CN105087554B - DNA phosphorothioate modifier clusters - Google Patents

Abstract

The invention discloses a kind of DNA phosphorothioates modifier cluster; the DNA phosphorothioates of gene cluster control are modified to single-stranded modification; selective sequence site is CpsC; whole phosphorothioate modifier cluster includes 4 genes; wherein; it is responsible for encoding aminothiopropionic acid desulfurization enzyme gene one, is responsible for the phosphosulfate reductase of 3 ' AMP 5 '（PAPS）Gene one, the albumen of two other gene code is under the jurisdiction of abc transport protein family and DUF4007 superfamilies respectively.Gene and its albumen that the present invention is provided, available for biopharmacy fields such as heterologous host DNA modification, epigenetics, acellular phosphorothioateization modification system construction, oligonucleotide drug modifications.

Description

DNA Phosphorylation Modifier Gene Cluster

technical field

The invention belongs to the technical field of bioengineering, and in particular relates to a gene cluster modified by phosphorothioation of DNA.

Background technique

It is well known that DNA is the carrier of genetic information, and various chemical modifications on DNA can cause changes in genetic information. Studies have found that there are many types of chemical modifications on DNA, which can be generally divided into two categories: one occurs on the bases of DNA, and the other occurs on the backbone of DNA. The first type of modification exists widely in organisms, and people have studied it thoroughly, the most famous of which is the DNA base methylation modification discovered in bacteria in 1965. On the one hand, this modification can be used by organisms to mark their own DNA to distinguish foreign DNA, thereby limiting its invasion and ensuring the stability of biological inheritance; The development has played a milestone contribution. The second type of modification is the most common DNA phosphorothioylation modification, which was originally an artificial modification used to modify oligonucleotides to stabilize their structure and prevent degradation by nucleases. In 1988, when Zhou Xiufen et al. were studying the electrophoresis of 66 genomic DNA of Streptomyces lividans, they found that the genomic DNA was degraded in ordinary gel electrophoresis or pulsed field gel electrophoresis experiments. Through trial and error, it was finally ruled out that this phenomenon was caused by nuclease contamination or human error. This phenomenon is named Dnd phenotype (DNA degradation phenotype). In 2007, Chinese scientists cooperated with British and American scientists to discover natural phosphorothioylation modification on the DNA backbone of Streptomyces lividans (Wang, LR; Chen, S.; Xu, TG; Taghizadeh, K.; Wishnok , JS; Zhou, XF; You, DL; Deng, ZX; Dedon, PC Nat . Chem . Biol . 2007, 3 , 709.), studies have confirmed that sulfur penetrates into the DNA backbone, so that the Dnd table is generated during DNA electrophoresis type.

The phosphorothioylation modification of DNA exists widely in the bacterial kingdom, ranging from animal and plant pathogenic bacteria and toxin-causing bacteria to antibiotic-producing bacteria living in various extreme environments, environmental repair microorganisms, symbiotic microorganisms, and psychrophilic, acidophilic, and psychrophilic bacteria. It is found in terrestrial or marine bacteria such as thermophilic or barophilic. It was determined that the chemical essence of the sulfur modification is that the DNA backbone undergoes phosphorothioylation to form a phosphorothioate bond, which exists in a specific R _p spatial conformation, and that this modification is sequence-selective (Wang, LR; Chen , S.; Xu, TG; Taghizadeh, K.; Wishnok, JS; Zhou, XF; You, DL; Deng, ZX; Dedon, PC Nat . Chem . Biol . 2007, 3 , 709.). The Dnd phenotype of Streptomyces lividans is controlled by the dnd gene (DNA degradation gene) through chemical induction reagent mutation, ultraviolet irradiation and gene knockout. The dnd gene is located on an 8kb DNA sequence and consists of five genes arranged in clusters, which are named dndA , dndB , dndC , dndD , and dndE , among which the dndBCDE gene forms an operon, which is opposite to the transcription direction of dndA (Zhou , XF; He, XY; Liang, JD; Li, AY; Xu, TG; Kieser, T.; Helmann, JD; Deng, ZX Mol. Microbiol. 2005, 57, 1428). In more than 20 representative bacterial and environmental DNAs that have been sequenced, the structure and sequence of the phosphorothioylation modification gene cluster are very similar to the dnd gene cluster, which also implies that this modification system is a common phenomenon. We call it It is a type I phosphorothioylation modification gene cluster, and its common modification sites are determined to be GpsA, GpsT and GpsG. These modification sites exist alone or in some strains, and present double-stranded modifications ( Liang, JD; Wang, ZJ; He, XY; Li, JL; Zhou, XF; Deng, ZX Nucleic Acids Res. 2007, 35, 2944.).

