CN113957077B - Sisal hemp cysteine proteinase inhibitor gene and application thereof - Google Patents
Sisal hemp cysteine proteinase inhibitor gene and application thereof Download PDFInfo
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- CN113957077B CN113957077B CN202111206967.1A CN202111206967A CN113957077B CN 113957077 B CN113957077 B CN 113957077B CN 202111206967 A CN202111206967 A CN 202111206967A CN 113957077 B CN113957077 B CN 113957077B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
- A01N47/44—Guanidine; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a sisal cysteine protease inhibitor gene and application thereof, and relates to the field of bioengineering, wherein the nucleotide sequence of the sisal cysteine protease inhibitor gene is shown as SEQ No.1, and the amino acid sequence of protein encoded by the gene is shown as SEQ No. 2. The sisal hemp cysteine proteinase inhibitor protein has antibacterial biological activity, can be used for preparing antibacterial products, is applied to crop disease prevention and control, and can inhibit the growth of phytophthora nicotianae, mango anthracnose and watermelon fusarium wilt.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a sisal cysteine protease inhibitor gene and application thereof.
Background
Sisal is a hard leaf fiber crop widely planted in tropical subtropical regions. The application is relatively wide, and mainly relates to industries such as fishery, navigation, aerospace, industrial and mining, transportation, oil fields, spinning and the like. The sisal hemp has more common diseases and insect pests in the growth process, mainly comprises sisal hemp zebra disease, purple leaf curl, stem rot, red spiders, new pineapple gray mealy bugs and the like, and the sisal hemp fiber yield reduction caused by disease and insect pest outbreak has great influence on the sisal hemp industry. Chemical control is still a main means of sisal hemp disease and pest control at present, but the chemical control has great influence on the environment ecology, so that the exploration of a green and efficient disease and pest control technology is important for sustainable development of sisal hemp industry.
Cysteine protease inhibitors (Cysteine proteinase inhibitor, CPI), also known as thiol protease inhibitors, bind specifically to cysteine proteases and protect disulfide bonds in proteins from destruction by inhibiting enzymatic activity, thereby preserving the functional integrity of the corresponding proteins to regulate life processes. Plant cysteine protease inhibitor genes have been reported in a variety of plants, such as rice, barley, wheat, maize, arabidopsis, pea, tomato, and the like. The expression of the CPI gene is affected by factors such as plant growth regulators, mechanical damage, stress, etc.
At present, CPI genes have proved to have important functions in improving plant disease resistance, and more researches on plant cysteine protease inhibitor genes are reported at home and abroad, but are not reported in sisal hemp. Therefore, it is necessary to invent a sisal cysteine protease inhibitor gene and application thereof to solve the above problems.
Disclosure of Invention
The invention aims to provide a sisal cysteine protease inhibitor gene and application thereof, so as to solve the problems in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a sisal cysteine proteinase inhibitor gene has a nucleotide sequence shown in SEQ No.1, and specifically comprises the following steps:
ATGAAGGCTAGTTCTCTTGTTCTTCTTCTTCTTGCTTCAACTTTCATGCTTGCCAATTTATGCTCTGCTTCAAGAGGCTCTGGTCCAATGGTTGGGGGATGGAGCACAATCAAGAACATGAGTGACCCACATATTGCAGAGATTGGGGAGTTTGCAATCTCTGAGCACAACAAGGAGACCAACTCCAGGCTTGCATTCAACAGAGTGATCAAGGGTAAGATCCAAGTTGTGGCTGGTTTCAATTACAAGCTTGTTATTGAGTCCAAGGATGGGAATAAAGTTAGGAAGTATGAGGCAGTTGTTTGGGAGAAAGTTTGGGAGAATTTCTTGAAGCTTACTTCCTTCAAGCCTCTTAAGATCTGA
a sisal cysteine proteinase inhibitor protein has an amino acid sequence shown in SEQ No.2, and specifically comprises the following steps:
MKASSLVLLLLASTFMLANLCSASRGSGPMVGGWSTIKNMSDPHIAEIGEFAISEHNKETNSRLAFNRVIKGKIQVVAGFNYKLVIESKDGNKVRKYEAVVWEKVWENFLKLTSFKPLKI
further, the sisal cysteine protease inhibitor protein is obtained by introducing the sequence of SEQ No.1 into a prokaryotic expression vector and performing prokaryotic expression.
