CN111087454B - Heat shock transcription factor 1 dominant negative effect mutant and application thereof - Google Patents
Heat shock transcription factor 1 dominant negative effect mutant and application thereof Download PDFInfo
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- CN111087454B CN111087454B CN202010121694.XA CN202010121694A CN111087454B CN 111087454 B CN111087454 B CN 111087454B CN 202010121694 A CN202010121694 A CN 202010121694A CN 111087454 B CN111087454 B CN 111087454B
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- hsf1
- fragment
- aspergillus flavus
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- dominant negative
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Abstract
The invention discloses a heat shock transcription factor 1 dominant negative effect mutant dn-Hsf1 and application thereof, belonging to the technical field of biology. Human and Aspergillus flavus by homology alignment (Aspergillus flavus) The heat shock transcription factor 1HSF1 protein in Aspergillus flavus is obtained by deleting 213 amino acid residues from 576 th site to 788 th site of the C-terminal of the Hsf1 protein to obtain the dominant negative effect mutant dn-Hsf1 protein in Aspergillus flavus with the amino acid sequence shown as SEQ ID No.1 and the coding nucleotide sequence shown as SEQ ID No. 2. The dominant negative mutant dn-Hsf1 can inhibit normal Hsf1 from playing a role in fungus bodies, so that the growth of the fungi is inhibited, and the dominant negative mutant dn-Hsf1 is applied to the aspect of preventing and controlling the pollution of the fungi.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a heat shock transcription factor 1 dominant negative effect mutant and application thereof.
Background
Fungi are a group of eukaryotes widely distributed in nature and are also harmful to human life. Many fungi are usProvides food sources, and has important application value in industrial production and medicine as important strains in the fermentation industry and the food processing industry. There are also many fungi that pose great harm to human health and socioeconomic performance. A plurality of fungi, e.g. Aspergillus fumigatus (Aspergillus fumigatus) And Candida albicans: (Cadida albicans) Can infect human body, cause local tissue organ or systemic fungal infection and even death. Many fungi also parasitize animals and plants and their processed products, and produce virulent mycotoxin contamination that seriously harms human and animal health, such as Aspergillus flavus and Aspergillus parasiticus ((R))Aspergillus paraciticus) Can produce aflatoxin, fusarium (f)Fusarium graminearum) Can produce fumonisins and vomitoxin, and the aspergillus fumigatus can produce gliotoxin. China has great loss caused by the pollution of agricultural products and products thereof by mycotoxin every year. Therefore, if the intervention can be carried out in advance to prevent or reduce the fungal pollution, the loss of agriculture and food processing industry can be effectively reduced, the food safety and public health can be better guaranteed, and the method has great social and economic significance.
In nature, the heat shock reaction is a mechanism for protecting organisms widely existing in bacteria, fungi, plants and animals, and has the main function of regulating the transcription expression of related genes in the organisms under the growth and development process and various stress conditions, thereby playing the roles of defense and protecting the organisms. Mammalian Heat Shock transcription Factor 1 (HSF 1) is a major regulator of cellular Heat Shock protein expression. HSF1 can be activated by heat stress, oxidative stress, chemical stimulus and physiological stress, etc., to form homotrimer, which is combined with heat shock element in heat shock protein promoter region to regulate synthesis of heat-conducting shock protein. The HSF1 can regulate the expression of heat shock protein and the expression of multidrug resistance gene MDR 1. Studies in human hepatoma cells showed that activation of HSF1 promotes doxorubicin resistance in HepG2 cells. Candida albicans and Phytophthora sojae: (Phytophthora sojae) HSF1 of (a) is also associated with the pathogenicity of the strain.
Expression of the mutant dn-cHSF1 with dominant negative effects in mammals inhibits the function of normal HSF1 in vivo. However, there is currently no application of a dominant negative mutant of Hsf1 in fungi to prevent and control fungal contamination. Therefore, the invention constructs a fungus-derived Hsf1 dominant negative mutant dn-Hsf1 to inhibit the normal Hsf1 from functioning in the fungus body, thereby inhibiting the growth of the fungus and achieving the purpose of preventing and controlling the fungal pollution.
Disclosure of Invention
The invention aims to provide a heat shock transcription factor 1 dominant negative effect mutant and application thereof. And (3) deleting 213 amino acid residues from 576 th to 788 th at the C terminal of the Aspergillus flavus Hsf1 protein to obtain the heat shock transcription factor 1 dominant negative effect mutant dn-Hsf1 in the Aspergillus flavus. The dominant negative mutant dn-Hsf1 can inhibit normal Hsf1 from playing a role in fungi, so that the growth of the fungi is inhibited, and the dominant negative mutant has a wide application prospect in the aspect of preventing and controlling fungal pollution.
In order to achieve the purpose, the invention adopts the following technical scheme:
a heat shock transcription factor 1 dominant negative effect mutant dn-Hsf 1:
(1) the amino acid sequence of the dn-Hsf1 protein is shown in SEQ ID NO. 1; or
(2) A dn-Hsf1 derived from (1) and having a sequence defined in (1) but formed by substitution, deletion or addition of one or more amino acid residues other than the above sequence and having substantially the function of dn-Hsf1 defined in (1).
