Barley stripe disease pathogenic gene Pg00778 and application thereof

Abstract

The barley stripe disease (Barley stripe disease) is an important disease in the worldwide range, which is caused by pyrenopora graminea, and the disease is one of the diseases which are commonly generated in barley producing areas in China and are seriously harmful to the barley producing areas. At present, breeding and utilization of disease-resistant varieties are the most economical, effective and safe means for controlling the disease. The invention provides screening and identification of the virulence gene Pg00778 of the barley stripe pathogen, a Pg00778 mutant strain is obtained through RNA interference and overexpression methods, and the influence of the Pg00778 gene on the growth, development and pathogenicity of the barley stripe pathogen is further proved. Technical support is provided for subsequent large-scale screening of barley streak pathogen pathogenesis related mutant strains, and separation, identification and pathogenesis research of barley streak pathogen pathogenesis related genes are promoted.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to the pathogenicity of barley stripe pathogen Pg00778 Gene, and its application in regulating hyphal growth rate and reducing and / or increasing the pathogenicity of barley stripe disease after silencing and / or overexpressing the gene. Background Art

[0002] barley( Hordeum vulgare Hordeum vulgare L. is an annual herbaceous plant of the genus Hordeum in the Poaceae family. It is one of the most important food crops in the world. Compared with other cultivated crops, its planting area ranks fourth after corn, rice and wheat. [1-3] In recent years, barley has not only been used as feed for livestock farming, but also as a key raw material for brewing beer, and can be used to make food and health products. [4-5] Therefore, promoting the stable development of the barley industry is of great significance to accelerating economic growth and improving people's living standards. However, during the growth cycle of barley, it faces the threat of various diseases, among which the fungus Pyricularia tauriformis ( Pyrenophora graminea Barley streak disease, caused by barley leaf stripes, is a common disease. It typically appears as brown or yellowish-brown stripes or spots on leaves. In severe cases, these stripes or spots can cover the entire leaf, affecting photosynthesis and reducing barley yield. [6-7] Studies have shown that a 1% increase in the incidence of barley stripe disease leads to a 0.9% to 1.0% reduction in barley yield. Once the disease becomes prevalent, it will inevitably cause a significant reduction in barley yield. [8] Therefore, it is particularly important to study the pathogenic mechanism of this pathogen.

[0003] The laboratory initially isolated the stripe pathogen from different barley-producing areas in Gansu Province and finally screened out the highly pathogenic strain QWC, which was sequenced using the Illumina Hiseq 2000 platform. The strain was then used to infect the barley variety 'Aleixs', and seed embryos were collected at 7, 14, and 18 days for transcriptomic analysis. The results showed that differentially expressed genes Pg00778 It showed high expression levels in all three periods, suggesting that it may be involved in regulating the pathogenicity of barley streak pathogen to the host.

[0004] This study identified genes from whole-genome data of sequenced strains of barley streak pathogen Pg00778 Bioinformatics analysis was also conducted to analyze the colony growth and pathogenicity of mutant and wild strains through gene interference and overexpression methods to clarify their pathogenic mechanisms. Pg00778It provides a basis for studying the molecular pathogenic mechanism of the pathogen and finding target genes to prevent and control barley stripe disease, and also provides a theoretical basis and technical support for molecular biological research on the pathogenic mechanism of the pathogen and functional analysis of genes related to pathogenicity.

[0005] Problems with the existing technology: Barley stripe disease is not found in the existing technology Pg00778 Reports on genes and their related applications. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention provides a barley stripe disease pathogenic gene Pg00778 To meet the needs of the target gene of the disease, RNA interference and overexpression technology were used to obtain the gene Pg00778 The invention further provides a mutant strain of the gene in growth, differentiation and pathogenicity. To solve the above technical problems, the present invention adopts the following technical solutions: 1. Barley stripe disease pathogenicity gene Pg00778 , Pg00778 The full-length sequence of the transcript is shown in SEQ ID NO: 1, wherein the CDS region is 51-623 bp.

[0007] 2. Barley stripe disease pathogenicity gene Pg00778 The acquisition method comprises: (1) culturing a wild strain of barley stripe disease QWC; (2) Pg00778 Gene cloning; (3) Pg00778 Bioinformatics analysis of genes.

[0008] 3. Barley stripe disease pathogenicity gene Pg00778 A vector construction method, comprising: obtaining a vector by RNA-seq sequencing results Pg00778 The CDS sequence of the gene is based on the multiple cloning site on the vector map and interferes with the vector pSilent-1: Pg00778 Construction selection Kpn I and Bgl II. Xho I and Hin d III restriction site, pBARGPE1-Hyg: Pg00778 Construction and selection of overexpression vectors Bam HI and Eco RI restriction site, and the target sequence was synthesized.

[0009] 4. Barley stripe disease pathogenicity gene Pg00778 A genetic transformation method comprising: (1) preparation of wild strain QWC protoplasts; (2) PEG-CaCl2-mediated genetic transformation; and (3) screening and identification of transformed strains.

[0010] 5. Interference with the pathogenicity gene of barley stripe disease Pg00778 The application of expression in regulating the nutritional growth of bacterial strains, Pg00778 The full-length sequence of the transcript is shown in SEQ ID NO: 1, wherein the CDS region is 51-623 bp.

[0011] 6. Interference with the pathogenicity gene of barley stripe disease Pg00778 The application of expressing in reducing the pathogenicity of the fungus Pyricularia taedifolia, Pg00778 The full-length sequence of the transcript is shown in SEQ ID NO: 1, wherein the CDS region is 51-623 bp.

