subsp. The invention realizes efficient amplification of transposons and their flanking regions, thereby greatly reducing the cost of preparing sequencing libraries. At the same time, because the reverse PCR amplification fragment can be determined by the PCR program, the flanking sequences are also relatively long, and the comparison The result is more accurate.

Technical scheme of the present invention is as follows:

A method for constructing a highly saturated transposon mutant library of X. citri, the steps are as follows:

S1: Construction of plasmids pLN2-Trans and pLLN2-Trans:

(1) Construct pLN2-Trans, and connect the lactose promoter Plac to pN2-Trans to obtain pLN2-Trans;

Preferably, primers are used to amplify the 270 bp nucleotide sequence upstream of the LacZ gene in the pBBR1MCS-2 vector to obtain a lactose promoter that drives gene expression; the lactose promoter is replaced by the lactose promoter in the known vector pMCS2-transposase using the seamless cloning assembly method The promoter fragment of the transposase itself is obtained to obtain the vector pLN2-Trans for constructing the transposon insertion mutant library;

(2) Construct pLLN2-Trans, and connect Plac at the 5' end of the transposase in pLN2-Trans obtained in step (1);

Preferably, use NdeI and KpnI double digestion to treat pLN2-Trans to obtain a linearized vector; use primers to amplify the 270bp nucleotide sequence upstream of the LacZ gene in the pBBR1MCS-2 vector; use the seamless cloning assembly method to connect the promoter fragment to Among the linearized vectors, the vector pLLN2-Trans used to construct the transposon insertion mutant library was obtained.

S2: A method for constructing a transposon mutant library based on plasmids pLN2-Trans and pLLN2-Trans, the steps are as follows:

(1) Prepare Xanthomonas competent cells;

Preferably, the Xanthomonas bacteria liquid obtained by single colony culture is prepared and resuspended with sterile water after centrifugation. Cells were washed several times with pre-cooled 10% glycerol to obtain Xanthomonas competent cells.

(2) Add the Xanthomonas competent cells obtained in step (1) to the plasmids pLN2-Trans and pLLN2-Trans and mix, both volumes are 90:2, and introduce the plasmids into Xanthomonas by electric shock; use additional The NB liquid medium with a mass fraction of 1% sucrose was added, and the NB solid medium supplemented with 50 μg/ml kanamycin was selected to obtain xanthomonads successfully transformed by the transposon pLN2-Trans and pLLN2-Trans, respectively bacteria;

Preferably, the Xanthomonas competent cells obtained in step (1) are added to the plasmids pLN2-Trans and pLLN2-Trans and mixed, and the transposon vector is introduced into Xanthomonas by 30 electric shocks; The NB liquid medium with a fraction of 1% sucrose was cultivated, and incubated in a 30°C incubator for 3 h; then plated on the NB solid medium supplemented with 50 μg/ml kanamycin, and cultured upside down at 30°C for 2 d; Xanthomonas successfully transformed with transposons pLN2-Trans and pLLN2-Trans were obtained.

(3) Scrape the Xanthomonas successfully transformed with the transposon pLN2-Trans obtained in step (2) and the Xanthomonas successfully transformed with the transposon pLLN2-Trans respectively, and then separate the obtained Xanthomonas The mutants were added to NB liquid medium supplemented with 50 μg/ml kanamycin to obtain mutant libraries transformed into pLN2-Trans and pLLN2-Trans, which were named T1 and T2, respectively. The mutant library was evenly divided and stored at -80°C.

The present invention also includes the highly saturated transposon mutant library of X. citri obtained by the above construction method.

Further, the present invention also includes a sequencing library and a preparation method, the steps are as follows:

The mutant library was expanded with NB liquid medium supplemented with 50 μg/ml kanamycin, and the genomic DNA of Xanthomonas was extracted using the CTAB method, and the restriction enzymes NcoI, EcoRI, BamHI, SphI, MfeI, Genomic DNA was digested with MluI and NotI, reprecipitated with 100% ethanol to extract the DNA, and then added T4 ligase for self-ligation. After DNA purification was performed using a gel recovery kit, reverse PCR was performed using restriction enzyme specific primers. The conditions for PCR were: : 98°C for 3 minutes, 98°C for 10s, 58°C for 15s, 72°C for 2 minutes, 33 cycles, 72°C for 10 minutes, and incubation at 12°C to obtain vector transposon flanking genome sequence fragments; then use the gel recovery kit again for DNA Purify to obtain the sequencing library.