Preferred amino acid residues are substituted, deleted or added with 1-20 or 1-15 or 1-3 or 1 amino acid.
More preferably, 15 amino acid residues from position 364 to position 378 of the amino acid sequence shown in SEQ ID NO.1 are deleted.
Preferably, the dn-Hsf1 is derived from Aspergillus flavus (A. flavus) ((A. flavus))Aspergillus flavus）。
In a further preferred embodiment, the amino acid sequence of dn-Hsf1 has 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96%, 97%, 98%, 99% homology with the amino acid sequence shown in SEQ ID NO. 1.
In a second aspect, the present invention provides a nucleotide sequence encoding dn-Hsf1 as described in the first aspect.
Preferably, the coding nucleotide sequence of dn-Hsf1 is shown in SEQ ID NO. 2.
In a third aspect, the present invention provides an expression vector comprising the nucleotide sequence encoding dn-Hsf1 of the first aspect.
The construction method of the expression vector containing the coding nucleotide sequence of dn-Hsf1 comprises the following steps: taking Aspergillus flavus NRRL3357 strain genome DNA as a template, and amplifying Hsf1 self promoter by PCRP hsf1 And Hsf1 terminatorT hsf1 A nucleotide fragment of (a); amplification of dn-Hsf1 encoding nucleotide fragment by using Aspergillus flavus NRRL3357 strain cDNA as templatedn-hsf1(ii) a Will be provided withP hsf1 、 dn-hsf1、AndT hsf1 the construction of a total of 3 fragments joined together by fusion PCR method compriseshsf1Self-promotersP hsf1 、dn-hsf1Coding nucleotide and Hsf1 terminatorT hsf1 The fusion nucleotide fragment ofP hsf1 -dn-hsf1-T hsf1 Inserting the plasmid into pPTR I vector, transforming, screening positive transformant, sequencing and verifying to obtain expression vector plasmid pPTR I-dn-hsf1。
In a fourth aspect, the present invention provides a fusion nucleotide fragment comprising the nucleotide sequence encoding dn-Hsf1 according to the first aspect.
The construction method of the fusion nucleotide fragment containing the coding nucleotide sequence of dn-Hsf1 comprises the following steps: PCR amplification dn-Hsf1 upstream recombinant fragment by taking Aspergillus flavus NRRL3357 strain genome DNA as templateup(ii) a PCR amplification from Aspergillus fumigatus AF293 strain genomic DNApyrGScreening marker gene segments; PCR amplification of xylose-inducible promoter from genomic DNA of Penicillium chrysogenum NRRL1951 StrainP xylP A fragment; from plasmid pPTR I-dn-hsf1In the method of PCR amplificationdn- hsf1-T hsf1 A nucleotide fragment; recombining the upstream fragmentsup、pyrGScreening marker gene fragment and xylose-induced promoterP xylP Fragments anddn-hsf1-T hsf1 the nucleotide fragments, 4 fragments in total, are connected together by a fusion PCR method to construct a fusion nucleotide fragmentup-pyrG-P xylP -dn-hsf1-T hsf1 。
The sequences of the primers used in the construction of the expression vectors and the fused nucleotide fragments are shown in Table 1.
TABLE 1 primer sequence Listing
In a fifth aspect, the invention provides the use of dn-Hsf1 according to the first aspect, or the coding nucleotide sequence according to the second aspect, or the expression vector according to the third aspect, or the fusion nucleotide fragment according to the fourth aspect, for controlling fungal contamination.
A heat shock transcription factor 1Hsf1 dominant negative effect mutant dn-Hsf 1; or the coding nucleotide sequence of the heat shock transcription factor 1Hsf1 dominant negative effect mutant dn-Hsf 1; or a vector carrying the coding nucleotide sequence of the dominant negative effect mutant dn-Hsf1 of the heat shock transcription factor 1Hsf1, or a fusion nucleotide fragment of the coding nucleotide sequence of the dominant negative effect mutant dn-Hsf1 of the heat shock transcription factor 1Hsf1 is introduced into the fungal cell, so that the fungal cell expresses dn-Hsf1, the normal Hsf1 in the cell is inhibited to play a function, and the growth of the fungus is inhibited.
The application and the advantages of the invention are as follows:
(1) the invention provides a dn-Hsf1 derived from fungi, which has low homology with human dn-cHSF1, and the dn-Hsf1 can inhibit the growth of fungi, particularly moulds;
(2) the dn-Hsf1 provided by the invention can inhibit the function of normal Hsf1 in fungal cells, and further inhibit the growth of fungi, so that the dn-Hsf1 does not need to consider the self function of the fungal cells during applicationhsf1The presence or absence and copy number of the gene;
(3) the dn-Hsf1 provided by the invention is more environment-friendly and safer to inhibit the growth of fungi, can avoid environmental pollution and human toxic and side effects caused by using chemical pesticides for bacteriostasis, and has wide application prospect in the aspect of preventing and controlling the pollution of fungi such as mold.
Drawings
FIG. 1 construction of Aspergillus flavus dn-Hsf1 expression vector. A: aspergillus flavus amplification by fusion PCR methodP hsf1 -dn-hsf1- T hsf1 Agarose gel electrophoresis pattern of the nucleotide fragments; b: expression vector pPTR I-dn-hsf1Positive E.coli transformant colony PCR amplificationP hsf1 -dn-hsf1-T hsf1 Agarose gel electrophoresis pattern of the fragments.
