CN113388625A - Sugarcane top rot effect factor Fs _00548 gene and application thereof - Google Patents
Sugarcane top rot effect factor Fs _00548 gene and application thereof Download PDFInfo
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Abstract
The invention discloses a sugarcane tip rot effect factor Fs _00548 gene and application thereof, wherein the sugarcane tip rot effect factor Fs _00548 gene is from common sugarcane tip rot pathogenic bacteria, and the sugarcane tip rot effect factor Fs _00548 gene has a nucleotide sequence shown as SEQ ID No. 1. The protein coded by the sugarcane top rot effect factor Fs _00548 gene has an amino acid sequence shown in SEQ ID NO. 2. Starting from the genome of pathogenic bacteria, the invention searches for a gene containing a conserved NPP1 structural domain, and preliminarily verifies that the gene can induce the immune response of tobacco leaves, which indicates that the protein can be an exciton capable of inducing plant immunity, and provides reference for green prevention and control of sugarcane diseases. The invention discovers the NPP1 gene highly expressed in the infection process, preliminarily analyzes that the gene can play an important role in the interaction between fungi and plants, and provides a basis for further exploring the interaction between pathogenic bacteria and plants.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a sugarcane tip rot effect factor Fs _00548 gene and application thereof.
Background
Sugarcane is an important sugar crop in China, and the sugar yield of the sugarcane is an important component of the total sugar yield of the whole country[1]And sugarcane is an important energy crop and can be used as a raw material for producing ethanol[2]. Sugarcane top rot (sugarcane pokkah boeng disease) is a global fungal disease and was reported in java as early as 1896. The disease mainly occurs at the tender leaf part of the tip, leaves at the position of the tip which is infected with the disease are twisted together, and the conditions of rotten growing points of the tip, necrosis of young heart leaves and withered whole sugarcane can occur in serious conditions. In recent years, the sugarcane area tip rot is increasingly serious, the morbidity of some susceptible varieties is as high as 70% -80%, and serious yield loss (about 10-40%) is caused in sugarcane production. Therefore, the sugarcane tip rot has gradually become one of the main diseases of sugarcane in China.
Bailey et al purified a 24kDa protein from a culture of Fusarium oxysporum that induced plant Necrosis and Ethylene production (Neocross-and Ethylene-inducing Peptide 1-like proteins, Nep1-like proteins) in 1995. It was subsequently found that such proteins are contained in phytopathogensThere is a conserved domain NPP1 (neocross-inducing Phytophthora protein). Staats et al experimentally verified that this class of proteins can induce necrosis only in dicotyledonous plants and not in monocotyledonous leaves. Subsequently, Odalys et al cloned 2 Nep (neocross and ethylene-inducing proteins) genes encoded by the pathogenic bacterium Monilinothora panicosa, and all of them induced cell death on tobacco cells. ZAFER and the like[8]Two genes SsNep1 and SsNep2 were cloned from Sclerotinia sclerotiorum and proved that they can induce the necrosis of tobacco cells. Parthasarath et al cloned 7 NLP genes in Verticillium dahliae and found that only 2 genes induced leaf death in Nicotiana benthamiana. Stan et al [ found that HaNLP3 in Hypalonospora arabidopsis is a novel MAMP that induces immune response in Arabidopsis thaliana. Chen et al cloned 10 NLPs of Phytophthora capsic of Phytophthora capsici but only three genes could induce cell death. Rebeca et al cloned 4 NLP genes in Botrytis viticola pathogen and successfully expressed the protein, and found that only DserNEP1 and DserNEP2 induced cell necrosis.
The NPP1 gene is divided into 2 types according to the cysteine content in the structural domain, 2 cysteines are contained as I type, 4 cysteines are contained as II type, the I type NPP1 is distributed in fungi, oomycetes and bacteria, and the II type NPP1 is only distributed in the fungi and bacteria. Thus, fungi have both type I NPP1 and type II NPP1, such as Magnaporthe grisea genome of Magnaporthe grisea, with two genes encoding type I and type II NPP1, respectively[13]. NPP1 has a conserved domain of peptide 7, "GHRHDWEN". Feng et al identified the active site D therein by means of point mutation112，H120，D123And E, and125. ZAFER et al found that two Nep genes in Sclerotinia sclerotiorum induced cellular necrosis depending on Ca2+A signal pathway and a cyclic-a pathway. Teh et al found that jasmonic acid and salicylic acid can increase the transcriptional abundance of the GbNEP gene in pathogenic bacteria Ganoderma boninense, and also that GbNEP induction of cell necrosis may depend on Ca2+And (4) signal path.
Sugarcane tip rot caused by Fusarium sacchari (f.sacchari) seriously affects sugarcane yield and quality. The pathogenic mechanism of pathogenic bacteria is analyzed, and the method has important significance for guiding the disease-resistant breeding and green prevention and control of the sugarcane. The invention finds that NLPs (Nep1-like proteins) genes play an important role in the fungal infection and colonization process.
At present, the sugarcane top rot effector factor Fs _00548 gene of fungal peptide capable of inducing plant autoimmunity and application thereof are lacked.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a sugarcane top rot effector Fs _00548 gene of fungal peptide capable of inducing plant autoimmunity and application thereof.
In order to solve the problems in the prior art, the invention provides the following technical scheme: the sugarcane tip rot effect factor Fs _00548 gene is from common sugarcane tip rot pathogenic bacteria, and the sugarcane tip rot effect factor Fs _00548 has a nucleotide sequence shown as SEQ ID NO. 1.
