A method for constructing a directed SNP mutant in neurospora crassa
By designing homologous arms and selection markers in Neurospora crassa, and combining fusion PCR and homologous recombination technologies, linear fragments containing mutation sites were constructed. This solved the problems of strong randomness of mutation sites and instability of the CRISPR/Cas9 system in existing technologies, and achieved efficient and accurate construction of directional SNP mutants.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING UNIV
- Filing Date
- 2026-02-02
- Publication Date
- 2026-06-23
AI Technical Summary
Existing technologies for constructing gene mutants in Neurospora crassa present problems such as strong randomness of mutation sites, large screening workload, difficulty in achieving precise substitution of specific bases, and unstable editing efficiency of the CRISPR/Cas9 system in this system, as well as the risk of off-target and random integration.
By designing homologous arms and selection markers, and combining fusion PCR and homologous recombination technologies, a complete linear fragment containing mutation sites, homologous arms, and selection markers was constructed. Homologous recombination mechanism was used to achieve efficient and accurate genome integration. The hygromycin resistance gene hph was used as a selection marker, and the accuracy of the mutation was verified by sequencing.
This study enables efficient and precise construction of targeted SNP mutants in Neurospora crassa, simplifies the operation process, improves the success rate and reproducibility of single-base editing, and provides a stable and controllable gene editing method.
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Figure CN122256399A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, and more specifically to a method for constructing directed SNP mutants in Neurospora crassa. Background Technology
[0002] Neurospora crassa ( Neurospora crassa As a classic model filamentous fungus, *Phyllostachys edulis* plays a vital role in genetics, epigenetics, biochemistry, and molecular biology research. Its short growth cycle, simple genetic system, and abundant mutant resources make it an ideal system for studying eukaryotic gene function, signal transduction, and metabolic regulation. In gene function research, constructing mutants at specific gene sites, especially single nucleotide polymorphism (SNP) mutants, is essential for elucidating the functions of key amino acid residues, resolving the relationship between protein structure and function, and studying the role of gene regulatory elements.
[0003] Currently, traditional methods for constructing gene mutants in *Neurospora crassa* mainly include chemical mutagenesis (such as using ethyl methanesulfonate EMS) and physical mutagenesis (such as ultraviolet irradiation). Although these methods can generate a rich mutant library, the mutation sites are random, making subsequent screening of target mutants time-consuming and laborious, and making it difficult to achieve precise substitution of specific bases. In recent years, the CRISPR / Cas9 system, as a highly efficient genome editing tool, has been successfully applied in animals, plants, and various fungi. However, when performing CRISPR / Cas9 editing in the *Neurospora crassa* system, it is usually necessary to simultaneously transform multiple plasmids carrying the Cas9 protein-coding gene, gRNA expression cassette, and donor DNA template sequence, respectively. The experimental process is complex and the editing efficiency is low, so it cannot yet become a standard tool for gene editing in *Neurospora crassa*. In addition, the random integration of plasmids or small DNA fragments into the genome during transformation may introduce unpredictable genetic background interference, posing certain potential risks. Therefore, developing an efficient, accurate, and easy-to-operate method for constructing directional SNP mutants to overcome the limitations of existing technologies and meet the practical needs of Neurospora crassa gene function research has significant scientific value and application prospects.
[0004] Currently, there is a lack of methods for constructing directed SNP mutants in Neurospora crassa. Summary of the Invention
[0005] The purpose of this invention is to provide a method for efficiently and accurately constructing targeted SNP mutants in Neurospora crassa. This method achieves single-base substitution at the target site by rationally designing homologous arms and selection markers, combined with fusion PCR and homologous recombination techniques, and ensures the accuracy of the mutation through resistance screening and sequencing verification.
[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In one aspect, this application provides a method for constructing directed SNP mutants in Neurospora crassa.
[0007] Secondly, this application provides a kit for implementing the method.