However, recently, in some halophilic Vibrio isolated from the ocean, we found another new modification site—CpsC phosphorothioation modification. Surprisingly, no common dnd gene cluster was found in its genome. Through the continuous deepening of research and exploration, it has now been determined that the gene cluster encoding CpsC phosphorothioylation is composed of four genes: vpt1, vpt2, vpt3 and vpt4 . In view of the fact that this is a completely different system from the dnd gene cluster in performing the function of DNA phosphorothioylation modification, it is called type II phosphorothioylation modification gene cluster. In addition, there is a difference from type I phosphorothioylation modification: type II phosphorothioylation modification is a single-chain modification.

In addition to the CpsC phosphorothioylation modification found in marine halophilic Vibrio FF75, we have found and identified CpsC-targeted sites in more and more strains such as Vibrio.beoganii 1C-10, Alteromonas variabilis ATCC29413, Streptomyces clavuligerus ATCC27064 Phosphorothioation modifier gene cluster. This newly discovered modification site and single-strand modification method will provide us with directional guidance and new ideas for studying the occurrence pathway, chemical nature and biological significance of DNA phosphorothioylation modification. The field of vaccine or molecular biology research provides different enzymatic methods to achieve the purpose of treating diseases.

Contents of the invention

The object of the present invention is to overcome the deficiencies in the prior art, and provides a novel DNA phosphorothioylation modification gene cluster, the DNA phosphorothioylation modification controlled by the gene cluster is a single-stranded modification, and the selective sequence site is CpsC, we call the type II phosphorothioation modifier gene cluster. The invention provides all gene and protein information related to this natural DNA phosphorothioylation modification, and provides a basis for genetic modification. It can be used in molecular biology research, gene therapy, gene vaccine and many other fields.

The purpose of the present invention is to be achieved by the following technical solutions:

The invention relates to a DNA phosphorothioylation modification gene cluster, and the DNA phosphorothioylation modification controlled by the gene cluster is a single-strand modification.

The invention relates to a DNA phosphorothioylation modification gene cluster, and the DNA phosphorothioylation modification selective sequence site controlled by the gene cluster is CpsC.

The DNA phosphorothioylation modification gene cluster involved in the present invention includes 4 genes, of which, one gene is responsible for coding cysteine desulfurase, one gene is responsible for 3' adenosine phosphate-5' phosphate sulfuric acid reductase, and the other two The proteins encoded by the genes belong to the ABC transporter family and the DUF4007 superfamily respectively.

The DNA phosphorothioylation modification gene cluster involved in the present invention comprises 4 genes: vpt1, vpt2, vpt3 and vpt4 , the nucleotide sequence of the gene cluster is as shown in SEQ ID NO:1 or formed by SEQ ID NO:1 complementary sequence.

The gene vpt4 is located at base 5146-6132 of the nucleotide sequence of the gene cluster and has a length of 987 base pairs. The encoded protein sequence is shown in SEQ ID NO: 2, and the gene encodes cysteine desulfurase.

The gene vpt3 is located at the 4300-5145th base of the gene cluster nucleotide sequence, with a length of 846 base pairs. The encoded protein sequence is shown in SEQ ID NO: 3, and the gene is encoded as 3'adenosine phosphate- 5' Phosphosulfate reductase (PAPS).