The protein coded by the sugarcane top rot effect factor Fs _00548 gene has an amino acid sequence shown in SEQ ID NO. 2.
The signal peptide of the protein coded by the sugarcane top rot effector Fs _00548 gene has secretory activity.
The invention relates to a primer pair for cloning a sugarcane top rot effect factor Fs _00548 gene sequence, which is characterized in that: the primer pair is a PVX-GFP primer pair, a PVX-Bax primer pair, a PVX-Fs-00548 primer pair, a PVX-Fs-03159 primer pair, a PVX-Fs-06646 primer pair, a PVX-Fs-06646 primer pair, a PVX-Fs-11062 primer pair, a PVX-Fs-00548 delta SP primer pair, a pSUC 2-Fs-00548 SP primer pair, a pSUC 2-Fs-03159 SP primer pair, a pSUC 2-Fs-11062 SP primer pair, a Nb _ EF-1 primer pair, a Fv _ Actin primer pair and a Fs-00548-RT primer pair;
the forward primer of the PVX-GFP has a nucleotide sequence shown as SEQ ID NO. 3;
the reverse primer of the PVX-GFP has a nucleotide sequence shown as SEQ ID NO. 4.
The forward primer of the PVX-Bax has a nucleotide sequence shown as SEQ ID NO. 5;
the reverse primer of the PVX-Bax has a nucleotide sequence shown in SEQ ID NO. 6;
the forward primer of the PVX-Fs _00548 has a nucleotide sequence shown in SEQ ID NO. 7;
the reverse primer of the PVX-Fs _00548 has a nucleotide sequence shown in SEQ ID NO. 8;
the forward primer of the PVX-Fs-03159 has a nucleotide sequence shown as SEQ ID NO. 9;
the reverse primer of the PVX-Fs-03159 has a nucleotide sequence shown as SEQ ID NO. 10;
the forward primer of the PVX-Fs-06646 has a nucleotide sequence shown as SEQ ID NO. 11;
the reverse primer of the PVX-Fs-06646 has a nucleotide sequence shown as SEQ ID NO. 12;
the forward primer of the PVX-Fs-11062 has a nucleotide sequence shown as SEQ ID NO. 13;
the reverse primer of the PVX-Fs-11062 has a nucleotide sequence shown as SEQ ID NO. 14;
the forward primer of the PVX-Fs _00548 delta SP has a nucleotide sequence shown as SEQ ID NO. 15;
the reverse primer of the PVX-Fs _00548 delta SP has a nucleotide sequence shown as SEQ ID NO. 16;
the forward primer of the pSUC2-Fs _00548SP has a nucleotide sequence shown in SEQ ID NO. 17;
the reverse primer of the pSUC2-Fs _00548SP has a nucleotide sequence shown in SEQ ID NO. 18;
the forward primer of the pSUC2-Fs _03159SP has a nucleotide sequence shown as SEQ ID NO. 19;
the reverse primer of pSUC2-Fs _03159SP has a nucleotide sequence shown as SEQ ID NO. 20;
the forward primer of pSUC2-Fs _11062SP has a nucleotide sequence shown in SEQ ID NO. 21;
the reverse primer of pSUC2-Fs _11062SP has a nucleotide sequence shown in SEQ ID NO. 22;
the forward primer of the Nb _ EF-1 has a nucleotide sequence shown as SEQ ID NO. 23;
the reverse primer of the Nb _ EF-1 has a nucleotide sequence shown as SEQ ID NO. 24;
the forward primer of the Fv _ Actin has a nucleotide sequence shown in SEQ ID NO. 25;
the reverse primer of the Fv _ Actin has a nucleotide sequence shown in SEQ ID NO. 26;
the forward primer of the Fs _00548-RT has a nucleotide sequence shown in SEQ ID NO. 27;
the reverse primer of the Fs _00548-RT has a nucleotide sequence shown in SEQ ID NO. 28;
the invention discloses application of an Fs _00548 gene in preparation of a sugarcane top rot fungal peptide resistant medicament.
Further, the Fs00548 gene induces cell death on the Nicotiana benthamiana leaf, and the qRT-PCR result shows that the gene is transcribed and expressed in the whole infection process.
Has the advantages that: the invention can induce the fungal peptide of plant autoimmunity, analyze the secretory protein in the pathogenic fusarium sacchari whole genome sequence (not published), and find 4 sugarcane tip rot effect factors Fs _00548, Fs _03159, Fs _06646 and Fs _11062 through Blastp. Through transient expression of tobacco leaves, only the Fs _00548 gene is found to induce cell death on the Nicotiana benthamiana leaves, and the translation condition of the transcription of the genes after infecting tobacco is verified through RT-PCR and Western bot. The qRT-PCR result shows that the gene is transcribed and expressed in the whole infection process. According to results, the Fs _00548 is probably a Nep1-like gene, and a signal peptide at the N terminal has secretory activity and can induce cell necrosis on tobacco cells.
Compared with the prior art, the invention has the following advantages:
(1) starting from the genome of pathogenic bacteria, the invention searches for a gene containing a conserved NPP1 structural domain, and preliminarily verifies that the gene can induce the immune response of tobacco leaves, which indicates that the protein can be an exciton capable of inducing plant immunity, and provides reference for green prevention and control of sugarcane diseases.
(2) The invention discovers the NPP1 gene highly expressed in the infection process, preliminarily analyzes that the gene can play an important role in the interaction between fungi and plants, and provides a basis for further exploring the interaction between pathogenic bacteria and plants.