[0008] The first aspect of this application provides a method for constructing directed SNP mutants in Neurospora crassa, comprising the following steps: (1) Preparation of gene fragment A containing SNP: Using Neurospora crassa genomic DNA as a template, forward primer AF and reverse primer AR were designed, where AF contains the target SNP site and AR is located downstream of the 3'-UTR of the gene; after synthesizing the primers, fragment A was obtained by PCR amplification and recovered, 20 μL PCR reaction system; (2) Preparation of upstream homologous fragment B: Design primers upstream of fragment A to amplify fragment B of 1000 bp, wherein the reverse primer BR contains the reverse complementary sequence of AF and the full length is not less than 40 bp; amplify fragment B using genomic DNA as a template and recover it; (3) Preparation of downstream homologous fragment C: Design primers at 500-1000 bp downstream of fragment A to amplify fragment C, which is 1000 bp long, using genomic DNA as a template for amplification and recovery; (4) Amplification of the hygromycin resistance gene hph: Using plasmid PCSN44 containing the hygromycin resistance gene hph, primers hph-F and hph-R were designed, wherein hph-F is inversely complementary to the reverse primer AR of fragment A, and hph-R is inversely complementary to the forward primer of fragment C; the hph gene fragment was obtained by PCR amplification and recovered; the nucleotide sequence of PCSN44 is shown in SEQ ID NO:11; (5) Construction of fusion fragment BA-hph-C: Fragment A, fragment B, fragment C and hph were mixed in equal amounts of 40 ng. Fusion PCR was performed using the forward primer BF of fragment B and the reverse primer CR of fragment C to obtain the fusion fragment. The PCR reaction system was 40 μL. The size was verified by agarose gel electrophoresis, and the SNP was correctly introduced after recovery, purification and sequencing. (6) Electroporation and resistance screening: The fusion fragment was transferred into Neurospora crassa by electroporation, and after cultivation, colonies were picked and screened for positive transformants in a medium containing hygromycin. (7) Mutation site verification: Extract genomic DNA from homozygous transformants, amplify the target fragment using primers BF and CR, and verify the correctness of the SNP by sequencing and exclude unexpected mutations.
[0009] Further, in step (1), the Neurospora crassa genomic DNA is the NCU02247 gene. The wild-type nucleotide sequence of the NCU02247 gene is shown in SEQ ID NO:1, and the amino acid sequence of the NCU02247 mutant is shown in SEQ ID NO:2. The nucleotide sequence of the forward primer AF is shown in SEQ ID NO:3, the nucleotide sequence of the reverse primer AR is shown in SEQ ID NO:4, the nucleotide sequence of the forward primer BF is shown in SEQ ID NO:5, the nucleotide sequence of the reverse primer BR is shown in SEQ ID NO:6, the nucleotide sequence of the forward primer CF is shown in SEQ ID NO:7, the nucleotide sequence of the reverse primer CR is shown in SEQ ID NO:8, the nucleotide sequence of the forward primer hph-F is shown in SEQ ID NO:9, and the nucleotide sequence of the reverse primer hph-R is shown in SEQ ID NO:10.
[0010] Further, in step (1), the PCR reaction system consists of: 2.6 μL DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, and 3.4 μL ddH2O; the forward primer AF is 18-21 bp in length, wherein the 3' end region contains artificially introduced specific base changes to introduce the target SNP. This includes artificially introduced specific base changes.
[0011] Furthermore, in step (2), the reverse primer BR is 40-60 bp in length, wherein the 3' end contains a sequence that is completely inversely complementary to the forward primer AF; in step (4), the vector containing the hygromycin resistance gene hph is plasmid pCSN44; 20 μL PCR reaction system: 2.6 μL plasmid DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, 3.4 μL ddH2O.
[0012] Further, in step (5), mixing refers to mixing fragment A, fragment B, fragment C with the hph gene fragment in an equimolar ratio; the extension time of the fusion PCR is 4-6 minutes; the PCR reaction system is: 1.36 μL A, 1.3 μL B, 1.3 μL C, 1.3 μL hph, 2 μL BF, 2 μL CR, 20 μL high-fidelity DNA polymerase Master Mix, and 6.8 μL ddH2O.
[0013] Furthermore, in step (6), the hygromycin screening concentration is 150-300 μg / mL.
[0014] Furthermore, in step (6), the hygromycin screening concentration is 200 μg / mL.
[0015] Furthermore, step (7) includes verifying by first-generation sequencing that there are no other unexpected base changes besides the target SNP.
[0016] A second aspect of this application provides a kit for implementing the method, comprising: Master Mix, a high-fidelity DNA polymerase for amplifying fragments A, B, and C; Hygromycin resistance gene hph or a vector containing the hph gene; High-fidelity DNA polymerase Master Mix for fusion PCR; The instruction manual describes the steps of the method as described.