The gene vpt2 is located at the 967th-4299th base of the gene cluster nucleotide sequence and has a length of 3333 base pairs. The encoded protein sequence is shown in SEQ ID NO: 4, and the protein encoded by the gene belongs to the ABC transporter family.

The gene vpt1 is located at bases 1-966 of the nucleotide sequence of the gene cluster, with a length of 966 base pairs. The encoded protein sequence is shown in SEQ ID NO: 5, and the protein encoded by the gene belongs to the DUF4007 superfamily .

The present invention also relates to a DNA phosphorothioylation modification gene cluster, the nucleotide sequence of the gene cluster is highly homologous to the DNA phosphorothioation modification gene cluster of SEQ ID NO: 1, and the DNA phosphorothioation modification gene cluster controlled by the gene cluster The acylation modification is a single-strand modification, and the optional sequence site is CpsC.

The DNA phosphorothioylation modification gene cluster with the nucleotide sequence of SEQ ID NO: 1 involved in the present invention is derived from the marine halophilic Vibrio cyclitrophicus FF75.

The DNA phosphorothioylation modification gene cluster with high homology to SEQ ID NO:1 sequence involved in the present invention is derived from Vibrio breoganii 1C-10 , Vibro Breoganii ZF29 , Escherichia coli 042 , Shewanella fridigimarina NCIMB400 , Alteromonas macleodii Deep ecotype, Vibro ordalii FF-93 , Methyllibium petroleumiphilum PM1 plasmamid RPME01, Yersinia bercovieri ATCC 43970 , Polaromonas naphthalenivorans CJ 2 plasmad pPNAP02 , Alteromonas variabilis ATCC29413 , Streptomyces clavuligerus ATCC2706 .

The present invention also provides the use of a DNA phosphorothioylation modified gene cluster, the nucleotide sequence of the gene cluster or the cloned gene or DNA fragment of a small part of the sequence is transferred into an exogenous Hosts, these exogenous hosts include Escherichia coli, Streptomyces, Bacillus, yeast, plants, animals and engineering bacteria, can endow the host with CpsC phosphorothioylation properties.

The invention also provides the use of a DNA phosphorothioylation modification gene cluster, which can be used in biopharmaceutical fields such as heterologous host DNA modification, epigenetics, and oligonucleotide drug modification.

The present invention also provides a use of a DNA phosphorothioylation modification gene cluster to find a nuclease with a new function through the DNA phosphorothioylation modification controlled by the gene cluster.

Compared with the prior art, the present invention has beneficial effects: the following purposes can be achieved by using the gene cluster of the present invention:

The nucleotide sequence can be modified or mutated by means of insertions or substitutions, polymerase chain reaction, error-mediated polymerase chain reaction, site-specific mutations, rejoining of different sequences, sequence differences Directed evolution (DNA shuffling) of homologous sequences from partial or other sources, or mutagenesis with ultraviolet light or chemical reagents, etc.

The amino acid sequence or at least a partial sequence of the single-chain modification, phosphorothioylation modification gene cluster with a specific CpsC modification site provided by the present invention can be used to isolate the desired protein and be used for antibody preparation.

The genes or gene clusters of nucleotide sequences or partial sequences provided by the present invention can be expressed in heterologous hosts, and their functions in host metabolism can be understood by DNA chip technology.

The amino acid sequence or partial sequence of the polypeptide modified by phosphorothioation in the present invention may still have biological activity or even new biological activity after removing or replacing a certain amino acid, or increase the yield or optimize protein dynamics characteristics or other properties that strive to be obtained.