Further, the sisal cysteine protease inhibitor gene or the sisal cysteine protease inhibitor protein is applied to an antifungal product.
Further, the sisal cysteine protease inhibitor gene or the sisal cysteine protease inhibitor protein is applied to prevention and control of crop fungal diseases.
Furthermore, the sisal cysteine protease inhibitor gene or the sisal cysteine protease inhibitor protein is applied to inhibiting the growth of phytophthora nicotianae, mango anthracnose and watermelon fusarium wilt.
Furthermore, an antifungal product or a product for preventing and controlling crop fungal diseases or a product for inhibiting growth of phytophthora nicotianae, colletotrichum gloeosporioides and fusarium wilt of watermelons contains the sisal cysteine protease inhibitor protein.
The invention also provides application of the protein in the technical scheme in preparing an antifungal product, wherein the antifungal product comprises an equilibrium buffer solution containing sisal cysteine protease inhibitor protein.
The invention has the technical effects and advantages that:
the gene and protein sequence related by the invention have obvious difference with the published plant cysteine protease inhibitor, the nucleotide sequence length is 363bp, and the amino acid sequence length is 120.
The sisal cysteine proteinase inhibitor protein has antibacterial biological activity, can inhibit the growth of phytophthora nicotianae, mango anthracnose and watermelon fusarium wilt, can be used for preparing antibacterial products, and is applied to prevention and control of crop diseases.
Drawings
FIG. 1 is a schematic diagram showing the results of agarose gel electrophoresis verification of a prokaryotic expression vector of a sisal cysteine protease inhibitor gene;
fig. 2 is a schematic diagram showing the inhibition effect of the sisal cysteine protease inhibitor protein on phytophthora nicotianae verified by the bacteriostasis experiment.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a sisal cysteine protease inhibitor gene shown in fig. 1-2, which is obtained by sequence alignment of a sisal transcriptome database, wherein the nucleotide sequence of the sisal cysteine protease inhibitor gene is shown as SEQ No.1, and is specifically as follows:
ATGAAGGCTAGTTCTCTTGTTCTTCTTCTTCTTGCTTCAACTTTCATGCTTGCCAATTTATGCTCTGCTTCAAGAGGCTCTGGTCCAATGGTTGGGGGATGGAGCACAATCAAGAACATGAGTGACCCACATATTGCAGAGATTGGGGAGTTTGCAATCTCTGAGCACAACAAGGAGACCAACTCCAGGCTTGCATTCAACAGAGTGATCAAGGGTAAGATCCAAGTTGTGGCTGGTTTCAATTACAAGCTTGTTATTGAGTCCAAGGATGGGAATAAAGTTAGGAAGTATGAGGCAGTTGTTTGGGAGAAAGTTTGGGAGAATTTCTTGAAGCTTACTTCCTTCAAGCCTCTTAAGATCTGA
the invention also provides a protein coded by the sisal cysteine protease inhibitor gene of the technical scheme, which is obtained by introducing a sequence of SEQ No.1 into a prokaryotic expression vector and taking escherichia coli as a host cell through prokaryotic expression, wherein the amino acid sequence of the protein is shown as SEQ No.2, and the protein is specifically as follows:
MKASSLVLLLLASTFMLANLCSASRGSGPMVGGWSTIKNMSDPHIAEIGEFAISEHNKETNSRLAFNRVIKGKIQVVAGFNYKLVIESKDGNKVRKYEAVVWEKVWENFLKLTSFKPLKI
the invention also provides application of the protein in the technical scheme in preparing antifungal products, wherein the antifungal products comprise balanced buffer solution containing sisal cysteine protease inhibitor protein.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of sisal cysteine protease inhibitor proteins
The invention relates to prokaryotic expression of a sisal cysteine protease inhibitor, which comprises the following steps:
step one: blast alignment was performed in sisal transcriptome database (SRA access: PRJNA 432160) using published asparagus cysteine protease inhibitor gene CPI1 (NCBI sequence number: XM_ 020388810.1) as a reference sequence to obtain a DNA fragment shown in SEQ No.1, and primers were designed using the DNA fragment shown in SEQ No.1 as a template to verify the expression pattern by real-time quantitative PCR, wherein the sequences of the primers are as follows:
forward primer: 5'-ATGACTGTGTTTATTTCTTGT-3';
reverse primer: 5'-TCAACAAGGTCTAGTGCAGAA-3'.