Drawings
FIG. 1 is a diagram showing the structure of 4 NPP1 domain-containing genes according to the present invention;
4 genes containing NPP1 structural domains are found in sugarcane top rot pathogenic bacteria Fusarium saccharophili through Blastp, gene structure drawing display is carried out by applying IBS 1.0 software, and a signal peptide area, a specific position of the structural domains, and cysteine content and position information in the structural domains are marked.
FIG. 2 is a diagram showing the alignment analysis of the protein sequences of the present invention;
protein sequence alignment analysis is carried out by using DNMAN software, and a conserved peptide segment 'GHRHDEN' is found to be contained in the structural domain of the protein sequence alignment analysis, and the starting and stopping positions of the conserved region are displayed. The peptide fragment of Fs 03159 differs from the others.
FIG. 3 is a diagram showing the transient expression of the recombinant Agrobacterium of the present invention in tobacco leaves by osmotic injection;
and performing osmotic injection on the recombinant agrobacterium on the back of the tobacco leaf, and marking each site. Observing the state of the leaves every day, photographing after 7d for recording, then putting the leaves into absolute ethyl alcohol for decoloring, and photographing for recording. Sampling each site after infection for 2d, extracting RNA, performing reverse transcription, and performing RT-PCR to verify whether the gene is transcribed; and extracting total protein, performing Western blot experiment, and verifying the protein translation condition.
FIG. 4 is a diagram of a method for verifying secretion function of a yeast signal peptide according to the present invention;
verifying the signal peptide secretion function of the candidate gene by using a yeast signal peptide secretion function verification method, and taking Avr1b in phytophthora sojae as a positive control; the non-secreted protein Mg87 in Magnaporthe grisea served as a negative control, and the results showed that the signal peptides of Fs _00548 and Fs _03159 have secretory activity.
FIG. 5 is a graph showing the expression of the gene of the present invention during fungal infection;
the expression condition of the gene in the fungal infection process is verified by a qRT-PCR method. Using the expression level of the gene in hyphae as a reference, Fs _00548 shows significant induced expression in 12 hours of sugarcane infection by Fusarium oxysporum till 72 hours reaches the expression peak (up to 48 times). Then the expression level gradually decreases.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example 1
Test example 1
1 materials and methods
1.1 Experimental materials
The test plants: no.1 sugarcane in the sugarcane variety, this laboratory provides, and the bucket is planted and is cultivateed in the warmhouse booth, and five leaves are opened up for subsequent use, and this variety shows the infection to F. Nicotiana benthamiana, provided in the laboratory, was cultured in a light incubator (16 h at 28 ℃ and 8h at 25 ℃ overnight). Test strains: escherichia coli Top10, purchased from Shanghai Weidi, was cultured at 37 ℃ at constant temperature; agrobacterium GV3101(pJIC SA _ Rep) purchased from Shanghai Diego, cultured at 28 ℃ at constant temperature; yeast strain YTK12, which is given by Liuwende teacher of Chinese academy of agricultural sciences, and is cultured at constant temperature of 30 deg.C. Test carrier: the PVX virus vector is presented by Kangzhen academy of science and technology university of agriculture and forestry, northwest; the vectors pSUC2, pSUC2-Mg87, pSUC2-Avr1b were gifted by the Master Liuwend, the institute of agricultural sciences, China.
1.2 Experimental methods
1.2.1 analysis of the sequence of the Gene of interest Conserved domains of the predicted gene are found in Conserved domains (https:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb. cgi)
Pfam (http:// Pfam. xfam. org/search). Prediction of the Signal peptide region of the Gene-encoded protein in SignalP
4.1(www.cbs.dtu.dk/services/SignalP /) and
SMART (http:// SMART. embl-heidelberg. de/SMART/set _ mode. cginormal ═ 1). The prediction of the transmembrane domain of the protein encoded by the gene was carried out on TMHMM (http:// www.cbs.dtu.dk/services/TMHMM /). Cloning of gene Primer design was performed on Primer-BLAST (https:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST /). The qRT-PCR primer design was performed on IDT (https:// www.ncbi.nlm.nih.gov/tools/primer-blast /). Primer sequences used in this study are shown in Table 1: a forward primer F; and a reverse primer R.
TABLE 1
1.2.2 cloning of target genes and construction of vectors PCR amplification is carried out by using primers in Table 1, RNA reverse transcription products of sugarcane No.1 leaves infected by F.sacchari are used as templates, and after amplification, a T vector is constructed, and sequencing verification is carried out. The PVX vector contains ClaI-SmaI-Not I-Sal I restriction sites, ClaI and Not I restriction sites are selected, HA tags are reserved, and the vector construction is carried out by using an In-fusion method. The Bax gene is synthesized by Shanghai. The signal peptide segment is predicted by software, and the region of the Fs _00548 gene from which the signal peptide is removed is amplified and constructed into a PVX vector. Based on the EcoRI-Xho I cleavage site of the pSUC2T7M13ORI (pSUC2) vector, a primer amplification signal peptide region was designed to be connected to the pSUC2 vector, pSUC2-Avr1b as a positive control and pSUC2-Mg87 as a negative control, and transformed into yeast strain YTK 12.