[0017] Beneficial Effects: This invention provides a method for efficiently constructing targeted SNP mutants in *Neurospora crassa*. This method has a clear operational procedure, low technical threshold, and enables precise, site-specific modification of specific gene loci. This method not only provides a direct and reliable technical tool for studying gene function and the role of key amino acid residues in proteins of *Neurospora crassa*, but also lays a crucial experimental foundation for in-depth analysis of the genetic regulatory network, metabolic pathway evolution, and adaptive mechanisms of filamentous fungi. Furthermore, the strategy of combining fusion PCR and homologous recombination employed in this method can also provide technical reference and process optimization ideas for the fine gene editing of other filamentous fungi or eukaryotic microorganisms.
[0018] Compared with existing technologies, this invention has the following advantages: This invention provides, for the first time, a homologous recombination editing scheme in *Neurospora crassa* that is independent of the CRISPR / Cas9 system and optimized for precise single-base substitution. In recent years, the application of CRISPR-based gene editing technology in fungi has increased, but existing methods in *Neurospora crassa* typically involve multi-plasmid co-transformation, unstable editing efficiency, and risks of off-target effects and random integration. Furthermore, while traditional chemical or radiation mutagenesis methods can generate mutations, the mutations are highly random and require a large screening workload, failing to meet the research needs for specific SNP sites. This invention, through the ingenious design of overlapping primers and fragment splicing strategies, achieves direct in vitro assembly of complete linear fragments containing mutation sites, homologous arms, and selection markers, utilizing homologous recombination to achieve efficient and precise genome integration. This method not only avoids the cumbersome steps of complex vector construction and multi-system coordination but also significantly improves the success rate and reproducibility of single-base editing, providing a more stable, controllable, and easily scalable gene editing method for *Neurospora crassa* genetic research. Attached Figure Description
[0019] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1 This is a schematic diagram of the process of the present invention; Figure 2 This is a schematic diagram illustrating the homologous recombination of the fusion fragment BA-hph-C with the genome sequence of the present invention; Figure 3 This is a schematic diagram of the hygromycin resistance screening plate results of the present invention; wherein, white single colonies are positive transformants.
[0021] Figure 4 This is a schematic diagram illustrating the sequencing verification of SNPs introduced in this invention; wherein, the variant site is a gene. NCU02247 The stop codon (TAG) was mutated by a single base substitution from A to G, resulting in a TAG mutation that replaced the TAG with tryptophan (TGG). Detailed Implementation
[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0023] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.
[0024] In this application, "~ one less" means one or more, and "more than" means two or more. "~ one less item (item) below" or similar expressions refer to any combination of these items, including any combination of single items (items) or multiple items (items). For example, "~ one less item (item) in a, b, or c", or "~ one less item (item) in a, b, and c", can all mean: a, b, c, a~b (i.e., a and b), a~c, b~c, or a~b~c, where a, b, and c can be single or multiple.
[0025] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.
[0026] The first aspect of this application provides a method for constructing a directed SNP mutant in Neurospora crassa, comprising the following steps: (1) Preparation of gene fragment A containing SNP: Using Neurospora crassa genomic DNA as a template, forward primer AF and reverse primer AR were designed, where AF contains the target SNP site and AR is located downstream of the 3'-UTR of the gene; after synthesizing the primers, fragment A was obtained by PCR amplification and recovered, 20 μL PCR reaction system; (2) Preparation of upstream homologous fragment B: Design primers upstream of fragment A to amplify fragment B of 1000 bp, wherein the reverse primer BR contains the reverse complementary sequence of AF and the full length is not less than 40 bp; amplify fragment B using genomic DNA as a template and recover it; (3) Preparation of downstream homologous fragment C: Design primers at 500-1000 bp downstream of fragment A to amplify fragment C, which is 1000 bp long, using genomic DNA as a template for amplification and recovery; (4) Amplification of the hygromycin resistance gene hph: Using plasmid PCSN44 containing the hygromycin resistance gene hph, primers hph-F and hph-R were designed, wherein hph-F is inversely complementary to the reverse primer AR of fragment A, and hph-R is inversely complementary to the forward primer of fragment C; the hph gene fragment was obtained by PCR amplification and recovered (20 μL PCR reaction system: 2.6 μL plasmid DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, 3.4 μL ddH2O); the nucleotide sequence of PCSN44 is shown in SEQ ID NO:11; (5) Construction of fusion fragment BA-hph-C: Fragment A, fragment B, fragment C and hph were mixed in equal amounts of 40 ng. Fusion PCR was performed using the forward primer BF of fragment B and the reverse primer CR of fragment C to obtain the fusion fragment. The PCR reaction system was 40 μL. The size was verified by agarose gel electrophoresis, and the SNP was correctly introduced after recovery, purification and sequencing. (6) Electroporation and resistance screening: The fusion fragment was transferred into Neurospora crassa by electroporation, and after cultivation, colonies were picked and screened for positive transformants in a medium containing hygromycin. (7) Mutation site verification: Extract genomic DNA from homozygous transformants, amplify the target fragment using primers BF and CR, and verify the correctness of the SNP by sequencing and exclude unexpected mutations.