Further, the data processing method of the sequencing library, the method is as follows:

Install Ubuntu on a Windows 10 system computer to build a Linux subsystem. The quality control of the original sequencing data was carried out by using fastp software, and the repeated sequence was removed by seqkit software, and the one-way specificity screening was carried out with the end sequence of the vector transposon. T1 reads were screened by requiring a 25 bp O-end sequence or I-end sequence exact match. After performing forward and reverse screening respectively, the reverse sequence was reversed and merged. The O-end sequence was identified using the bwa-based TPP software, and the results were mapped to the whole genome of Xanthomonas (CQ13 strain chromosomal genome and its two plasmids) to obtain the insertion site. After the data of the insertion site is extracted, screening is performed to delete the incomplete match between the sequenced sequence and the genome sequence, and effective data is extracted for subsequent analysis.

Compared with the prior art, the present invention has the following advantages:

(1) The T1 transposon used in the present invention realizes the mutation of more than 700,000 random sites in the whole genome, and establishes a highly saturated mutant library with a resolution of 8 bp.

(2) The present invention achieves efficient amplification of transposons and amplification of their flanking regions through the combination of inverse PCR and various enzyme cuts, thereby greatly reducing the cost of preparing sequencing libraries, and at the same time due to inverse PCR amplification The fragment can be determined by the PCR program, and the flanking sequence is also longer, so the comparison result is more accurate.

Description of drawings

Figure 1 is a graph showing the polarity effect of transposon insertion distribution on each gene of pLN2-Trans and pLLN2-Trans plasmids in the whole genome;

Figure 2 is a graph showing the polarity effect of insertion differences between pLN2-Trans and pLLN2-Trans in each gene;

Fig. 3 is a diagram of the polarity effect of the transposon insertion of the operon;

Figure 4 is a clustering study diagram of each category in the essential genome based on the COG functional class;

Figure 5 is an enrichment map of COG classification;

Figure 6 is a KEGG-based essential genome clustering study;

Figure 7 shows the distribution of essential genes on the whole genome.

Detailed ways

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

Example 1: X. citri highly saturated transposon mutant library and construction method

The build steps are as follows:

S1: Construction of plasmids pLN2-Trans and pLLN2-Trans:

(1) Use primers to amplify the 270 bp nucleotide sequence upstream of the LacZ gene in the pBBR1MCS-2 vector to obtain the lactose promoter driving gene expression; use the seamless cloning assembly method to replace the lactose promoter in the known vector pMCS2-transposase The promoter fragment of the transposase itself is obtained to obtain the vector pLN2-Trans for constructing the transposon insertion mutant library;

(2) Construct pLLN2-Trans, and use NdeI and KpnI double digestion to treat pLN2-Trans to obtain a linearized vector; use primers to amplify the 270bp nucleotide sequence upstream of the LacZ gene in the pBBR1MCS-2 vector; use the seamless cloning assembly method to The promoter fragment was ligated into the linearized vector to obtain the vector pLLN2-Trans for constructing the transposon insertion mutant library.

S2: Construction of transposon mutant libraries based on plasmids pLN2-Trans and pLLN2-Trans

The build steps are as follows:

(1) To prepare Xanthomonas competent cells, prepare 300 ml of Xanthomonas bacterial liquid obtained from single colony culture, resuspend in 6 ml of sterile water after centrifugation. Use pre-cooled 10% glycerol to wash the cells several times to obtain Xanthomonas competent cells;

(2) Add the Xanthomonas competent cells obtained in step (1) to the plasmids pLN2-Trans and pLLN2-Trans and mix, the volume is 90:2, and the transposon vector is introduced into Xanthomonas by 30 electric shocks Bacteria; cultured in NB liquid medium with 1% sucrose added, and incubated in a 30°C incubator for 3 h; then plated on NB solid medium added with 50 μg/ml kanamycin at 30°C Xanthomonas successfully transformed by transposon pLN2-Trans and pLLN2-Trans were obtained by inverting culture for 2 days;

(3) Add 50 μg/ml kanamycin to the Xanthomonas successfully transformed by the transposon pLN2-Trans obtained in step (2) and the Xanthomonas successfully transformed by the transposon pLLN2-Trans respectively In the NB liquid culture medium, the mutant libraries transformed into pLN2-Trans and pLLN2-Trans were obtained, respectively named as T1 and T2. The mutant library was evenly divided and stored at -80°C.