1.2.3 Agrobacterium-mediated transient transformation of tobacco all recombinant PVX vectors constructed in this experiment were transformed into Agrobacterium GV3101 by freeze-thaw method. Liquid LB medium (50 mg. mL) for positive recombinant Agrobacterium-1Kan，20mg·mL- 1Rif，50mg·mL-1Gen), shaking the culture medium, and culturing at 28 ℃ and 220rpm for 24h with shaking. 40Cells were collected at 00rpm for 5min and 10ml of 10mM MgCl was used2Washing the bacterial liquid, repeating for three times, and adjusting OD6000.4, standing at 28 ℃ for 2h in the dark. Selecting tobacco leaves with the size of one month, performing osmotic injection on the back of the leaves by using a syringe with a needle removed, and taking a site inoculated with an empty vector and PVX-GFP as a negative control and a site inoculated with PVX-Bax as a positive control. Each treatment was repeated 3 times, and the inoculated plants were placed in a greenhouse for cultivation, and the change in symptoms was observed and recorded every day, and photographed and recorded after 7 days.
1.2.4 Western blot As the PVX carrier contains a section of HA tag protein, the HA tag protein sequence is reserved when the carrier is constructed. The recombinant vector is transformed into agrobacterium GV3101, positive clones are identified and then delivered to Shanxi Eryou millet company for Western blot experiment.
1.2.5 Signal peptide secretion function verification the signal peptide secretion function of Fs _00548 protein was verified using the yeast signal peptide secretion system. Yeast strain YTK12, no tryptophan synthesis gene, no sucrose invertase gene; pSUC2 tryptophan synthesis gene, a sucrose invertase gene with deletion of ATG and signal peptide; CMD-W medium (CMD-W medium: 0.67% yeast N base without amino acids, 0.075% tryptophan drop supplement, 2% sucrose, 0.1% glucose, and 2% agar), containing no tryptophan, using sucrose and glucose as carbon sources; YPRAA medium (1% yeast extract, 2% peptone, 2% raffinose, and 2 mg. mL-1 antimycin A) contained no tryptophan and had raffinose as a carbon source. Untransformed YTK12 strain, unloaded pSUC2 and pSUC2-Mg87 were used as negative controls, and pSUC2-Avr1b was used as positive control. If the predicted signal peptide has a secretion function, the sucrose invertase can be secreted to the outside of cells, 2, 3, 5-triphenyltetrazolium chloride (TTC) is reduced to red 1, 3, 5-triphenylformazan, and the secretion function of the signal peptide can be further determined by carrying out TTC staining on the transformed yeast. Inoculating the positive clone into 10ml YPDA medium, shake culturing at 30 deg.C for 2d, 12000rpm, collecting thallus for 1min, and collecting ddH2O washes twice, and finally resuspends the cells with 750. mu.L sterile water. Adding 250 μ L10 mmol. multidot.L-1Acetic acid-sodium acetate buffer (pH 4.7), 500. mu.L of 10% sucrose solution, incubation of 10mi at 37 ℃n; centrifuging at 12000rpm for 1min, adding 900 μ L0.1% TTC into 100 μ L supernatant, standing at room temperature for 5min, and observing color change, wherein if the color changes to red, signal peptide has secretion function.
1.2.6 analysis of expression Pattern of target Gene Fusarium sacchari F.saccharophil was grown on PDA plates for 7d, cake was punched out on the plates, inoculated on leaf of sugarcane No.1, and moisturized for 24 h. Samples were taken at 0h, 12h, 24h, 48h, 72h, 120h, 144h, 216h post inoculation, respectively. The total RNA was extracted from the time point samples and the mycelia with reference to TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa), and then reverse transcription was performed to obtain cDNA. A LightCycler 96 quantitative PCR instrument is used, cDNA obtained through reverse transcription is used as a template, a Fusarium Verticillioides (F. Verticillioides) Actin gene is selected as an internal reference gene, a TB Green (TaKaRa) dye method is used for PCR amplification, and each reaction biology is repeated for 3 times. After the reaction is complete, according to 2-△△CtThe method analyzes the relative expression quantity of the genes at different time points of sugarcane infection by fusarium oxysporum.
2 results and analysis
2.1 sequence analysis results
The full length of the Fs _00548ORF is 708bp, the ORF codes 235 amino acids, the N end of the ORF has a 19aa signal peptide, and a transmembrane region is absent. Fs-03159 is 807bp in full length, encodes 268 amino acids, has an 18aa signal peptide at the N-terminal, and has no transmembrane region. Fs-06646 has a full length of 906bp, codes 302 amino acids, and has an N-terminal 18aa signal peptide without a transmembrane region. Fs-11062 has a full length of 771bp, encodes 256 amino acids, has a 20aa signal peptide at the N terminal, and has no transmembrane region. The protein is divided into type I and type II according to the cysteine content in the NPP1 structural domain (figure 1), wherein the Fs _00548 protein structural domain contains 2 cysteines, and belongs to the type I NPP1 gene; the protein domains Fs _03159, Fs _06646 and Fs _11062 contain 4 cysteines, and belong to the II type NPP1 gene. The domain of the 4 genes contained a conserved motif "GHRHDWEN", but "GHTHDWEN" in Fs _03159 (fig. 2).
FIG. 14 genes are classified into two types, type I and type II, according to the cysteine content in the NPP1 domain. FIG. 24 shows that the structural domain of the gene contains a conserved motif "GHRHDEN", and Fs-03159 shows "GHTHDWEN".