[0027] In some embodiments, in step (1), the Neurospora crassa genomic DNA is the NCU02247 gene, the wild-type nucleotide sequence of the NCU02247 gene is shown in SEQ ID NO:1, and the amino acid sequence of the NCU02247 mutant is shown in SEQ ID NO:2; the nucleotide sequence of the forward primer AF is shown in SEQ ID NO:3, the nucleotide sequence of the reverse primer AR is shown in SEQ ID NO:4, the nucleotide sequence of the forward primer BF is shown in SEQ ID NO:5, the nucleotide sequence of the reverse primer BR is shown in SEQ ID NO:6, the nucleotide sequence of the forward primer CF is shown in SEQ ID NO:7, the nucleotide sequence of the reverse primer CR is shown in SEQ ID NO:8, the nucleotide sequence of the forward primer hph-F is shown in SEQ ID NO:9, and the nucleotide sequence of the reverse primer hph-R is shown in SEQ ID NO:10.
[0028] In some embodiments, in step (1), the PCR reaction system consists of: 2.6 μL DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, and 3.4 μL ddH2O; the forward primer AF is 18-21 bp in length, wherein the 3' end region contains artificially introduced specific base changes to introduce the target SNP. This includes artificially introduced specific base changes.
[0029] In some embodiments, in step (2), the reverse primer BR is 40-60 bp in length, wherein the 3' end contains a sequence that is completely inversely complementary to the forward primer AF; in step (4), the vector containing the hygromycin resistance gene hph is plasmid pCSN44; 20 μL PCR reaction system: 2.6 μL plasmid DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, 3.4 μL ddH2O.
[0030] In some embodiments, in step (5), mixing refers to mixing fragment A, fragment B, fragment C with the hph gene fragment in an equimolar ratio; the extension time of the fusion PCR is 4-6 minutes; the PCR reaction system is: 1.36 μL A, 1.3 μL B, 1.3 μL C, 1.3 μL hph, 2 μL BF, 2 μL CR, 20 μL high-fidelity DNA polymerase Master Mix, and 6.8 μL ddH2O.
[0031] In some embodiments, in step (6), the hygromycin screening concentration is 150-300 μg / mL.
[0032] In some embodiments, in step (6), the hygromycin screening concentration is 200 μg / mL.
[0033] Furthermore, step (7) includes verifying by first-generation sequencing that there are no other unexpected base changes besides the target SNP.
[0034] A second aspect of this application provides a kit for implementing the method, comprising: Master Mix, a high-fidelity DNA polymerase for amplifying fragments A, B, and C; Hygromycin resistance gene hph or a vector containing the hph gene; High-fidelity DNA polymerase Master Mix for fusion PCR; The instruction manual describes the steps of the method as described.
[0035] The present invention will be further described below by way of examples, but these are not intended to limit the scope of application of the present invention.
[0036] Example 1 The present invention provides a method for constructing directed SNP mutants in Neurospora crassa, comprising the following steps: (1) Preparation of gene fragment A containing the SNP: Using Neurospora crassa genomic DNA as a template, forward primer AF and reverse primer AR were designed, where AF contains the target SNP site and AR is located downstream of the 3'-UTR of the gene; after synthesizing the primers, fragment A was obtained by PCR amplification and recovered (20 μL PCR reaction system: 2.6 μL DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, 3.4 μL ddH2O); the forward primer AF is 18 bp in length, and the 3' end region contains a specific base change introduced artificially to introduce the target SNP.
[0037] (2) Preparation of upstream homologous fragment B: Design primers upstream of fragment A to amplify fragment B of 1000 bp, wherein the reverse primer BR contains the reverse complementary sequence of AF and the full length is not less than 40 bp; amplify fragment B using genomic DNA as a template and recover it; the reverse primer BR is 40 bp in length, wherein the 3' end contains a sequence that is completely reverse complementary to the forward primer AF.