The puncture inoculation method is adopted to inoculate phytophthora nicotianae which is the pathogen of sisal zebra stripe, the control treatment is only puncture without inoculation of pathogen, and sampling is carried out after 5 days of inoculation. Sample RNA was extracted by the total Tiangen plant RNA extraction kit (Tiangen Biochemical technology, beijing, china) and reverse transcribed into cDNA by GoScript Reverse Transcription System (Promega, USA). Real-time quantitative PCR validation of the sisal cysteine protease inhibitor gene was performed by the Quantum studio 6 real-time fluorescent quantitative PCR System (Sieimer, USA).
The amplification system was 20. Mu.L, which included 0.5. Mu.L forward primer, 0.5. Mu.L reverse primer, 1. Mu.L cDNA template, 10. Mu. L TransStart Tip Green qPCR Supermix (full gold, china), 0.4. Mu.L positive Ref-interference Dye (full gold, china) and 7.6. Mu.L double distilled water.
The reaction procedure was 94℃for 30s;94 ℃ for 3min,94 ℃ for 10s and 58 ℃ for 30s, and 40 cycles are total; the dissolution profile is cycled.
Sisal hemp PP2A gene (protein phosphatase 2A) was used as an internal gene, and each sample was repeated 3 times as a technical repeat, resulting in 2 -ΔΔCt The relative quantitative analysis is carried out by the method, and the result shows that the sisal cysteine protease inhibitor gene is obviously up-regulated after the phytophthora nicotianae infection, which indicates that the sisal cysteine protease inhibitor gene participates in sisal disease resistance response.
Step two: then, the sisal hemp cysteine proteinase inhibitor gene is synthesized completely and introduced into
The prokaryotic expression vector of the Blunt E1 Expression Vector is introduced into BL21 (DE 3) Chemically Competent Cell competent cells by adopting a heat shock method, and after incubating for 12 hours on a LB solid medium plate, a monoclonal is selected for PCR detection and LB liquid medium culture.
Step three: whether the primer sequence is inserted into the prokaryotic expression vector is detected by PCR, and the primer sequence is the same as that in the first step.
The 20. Mu.l reaction system comprises: 10 μl of
PCR SuperMix (full gold,china), 10pmol of each forward and reverse specific primer, 1 μl of bacterial liquid containing the prokaryotic expression vector, and 7 μl of ddH2O.
The reaction procedure is: firstly, denaturation at 95 ℃ for 5min, then, cyclic amplification is carried out for 30 times under the conditions of denaturation at 95 ℃ for 15s, denaturation at 60 ℃ for 15s and denaturation at 72 ℃ for 15s, and finally, denaturation at 72 ℃ for 2min.
As shown in FIG. 1, agarose gel electrophoresis detection shows that the PCR product is about 360bp in length, and the PCR product is subjected to Sanger sequencing according to expectations, and the sequencing result is consistent with the sequence shown in SEQ No. 1.