Embodiment 2: TnSeq sequencing library and preparation method

Proceed as follows:

The mutant library T1 and T2 were expanded with 100 ml of NB liquid medium supplemented with 50 μg/ml kanamycin, and the genomic DNA of Xanthomonas was extracted using the CTAB method, and the restriction enzymes NcoI, EcoRI, BamHI , SphI, MfeI, MluI, and NotI to digest and process genomic DNA, reprecipitate and extract DNA with 100% ethanol, then add T4 ligase for self-ligation, use gel recovery kit (omega) for DNA purification; use gel recovery kit for DNA After purification, reverse PCR was performed using restriction enzyme specific primers. The PCR conditions were: 98°C for 3 min, 98°C for 10 s, 58°C for 15 s, 72°C for 2 min, 33 cycles, 72°C for 10 min, and incubation at 12°C to obtain Carrier transposon flanking genome sequence fragments; followed by DNA purification using the gel recovery kit (Omega) again to obtain a TnSeq sequencing library.

The data processing method of described TnSeq sequencing library, described method is as follows:

Install Ubuntu on a Windows 10 system computer to build a Linux subsystem. The quality control of the original sequencing data was carried out by using fastp software, and the duplication was removed by using seqkit software, and the one-way specificity screening was carried out with the vector transposon end sequence. T1 reads were screened by requiring a 25 bp O-end sequence or I-end sequence exact match. After performing forward and reverse screening respectively, the reverse sequence was reversed and merged. The O-end sequence was identified using the bwa-based TPP software, and the results were mapped to the whole genome of Xanthomonas (CQ13 strain chromosomal genome and its two plasmids) to obtain the insertion site. After data extraction at the insertion site, screening is performed to delete incomplete matches, and valid data is extracted for subsequent analysis.

Test example:

1. Establishment of highly saturated insertion mutant library

According to Example 1, about 400,000 mutant single colonies were collected by pLN2-Trans plasmid transformation, and the transposon mutation library T1 was constructed; about 500,000 mutant single colonies were collected by pLLN2-Trans plasmid transformation, and transposition was constructed. Submutation library T2.

The mutant libraries T1 and T2 obtained twice were pooled and cultured respectively, genomic DNA was extracted, genomic DNA was digested with restriction enzymes, and the library was constructed by inverse PCR. A total of 123,958,517 sequencing sequences were obtained by high-throughput sequencing, and 737,899 independent insertion sites were obtained by mapping to the genome. This data shows that the insertion abundance has reached a high saturation resolution of a single insertion per 8 bp.

Identification of essential genes

Essential genes refer to genes that are indispensable to maintain the normal survival of living organisms under certain conditions. Different species have different genome sizes and genetic compositions, but these vastly different genomes all contain a set of essential genes for key cellular functions. Most of the essential genes are housekeeping genes responsible for the indispensable life reactions of DNA replication, transcription, translation, and protein folding in this kind of life. People understand the origin of life and the connections between different species during evolution.

Essentiality identification of all genes was performed using Tn5Gaps in TRANSIT. According to the p value calculated by Tn5Gaps (p value<0.05), a total of 525 essential genes and 181 high-fitness genes were screened, and 48 proteins with unknown functions were included in the essential genes.

Polarity effects on adjacent genes within an operon

By comparing the mutant library T1 and T2, the distribution of transposons in all genes on the whole genome was counted, and the ratio of the number of transposons inserted in a single gene to the number of transposons in all genes was calculated and The gap between the two, the gene XCQ_RS1890 is an essential gene, and the gene XCQ_RS1885 upstream of the gene and pcaF downstream are non-essential genes; the results are shown in Figure 1 and Figure 2, the insertion of T1 and T2 in the whole genome The trend is similar, which is also consistent with the conclusion that the polarity effect only affects adjacent genes in a small number of cases in the literature. In Figure 2, if the insertion abundance of T1 is greater than T2, the difference between the two is positive; if the insertion abundance of T1 is less than T2, the difference between the two is negative.