FIG. 2. pPTR I-dn-Hsf1The expression vector transforms Aspergillus flavus. WT means the starting strain, "-" means protoplast without transformed nucleic acid, "+ pPTR I" means empty vector for transforming pPTR I, "+ pPTR I-dn-hsf1"indicates the transformation of pPTR I-dn-hsf1The plasmid, "+ Pyrithiamine" indicates that Pyrithiamine was added to the medium, and "-Pyrithiamine" indicates that Pyrithiamine was not added to the medium.
FIG. 3 is a schematic diagram of a construction strategy for integrating an inducible dn-Hsf1 expression fragment into an Aspergillus flavus genome. A: construction of recombinant sequences comprising upstream sequences by fusion PCR methodupDerived from Aspergillus fumigatus (Aspergillus fumigatus) Is/are as followspyrGNutrition screening marker gene and gene derived from penicillium chrysogenumP xylP Xylose inducible expression promoter, coding nucleotide and terminator of dn-Hsf1T hsf1 The fusion fragment of (1), i.eP xylP -dn-hsf1-T hsf1 Fragment, turnDissolving and integrating into Aspergillus flavus genome to obtain induced typeP xylP -dn-hsf1A strain; b: dn-Hsf1 Var: 15 amino acid residues from the central 364 th to 378 th of the dn-Hsf1 protein are deleted.
FIG. 4 shows that the induction expression of dn-Hsf1 and its active fragment dn-Hsf1Var can inhibit the growth of Aspergillus flavus. T1 and T2 in the figure represent different transformants, YGT and GMM are non-induction medium containing glucose, YXT and XMM are induction medium containing xylose and xylan, and the colony growth after 3 days of culture at 37 ℃ is shown.
Detailed Description
The inventor finds that a mutant dn-Hsf1 with dominant negative effect can be obtained by deleting 213 amino acid residues from 576 th to 788 th at the C terminal of the Aspergillus flavus heat shock transcription factor 1Hsf1 protein, and the amino acid sequence of the mutant is shown as SEQ ID NO. 1. Further research shows that the dn-Hsf1 protein fragment obtained by deleting 15 amino acid residues from the 364 th site to the 378 th site of the dn-Hsf1 protein still has dominant negative effect activity. The compounds can inhibit normal Hsf1 from playing a role in fungal bodies, obviously inhibit the growth of fungi and have application value of preventing and controlling fungal pollution.
As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in its native state in a living cell is not isolated or purified, the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in its native state.
The term "native state" as used herein refers to an activity of a polypeptide in a microorganism in an unmodified state, i.e., an activity in the native state.
As for the present invention, dn-Hsf1 may or may not be a protein corresponding to the amino acid sequence shown in SEQ ID NO. 1; in other words, dn-Hsf1 of the present invention may have high homology or low homology with the amino acid sequence shown in SEQ ID NO. 1. dn-Hsf of the invention1 may be derived from various species, including but not limited to Aspergillus flavus (A. flavus) ((A. flavus))Aspergillus flavus) Aspergillus oryzae (A. oryzae)Aspergillus oryzae) Aspergillus parasiticus (A. parasiticus)Aspergillus parasiticus) Aspergillus nigerAspergillus niger) Aspergillus terreus (A.terreus)Aspergillus terreus) Aspergillus fumigatus (Aspergillus fumigatus) Aspergillus clavatus (A. clavatus)Aspergillus clavatus) Aspergillus nidulans (Aspergillus nidulans) Neurospora crassa (A), (B), (C)Neurospora crassa) Fusarium graminearum (F.), (Fusarium graminearum) It is also within the scope of the present invention that normal Hsf1 can be inhibited from functioning in the body of the fungus to inhibit the growth of the fungus, so long as it has the activity of dn-Hsf 1.
In a specific embodiment, the homology or sequence identity may be 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96%, 97%, 98%, 99% homology. Therefore, dn-Hsf1 having 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96%, 97%, 98%, 99% sequence identity or homology to the specific dn-Hsf1 of the present invention is also included in the scope of the present invention.
The term "active fragment" as used herein is the same or similar in meaning as conventionally understood by those skilled in the art, and means that the amino acid sequence of the fragment is a portion of the amino acid sequence of the complete protein or polypeptide, but that the fragment has the same or similar function or activity as the complete protein or polypeptide. Specifically, in the present invention, "active fragment" means any amino acid sequence having the activity of dn-Hsf 1.
Furthermore, it will be appreciated by those of ordinary skill in The art that altering a small number of amino acid residues in certain regions of a polypeptide does not substantially alter The biological activity, e.g., that The sequence resulting from appropriate substitution of certain amino acids does not affect its activity (see Watson et al, Molecular Biology of The Gene, fourth edition, 1987, The Benjamin/Cummingspub. Co. P224). Thus, one of ordinary skill in the art would be able to perform such substitutions (including substitutions, deletions, insertions, additions of residues) and ensure that the resulting polypeptide retains its original function. Thus, it is apparent that further mutations in dn-Hsf1 of the invention result in further mutants that still possess the function and activity of dn-Hsf 1. For example, the inventors deleted the amino acid residues 364 to 378 of dn-Hsf1 protein to obtain a dn-Hsf1 protein fragment with dominant negative effects.