In a word, the information of all the genes and proteins of the phosphorothioylation modification gene cluster with single-strand modification characteristics and specific CpsC modification sites provided by the present invention will help to further clarify and understand the relationship between DNA phosphorothioylation modification in microorganisms. chemical nature and biological origin. The phosphorothioylation modification of DNA has the characteristics of resisting hydrolysis of various nucleases, so the CpsC phosphorothioylation modified gene and encoded protein provided by the present invention are of great significance for the development of antisense oligonucleotide drugs.

Description of drawings

Figure 1 is a schematic diagram of the structure of a DNA phosphorothioation modification gene cluster;

Figure 2 is a comparison of the genotypes of some in-frame deletion mutants and wild-type marine halophilic Vibrio FF75,

Arrows indicate a gene and the direction of transcription, and the blank area is the missing fragment in the mutant strain. XXL-2, XXL-4, XXL-5, and XXL-7 are single-gene in-frame deletion strains of vpt2, vpt1, vpt3, and vpt4, respectively;

Figure 3 is the analysis chart of the artificial standard CpsC phosphorothioylation modified high performance liquid chromatography-secondary mass spectrometry (LC-MS/MS),

The arrow in A shows the precursor ion mass spectrum of the artificial standard CpsC phosphorothioylation modified primary mass spectrometry, m/z=533; B the secondary mass spectrometry product ion spectrum, m/z are 422,112 respectively;

Figure 4 is the LC-MS/MS analysis diagram of the mutant strain DNA generated by in-frame deletion of each gene in the marine halophilic Vibrio cyclitrophicus FF75 DNA phosphorothioylation modification gene cluster,

A is the LC-MS/MS precursor ion map of marine halophilic Vibrio wild-type FF75. The peaks before 10 min are all solvent or impurity peaks. The peak elution time of the target product CpsC is about 16 minutes, which is the same as that of the artificial standard;

B is the LC-MS/MS product ion map of the marine halophilic Vibrio Vibrio wild-type FF75, m/z are 422,122 respectively;

C is the same frame deletion vpt1 mutant; D is the same frame deletion vpt2 mutant; E is the same frame deletion vpt3 mutant; F is the same frame deletion vpt4 mutant;

Figure 5 is the LC-MS/MS diagram of XXL-1 (knockout 1585),

A is the parent ion mass spectrum of the mutant strain XXL-1 first-order mass spectrometry, m/z=533,

B is the secondary mass spectrometry product ion spectrum, m/z are 422,112 respectively;

Figure 6 is the LC-MS/MS diagram of the mutant strain XXL-3 (knockout 1587-1590),

A is the precursor ion mass spectrum of the mutant strain XXL-3, m/z=533;

B is the secondary mass spectrometry product ion spectrum, m/z are 422,112 respectively;

Fig. 7 is the LC-MS/MS figure of mutant strain XXL-6 (deletion 1594),

A is the precursor ion mass spectrum of the mutant strain XXL-6, m/z=533;

B is the secondary mass spectrometry product ion spectrum, m/z are 422,112 respectively;

Figure 8 is a DNA single molecule real time sequencing (Single molecule real time sequencing, SMRT) diagram,

A is part of the SMRT map of marine halophilic Vibrio cyclitrophicus FF75, PT represents phosphorothioylation modification,

B is the SMRT map of Escherichia coli B7A part;

Figure 9 is a schematic diagram of the structure of the heterologous expression plasmid pWHU732;

Figure 10 is the LC-MS/MS analysis diagram of type II DNA phosphorothioylation modification gene cluster in Escherichia coli E.coli ER2796 heterologously expressed DNA

A and B are the precursor ion and product ion LC-MS/MS diagrams of the control ER2796 (pBluescriptⅡSK(+)), respectively;

C and D are the precursor ion and product ion LC-MS/MS diagrams of ER2796 (pWHU732), respectively.

detailed description

The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings. The following examples are implemented on the premise of the technical solutions of the present invention, and detailed implementation methods and specific operation processes are provided, but the protection scope of the present invention is not limited to the following examples. Those who do not indicate specific techniques or conditions in the embodiments, according to the techniques or conditions described in the literature in this field (for example, refer to "Molecular Cloning Experiment Guide" translated by J. Sambrook et al., Huang Peitang et al., third edition, Science Press) or follow the product instructions. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Example 1: Preliminary deduction of CpsC phosphorothioation gene cluster