2.2 Fs _00548 can induce the necrosis of tobacco cells
The recombinant plasmid is transformed into agrobacterium GV3101 by a freeze-thaw method, and the recombinant plasmid is transiently expressed in tobacco leaves by an osmotic injection method. Bax is a mouse pro-apoptotic protein that can induce cell necrosis on Nicotiana benthamiana leaves, similar to the HR response induced by pathogenic bacteria. No load, GFP was used as a negative control, and Bax was used as a positive control. After 7d, the leaves injected with Fs _00548 showed necrosis, consistent with the positive control results, and the necrotic spots were more evident after alcohol bleaching (fig. 3a 1). While the site inoculated with PVX-Fs-03159 and PVX-Fs-11062 showed no significant change (FIGS. 3A2 and A3). The Fs _00548 gene without signal peptide was used to construct a vector, which was transiently expressed in tobacco leaves and markedly reduced the degree of necrosis (FIG. 3A 4). The transcription of the gene after Agrobacterium infection of tobacco leaves was verified by RT-PCR (FIG. 3B). The expression of the protein was verified by Western blot experiments (FIG. 3C).
FIG. 3A. Fs _00548 induced The programmed cell depth in bacterial cell for function (A1) Fs _00548 gene induced programmed cell depth BAX (A2, A3) Fs _03159 and Fs _11062 d not induced The cell depth (A4) Fs _00548 induced cell depth with signal peptide B.RNA extracted from bacterial cell after fed for 2 days, Detection of gene conversion; nb _ EF-1 water used as a reference gene.C. proteins expressed power fixed by western blot.
2.3 Fs _00548 Signal peptide with secretory function
The plasmid pSUC2 contains a tryptophan synthase gene and a sucrose invertase gene (SUC2) lacking the signal peptide and the initiation codon ATG. Therefore, yeast strain YTK12 containing pSUC2 plasmid can grow on CMD-W medium, but cannot grow on YPRAA medium with raffinose as carbon source. When the signal peptide containing the initiation codon ATG was inserted into the sucrose invertase gene of the pSUC2 vector, growth was achieved on YPRAA medium. Avr1b is an effector protein in phytophthora sojae, and its signal peptide has secretory activity and is used as a positive control in this experiment; mg87 is a non-secreted protein in Magnaporthe grisea, and its signal peptide has no secretory activity and serves as a negative control in this experiment. The results showed that yeast strains transformed with pSUC2, pSUC2-Mg87sp, pSUC2-Avr1bsp, pSUC2-Fs _00548sp, pSUC2-Fs _03159sp, pSUC2-Fs _11062sp all grew on CMD-W medium, indicating that the recombinant plasmid had been transformed into yeast. However, only the transformed yeast strains pSUC2-Avr1bsp, pSUC2-Fs _00548sp and pSUC2-Fs _03159sp could grow on YPRAA medium, indicating that the signal peptides of Fs _00548 and Fs _03159 have secretory activity, and TTC staining further confirmed the experimental results.
FIG. 4 Functional identification of the signal peptide of Fs _00548 by second layer signal peptide derived trap, the Fs _00548 signal peptide derived from the inner of the pSUC2 vector and transformed into the third layer YTK12 vector, the predicted signal peptide of pSUC2-Avr1b wave used as a positive control, the third layer YTK12, YTK12 carrying pSUC2 vector and YTK12 carrying the pSUC2-Mg87 used as a negative control, Yeast growing on CMD-W medium derived from the inner of the channel C transformed into the third layer signal of the channel C.
2.4 Fs _00548 inducible expression in Fusarium saccharum infection process
The expression mode of the Fs _00548 gene in the sugarcane leaf infection process by the fusarium saccharum is analyzed by extracting RNA of the sugarcane leaf at each time point in the hypha and the infection process, carrying out reverse transcription to obtain cDNA and taking the expression level in the fungal hypha as a control (the relative expression level is 1). The qRT-PCR results show: compared with hyphae, Fs _00548 shows significant inducible expression at 12h when sugarcane is infected by Fusarium saccharum, until 72h reaches the expression peak (up to 48 times). Then the expression level gradually decreases. FIG. 5 Expression part of Fs _00548 in the interaction between genetic and genetic proteins, the Expression specificity differential to mycelium. my. hpi. our passage infection, ("﹡" indicators p <0.05, "﹡ ﹡" indicators p <0.01)
3 conclusion and discussion
NLP genes are found in the genomes of numerous fungi, bacteria and oomycetes which are published at present, and the genes are proved to be widely existed in various pathogenic bacteria and play important functions in the interaction of the pathogenic bacteria and plants. In 1995, the NPP1 gene in Fusarium was reported, and the gene containing the NPP1 conserved domain was found in prokaryotic and eukaryotic pathogenic bacteria such as Sclerotinia sclerotiorum, Bacillus, Verticillium dahliae, Phytophthora infestans, Magnaporthe oryzae, and the like. In the experiment, 4 Nep1-like genes containing NPP1 conserved structural domains are analyzed from the genome, but only 3 genes (Fs _00548, Fs _03159 and Fs _11062) are cloned. By transiently expressing nicotiana benthamiana leaves, it was found that only Fs _00548 could induce cell necrosis on tobacco leaves. Unlike the "GHRHDEN" of Fs _00548 protein, the conserved peptide of Fs _03159 protein is "GHTHDWEN", and the signal peptide of Fs _03159 protein has secretory activity, and it is presumed that the failure of the gene to induce necrosis may be caused by mutation, but further amino acid mutation experiments are required for verification. The invention of Parthasarath et al finds that pathogenic bacteria causing blight V.dahliae contain 7 NLP genes, but only 2 genes can induce the leaf necrosis of the Nicotiana benthamiana. The invention of Chen et al finds that phytophthora capsici pathogenic bacteria P.capsic contains 10 NLP genes, but only 3 genes can induce the necrosis of tobacco cells and pepper leaves. Based on these results, the NLP gene of the same pathogen may play different functions in the fungal infestation of plants.