Based on the teachings of the present invention and the specific derivation of dn-Hsf1 of the present invention, one skilled in the art would not have difficulty obtaining active fragments having the same or similar activity or function, and such active fragments would, of course, fall within the scope of the present invention.
The corresponding positions of any amino acid sequence to the amino acid sequence shown in SEQ ID NO.1 can be determined by alignment between the amino acid sequences. Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: computer Molecular Biology (computerized Molecular Biology), Lesk, a. M, eds., oxford university press, new york, 1988; biological calculation: informatics and genomic Projects (Biocomputing: information and Genome Projects), Smith, d.w. eds, academic press, new york, 1993; computer Analysis of Sequence Data (Computer Analysis of Sequence Data), first part, Griffin, A.M, and Griffin, eds H.G., Humana Press, New Jersey, 1994; sequence Analysis in Molecular Biology (Sequence Analysis in Molecular Biology), von Heinje, g., academic Press, 1987 and Sequence Analysis primers (Sequence Analysis Primer), Gribskov, m. and Devereux, j. eds M Stockton Press, New York, 1991 and Carllo, h. and Lipman, d.s., SIAM j. Applied Math., 48:1073 (1998). The preferred method of determining identity is to obtain the greatest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG program package (Devereux, J. et al, 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al, 1990). BLASTX programs are publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al, NCBI NLM NIH Bethesda, Md.20894; Altschul, S. et al, 1990). The well-known Smith Waterman algorithm can also be used to determine identity.
The invention also provides a polynucleotide (shown as SEQ ID No. 2) for encoding the polypeptide. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.dn-hsf1The coding nucleotide of (a) may be a DNA which hybridizes with a probe prepared from the nucleotide sequence shown in SEQ ID NO.2 (e.g., a sequence complementary to a part or the entire sequence of the nucleotide sequence shown in SEQ ID NO. 2) under stringent conditions as long as the original function thereof is maintained. The "stringent conditions" refer to conditions under which so-called specific hybridization can be formed and non-specific hybridization is not formed. For example, the conditions for hybridization of DNAs having high homology, for example, DNAs having homology of 80% or more, and DNAs having homology of less than 80% do not hybridize with each other, or the washing conditions for ordinary Southern hybridization, that is, the conditions for washing 1 time, preferably 2 to 3 times at a salt concentration and temperature equivalent to 60 ℃, 1 XSSC, 0.1% SDS, preferably 60 ℃, 0.1 XSSC, 0.1% SDS, more preferably 68 ℃, 0.1 XSSC, 0.1% SDS.
In addition, since the degeneracy of codons varies depending on the host,dn-hsf1any codon in the coding nucleotide of (a) may be replaced with a corresponding equivalent codon, that is,dn-hsf1the coding nucleotide of (A) may be any due to the degeneracy of the genetic codedn-hsf1The coding nucleotide of (1) above. For example,dn-hsf1the encoding nucleotide of (a) may be a modified nucleotide such that it has an optimum codon according to the codon frequency in the host to be used.
The term "host" as used herein is a host having the meaning commonly understood by one of ordinary skill in the art, i.e., capable of producing the dn-Hsf1 of the present invention. In other words, the present invention can be applied to any host as long as dn-Hsf1 of the present invention can be expressed in the host.
For example, a host suitable for use in the present invention is derived from, but not limited to, Aspergillus flavus (A), (B), (C), (D), (C), (D) and D) aAspergillus flavus) Aspergillus oryzae (A. oryzae)Aspergillus oryzae) Parasitic elementAspergillus (A), (B)Aspergillus parasiticus) Aspergillus nigerAspergillus niger) Aspergillus terreus (A.terreus)Aspergillus terreus) Aspergillus fumigatus (Aspergillus fumigatus) Aspergillus clavatus (A. clavatus)Aspergillus clavatus) Aspergillus nidulans (Aspergillus nidulans) Neurospora crassa (A), (B), (C)Neurospora crassa) Fusarium graminearum (F.), (Fusarium graminearum) (ii) a More preferably Aspergillus flavus, Aspergillus oryzae, Aspergillus parasiticus, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Aspergillus clavatus, Aspergillus nidulans.
The nucleotide fragment or expression vector prepared according to the invention can be introduced into fungal cells by protoplast-based genetic transformation, e.g., PEG/CaCl2The mediated protoplast transformation method, the liposome-protoplast fusion method, the restriction enzyme-mediated fusion (REMI) method, etc., may be genetic transformation methods based on physical methods such as electroporation technique transformation method, gene gun technique transformation method and recently implemented nanoparticle spray transformation method (DOI: 10.1101/805036), or genetic transformation methods using biological delivery such as Agrobacterium (R) andAgrobacterium) Mediated genetic transformation methods, and the like.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
I. Materials and methods
The information of the strains used in the present invention is as follows: aspergillus flavus (Aspergillus flavus) Strain NRRL3357 (ATCC 200026), Aspergillus fumigatus (Aspergillus fumigatus)Aspergillus fumigatus) AF293 strain (ATCC MYA-4609), Penicillium chrysogenum (Penicillium chrysogenum) NRRL1951 strain (ATCC 28089) was purchased from this laboratory; aspergillus flavus WT strain (∆ku70) And CA14PTs (Δ)pyrG, ∆ku70) The strain is a gift of professor Perng-Kuang Chang of southern research center of the department of agriculture of America (DOI: 10.1016/j. mimet.2010.03.010.); coli DH 5. alpha. chemocompetent cells were purchased from TransGen.