Step three: BL21 competent cells containing the prokaryotic expression vector are cultured in LB liquid medium for 24 hours, and then are crushed by ultrasonic waves
The Ni-NTA Resin protein purification kit (full gold, china) extracts sisal cysteine protease inhibitor protein.
Example two
Application of sisal hemp cysteine proteinase inhibitor protein in preparation of antifungal products
Respectively inoculating phytophthora nicotianae, mango anthracnose and watermelon fusarium wilt on a PDA culture medium, inoculating a fungus block at the center of a culture dish, simultaneously placing oxford cups around the fungus block, respectively adding balance buffer solution (300 mM NaCl, 50mM NaH2PO4, 10mM imidozole and 10mM Tris base,pH8.0) and balance buffer solution containing sisal cysteine protease inhibitor protein (100 mug/mL) into the oxford cups inoculated with three fungus culture dishes, and observing colony growth after culturing for 4 days at 28 ℃.
As shown in FIG. 2, the balance buffer (left side of FIG. 2) has no inhibition effect on Phytophthora nicotianae, and the balance buffer containing the sisal cysteine protease inhibitor protein enables Phytophthora nicotianae to fall on the edge of the oxford cup to generate an arc-shaped inhibition zone, so that the sisal cysteine protease inhibitor protein has inhibition effect on Phytophthora nicotianae. The antibacterial effect of the sisal cysteine protease inhibitor protein is quantitatively counted, and the results show that the antibacterial rates of the sisal cysteine protease inhibitor protein on phytophthora nicotianae, mango anthracnose and watermelon fusarium wilt are 61.75%, 50.65% and 56.05%, respectively, and the sisal cysteine protease inhibitor protein has an inhibitory effect on three fungi.
In conclusion, the sisal cysteine protease inhibitor protein can be used for preparing antibacterial products and is applied to prevention and control of crop diseases.
Finally, it should be noted that: the foregoing description is only illustrative of the preferred embodiments of the present invention, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements or changes may be made without departing from the spirit and principles of the present invention.
Sequence listing
<110> national academy of Tropical agriculture Environment and plant protection institute
<120> sisal cysteine proteinase inhibitor gene and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 363
<212> DNA/RNA
<213> sisal cysteine protease inhibitor Gene
<400> 1
atgaaggcta gttctcttgt tcttcttctt cttgcttcaa ctttcatgct tgccaattta 60
tgctctgctt caagaggctc tggtccaatg gttgggggat ggagcacaat caagaacatg 120
agtgacccac atattgcaga gattggggag tttgcaatct ctgagcacaa caaggagacc 180
aactccaggc ttgcattcaa cagagtgatc aagggtaaga tccaagttgt ggctggtttc 240
aattacaagc ttgttattga gtccaaggat gggaataaag ttaggaagta tgaggcagtt 300
gtttgggaga aagtttggga gaatttcttg aagcttactt ccttcaagcc tcttaagatc 360
tga 363
<210> 2
<211> 120
<212> PRT
<213> sisal cysteine protease inhibitor protein
<400> 2
Met Lys Ala Ser Ser Leu Val Leu Leu Leu Leu Ala Ser Thr Phe Met
1 5 10 15
Leu Ala Asn Leu Cys Ser Ala Ser Arg Gly Ser Gly Pro Met Val Gly
20 25 30
Gly Trp Ser Thr Ile Lys Asn Met Ser Asp Pro His Ile Ala Glu Ile
35 40 45
Gly Glu Phe Ala Ile Ser Glu His Asn Lys Glu Thr Asn Ser Arg Leu
50 55 60
Ala Phe Asn Arg Val Ile Lys Gly Lys Ile Gln Val Val Ala Gly Phe
65 70 75 80
Asn Tyr Lys Leu Val Ile Glu Ser Lys Asp Gly Asn Lys Val Arg Lys
85 90 95
Tyr Glu Ala Val Val Trp Glu Lys Val Trp Glu Asn Phe Leu Lys Leu
100 105 110
Thr Ser Phe Lys Pro Leu Lys Ile
115 120
<210> 3
<211> 21
<212> DNA/RNA
<213> sisal cysteine protease inhibitor Gene
<400> 3
atgaaggcta gttctcttgt t 21
<210> 4
<211> 21
<212> DNA/RNA
<213> sisal cysteine protease inhibitor Gene
<400> 4
tcagatctta agaggcttga a 21
Claims (7)
1. A sisal cysteine protease inhibitor gene, characterized in that: the nucleotide sequence of the sisal hemp cysteine protease inhibitor gene is shown as SEQ No. 1.