Counting the insertion of non-essential genes upstream of essential genes in the same operon in T1 and T2, and focusing on non-essential genes that are not inserted in T1, a total of 86 non-essential genes were counted, and obtained Of the 28 non-essential genes that were not inserted under T1 conditions, 25 of them were shown to tolerate transposon insertions under T2 conditions, as shown in Figure 3 in an operon, where the non-essential gene upstream of the essential gene XCQ_RS1890 is in T1 was not inserted by transposons, but transposon insertions were allowed in T2, and the downstream transposon insertions were not significantly affected. The data indicated that the outward-facing promoter in pLN2-Trans effectively reduced the impact of polarity effects on non-essential genes around essential genes, and improved the accuracy of identification of essential genes.

Functional characterization of essential genes

The essential genomes of Xanthomonas are functionally classified by COG (Cluster of Orthologous Groups) or KEGG. COG (as shown in Figure 4, the number of genes next to the bar graph) shows that about one-third of the essential genes are concentrated in Among the three functional categories: 15% of the genes are related to gene translation, ribosome structure and biological synthesis (J), 9.3% of the genes are related to energy production and conversion (C), 7.6% are related to the biology of the cell wall/cell membrane Learning Synthetic Correlation (M). Although the enrichment levels of the essential genomes of different Xanthomonas were different, their enrichment trends were similar: the essential genes were mostly concentrated in basic functions such as translation and energy metabolism. The analysis revealed 62 essential genes of unknown function. Another COG analysis shows the richness of different functional categories in the whole genome, as shown in Figure 5, which makes a more accurate assessment of the importance of functions, and the calculation method is the relationship between a specific gene class in the essential genome and its presence in the genome. The proportion of the total proportion. Assessment showing essential gene enrichment cluster categories: Gene Translation, Ribosome 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 allocation (D), intracellular trafficking, secretion and vesicular trafficking (U), DNA replication recombination and repair (L), cell membrane biogenesis (M) and post-translational modifications, protein turnover and Molecular chaperones, etc. (O).

In addition to COG, a more detailed clustering of genomic functions was performed using the KEGG pathway, as shown in Figure 6. The figure shows that the number of essential genes related to tRNA-amino acid transport, ribosome and photosynthesis is significantly more than others, and the proportion of essential genes in tRNA-amino acid transport and cell cycle-related genes is also much higher than that of other functions (respectively 87.5 % and 88.3%).

Genome-wide distribution and comparison of essential genes

Analyze and map the distribution of essential genes in X. citri on the whole genome. With 500,000 bases as the moving window and 10,000 bases as the step distance, the hypergeometric distribution is used to calculate the probability of essential genes appearing in this window And specify the p value, and plot the p value on the whole genome after processing, as shown in Figure 7.

Figure 7 shows the enrichment of essential genes in the whole genome. The enrichment of essential genes reaches the highest value at about 0.2 (1,000,000 bases) in the whole genome. After inspection, it is found that this is the concentration of proteins encoded by the ribosome structure In addition, it was also found that the enrichment of the chromosomal DNA replication start site and terminal site of X. Concentrated in the central and start-end regions, E. coli had a higher enrichment of essential genes in the region near the start site.

Claims (5)