[0038] (3) Preparation of downstream homologous fragment C: Primers were designed 800 bp downstream of fragment A to amplify fragment C, which was 1000 bp long. Genomic DNA was used as a template for amplification and recovery. (4) Hygromycin resistance gene hphAmplification: Using a vector containing the hygromycin resistance gene hph, primers hph-F and hph-R were designed, where hph-F is inversely complementary to the reverse primer AR of fragment A, and hph-R is inversely complementary to the forward primer C of fragment C. The hph gene fragment was obtained by PCR amplification and recovered (20 μL PCR reaction system: 2.6 μL plasmid DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, 3.4 μL ddH2O). The vector containing the hygromycin resistance gene hph was plasmid pCSN44. The nucleotide sequence of PCSN44 is shown in SEQ ID NO:11.
[0039] (5) Construction of the fusion fragment BA-hph-C: Fragment A, fragment B, fragment C and hph were mixed in equal amounts (40 ng). Fusion PCR was performed using the forward primer BF of fragment B and the reverse primer CR of fragment C to obtain the fusion fragment (40 μL PCR reaction system: 1.36 μL A, 1.3 μL B, 1.3 μL C, 1.3 μL hph, 2 μL BF, 2 μL CR, 20 μL high-fidelity DNA polymerase Master Mix, 6.8 μL ddH2O). The size was verified by agarose gel electrophoresis, and the SNP was correctly introduced after recovery, purification and sequencing. The mixing refers to mixing fragment A, fragment B, fragment C and hph gene fragment in an equimolar ratio. The extension time of the fusion PCR was 6 minutes.
[0040] (6) Electroporation and resistance screening: The fusion fragment was transferred into Neurospora crassa by electroporation. After cultivation, colonies were picked and screened for positive transformants in a culture medium containing hygromycin. The concentration of hygromycin for screening was 150 μg / mL.
[0041] (7) Mutation site verification: Genomic DNA of homozygous transformants is extracted, and the target fragment is amplified using primers BF and CR. The correctness of the SNP is verified by sequencing, and unexpected mutations are excluded. This includes verifying by first-generation sequencing that there are no other unexpected base changes besides the target SNP.
[0042] Example 2 The difference between Example 2 and Example 1 is that: the method for constructing a directed SNP mutant in Neurospora crassa according to the present invention includes the following steps: In step (1), the preparation of gene fragment A containing the SNP: using Neurospora crassa genomic DNA as a template, forward primer AF and reverse primer AR were designed, wherein AF contains the target SNP site and AR is located downstream of the gene's 3'-UTR; after synthesizing the primers, fragment A was obtained by PCR amplification and recovered (20 μL PCR reaction system: 2.6 μL DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, 3.4 μL ddH2O); the forward primer AF is 21 bp in length, wherein the 3' end region contains a specific base change artificially introduced to introduce the target SNP.
[0043] In step (2), the upstream homologous fragment B is prepared: a primer is designed upstream of fragment A to amplify fragment B of 1000 bp, wherein the reverse primer BR contains the reverse complementary sequence of AF and the full length is not less than 40 bp; fragment B is amplified and recovered using genomic DNA as a template; the reverse primer BR is 60 bp in length, wherein the 3' end contains a sequence that is completely reverse complementary to the forward primer AF.
[0044] In step (3), the preparation of downstream homologous fragment C: primers are designed 1000 bp downstream of fragment A to amplify fragment C, which is 1000 bp long, using genomic DNA as a template for amplification and recovery; In step (4), the hygromycin resistance gene hph Amplification: Using a vector containing the hygromycin resistance gene hph, primers hph-F and hph-R were designed, wherein hph-F is inversely complementary to the reverse primer AR of fragment A, and hph-R is inversely complementary to the forward primer of fragment C; the hph gene fragment was obtained by PCR amplification and recovered (20 μL PCR reaction system: 2.6 μL plasmid DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, 3.4 μL ddH2O); the vector containing the hygromycin resistance gene hph was plasmid pCSN44.
[0045] In step (5), the fusion fragment BA-hph-C is constructed as follows: Fragment A, fragment B, fragment C and hph are mixed in equal amounts (40 ng each). Fusion PCR is performed using the forward primer BF of fragment B and the reverse primer CR of fragment C to obtain the fusion fragment (40 μL PCR reaction system: 1.36 μL A, 1.3 μL B, 1.3 μL C, 1.3 μL hph, 2 μL BF, 2 μL CR, 20 μL high-fidelity DNA polymerase Master Mix, 6.8 μL ddH2O). The size is verified by agarose gel electrophoresis, and the SNP is confirmed to be correctly introduced after recovery, purification and sequencing. The mixing refers to mixing fragment A, fragment B, fragment C and hph gene fragment in an equimolar ratio. The extension time of the fusion PCR is 6 minutes.