Secretory proteins of pathogenic bacteria are all likely to function as effector proteins, and since effector proteins proceed through the secretory pathway of the endoplasmic reticulum-golgi apparatus, in order to ensure the normal progress of secretory functions, it is necessary for a signal peptide at the N-terminal to function as a secretory protein. Since the signal peptide can be recognized by the analysis software, the scope can be narrowed when the software analyzes the effector protein. The N ends of the sequences Fs _00548, Fs _03159 and Fs _11062 are all provided with a segment of signal peptide, and through a yeast signal peptide secretion function verification system, the signal peptides of Fs _00548 and Fs _03159 have a secretion function, and the signal peptide region of Fs _11062 has no secretion activity, so that Fs _00548 and Fs _03159 are typical secretory protein genes. The lack of secretory activity of the signal peptide of the Fs _11062 protein may be one of the reasons that the gene cannot induce cell necrosis on tobacco leaves, and further experimental exploration is required. When the Fs _00548 protein lacks a signal peptide, the necrosis-inducing function of the gene is found to be weakened, which indicates that a signal peptide region has an important function on the function of the protein, and since the signal peptide is an important component of a secreted protein, the deletion of the signal peptide is presumed to influence the recognition of the protein by plants, and further experimental exploration is needed.
In the experiment, a sugarcane leaf infection method is adopted, and the expression condition of the Fs _00548 gene in the infection process is invented through qRT-PCR. The results show that the Fs _00548 gene has expression level in the whole infection process, the expression level is the highest in 72h and is 48 times higher than that in hyphae, and then the expression level is continuously reduced. The expression mode of the NLP gene is also reported before, the expression quantity of the Ps NLP1 of the phytophthora sojae is obviously up-regulated 48 hours after the phytophthora sojae is inoculated, and the phytophthora infestans PinPP1.1 can be detected only at the later stage of infection. After the potato leaves are inoculated with phytophthora infestans, the expression level of PITG _10839 shows different up-regulation trends along with different inoculation times, and the up-regulation times are highest at 48h and reach about 350 times [25 ]. Mg NLP in the pathogen Mycosphaerella graminicola was detected to be highly expressed at the end of infection. Indicating that this class of genes may be expressed throughout the fungal infestation, but function primarily at a later stage. Fungi secrete proteins that induce cell necrosis during the infective stage, which not only can disrupt the host defense system, help pathogenic bacteria successfully colonize sugarcane, but also may transform their trophic patterns from parasitism to saprophytic. Although this class of genes is also expressed early in infection, its activity may be inhibited by other effector proteins. The specific function of the gene in the infection process needs further experimental exploration.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
<110> Guangxi university
<120> sugarcane tip rot effect factor Fs _00548 gene and application thereof
<130> 2020
<160> 28
<170> SIPOSequenceListing 1.0
<210> 1
<211> 708
<212> DNA
<213> Artificial sequence (Fs _00548 nucleotide sequence)
<400> 1
atggtgctaa ttactcaact tctctcagcc ttgacccttg cttcaggtat ccttgcatca 60
cctattgagc gccgtgctgt tattaaccac gatgctgttg ttgggtttcc gcaaactgtt 120
ccatccaata cagctggctc actctatctg aagtacaagc cttacctcaa ggtcttcaac 180
ggatgtgtgc cctttcccgc ggttgatagt aacggaaaca ctggtggagg cctagccaca 240
agtggctcat cgaatggagg ttgcagtagc agtacgggcc aggtctacgt gcgaggcggg 300
acttacaatg gccgctacgg cattatgtac tcttggtata tgccaaagga ttcaccctct 360
cccggactag gacatcgcca cgactgggaa aacgcggtga tatggttgtc tggtgaaagc 420
acaagtgcta caattgttgg catggcagtt tctcagcacg gaggatatga caagagaacg 480
tctggtacat tctccggaaa tagtccttta gtgggatata ccgccatctg gcccactaac 540
caccaaatga tctttaccaa cgacaaaggt ggccagcagc cgctgattgc ctgggagagt 600
ttgaccgctg cggctcgtac agctttgaca aacactgact tcggcagtgc caacgtccca 660
ttcaaggacg gaagctttga gtcaaatcta gaaaaggcag ctgtctaa 708
<210> 2
<211> 235
<212> PRT
<213> Artificial sequence (Fs _00548 amino acid sequence)
<400> 2
Met Val Leu Ile Thr Gln Leu Leu Ser Ala Leu Thr Leu Ala Ser Gly
1 5 10 15
Ile Leu Ala Ser Pro Ile Glu Arg Arg Ala Val Ile Asn His Asp Ala
20 25 30
Val Val Gly Phe Pro Gln Thr Val Pro Ser Asn Thr Ala Gly Ser Leu
35 40 45
