阅读说明：本技术 来源于大刍草的抗菌肽sm-985及其应用 (Teosinte-derived antimicrobial peptide SM-985 and application thereof ) 是由 马龙 董五辈 于 2020-04-08 设计创作，主要内容包括：本发明属于生物技术领域,具体公开了来源于大刍草的抗菌肽SM-985及其应用,所述抗菌肽的序列为SEQ ID NO.2所示。SM-985可增加细菌细胞膜的渗透性,SM-985与细菌膜(革兰氏阴性或革兰氏阳性)结合的亲和力较高。用SM-985处理过的细菌SEM和TEM图像显示,细菌细胞膜出现损伤,细胞发生溶解,具有广谱、较强的抗菌活性。体内抗菌活性表明,SM-985可预防由丁香假单胞菌DC3000引起的番茄叶斑病感染。(The invention belongs to the technical field of biology, and particularly discloses teosinte SM-985 derived from teosinte and application thereof, wherein the sequence of the antimicrobial peptide is shown in SEQ ID NO. 2. SM-985 can increase the permeability of the bacterial cell membrane, and SM-985 has higher binding affinity with the bacterial membrane (gram negative or gram positive). SEM and TEM images of the bacteria treated by SM-985 show that the bacterial cell membrane is damaged, the cells are dissolved, and the antibacterial activity is broad-spectrum and strong. The in vivo antibacterial activity indicates that SM-985 can prevent tomato leaf spot infection caused by Pseudomonas syringae DC3000.)

1. An isolated antibacterial peptide derived from teosinte, wherein the amino acid sequence of the antibacterial peptide is shown in SEQ ID No. 2.

2. A nucleotide sequence encoding the antimicrobial peptide of claim 1.

3. The sequence of claim 2, which is represented by SEQ ID NO. 1.

4. Use of the antimicrobial peptide of claim 1 or the nucleotide sequence of claim 2 in the preparation of a bacterial bacteriostatic agent.

5. Use of the antimicrobial peptide of claim 1 or the nucleotide sequence of claim 2 for the preparation of a plant bacterial disease biocontrol agent.

6. The use according to claim 4 or 5, wherein said bacterium is Rhizoctonia cerealis (ClavibacterfangiiBacterial canker of tomato (C.elegans)Clavibactermichiganesissubsp.michiganesisBacillus subtilis (B.subtilis) (B.subtilis)Bacillus subtilis) Pseudomonas syringae tomato pathogenic variants (A)Pseudomonas syringaepv.tomato) Ralstonia solanacearum (L.), (B.), (C.), (Ralstonia solanacearum.）、Xanthomonas campestris Hippocampus pathogenic variety (Xanthomonas campestrispv.holcicola.）、Bacterial blight of rice (1)Xanthomonas oryzaepv. oryzae) Or Escherichia coli (Escherichia coli）。

7. The use according to claim 5, wherein the plant is tomato or tobacco.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to teosinte SM-985 derived from teosinte and application thereof.

Background

Antimicrobial peptides (AMPs) are natural small molecules that are considered as one of the best alternatives to conventional antibiotics because they are small molecules, and they generally have broad antimicrobial activity and relatively low cytotoxicity. Antimicrobial peptides are part of the innate immune system, which can be produced by all microorganisms, including bacteria (Hassan et al, 2012), animals (Hanco ck and Scott,2000) and plants (Benko-Iseppon et al, 2010). Antibacterial peptides are the first line of defense against bacterial infection (Ebbensgaard et al, 2015), plant antibacterial peptides are the model of the plant defense system, can be isolated from a wide variety of flowers, seeds, leaves, stems and roots, and have resistance to plant pathogensActivity (Nawrot et al, 2014). Teosinte is not only a plant, but also includes four perennial and one-year-old maize species (Galinat, 1969). Teosinte and maize share a common ancestor (Doebley, 1992). George bean proposed that maize is a domesticated form of teosinte, and in the past 10000 years, some of the major genes selected by mexicans might have converted teosinte into maize (bead, 1939) (bead, 1972). Breeding programs and germplasm studies have shown that cultivated plants have a relatively low level of resistance to biotic and abiotic stress compared to wild ancestors (Rosenthal and Dirzo, 1997). cDNA is a complementary DNA copy of mrna produced by reverse transcriptase, and the construction of cDNA libraries is a powerful tool for determining cell-and tissue-specific gene expression. cDNA prepared from mRNA has no inverted sequence such as intron. Thus, the cDNA reflects both expressible RNA and gene products (proteins) (Ying, 2004). Because of the large size of these libraries, which can reach thousands, screening of cDNA libraries for antimicrobial peptides is very challenging. Antimicrobial peptides were screened by the bacillus subtilis expression system in the cDNA library of our laboratory (Kong et al, 2018) (Wu et al, 2020), but this approach is time consuming and expensive. Computerized prediction is a time-saving, money-saving method for large-scale screening and identification of novel potential antimicrobial peptides (Liu et al, 2017). Well-structured antimicrobial peptide databases provide a good basis for developing antimicrobial peptide predictions, and many prediction methods have been proposed in the past few years (f.et al, 2012), and these servers use several algorithms based on different parameters (Liu et al, 2017). For example, CAMP_R3A prediction tool of the antibacterial peptide is developed based on supporting algorithms such as Support Vector Machines (SVM), Random questions (RF) and cognitive Analysis (DA). (Thomaset al., 2010).