Download the genome file of Vibrio halophilus FF75 from the NCBI website, and upload it to the Rapid annotation using subsystem technology website for genome annotation. After the analysis, all about 4500 genes were searched with keywords of thioredoxin (PAPS reductase) and cysteine desulfurase (cysteine desulfurase). The obtained candidate genes were compared one by one, and a gene cluster composed of three genes with a length of about 6 kb was found, and the third gene was PAPS reductase. At the same time, a gene encoding cysteine desulfurase was found about 7 kb upstream of the gene cluster. The other genes in and around this region do not belong to any sulfur metabolism system, so it is inferred that this region is very likely to be a gene cluster encoding CpsC phosphorothioylation modification of DNA. These four genes are named vpt1 , vpt2 , vpt3 and The structure of vpt4 is shown in Figure 1.

Example 2: Gene knockout proves that vpt1, vpt2, vpt3 and vpt4 are essential genes for marine halophilic Vibrio cyclitrophicus FF75 DNA phosphorothioylation modification

The primers corresponding to the four genes were designed according to the genome sequence of the marine halophilic Vibrio FF75. Using the extracted total DNA of Vibrio FF75 as a template, the left and right arms of each gene were PCRed, and the left and right arms were synthesized by the second PCR. The left and right arms of the TA clone were sequenced. After the result is correct, use the SpeI and SacI double enzyme digestion system to treat the recombined T vector to obtain the fragment of the conjoined left and right arms; use the same combination of endonucleases to treat the suicide vector pJC4 plasmid to obtain a double-nicked linear plasmid band. Or ligated by T4 ligase.

The ligation product was electrotransformed into E. coli WM6024 (the strain is DAP auxotroph), and spread on a plate containing chloramphenicol and DAP. Pick a single clone and culture it in LB containing chloramphenicol and DAP, extract the plasmid, and use the SpeⅠ and SacⅠ double enzyme digestion system to verify the enzyme digestion. The correct strain can be used as the donor bacteria for the conjugation transfer experiment, and Vibrio FF75 is used as the recipient. body bacteria. Take a single clone of Vibrio FF75 and E. coli WM6024 with the left and right arm sequences of the corresponding gene and culture them overnight in the corresponding liquid medium, and then carry out the binding transfer experiment on the plate containing DAP without antibiotics (using the patch mating method combined transfer). The zygote was screened with DAP-free chloramphenicol-resistant medium, and the screened zygote could be used for the next step of sucrose-guided double crossover experiment. Mutant strains can be screened by culturing in a medium containing 5% sucrose for two rounds, and then the screened strains are verified by PCR and sequenced to finally determine that they are mutant strains of the corresponding gene.

Experiments have shown that the knockout mutant strains XXL-4, XXL-2, XXL-5, and XXL7 obtained by the above method (respectively, the deletion genes vpt1, vpt2, vpt3, vpt4 ) (see Figure 2), extract genomic DNA, and LC-MS/MS detection was carried out separately, and the peak eluting time of the target product CpsC was generally about 16 minutes. Compared with the artificial standard CpsC phosphorothioylation modified LC-MS/MS (see Figure 3), it can be seen that Vibrio wild-type FF75 There are obvious CpsC peaks in (see Figure 4A, 4B). Fig. 4 CDEF is the same frame mutant strains XXL-4, XXL-2, XXL-5, XXL-7 respectively, that is, the genes vpt1.vpt2.vpt3.vpt4 are deleted respectively. It can be seen from the figure that none of the characteristic peaks of CpsC appear, proving that the Individual genes in the gene cluster are indeed responsible for DNA phosphorothioylation in Vibrio cyclitrophicus FF75.