Tyr Leu Lys Tyr Lys Pro Tyr Leu Lys Val Phe Asn Gly Cys Val Pro
50 55 60
Phe Pro Ala Val Asp Ser Asn Gly Asn Thr Gly Gly Gly Leu Ala Thr
65 70 75 80
Ser Gly Ser Ser Asn Gly Gly Cys Ser Ser Ser Thr Gly Gln Val Tyr
85 90 95
Val Arg Gly Gly Thr Tyr Asn Gly Arg Tyr Gly Ile Met Tyr Ser Trp
100 105 110
Tyr Met Pro Lys Asp Ser Pro Ser Pro Gly Leu Gly His Arg His Asp
115 120 125
Trp Glu Asn Ala Val Ile Trp Leu Ser Gly Glu Ser Thr Ser Ala Thr
130 135 140
Ile Val Gly Met Ala Val Ser Gln His Gly Gly Tyr Asp Lys Arg Thr
145 150 155 160
Ser Gly Thr Phe Ser Gly Asn Ser Pro Leu Val Gly Tyr Thr Ala Ile
165 170 175
Trp Pro Thr Asn His Gln Met Ile Phe Thr Asn Asp Lys Gly Gly Gln
180 185 190
Gln Pro Leu Ile Ala Trp Glu Ser Leu Thr Ala Ala Ala Arg Thr Ala
195 200 205
Leu Thr Asn Thr Asp Phe Gly Ser Ala Asn Val Pro Phe Lys Asp Gly
210 215 220
Ser Phe Glu Ser Asn Leu Glu Lys Ala Ala Val
225 230 235
<210> 3
<211> 48
<212> DNA
<213> Artificial sequence (forward primer for PVX-GFP)
<400> 3
agcaccagct agcatcgata tgagtaaagg agaagaactt ttcactgg 48
<210> 4
<211> 52
<212> DNA
<213> Artificial sequence (reverse primer of PVX-GFP)
<400> 4
atcgtatggg tacgcggccg ctttgtatag ttcatccatg ccatgtgtaa tc 52
<210> 5
<211> 36
<212> DNA
<213> Artificial sequence (forward primer of PVX-Bax)
<400> 5
agcaccagct agcatcgata tggacgggtc cgggga 36
<210> 6
<211> 43
<212> DNA
<213> Artificial sequence (reverse primer of PVX-Bax)
<400> 6
atcgtatggg tacgcggccg cgcccatctt cttccagatg gtg 43
<210> 7
<211> 46
<212> DNA
<213> Artificial sequence (forward primer of PVX-Fs _ 00548)
<400> 7
agcaccagct agcatcgata tggtgctaat tactcaactt ctctca 46
<210> 8
<211> 46
<212> DNA
<213> Artificial sequence (reverse primer of PVX-Fs _ 00548)
<400> 8
atcgtatggg tacgcggccg cgacagctgc cttttctaga tttgac 46
<210> 9
<211> 38
<212> DNA
<213> Artificial sequence (forward primer of PVX-Fs _ 03159)
<400> 9
agcaccagct agcatcgata tgcacgcctt cacaatcg 38
<210> 10
<211> 40
<212> DNA
<213> Artificial sequence (reverse primer of PVX-Fs _ 03159)
<400> 10
atcgtatggg tacgcggccg cctcgtcctt ggcgggatcg 40
<210> 11
<211> 45
<212> DNA
<213> Artificial sequence (forward primer of PVX-Fs _ 06646)
<400> 11
agcaccagct agcatcgata tgcatatccc atctgtgcta ttctc 45
<210> 12
<211> 52
<212> DNA
<213> Artificial sequence (reverse primer of PVX-Fs _ 06646)
<400> 12
atcgtatggg tacgcggccg ctatcaattt cttctcagaa ccattacctt tc 52
<210> 13
<211> 42
<212> DNA
<213> Artificial sequence (forward primer of PVX-Fs-11062)
<400> 13
agcaccagct agcatcgata tgcagaacaa gatcatcacc gc 42
<210> 14
<211> 45
<212> DNA
<213> Artificial sequence (reverse primer of PVX-Fs-11062)
<400> 14
atcgtatggg tacgcggccg cctcgtcctt ataaggatcg aagcc 45
<210> 15
<211> 39
<212> DNA
<213> Artificial sequence (forward primer of PVX-Fs _ 00548. DELTA. SP)
<400> 15
agcaccagct agcatcgata tgtcacctat tgagcgccg 39
<210> 16
<211> 46
<212> DNA
<213> Artificial sequence (reverse primer of PVX-Fs _ 00548. DELTA. SP)
<400> 16
atcgtatggg tacgcggccg cgacagctgc cttttctaga tttgac 46
<210> 17
<211> 35
<212> DNA
<213> Artificial sequence (forward primer of pSUC2-Fs _00548 SP)
<400> 17
ccggaattca tggtgctaat tactcaactt ctctc 35
<210> 18
<211> 31
<212> DNA
<213> Artificial sequence (reverse primer of pSUC2-Fs _00548 SP)
<400> 18
ccgctcgagt gcaaggatac ctgaagcaag g 31
<210> 19
<211> 28
<212> DNA
<213> Artificial sequence (forward primer of pSUC2-Fs _03159 SP)
<400> 19
ccggaattca tgcacgcctt cacaatcg 28
<210> 20
<211> 27
<212> DNA
<213> Artificial sequence (reverse primer of pSUC2-Fs _03159 SP)
<400> 20
ccgctcgaga gcttcgacgg tgagagc 27
<210> 21
<211> 32
<212> DNA
<213> Artificial sequence (forward primer of pSUC2-Fs _11062 SP)
<400> 21
ccggaattca tgcagaacaa gatcatcacc gc 32
<210> 22
<211> 27
<212> DNA
<213> Artificial sequence (reverse primer of pSUC2-Fs _11062 SP)
<400> 22
ccgctcgagg aggccatggc actcagg 27
<210> 23
<211> 21
<212> DNA
<213> Artificial sequence (forward primer of Nb _ EF-1)
<400> 23
tggtgtcctc aagcctggta t 21
<210> 24
<211> 21
<212> DNA
<213> Artificial sequence (reverse primer of Nb _ EF-1)
<400> 24
acgcttgaga tccttaaccg c 21
<210> 25
<211> 26
<212> DNA
<213> Artificial sequence (forward primer of Fv _ Actin)
<400> 25
gagaacgagc gtgtcttgat tgagcc 26
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence (reverse primer of Fv _ Actin)
<400> 26
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence (Forward primer of Fs _ 00548-RT)
<400> 27
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence (reverse primer of Fs _ 00548-RT)
<400> 28
Claims (6)
1. A sugarcane top rot effector Fs _00548 gene is from common sugarcane top rot pathogenic bacteria, and is characterized in that: the sugarcane top rot effect factor Fs _00548 has a nucleotide sequence shown in SEQ ID NO. 1.