High fidelity DNA polymerase KOD Plus was obtained from Toyobo company (Code number KOD-201), rTaq DNA polymerase, restriction enzyme from Invitrogen, pyrithione from Sigma Aldrich, Yeast extract and agarose from Oxiod, glucose, xylose, xylan, ampicillin from Solambio, plasmid vector pPTR I from Takara (Code number 3621), RNA extraction reagent TRIzol (Code number 15596026) and reverse transcription kit (Code number K1622) from Thermo Fisher Scientific. Other conventional chemicals were purchased from the Sangon corporation and the national drug group.
The specific components of the various solutions and media used in this embodiment are as follows:
1000 × microelement mother liquor (100 mL): ZnSO4·7H2O 2.2 g；H3BO3 1.1 g；MnCl2·4H2O 0.5 g；FeSO4·7H2O 0.16 g；CoCl2·5H2O 0.16 g；CuSO4·5H2O 0.16 g； (NH4)6Mo7O24·4H2O 0.11 g； Na4EDTA 5g is dissolved in deionized water and filtered to sterilize.
YGT liquid medium (1L): yeast extract 5 g; glucose 20 g; 1000X 1 mL of a mother solution of trace elements. The YGTUU liquid medium was prepared by adding Uridine (Uridine 0.001 g/L) and Uracil (Uracil 0.001 g/L) to the YGT liquid medium. Agar (Agar 20 g/L) is added into the two liquid culture media to obtain a solid culture medium. YXT solid culture medium is obtained by replacing Glucose (Glucose 20 g/L) in YGT culture medium with xylose and xylan (xylose 5g/L, xylon 5 g/L).
Resuscitation medium (1L): (iii) Sucrose 342.3 g; NaNO3 3 g；KCl 0.5 g；MgSO4 0.5 g；K2HPO41 g；FeSO4 001 g; agar 5g (upper layer) or 15 g (lower layer).
GMM solid medium (1L): NaNO3 6 g；KCl 0.52 g；MgSO4·7H2O 0.52 g；KH2PO41.52 g; 1000 times 1 mL of microelement mother liquor; 10 g of Glucose; agar 20 g. Replacing Glucose (Glucose 10 g/L) in the formula of the GMM solid culture medium with xylose and xylan (xylose 5g/L, xylon 5 g/L) to obtain the XMM solid culture medium.
The working concentration of the pyrithione amine was 0.2. mu.g/mL, and the solution was added to different media as needed.
Aspergillus flavus genome DNA extraction, total RNA extraction and reverse transcription, Aspergillus flavus protoplast preparation and transformation methods are all referred to published documents (DOI: 10.1016/j.mimet.2010.03.010; DOI: 10.1021/acs.jafc.6b02199).
The primers used in the specific implementation are shown in Table 1.
Example 1 construction of dn-Hsf1 expression vector
Studies in mammals have shown that deletion of the catalytic domain of about 150 amino acid residues from the C-terminus of the heat shock transcription factor 1HSF1 protein results in a dominant negative mutant dn-cHSF1 that inhibits the function of normal HSF1 in animals (DOI: 10.1021/acschembio.5b00740). Therefore, the inventor constructs a fungus-derived Hsf1 dominant negative mutant dn-Hsf1 to inhibit the normal Hsf1 from functioning in fungi, thereby inhibiting the growth of the fungi and achieving the purpose of preventing and controlling the fungal pollution.
The invention utilizes the amino acid sequence of the human heat shock transcription factor 1HSF1 protein to find the homologous protein Hsf1 (with the number of XP _ 002374014) and the encoding gene (with the number of AFLA _ 025030) predicted in the aspergillus flavus by an NCBI BLASTP homologous comparison method. The amino acid sequence similarity of the aspergillus flavus Hsf1 protein (containing 788 amino acid residues) and the main structural domain of the human HSF1 protein (containing 529 amino acid residues) is only 21%, and the similarity of the full-length protein is lower. Therefore, after the Lasergene MegAlign software is compared and analyzed, the inventor deletes 213 amino acid residues from 576 th to 788 th at the C terminal of the Aspergillus flavus Hsf1 protein to construct a mutant dn-Hsf1 with dominant negative effect, and the amino acid sequence of the mutant is shown as SEQ ID NO. 1.