This provides a method for examining and determining the genes and functions involved in the CpsC phosphorothioylation modification of Vibrio cyclitrophicus FF75 by knocking out individual genes in the Vibrio cyclitrophicus FF75 DNA phosphorothioylation gene cluster.

Example 3: Determination of the boundary of the marine halophilic Vibrio cyclitrophicus FF75 DNA phosphorothioylation modification gene cluster

In Example 2, the mutant strains XXL-4, XXL-2, XXL-5, and XXL-7 (knockout of vpt1 , vpt2 , vpt3 , and vpt4 respectively ) lost DNA phosphorothioation modification, so the gene knockout according to Example 2 In addition, the upstream and downstream were knocked out, and the mutant strain XXL-1 (knockout 1585), mutant strain XXL-3 (knockout 1587-1590), and XXL-6 (knockout 1594) were obtained respectively. Through LC- MS/MS detection, experiments show that the mutant strains XXL-1, XXL-3 and XXL-6 still have DNA phosphorothioylation modification (see Figures 5, 6, and 7 below, respectively). Therefore, Vibrio cyclitrophicus FF75 DNA phosphorosulfur The upstream boundary of the acylation modification gene cluster was determined between 1585 and vpt4 , the downstream boundary was determined between vpt3 and 1594, and the interval between vpt4 and vpt1 was 1587-1590. However, when any one of vpt1 , vpt2 , vpt3 , and vpt4 was knocked out in Example 2, the DNA phosphorothioylation modification disappeared, further illustrating that vpt1 , vpt2 , vpt3 , and vpt4 are CpsC phosphorothioylation gene clusters.

Example 4: Functional Analysis of Vibrio cyclitrophicus FF75DNA Phosphorylation Modified Gene Cluster

Through the determination of the boundaries of the DNA phosphorothioylation modified gene clusters, the knockout of each gene and the use of bioinformatics provided the relevant effects of some genes in the DNA phosphorothioylation modified gene clusters.

The function of vpt3 : Through the in-frame knockout of the vpt3 gene in Vibrio FF75, it was found that it must be associated with CpsC phosphorothioation. Input the protein sequence of vpt3 into NCBI's BLAST website for homology comparison and conserved domain analysis (using PSI-BLAST), and found that it has two conserved domains, one is the PAPS reductase domain, and the other is the dihydrogen Domains of orotase. This indicates that vpt3 may possess the same PAPS reductase activity as the DndC protein in the type I DNA phosphorothioation gene cluster. At the same time, vpt3 also has the ability to convert carbamoylaspartate to dihydroorotic acid, which is also a key reaction in the biosynthesis of pyrimidines. However, only 31% of the identities and 43% of the positives were compared with the DndC protein sequence of the typical type I phosphorothioylating bacteria E. coli B7A. In addition, the conserved domain analysis of DndC in large intestine B7A only found the domain of PAPS reductase, but not the domain of dihydroorotase, indicating that there are still many differences in the biological functions of the two.

The function of vpt4 : Through knocking out the vpt4 gene in Vibrio FF75 in-frame, the gene necessary for its phosphorothioation with CpsC was found, and the protein sequence of vpt4 was entered into the BLAST website of NCBI for homologous alignment and conserved structure Domain analysis (using PSI-BLAST) revealed that it has a cysteine desulfurase domain, NifS. This indicates that vpt4 may have the same function as the Dnd A protein of the type I DNA phosphorothioation gene cluster. Previous studies have found that DndA in bacteria is often replaced by the IscS protein, so that some DNA phosphorothioation species did not find the dndA gene. In addition, in-frame deletion experiments proved that the role of vpt4 in Vibrio FF75 is irreplaceable.