2. The sugarcane top rot effector Fs _00548 gene of claim 1 encoding a protein having an amino acid sequence shown in SEQ ID No. 2.
3. The sugarcane top rot effector Fs _00548 gene of claim 1 encoding a protein with secretory activity.
4. Primer pair for cloning the sugarcane top rot effector Fs _00548 gene sequence as claimed in claim 1, characterized in that: the primer pair is a PVX-GFP primer pair, a PVX-Bax primer pair, a PVX-Fs-00548 primer pair, a PVX-Fs-03159 primer pair, a PVX-Fs-06646 primer pair, a PVX-Fs-06646 primer pair, a PVX-Fs-11062 primer pair, a PVX-Fs-00548 delta SP primer pair, a pSUC 2-Fs-00548 SP primer pair, a pSUC 2-Fs-03159 SP primer pair, a pSUC 2-Fs-11062 SP primer pair, a Nb _ EF-1 primer pair, a Fv _ Actin primer pair and a Fs-00548-RT primer pair;
the forward primer of the PVX-GFP has a nucleotide sequence shown as SEQ ID NO. 3;
the reverse primer of the PVX-GFP has a nucleotide sequence shown as SEQ ID NO. 4.
The forward primer of the PVX-Bax has a nucleotide sequence shown as SEQ ID NO. 5;
the reverse primer of the PVX-Bax has a nucleotide sequence shown in SEQ ID NO. 6;
the forward primer of the PVX-Fs _00548 has a nucleotide sequence shown in SEQ ID NO. 7;
the reverse primer of the PVX-Fs _00548 has a nucleotide sequence shown in SEQ ID NO. 8;
the forward primer of the PVX-Fs-03159 has a nucleotide sequence shown as SEQ ID NO. 9;
the reverse primer of the PVX-Fs-03159 has a nucleotide sequence shown as SEQ ID NO. 10;
the forward primer of the PVX-Fs-06646 has a nucleotide sequence shown as SEQ ID NO. 11;
the reverse primer of the PVX-Fs-06646 has a nucleotide sequence shown as SEQ ID NO. 12;
the forward primer of the PVX-Fs-11062 has a nucleotide sequence shown as SEQ ID NO. 13;
the reverse primer of the PVX-Fs-11062 has a nucleotide sequence shown as SEQ ID NO. 14;
the forward primer of the PVX-Fs _00548 delta SP has a nucleotide sequence shown as SEQ ID NO. 15;
the reverse primer of the PVX-Fs _00548 delta SP has a nucleotide sequence shown as SEQ ID NO. 16;
the forward primer of the pSUC2-Fs _00548SP has a nucleotide sequence shown in SEQ ID NO. 17;
the reverse primer of the pSUC2-Fs _00548SP has a nucleotide sequence shown in SEQ ID NO. 18;
the forward primer of the pSUC2-Fs _03159SP has a nucleotide sequence shown as SEQ ID NO. 19;
the reverse primer of pSUC2-Fs _03159SP has a nucleotide sequence shown as SEQ ID NO. 20;
the forward primer of pSUC2-Fs _11062SP has a nucleotide sequence shown in SEQ ID NO. 21;
the reverse primer of pSUC2-Fs _11062SP has a nucleotide sequence shown in SEQ ID NO. 22;
the forward primer of the Nb _ EF-1 has a nucleotide sequence shown as SEQ ID NO. 23;
the reverse primer of the Nb _ EF-1 has a nucleotide sequence shown as SEQ ID NO. 24;
the forward primer of the Fv _ Actin has a nucleotide sequence shown in SEQ ID NO. 25;
the reverse primer of the Fv _ Actin has a nucleotide sequence shown in SEQ ID NO. 26;
the forward primer of the Fs _00548-RT has a nucleotide sequence shown in SEQ ID NO. 27;
the reverse primer of the Fs _00548-RT has a nucleotide sequence shown in SEQ ID NO. 28;
5. the use of the Fs _00548 gene of claim 1 in the preparation of a fungal peptide drug resistant to sugarcane top rot.
6. Use according to claim 5, characterized in that: the Fs00548 gene induces cell death on the Nicotiana benthamiana leaf, and qRT-PCR results show that the gene is transcribed and expressed in the whole infection process.
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| CN116024234B (en) * | 2022-12-12 | 2023-07-21 | 南京林业大学 | Poplar aschersonia aleyrodis effector protein SmCSEP3 and application thereof |
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