Next, the invention constructs a truncated mutant of aspergillus flavus Hsf1 protein with 213 amino acid residues deleted at the C terminal by a fusion PCR method. The specific method comprises the following steps:
primers SEQ ID NO.3 and SEQ ID NO.4 were used; the DNA of Aspergillus flavus genome is PCR amplified to 1329 bp long DNAhsf1Self-promotersP hsf1 The nucleotide fragment of (Region 11703 to 13031 on AAIH02000003.1, GenBank);
using primer SEQ ID NO.5 and primer SEQ ID NO. 6; PCR method for amplifying 1728bp fragment from Aspergillus flavus cDNAdn-hsf1The fragment is a coding nucleotide fragment of Hsf1 protein C terminal deletion 213 amino acid residues (reserving 1 st to 575 th amino acid residues)dn-hsf1(shown as SEQ ID NO. 2);
using primer SEQ ID NO.7 and primer SEQ ID NO. 8; PCR method for amplifying 1491 bp DNA containing DNA from Aspergillus flavus genomehsf1TerminatorT hsf1 The nucleotide fragment of (Region 7786 to 9279 on AAIH02000003.1, GenBank);
the 3 fragments were ligated together by fusion PCR to construct a fusion product containinghsf1Self-promotersP hsf1 、dn-hsf1Coding nucleotides andhsf1fusion nucleotide fragment of 4548 bp in length of terminatorP hsf1 -dn-hsf1-T hsf1 (FIG. 1A), inserting into pPTR I vector by LIC seamless connection cloning method, transforming Escherichia coli DH5 competent cell, selecting single clone to carry out colony PCR verification, screening positive transformant and sequencing, wherein the positive transformant can amplify a band with molecular weight of 4548 bpP hsf1 -dn-hsf1-T hsf1 (FIG. 1B), and sequencing was performed to confirm that pPTR I was obtained-dn-hsf1An expression vector plasmid.
Example 2 expression vector pPTR I-dn-hsf1Introduction of Aspergillus flavus
Will expressVector plasmid pPTR I-dn-hsf1And (3) introducing an aspergillus flavus WT strain protoplast, and screening an aspergillus flavus transformant on a recovery culture medium containing pyrithione. After 5 days of culture at 37 ℃ of the transformation plates, the results are shown in FIG. 2: the protoplasts without transformed nucleic acid were grown in both the first and second plates, since the first plate contained no pyrithione selection pressure, many colonies forming lawn were grown on the plate, indicating that the amount of protoplast for transformation was sufficient, while the second plate added pyrithione, no colonies were grown, indicating that the pyrithione selection pressure was effective. The third plate was protoplast transformed with empty vector pPTR I, and 10 clones were grown under selection of pyrithione, indicating that the transformation efficiency was adequate. The fourth plate was transformed with pPTR I-dn-hsf1No clone is grown in plasmid protoplast under the screening of pyrithione, which shows that the expression of dn-Hsf1 can obviously inhibit the growth of aspergillus flavus. The results show that the truncated mutant dn-Hsf1 of the Aspergillus flavus Hsf1 protein with 213 amino acid residues deleted at the C terminal is a mutant with dominant negative effect activity.
Example 3 inhibition of Aspergillus flavus growth by inducible expression of dn-Hsf1 with xylose promoter
In order to confirm the inhibition effect of dn-Hsf1 on the growth of aspergillus flavus, the invention further expresses the dn-Hsf1 protein by aspergillus flavushsf1Gene self-promoterP hsf1 Control changes to use of xylose-inducible promotersP xy1P To control (the construction strategy is shown in FIG. 3A), the upstream homologous sequence is constructed by the fusion PCR methodupDerived from Aspergillus fumigatuspyrGNutrition screening marker gene from penicillium chrysogenumP xylP Xylose inducible expression promoter, coding nucleotide of dn-Hsf1 and terminatorT hsf1 The fusion fragment of (1), i.eP xylP -dn-hsf1-T hsf1 Fragment, transformation and integration into Aspergillus flavus genome to obtain the induction typeP xylP -dn- hsf1And (3) strain. The influence of two states of non-expression and induced expression of dn-Hsf1 on the growth of aspergillus flavus is observed. The specific method comprises the following steps:
primers SEQ ID NO.9 and SEQ ID NO.10 were used; PCR method is used for amplifying upstream recombination fragment with length of 1474 bp from genome DNA of aspergillus flavus NRRL3357 strainup（Region 5751637 to 5753110 on CP044622.1，GenBank）；
Amplification of 1890 bp long DNA from the genomic DNA of A.fumigatus AF293 strain by PCR with the primers SEQ ID No.11 and SEQ ID No.12pyrGSelecting marker gene fragment (DOI: 10.1021/acs.jafc.6b02199);
PCR method for amplifying 1632 bp-long xylose-induced promoter from Penicillium chrysogenum NRRL1951 genome DNA by using primers SEQ ID NO.13 and SEQ ID NO.14P xylP Fragment (DOI: 10.1128/aem.66.11.4810-4816.2000);
expression plasmid pPTR I constructed from example 1 using primers SEQ ID NO.15 and SEQ ID NO.16-dn- hsf1The length of 3219 bp amplified by PCRdn-hsf1-T hsf1 A nucleotide fragment;
repeating the above-mentioned upstream to form segmentsup、pyrGScreening marker gene fragment and xylose-induced promoterP xylP Fragments anddn- hsf1-T hsf1 the nucleotide fragments, 4 fragments in total, are connected together by a fusion PCR method to construct a fusion nucleotide fragment with the length of 8215 bpup-pyrG-P xylP -dn-hsf1-T hsf1 Transforming Aspergillus flavus CA14PTs strain to integrate the fusion nucleotide fragment into Aspergillus flavus genomehsf1With replacement of one copy at the locushsf1Genes (FIG. 3A). After 5 days of culture at 37 ℃, 5 are obtained by screening and identificationP xylP -dn-hsf1And (4) positive transformant strains.