2. A sisal cysteine protease inhibitor protein, characterized in that: the amino acid sequence of the sisal cysteine proteinase inhibitor protein is shown as SEQ No. 2.
3. The sisal cysteine protease inhibitor protein according to claim 2, wherein: the sisal cysteine proteinase inhibitor protein is obtained by introducing the sequence of SEQ No.1 as claimed in claim 1 into a prokaryotic expression vector and carrying out prokaryotic expression.
4. Use of the sisal cysteine protease inhibitor gene according to claim 1 or the sisal cysteine protease inhibitor protein according to claim 2 in an antifungal product.
5. The use of the sisal cysteine protease inhibitor gene according to claim 1 or the sisal cysteine protease inhibitor protein according to claim 2 for controlling fungal diseases of crops.
6. The use of the sisal cysteine protease inhibitor gene according to claim 1 or the sisal cysteine protease inhibitor protein according to claim 2 for inhibiting the growth of phytophthora nicotianae, colletotrichum mangiferum and fusarium wilt of watermelon.
7. An antifungal product or a product for preventing and controlling crop fungal diseases or a product for inhibiting growth of phytophthora nicotianae, anthracnose of mango and fusarium wilt of watermelon, which is characterized in that: a sisal cysteine protease inhibitor protein comprising the sisal cysteine protease inhibitor protein of claim 2.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997014797A2 (en) * | 1995-10-20 | 1997-04-24 | Dana-Farber Cancer Institute | Cystatin m, a novel cysteine proteinase inhibitor |
| CN101186916A (en) * | 2007-11-20 | 2008-05-28 | 青岛大学 | Gene sequence of coding perinereis albuhitensis grube cysteine protease inhibitor and its amino acid sequence and application |
| CN106636122A (en) * | 2017-01-03 | 2017-05-10 | 内蒙古农业大学 | Clone and recombination expression method and application of cysteine proteinase inhibitor gene Pj_CPI of parabronema skrjabini |
-
2021
- 2021-10-18 CN CN202111206967.1A patent/CN113957077B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997014797A2 (en) * | 1995-10-20 | 1997-04-24 | Dana-Farber Cancer Institute | Cystatin m, a novel cysteine proteinase inhibitor |
| CN101186916A (en) * | 2007-11-20 | 2008-05-28 | 青岛大学 | Gene sequence of coding perinereis albuhitensis grube cysteine protease inhibitor and its amino acid sequence and application |
| CN106636122A (en) * | 2017-01-03 | 2017-05-10 | 内蒙古农业大学 | Clone and recombination expression method and application of cysteine proteinase inhibitor gene Pj_CPI of parabronema skrjabini |
Non-Patent Citations (3)
| Title |
|---|
| A new Piper nigrum cysteine proteinase inhibitor, PnCPI, with antifungal activity: molecular cloning, recombinant expression, functional analyses and molecular modeling;Aline Medeiros Lima;《Planta》;第252卷(第2期);编号16 * |
| Genome-wide identification and structure-function studies of proteases and protease inhibitors in Cicer arietinum (chickpea);Ranu Sharma;《Comput Biol Med》;第56卷;第67-81页 * |
| Peptide-based protease inhibitors from plants;Roland Hellinger;《Drug Discov Today》;第24卷(第9期);第1877-1889页 * |
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