The function of vpt1 : Through the in-frame knockout of the vpt1 gene in Vibrio FF75, it was found that it must be associated with CpsC phosphorothioation. The protein sequence of vpt1 was entered into NCBI's BLAST website for homology alignment and conserved domain analysis (using PSI-BLAST), and it was found that it belonged to the DUF4007 superfamily. The proteins of this family are about 300 amino acids, similar to vpt1 . In some proteins, this domain associates with that of PAPS reductase, suggesting a possible common role. In this gene cluster, it shows that the functional relationship between vpt1 and vpt3 may be very close.

The function of vpt2 : Through the in-frame knockout of the vpt2 gene in Vibrio FF75, it was found that it must be associated with CpsC phosphorothioation. Then input the protein sequence of vpt2 into NCBI's BLAST website for homology comparison and conserved domain analysis (using PSI-BLAST), and found that less than 5% of its amino acid sequence was found as a conserved domain of NHLM, This structure belongs to the ABC transporter family of the bacteriocin system. The characteristic is that vpt2 has a propeptide sequence conserved in this family and is homologous to a region of nitrile hydratase.

Example 5: DNA single molecule real time sequencing (Single molecule real time sequencing, SMRT) detection of single-strand phosphorothioylation modification of CpsC

Through SMRT sequencing of the wild-type genomic DNA of the marine halophilic Vibrio FF75, it was found that its specific modification site was CpsC phosphorothioylation modification, but its modification site was not a palindromic sequence, and phosphorothioation modification only occurred Phosphorothioylation was not found on the side chain between bases CC and its complementary chain (see Figure 8A). This is different from some traditional methylation modifications and GpsG, GpsA, GpsT phosphorothioylation modifications to recognize reverse complementary sequences for double-strand modification, such as SMRT sequencing of Escherichia coli B7A genomic DNA, phosphorothioylation modification Not only occurred on the side chain between the bases GA, but also found GT phosphorothioylation modification on the complementary chain (see Figure 8B).

Example 6: Heterologous expression of the marine halophilic Vibrio cyclitrophicus FF75 DNA phosphorothioylation modification gene cluster in Escherichia coli ER2796

After the total DNA of Vibrio FF75 was extracted by phenol-chloroform extraction, the fragments of vpt123 and vpt4 (both including the promoter region) were obtained by PCR respectively, and the two ends of the fragment (5828 bp) containing vpt123 were respectively added with KpnⅠ and BamHI restriction sites point, and recombined to the corresponding cloning site of pBluescriptⅡSK (+) by restriction enzyme digestion and enzyme ligation. cutting, enzyme-ligating and recombining to the corresponding cloning site of pWHU730, and the recombined vector was named pWHU732 (see Figure 9). Then the recombinant vector pWHU732 was transformed into the expression host E.coli ER2796 without CpsC phosphorothioylation modification, and the transformed strain was called ER2796 (pWHU732). At the same time, the original vector pBluescriptⅡSK(+) was also introduced into ER2796 to obtain ER2796 (pBluescriptⅡSK(+)), which was used as negative control bacteria.

Example 7: LC-MS/MS Detection of DNA Phosphorylation in E. coli ER2796 (pWHU732)

The DNA sample of E. coli ER2796 (pWHU732) was extracted by phenol-chloroform method and digested with nuclease P1. The system was: 100 μl system containing sodium acetate (pH5.2) with a final concentration of 30 mM, 0.5 mM zinc chloride and one unit Nuclease P1, react at 50°C for 2 hours. Then 10 ul of Tris-HCl (pH 8.0) and 4 U of alkaline dephosphatase CIP were added to the system, and reacted at 37°C for 4 hours. Finally, it was purified with a purification column (PALL Company), and the solution in the collection tube of the lower layer was spin-dried for storage. Before loading the mass spectrometer, dissolve it with 22 ul of ddH2O and add it to the inner liner of the mass spectrometry vial. Genomic DNA of Escherichia coli E. coli ER2796 (pWHU732) into which the DNA phosphorothioation modification gene cluster was transferred was extracted and used for LC-MS/MS detection.