Randomly selecting two of the above Aspergillus flavusP xylP -dn-hsf1Fresh spores of positive transformants (T1, T2) were inoculated 10 separately3Spores were cultured at the center of YGT, YXT, GMM and XMM solid medium plates for 3 days at 37 ℃. As shown in FIG. 4A, in the presence of glucose (YGT and GMM medium), the xylose promoter was inhibited by glucose and did not transcriptionally express dn-Hsf1, when the aspergillus flavus can normally grow; when glucose does not exist (YXT and XMM culture medium), the xylose promoter can be induced and activated by xylose and xylan, the expression of dn-Hsf1 is transcribed, the growth of aspergillus flavus is obviously inhibited, and the diameter of a colony is obviously reduced. This result indicates that the transcriptional expression of dn-Hsf1 is detrimental to Aspergillus flavus growth, and that dn-Hsf1 has significant dominant negative effect activity.
Example 4 inhibition of Aspergillus flavus growth by dn-Hsf1 active fragment
The invention further designs that the promoter will be induced by xyloseP xy1P To control the deletion of 15 amino acid residues from the central 364 th to 378 th of the expressed dn-Hsf1 protein, and to test whether the dn-Hsf1 protein fragment dn-Hsf1Var still has dominant negative activity (the construction strategy is shown in FIG. 3B). The specific method comprises the following steps:
aspergillus flavus obtained from example 3 above using primer SEQ ID NO.9 and primer SEQ ID NO.17P xylP - dn-hsf1Amplifying a fragment containing upstream recombination with the length of 6085 bp in strain genome DNA by using a PCR methodupScreening marker genespyrGXylose inducible promoterP xylP Anddn-hsf1a fragment of nucleotides encoding amino acid residues 1 to 363; aspergillus flavus obtained from example 3 above using primers SEQ ID NO.18 and SEQ ID NO.16P xylP -dn-hsf12085 bp long inclusion complex amplified by PCR method in strain genome DNAdn-hsf1Nucleotides encoding amino acid residues 379 to 575 andT hsf1 a fragment of (a);
the 2 fragments are connected together by a fusion PCR method to construct a fusion nucleotide fragment with the length of 8170 bpup-pyrG-P xylP -dn-hsf1Var-T hsf1 -downTransforming Aspergillus flavus CA14PTs strain, and finally screening to obtain 4 strainsP xylP - dn-hsf1VarAnd (4) positive transformant strains.
Randomly selecting two of the above Aspergillus flavusP xylP -dn-hsf1VarFreshness of Positive transformants (T1, T2)Respectively inoculating 10 spores of3Spores were cultured at the center of YGT, YXT, GMM and XMM solid medium plates for 3 days at 37 ℃. As shown in FIG. 4B, in the presence of glucose (YGT and GMM medium), the xylose promoter was inhibited by glucose and could not transcribe and express dn-Hsf1Var, at which time Aspergillus flavus could grow normally; when glucose does not exist (YXT and XMM culture medium), the xylose promoter can be induced and activated by xylose and xylan, and the dn-Hsf1Var is expressed by transcription, so that the growth of the aspergillus flavus is obviously inhibited, and the diameter of a colony is obviously reduced. This result indicates that the transcriptional expression of dn-Hsf1Var is detrimental to Aspergillus flavus growth, and that the dn-Hsf1 protein fragment dn-Hsf1Var also has dominant negative effect activity.