APD3 provides valuable information about peptide fragment discovery schedules, classifications, terminology, vocabularies, statistics and computational tools. APD can effectively search, design and predict antimicrobial peptides (Zhou and Huang, 2015). DBAASP developed a new simple predictive algorithm based on the physicochemical properties (e.g. hydrophobicity, amphiphilicity, net charge) of the peptide's interaction with the anionic membrane (Vishnepolsky and pirkskhalava, 2014). Antimicrobial peptides with high levels of positively charged amino acids (e.g., arginine) have high net charge values and are known as cationic antimicrobial peptides (CAMPs). Cationic antimicrobial peptides (CA MPs) have a number of beneficial biological properties, including broad spectrum antimicrobial activity, slow development of resistance and rapid action (cimmac et al, 2019). Antibacterial peptides with alpha-helices are important factors in mediating plant protection (Montesinos,2007) (Keymanesh et al, 2009). Their main mechanism of action is interference with the outer and plasma membranes of the pathogen, membrane rupture or pore formation leading to cell lysis (Holaskova et al, 2014). Bacterial plant diseases are responsible for the massive losses of agricultural crops and agricultural products, and their control relies mainly on chemical pesticides (Agrios, 2004). Ralstonia solanacearum causes tomato blight (Murthy et al, 2019), clavibacterium solanacearum causes tomato bacterial canker (Tancos et al, 2013), Xanthomonas oryzae causes bacterial blight (Sharma et al, 2017), Xanthomonas campestris causes sorghum bacterial red streak (Navi et al, 2002). Tomato bacterial spot disease is caused by Pseudomonas syringaepv. tomato DC3000 (Xin and He, 2013). Various pesticides have been banned from use due to adverse effects on the environment. However, some economically important plant diseases face management difficulties due to the lack of effective compounds.

Disclosure of Invention

The invention aims to provide an antibacterial peptide SM-985 derived from teosinte, wherein the amino acid sequence of the antibacterial peptide is shown in SEQ ID NO. 2.

The invention also aims to provide application of the antibacterial peptide SM-985, which can be used for preparing bacterial bacteriostat, in particular for preventing and treating plant bacterial diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

a teosinte (Zea mexicana (Schrad.) Kuntze) cDNA library was constructed using Bacillus subtilis SCK6 competent cells. And finally screening the SM-985 as the antibacterial peptide through computer prediction and manual screening.

The amino acid sequence of the antibacterial peptide SM-985 is shown in SEQ ID NO. 2; the nucleotide sequence for coding the polypeptide is preferably the sequence shown in SEQ ID NO. 1.

The sequence shown in SEQ ID NO.2 or the nucleotide sequence for coding the sequence is applied to the preparation of bacterial inhibitors, and the bacteria comprise gram-positive bacteria and gram-negative bacteria.

In the above-mentioned applications, preferably, the bacterium is Rhizoctonia cerealis (Clavibacterfangii.), bacterial canker of Lycopersicon esculentum (Clavibactericoides subsp., Microbacterium), Bacillus subtilis (Bacillus subtilis), Pseudomonas syringae (Pseudomonas syringaepv. tomato pathogenic variant), Ralstonia solanacearum (Ralstonia solanacearum), Xanthomonas campestris (Xanthomonas campestris pathogenic variant of Choristotheca, Xanthomonas oryzae pv. oryzae), or Escherichia coli (Escherichia coli).