[0046] In step (6), electroporation and resistance screening: the fusion fragment is transferred into Neurospora crassa by electroporation, and after cultivation, colonies are picked and screened for positive transformants in a culture medium containing hygromycin; the hygromycin screening concentration is 300 μg / mL.
[0047] In step (7), mutation site verification: Genomic DNA of homozygous transformants is extracted, and the target fragment is amplified using primers BF and CR. The correctness of the SNP is verified by sequencing, and unexpected mutations are excluded. This includes verifying through first-generation sequencing that there are no other unexpected base changes besides the target SNP. Example
[0048] The present invention provides a method for constructing directed SNP mutants in Neurospora crassa, comprising the following steps: In step (1), the preparation of gene fragment A containing the SNP: using Neurospora crassa genomic DNA as a template, forward primer AF and reverse primer AR were designed, wherein AF contains the target SNP site and AR is located downstream of the gene's 3'-UTR; after synthesizing the primers, fragment A was obtained by PCR amplification and recovered (20 μL PCR reaction system: 2.6 μL DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, 3.4 μL ddH2O); the forward primer AF is 20 bp in length, wherein the 3' end region contains a specific base change artificially introduced to introduce the target SNP.
[0049] In step (2), the upstream homologous fragment B is prepared: a primer is designed upstream of fragment A to amplify fragment B of 1000 bp, wherein the reverse primer BR contains the reverse complementary sequence of AF and the full length is not less than 40 bp; fragment B is amplified and recovered using genomic DNA as a template; the reverse primer BR is 50 bp in length, wherein the 3' end contains a sequence that is completely reverse complementary to the forward primer AF.
[0050] In step (3), the preparation of downstream homologous fragment C: primers are designed 500 bp downstream of fragment A to amplify fragment C, which is 1000 bp long, using genomic DNA as a template for amplification and recovery; In step (4), the hygromycin resistance gene hph Amplification: Using a vector containing the hygromycin resistance gene hph, primers hph-F and hph-R were designed, wherein hph-F is inversely complementary to the reverse primer AR of fragment A, and hph-R is inversely complementary to the forward primer of fragment C; the hph gene fragment was obtained by PCR amplification and recovered (20 μL PCR reaction system: 2.6 μL plasmid DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, 3.4 μL ddH2O); the vector containing the hygromycin resistance gene hph was plasmid pCSN44.
[0051] In step (5), the fusion fragment BA-hph-C is constructed as follows: Fragment A, fragment B, fragment C and hph are mixed in equal amounts (40 ng each). Fusion PCR is performed using the forward primer BF of fragment B and the reverse primer CR of fragment C to obtain the fusion fragment (40 μL PCR reaction system: 1.36 μL A, 1.3 μL B, 1.3 μL C, 1.3 μL hph, 2 μL BF, 2 μL CR, 20 μL high-fidelity DNA polymerase Master Mix, 6.8 μL ddH2O). The size is verified by agarose gel electrophoresis, and the SNP is confirmed to be correctly introduced after recovery, purification and sequencing. The mixing refers to mixing fragment A, fragment B, fragment C and hph gene fragment in an equimolar ratio. The extension time of the fusion PCR is 4 minutes.
[0052] In step (6), electroporation and resistance screening: the fusion fragment is transferred into Neurospora crassa by electroporation, and after cultivation, colonies are picked and screened for positive transformants in a culture medium containing hygromycin; the hygromycin screening concentration is 200 μg / mL.
[0053] In step (7), mutation site verification: Genomic DNA of homozygous transformants is extracted, and the target fragment is amplified using primers BF and CR. The correctness of the SNP is verified by sequencing, and unexpected mutations are excluded. This includes verifying through first-generation sequencing that there are no other unexpected base changes besides the target SNP.