The combination of the above results shows that the Hsf1 mutant dn-Hsf1 and the active fragment dn-Hsf1Var constructed by the invention can obviously inhibit the growth of aspergillus flavus when being expressed, and the dn-Hsf1 and the active fragment thereof really have dominant negative effect activity and have application values of inhibiting the growth of fungi and preventing and controlling fungal pollution.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> heat shock transcription factor 1 dominant negative effect mutant and application thereof
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<170> PatentIn version 3.3
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atgagtcctc aaggcttggc gtctcgcaaa agacctgctc ccggcacatc tcctatcgtt 60
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gaccagttcc tgcaatgggg tcagaacact tcctcgaatg ttgtcagccc ggcttccttt 180
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acggcaactg catctacaca actggcacgt agacagacac caaatcaact ggtcagccgg 300
aatcgcggat atgaacagac accgtcctcc atgtcggacc atgggagtaa taccggagag 360
cctgggggct ggggggaaag tttggacgag ctctaccagc gagcgttggt tgcaaagagg 420
gaggtgcagg ctaagaggaa acaaatccct ccgtttgttc aaaagctaag cagtttcctg 480
gacgagtcta agaacactga cctgattcgg tggtccgatg acggaaactc ctttatcgtg 540
ttagacgagg acgagttcgc aaagaccctt attcccgaac ttttcaagca taacaactac 600
gcttcctttg tccgccagtt gaacatgtac gggtttcata agaaggtggg gctctcggat 660
aattcgatgc gcgccagtga acgaaagaac aagagcccta gcgagtatgc gaacccatac 720
ttcaagcgtg gacaccccga tttgctgtgg ttgatacaga aacctaagaa tacggcaggg 780
caagggagca agtcagggaa agccaatgta cgcgtgaaaa ccgaagaagt ggatgaacat 840
gacaatgatg actacgatga tgtccctggt gcacgagacg accgatcccg aaaccgacaa 900
ttatccctaa ttcaaggagg gagtattatg ccgaaggatc aactcgcggg ggtttaccgg 960
gagttgcagg ctattcgaca gcaacagcag gttatctcca acacaattac caagctgcgg 1020
agagaacacg agcaactcta cgcacaggcc gccaacttcc aagagcagca cactcgtcac 1080
gagaattcca tcaatgcgat tctcaccttt ctggcgactg tctataatcg cagccttcag 1140
ggacaggaag ggcctcagaa tctcgccaat tcctttgctg gagccatctc acaggatcaa 1200
ggcaacgtag ttgacatggg agacgactat tcactgagca ccttgggtgc tcagcatatg 1260
aacagccctg ggggaccgcg agcaatgaag aagcaaccct tactactaaa agctgcgccg 1320
tcagaacgac aaagtcgcgc aacaaccctc tcaccggccg ccagtgcgta tgatggccca 1380
caaccgcggg gccatgctcg ccacccgagc gctccccaac acggtcatgt tgaagaagtc 1440
tttgatacca gtccccagcc gaaagaggcg caaccacccc aaaccgagca gtttcctcag 1500
cgggatatca tgtccgtcat tcaaaattcc aatgcgcgaa atggagttcc cccaacaagc 1560
tttgcggact ttcccaatgt attgtcgtcg ctggaaacct caaatggcaa tgttcctctt 1620
actccaaacc aacgcgcgga tatgctccgt ctcatggcca acgaaactag cgcgggtgac 1680
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<400> 12
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Claims (8)
1. A heat shock transcription factor 1 dominant negative effect mutant dn-Hsf1, comprising:
the amino acid sequence of the dn-Hsf1 protein is shown in SEQ ID NO. 1; or
15 amino acid residues from the 364 th to the 378 th in the amino acid sequence shown in SEQ ID NO.1 are deleted.
2. A nucleotide sequence encoding the heat shock transcription factor 1 dominant negative mutant dn-Hsf1 of claim 1.
3. The nucleotide sequence of claim 2, wherein the sequence is as shown in SEQ ID No. 2.
4. An expression vector comprising a nucleotide sequence encoding the heat shock transcription factor 1 dominant negative mutant dn-Hsf1 of claim 1.
5. The method for constructing the expression vector according to claim 4, wherein: aspergillus flavus NRRL3357 strain genome DNA is taken as a template, and the Aspergillus flavus NRRL3357 heat shock transcription factor 1 (Hsf 1) self promoter is amplified by PCRP hsf1 And a terminatorT hsf1 (ii) a Amplifying nucleotide fragment for coding dn-Hsf1 in claim 1 by using Aspergillus flavus NRRL3357 strain cDNA as templatedn-hsf1(ii) a Will be provided withP hsf1 、dn-hsf1、AndT hsf1 the construction of a total of 3 fragments joined together by fusion PCR method compriseshsf1Self-promotersP hsf1 、dn-hsf1Coding nucleotide and Hsf1 terminatorT hsf1 The fusion nucleotide fragment ofP hsf1 -dn-hsf1-T hsf1 Inserting the plasmid into pPTR I vector, transforming, screening positive transformant, sequencing and verifying to obtain expression vector plasmid pPTR I-dn-hsf1。
6. A fusion nucleotide fragment comprising a nucleotide sequence encoding the heat shock transcription factor 1 dominant negative effect mutant dn-Hsf1 of claim 1.
7. The method of constructing a fusion nucleotide fragment according to claim 6, wherein: PCR amplification of upstream recombination fragment of Aspergillus flavus NRRL3357 heat shock transcription factor 1 by using Aspergillus flavus NRRL3357 strain genome DNA as templateup(ii) a PCR amplification from Aspergillus fumigatus AF293 strain genomic DNApyrGScreening marker gene segments; PCR amplification of xylose-inducible promoter from genomic DNA of Penicillium chrysogenum NRRL1951 StrainP xylP A fragment; expression vector plasmid pPTR I obtained from the method according to claim 5-dn-hsf1Amplification by PCRdn-hsf1-T hsf1 A nucleotide fragment; combining the upstream recombinant fragmentsup、pyrGScreening marker gene fragment and xylose-induced promoterP xylP Fragments anddn-hsf1-T hsf1 the nucleotide fragments, 4 fragments in total, are connected together by a fusion PCR method to construct a gene containing an upstream recombination fragment and a screening marker genepyrGXylose inducible promoterP xylP Anddn-hsf1-T hsf1 fusion nucleotide fragments of fragmentsup-pyrG-P xylP -dn-hsf1-T hsf1 。
8. Use of dn-Hsf1 according to claim 1, or the coding nucleotide sequence according to claim 2, or the expression vector according to claim 4, or the fused nucleotide fragment according to claim 6, for the preparation of a formulation for controlling aspergillus flavus contamination.
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