LC-MS detection conditions are as follows: HPLC column adopts Thermo Hypersil Gold aQ column (150×2.1mm, 3um), flow rate is 0.2 ml/min, elution adopts 97% buffer A (0.1% acetic acid water, HPLC grade, Honeywell company), 3% buffer C (0.1% acetic acid acetonitrile, Honeywell company) for 5 min, 94% buffer A, 6% buffer C for 35 min, 2% buffer A, 98% buffer C for 15 min, and finally 97% buffer A, eluted with 3% buffer C for 20 min.

The Thermo TQU 03390 Triple Quad LC/MS used in the mass spectrometry was performed in positive ion mode, and the ion source was HESI (Hard Electron-spray Ionization);

The series of parameters used are: atomizer pressure: 40psi; atomizer temperature: 30°C; drying temperature (drying gas temperature): 320°C; capillary voltage (capillary voltage): 3500 V; SRM mode is used for detection of modified bases The dCpsC in the base was compared with the synthetic standard substance (purchased from IBA GmbH) modified by phosphorothioation of CpsC. The results are shown in Figure 3. The optimized parameters (retention time, mass spectrometry m/z, , collision energy) as follows, d(CpsC): 16, 533, 422, 112, 30. The LC-MS/MS detection results are shown in Figure 10. Figures A and B are the precursor ion and product ion LC-MS/MS images of the control ER2796 (pBluescriptⅡSK(+)), respectively, without CpsC modification; Figures C and D are ER2796 (pWHU732) parent ion and product ion LC-MS/MS diagram, obvious CpsC phosphorothioylation modification target peaks can be seen, indicating that the CpsC phosphorothioation modification gene cluster can be successfully reproduced in heterologous hosts.

Claims (4)

1. A DNA phosphorothioylation modifying gene cluster, characterized in that the DNA phosphorothioylation controlled by the gene cluster is modified as a single-strand modification, and the modification selective sequence site is CpsC, and the gene cluster comprises 4 genes , are respectively vpt1, vpt2, vpt3 and vpt4, the nucleotide sequence is as shown in SEQ ID NO:1 or the complementary sequence formed by SEQ ID NO:1, wherein: (1) The gene vpt4 is located at base 5146-6132 in the nucleotide sequence of the gene cluster, with a length of 987 base pairs. The encoded protein sequence is shown in SEQ ID NO: 2. The gene encodes cysteine desulfurase ; (2) The gene vpt3 is located at base 4300-5145 in the nucleotide sequence of the gene cluster, with a length of 846 base pairs. The encoded protein sequence is shown in SEQ ID NO: 3, and the gene is encoded as 3' adenosine phosphate - 5' phosphosulfate reductase (PAPS); (3) The gene vpt2 is located at the 967th-4299th base of the nucleotide sequence of the gene cluster, with a length of 3333 base pairs. The encoded protein sequence is shown in SEQ ID NO: 4, and the protein encoded by the gene belongs to ABC transport protein family; (4) Gene vpt1 is located at bases 1-966 of the nucleotide sequence of the gene cluster, with a length of 966 base pairs. The encoded protein sequence is shown in SEQ ID NO: 5. The protein encoded by this gene belongs to the DUF4007 super family. 2. The DNA phosphorothioation modification gene cluster according to claim 1, characterized in that it is derived from the marine halophilic Vibrio cyclitrophicus FF75. 3. The use of the DNA phosphorothioylation modified gene cluster according to claim 1 or 2, characterized in that: the cloned gene or DNA fragment of the nucleotide sequence or a small part of the sequence of the gene cluster is transduced, transformed, The method of conjugative transfer is to transfer to exogenous hosts, which include Escherichia coli, Streptomyces, Bacillus, yeast, plants, animals and engineering bacteria, which can endow the host with CpsC phosphorothioylation modification characteristics. 4. The use of the DNA phosphorothioylation modification gene cluster according to claim 1 or 2, characterized in that it can be used for heterologous host DNA modification, epigenetics, and oligonucleotide drug modification.