[0054] Example 4 The difference between Example 4 and Example 1 is as follows: the genomic DNA of Neurospora crassa is the NCU02247 gene. The wild-type nucleotide sequence of the NCU02247 gene is shown in SEQ ID NO:1, and the amino acid sequence of the NCU02247 mutant is shown in SEQ ID NO:2. The nucleotide sequence of the forward primer AF is shown in SEQ ID NO:3, the nucleotide sequence of the reverse primer AR is shown in SEQ ID NO:4, the nucleotide sequence of the forward primer BF is shown in SEQ ID NO:5, the nucleotide sequence of the reverse primer BR is shown in SEQ ID NO:6, the nucleotide sequence of the forward primer CF is shown in SEQ ID NO:7, the nucleotide sequence of the reverse primer CR is shown in SEQ ID NO:8, the nucleotide sequence of the forward primer hph-F is shown in SEQ ID NO:9, and the nucleotide sequence of the reverse primer hph-R is shown in SEQ ID NO:10. Example
[0055] A kit for implementing the method according to the present invention comprises: Master Mix, a high-fidelity DNA polymerase for amplifying fragments A, B, and C; Hygromycin resistance gene hph or a vector containing the hph gene; High-fidelity DNA polymerase Master Mix for fusion PCR; The instruction manual describes the steps of the method as described.
[0056] Table 1 Example
[0057] Neurospora crassa gene NCU02247 Constructing site-directed SNP mutations 1. Fragment A preparation: targeting the gene NCU02247To target the mutation site, a forward primer AF containing an SNP (a single base substitution from A to G) and a downstream reverse primer AR were designed. PCR was performed using Neurospora crassa genomic DNA as a template. The 20 μL PCR reaction mixture consisted of: 2.6 μL plasmid DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, and 3.4 μL ddH2O. The reaction conditions were: 94℃ for 5 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min, for 30 cycles; 72℃ for 10 min. Fragment A, 664 bp, was recovered.
[0058] 2. Preparation of fragments B and C: Design primers BF and BR upstream of fragment A, and primers CF and CR downstream to amplify fragment B (1051 bp) and fragment C (817 bp), respectively. PCR conditions are the same as before, and the products are recovered.
[0059] 3. hph Gene amplification: Using pCSN44 plasmid as a template, primers hph-F and hph-R were designed to be reverse complementary to AR and CF, respectively, for amplification. hph Gene fragments were extracted and recovered. 20 μL PCR reaction system: 2.6 μL plasmid DNA, 2 μL AF, 2 μL LA-R, 5 μL high-fidelity DNA polymerase Master Mix, 3.4 μL ddH2O; reaction conditions: 94℃ for 5 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 3 min, 30 cycles; 72℃ for 10 min.
[0060] 4. Fusion PCR: The four fragments A, B, C, and hph were mixed in equimolar proportions (40 ng). Fusion PCR was performed using BF and CR as primers. The 40 μL PCR reaction mixture consisted of: 1.36 μL A, 1.3 μL B, 1.3 μL C, 1.3 μL hph, 2 μL BF, 2 μL CR, 20 μL high-fidelity DNA polymerase Master Mix, and 6.8 μL ddH2O. The program was: 94℃ for 5 min; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 5 min, 30 cycles; 72℃ for 10 min. A fusion fragment of approximately 5 kb was obtained, verified by 1% agarose gel electrophoresis, and then excised and recovered from the gel.
[0061] 5. Electroporation and Screening: The purified fusion fragment was electroporated into Neurospora crassa conidia, spread on Vogel's medium, and incubated at 25°C for 2-3 days. Colonies were picked and transferred to medium containing 0.3% hygromycin B (200 μg / mL) for screening of positive transformants.
[0062] 6. Mutation Validation: Genomic DNA was extracted from 40 resistant strains and amplified by PCR using BF and CR methods, followed by sequencing. Results showed that the target SNPs were correctly introduced in approximately 68% (27 / 40) of the strains, with no other unexpected base changes, successfully obtaining the gene. NCU02247 The site-directed SNP mutant strain was obtained. This indicates that it has the characteristics of high mutation efficiency and accurate site, providing a reliable technical means for gene function research of Neurospora crassa.
[0063] The method described in this invention is not only applicable to single gene mutations, but can also be extended to simultaneous mutations at multiple gene loci, exhibiting high application flexibility and reliability.
[0064] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.