1. the construction method of X. citri highly saturated transposon mutant library, it is characterized in that, the steps of described construction method are as follows: S1: Construction of plasmids pLN2-Trans and pLLN2-Trans: (1) Construct pLN2-Trans, and connect the lactose promoter Plac to pN2-Trans to obtain pLN2-Trans; (2) Construct pLLN2-Trans, and connect Plac at the 5' end of the transposase in pLN2-Trans obtained in step (1); S2: A method for constructing a transposon mutant library based on plasmids pLN2-Trans and pLLN2-Trans, the steps are as follows: (1) Prepare Xanthomonas competent cells; (2) Add the Xanthomonas competent cells obtained in step (1) to the plasmids pLN2-Trans and pLLN2-Trans and mix, both volumes are 90:2, and introduce the plasmids into Xanthomonas by electric shock; use additional The NB liquid medium with a mass fraction of 1% sucrose was added, and the NB solid medium supplemented with 50 μg/ml kanamycin was selected to obtain xanthomonads successfully transformed by the transposon pLN2-Trans and pLLN2-Trans, respectively bacteria; (3) Scrape the Xanthomonas successfully transformed with the transposon pLN2-Trans obtained in step (2) and the Xanthomonas successfully transformed with the transposon pLLN2-Trans respectively, and then separate the obtained Xanthomonas The mutants were added to the NB liquid medium supplemented with 50 μg/ml kanamycin to obtain the mutant libraries transformed into pLN2-Trans and pLLN2-Trans respectively, which were named T1 and T2 respectively; after the mutant libraries were evenly distributed, all Store in -80 ℃ refrigeration; The S1 step (1) uses primers to amplify the 270 bp nucleotide sequence upstream of the LacZ gene in the pBBR1MCS-2 vector to obtain the lactose promoter driving gene expression; use the seamless cloning assembly method to replace the lactose promoter with the known vector pMCS2 -The transposase self-promoter fragment in the transposase is obtained for constructing the vector pLN2-Trans of the transposon insertion mutant library; In the step (2) of S1, use NdeI and KpnI double digestion to treat pLN2-Trans to obtain a linearized vector; use primers to amplify the 270 bp nucleotide sequence upstream of the LacZ gene in the pBBR1MCS-2 vector; use the seamless cloning assembly method to The promoter fragment was ligated into the linearized vector to obtain the vector pLLN2-Trans for constructing the transposon insertion mutant library. 2. The construction method according to claim 1, characterized in that the Xanthomonas bacterial liquid obtained from the single colony culture is prepared in the S2 step (1), resuspended with sterile water after centrifugation; The cells were washed several times with 10% glycerol to obtain Xanthomonas competent cells. 3. The construction method according to claim 1, characterized in that, in the S2 step (2), the Xanthomonas competent cells obtained in the S2 step (1) are added to the plasmids pLN2-Trans and pLLN2-Trans respectively After mixing, the transposon carrier was introduced into Xanthomonas by 30 electric shocks; cultured in NB liquid medium with 1% sucrose added, and incubated in a 30 °C incubator for 3 h; then plated at 50 On NB solid medium with μg/ml kanamycin, invert culture at 30℃ for 2 days; Xanthomonas successfully transformed with transposon pLN2-Trans and pLLN2-Trans were obtained respectively. 4. A method for preparing a sequencing library, characterized in that, the steps of the preparation method are as follows: Utilize the construction method of the highly saturated transposon mutant library of X. citri as described in any one of claims 1-3 to obtain the highly saturated transposon mutant library of X. citri, and then mutate the highly saturated transposon of X. citri The body bank was expanded with NB liquid medium supplemented with 50 μg/ml kanamycin, and the genomic DNA of Xanthomonas was extracted using the CTAB method, and the restriction enzymes NcoI, EcoRI, BamHI, SphI, MfeI, MluI, Genomic DNA was digested with NotI, re-precipitated with 100% ethanol to extract the DNA, then added T4 ligase for self-ligation, and purified DNA using a gel recovery kit; then used endonuclease-specific primers for inverse PCR to obtain vector transposon flanks Genome sequence fragments, PCR conditions are: 98°C for 3 minutes, 98°C for 10s, 58°C for 15s, 72°C for 2 minutes, 33 cycles, 72°C for 10 minutes, and 12°C for incubation; then use the gel recovery kit again for DNA purification, The sequencing library is obtained. 5. the data processing method of the sequencing library that preparation method obtains as claimed in claim 4, it is characterized in that, the method for data processing is as follows: Install Ubuntu on a Windows 10 system computer to build a Linux subsystem; use fastp software to perform quality control on the original sequencing data; use seqkit software to remove repetitive sequences, and perform unidirectional specificity screening with the vector transposon end sequence; the pass requires 25 bp O-end sequence or I-end sequence is completely matched, screened to obtain T1 read sequence; after forward and reverse screening respectively, the reverse sequence is reversed and merged; use bwa-based TPP software to identify O-end The sequence was mapped to the whole genome of Xanthomonas to obtain the insertion site; the data of the insertion site was extracted and screened to delete the incomplete match between the sequenced sequence and the genome sequence, and valid data was extracted for subsequent analysis.

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