Application of sequence shown in SEQ ID NO.2 or nucleotide sequence for coding sequence in preparation of plant bacterial disease biocontrol agent

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

SM-985 is a novel antimicrobial peptide rich in cationic arginine isolated from teosinte. The SM-985 antibacterial peptide has broad-spectrum and strong antibacterial activity on various gram-positive bacteria and gram-negative bacteria, and inhibits bacteria by destroying the permeability of bacterial cell membranes. Due to its broad spectrum resistance, SM-985 may have antibacterial activity against other pathogenic bacteria, including human pathogenic bacteria. SM-985 inhibits the infection of P.syringae tomato pathogenic variety DC3000 on B.benthamiana and tomato, making SM-985 suitable for use as an antimicrobial agent.

Drawings

FIG. 1: MLC results for SM-985

After all indicator bacteria (gram-negative and gram-positive bacteria) were treated with SM-985 in sodium phosphate buffer at a concentration of 2. mu.M for 4 hours, no bacterial growth was observed. However, after treatment with sterile water, the indicator bacteria grew well in the control.

FIG. 2: cell membrane integrity test (PI staining)

Concentration 1 × 10⁷CFU/ml ofIndicator bacteria (gram positive and gram negative) after 4 hours of treatment with 10 μ M SM-985, the cell membrane was destroyed and the control did not fluoresce, indicating that the cell membrane is intact and the red fluorescence of the PI dye indicates that the membrane has disintegrated. The absorption of PI was observed with Olympus BX61 laser scanning confocal microscope. Scale bar: 20 μm.

FIG. 3: quantitative analysis of cell Membrane integrity (PI staining)

After 4 hours of treatment with 10 μ M SM-985, the PI absorption height of the indicator bacteria (gram positive and gram negative) increased. Blue dots indicate unstained cells and red dots indicate stained cells. NC denotes a negative control, and PC denotes a positive control. Cytoflex lx measured PI uptake and analyzed the data by CyExpret 2.4 software.

FIG. 4: localization of FITC-labeled SM-985

SM-985 had a high affinity for the indicator membrane (gram positive and gram negative) after 4 hours of treatment with 4. mu.M FITC-SM-985. The green fluorescence of the FITC label indicates that there is an interaction between SM-985 and the bacterial membrane. Results were observed using an Olympus BX61 laser scanning confocal microscope. Scale bar: 30 and 3 μm.

FIG. 5: bacterial Membrane Permeability of FITC-SM-985

The indicator cell membranes (gram positive and gram negative) lost integrity after treatment with 10. mu.M FITC-SM-985 for 4 h. The red fluorescence of the PI dye indicated that the membrane had been destroyed. The green fluorescence of the FITC label indicates that there is an interaction between SM-985 and the bacterial membrane. Results were observed using a Leica TCS SP5 confocal microscope. Scale bar: 3. 5 and 10 μm.

FIG. 6: determination of in vivo antibacterial Activity by permeation

After 4 hours of treatment with 5. mu.M SM-985, the concentration was 1 × 10⁶CFU/ml PstDC3000 lost virulence on Nicotiana benthamiana and on tomato. (A) Benstonia tabacum treated with sterile water (control). (B) Bentonium japonicum treated with SM-985. (C) Tomatoes treated with sterile water (control). (D) Tomatoes treated with SM-985. Observations of nicotiana benthamiana after 48 hours and tomatoes after 96 hours.

FIG. 7: SM-985 can prevent leaf spot infection of tomato

Concentration 1 × 10 was treated with 5. mu.M SM-985⁶CFU/ml Pst DC3000, while the control was treated with sterile water. Both SM-985 treatment and control were sprayed directly onto tomato leaves (front and back) and the results were observed after 6 days. (A) Control group leaf front. (B) The control group had the back of the leaf. (C) SM-985 treats the blade front. (D) SM-985 handles the blade back. Red arrows point to leaf spots.

FIG. 8: investigation of cell Membrane Damage by SEM and TEM

Concentration 1 × 10 was treated with 15. mu.M SM-9895⁷CFU/ml bacterial suspension of the bacterial species of the species Leptosphaeria solani for 4 hours. (A, C, E and G) the plasma membrane of the cells was intact and the cells were normal in the control. (B, D, F and H) SM-985 causes damage to the plasma membrane of cells and cell lysis. Hitachi SU8010 scanning electron microscope and Hitachi H-7650 transmission electron microscope.

FIG. 9: SM-985 to CaCl₂Determination of salt sensitivity.

Concentration 1 × 10 was treated with 5. mu.M SM-985⁶CFU/ml bacterial suspension for 4h, adding CaCl at different concentrations₂. The antibacterial activity of SM-985 was significantly reduced after the addition of CaCl 2. CaCl₂And SM-985 is an inverse relationship (A) tomato canker pathogen (B) Pst DC3000.

Detailed Description

The technical scheme of the invention is the conventional technology in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.