Claims
1. A method of constructing a directed SNP mutant in Neurospora crassa, comprising Includes the following steps: (1) Preparation of gene fragment A containing SNP: Using Neurospora crassa genomic DNA as a template, forward primer AF and reverse primer AR were designed, where AF contains the target SNP site and AR is located downstream of the 3'-UTR of the gene; after synthesizing the primers, fragment A was obtained by PCR amplification and recovered, 20 μL PCR reaction system; (2) Preparation of upstream homologous fragment B: Design primers upstream of fragment A to amplify fragment B of 1000 bp, wherein the reverse primer BR contains the reverse complementary sequence of AF and the full length is not less than 40 bp; amplify fragment B using genomic DNA as a template and recover it; (3) Preparation of downstream homologous fragment C: Design primers at 500-1000 bp downstream of fragment A to amplify fragment C, which is 1000 bp long, using genomic DNA as a template for amplification and recovery; (4) Amplification of the hygromycin resistance gene hph: Using plasmid PCSN44 containing the hygromycin resistance gene hph, primers hph-F and hph-R were designed, wherein hph-F is inversely complementary to the reverse primer AR of fragment A, and hph-R is inversely complementary to the forward primer of fragment C; the hph gene fragment was obtained by PCR amplification and recovered; the nucleotide sequence of PCSN44 is shown in SEQ ID NO:11; (5) Construction of fusion fragment BA-hph-C: Fragment A, fragment B, fragment C and hph were mixed in equal amounts of 40 ng. Fusion PCR was performed using the forward primer BF of fragment B and the reverse primer CR of fragment C to obtain the fusion fragment. The PCR reaction system was 40 μL. The size was verified by agarose gel electrophoresis, and the SNP was correctly introduced after recovery, purification and sequencing. (6) Electroporation and resistance screening: The fusion fragment was transferred into Neurospora crassa by electroporation, and after cultivation, colonies were picked and screened for positive transformants in a medium containing hygromycin. (7) Mutation site verification: Extract genomic DNA from homozygous transformants, amplify the target fragment using primers BF and CR, and verify the correctness of the SNP by sequencing and exclude unexpected mutations.
2. The method of claim 1, wherein: In step (1), the genomic DNA of Neurospora crassa is the NCU02247 gene. The wild-type nucleotide sequence of the NCU02247 gene is shown in SEQ ID NO:1, and the nucleotide sequence of the NCU02247 mutant is shown in SEQ ID NO:
2. The nucleotide sequence of the forward primer AF is shown in SEQ ID NO:3, the nucleotide sequence of the reverse primer AR is shown in SEQ ID NO:4, the nucleotide sequence of the forward primer BF is shown in SEQ ID NO:5, the nucleotide sequence of the reverse primer BR is shown in SEQ ID NO:6, the nucleotide sequence of the forward primer CF is shown in SEQ ID NO:7, the nucleotide sequence of the reverse primer CR is shown in SEQ ID NO:8, the nucleotide sequence of the forward primer hph-F is shown in SEQ ID NO:9, and the nucleotide sequence of the reverse primer hph-R is shown in SEQ ID NO:
10.
3. The method of claim 1, wherein: In step (1), the PCR reaction system consists of 2.6 μL DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, and 3.4 μL ddH2O. The forward primer AF is 18-21 bp in length, wherein the 3' end region contains artificially introduced specific base changes to introduce the target SNP, which includes artificially introduced specific base changes.
4. The method of claim 1, wherein: In step (2), the reverse primer BR is 40-60 bp in length, wherein the 3' end contains a sequence that is completely inversely complementary to the forward primer AF; in step (4), the vector containing the hygromycin resistance gene hph is plasmid pCSN44; 20 μL PCR reaction system: 2.6 μL plasmid DNA, 2 μL AF, 2 μL AR, 5 μL high-fidelity DNA polymerase Master Mix, 3.4 μL ddH2O.
5. The method of claim 1, wherein: In step (5), the mixing refers to mixing fragment A, fragment B, fragment C with the hph gene fragment in an equimolar ratio; the extension time of the fusion PCR is 4-6 minutes; the PCR reaction system is: 1.36 μL A, 1.3 μL B, 1.3 μL C, 1.3 μL hph, 2 μL BF, 2 μL CR, 20 μL high-fidelity DNA polymerase Master Mix, and 6.8 μL ddH2O.
6. The method of claim 1, wherein: In step (6), the hygromycin screening concentration is 150-300 μg / mL.
7. The method of claim 6, wherein: In step (6), the hygromycin screening concentration is 200 μg / mL.
8. The method according to claim 7, characterized in that: Step (7) includes verifying, through first-generation sequencing, that there are no other unexpected base changes besides the target SNP.
9. A kit for implementing the method according to any one of claims 1-8, characterized in that: Include: Master Mix, a high-fidelity DNA polymerase for amplifying fragments A, B, and C; Hygromycin resistance gene hph or a vector containing the hph gene; High-fidelity DNA polymerase Master Mix for fusion PCR; The instruction manual describes the steps of the method as described.