[0012] Beneficial effect: The present invention successfully constructed the gene by using the technology of gene interference and overexpression Pg00778 The interference vector and overexpression vector were used to transform the gene. After PEG-CaCl2-mediated genetic transformation, two interference transformation strains and three overexpression transformation strains were obtained. The results showed that the colony growth rate, relative gene expression level, incidence rate and staining results of the interference transformation strains and overexpression transformation strains were significantly different from those of the wild strain, indicating that Pg00778 Genes associated with pathogenicity of barley stripe pathogen. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The primer sequences used in the present invention are as follows: the underlined portion is the 5-UTR and 3-UTR, and the ununderlined portion is the CDS.

[0014] Figure 2 Figure 1 shows the bioinformatics analysis of Pg00778. A shows the predicted secondary structure of Pg00778, B shows the predicted hydrophilicity of Pg00778, and C shows the predicted phosphorylation sites of Pg00778.

[0015] Figure 3 for Pg00778 Schematic diagram of gene interference and overexpression vector. Pg00778 Schematic diagram of gene interference vector, B is Pg00778 Schematic diagram of gene overexpression vector.

[0016] Figure 4 For the verification of transformants. In Figure A, M is DL 2000 DNA Marker; lanes 1-2 are different barley stripe pathogens transformed with pSilent-1: Pg00778 The Hyg gene bands amplified from a single colony of the recombinant vector, lane 3 is the Hyg gene band amplified from the pSilent-1 empty vector; Figure B: M is a DL 2000 DNA Marker; lanes 1-3 are different barley stripe pathogens transformed with pBARGPE1-Hyg: Pg00778Lane 4 is the Hyg gene band amplified from a single colony of the recombinant vector, and lane 5 is the Hyg gene band amplified from the empty vector pBARGPE1-Hyg.

[0017] Figure 5 is the relative expression level of the transformed strain. Pg00778-RNAi-3 and Pg00778- RNAi-7 The relative expression level of B is the overexpression transformed strain Pg00778-OE-D 、 Pg00778-OE-E and Pg00778-OE-I The relative expression level.

[0018] Figure 6 The growth morphology of the transformant on PDA medium is shown in Figure 1. A represents the growth morphology of the transformant on PDA medium, and B represents the diameter of the transformant on PDA medium.

[0019] Figure 7 Figure 1 shows the pathogenicity of potted barley leaves. A shows the potted image of the transformed strain, B shows the staining analysis of the transformed strain, C shows the relative chlorophyll content of the transformed strain, and D shows the incidence of the strain.

[0020] Figure 8 for Pg00778 Gene structure, underlined is UTR, bold is start codon and stop codon, between bold and including bold is CDS. DETAILED DESCRIPTION

[0021] The methods and apparatus used in the following embodiments of the present invention are conventional methods and apparatus unless otherwise specified; the equipment and reagents used are conventional equipment and reagents purchased from a reagent company. In order to make the purpose, technical solutions and advantages of the present invention clearer, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments. Examples of these preferred implementations are illustrated in the specific embodiments. It should also be noted that in order to avoid obscuring the technical solutions of the present invention due to unnecessary details, only technical solutions and / or processing steps closely related to the solutions of the present invention are shown in the embodiments, and other details that are not very relevant are omitted.

[0022] Example 1: This example provides a barley stripe disease pathogenicity gene Pg00778 , Pg00778 The full-length transcript sequence is shown in SEQ ID NO: 1 and Figure 8 As shown, the CDS region is 51-623 bp.

[0023] Example 2: This example provides a barley stripe disease pathogenicity gene Pg00778 Acquisition methods include: (1) Cultivation of barley stripe disease strains Weigh 200 g of fresh potatoes, peel them, remove the eyes, and cut them into 2 cm pieces. 2 1.2 L of distilled water was added to the small cubes and boiled for 20 minutes. The residue was filtered to remove the residue. 20 g of glucose and 18 g of agar were added to the filtrate. Stir with a glass rod to mix thoroughly. The volume was then brought to 1 L with distilled water. Autoclave at 121°C for 20 minutes before use (preparation of PDA medium). Barley stripe pathogen strain QWC, preserved in paraffin oil, was streaked onto PDA medium. After 3 days, a 5-10 mm diameter borer was used to punch the edge of the plate to obtain a cake. The cake was then plated onto a PDA medium plate and grown for 7 days. The plate was then stored at 4°C until use.

[0024] (2) Pg00778 Gene cloning Cloning using QWC genomic cDNA as template Pg00778 Gene, using primers Pg00778 -F / Pg00778 -R (with Figure 1 ) was obtained by PCR amplification Pg00778 The amplified product was purified and ligated into the cloning vector pMD19-T Vector: 1 μL of pMD19-T Vector (Simple), 4 μL of the target fragment, and 5 μL of Solution I. The ligation was incubated in a 16°C water bath overnight and then transformed into Escherichia coli DH5α competent cells. Single colonies were picked for PCR amplification and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The bacterial solution suitable for sequencing was preserved.

[0025] The PCR amplification system and reaction procedures are as follows: System: 1 μL template, 1 μL each of upstream and downstream primers (10 μM), 25 μL of high-fidelity Premix Taq enzyme, and ddH2O to 50 μL. Reaction procedure: initial denaturation: 95°C, 5 min; denaturation: 94°C, 40 s; annealing: 56°C, 40 s; extension: 72°C, 1 min; final extension: 10 min; store at 4°C. After amplification, products were detected by 1% agarose gel electrophoresis.

[0026] (3) Pg00778 Gene sequence and bioinformatics analysis The sequencing results were found using NCBI ORF finder and sequence translation was performed. The primary structure of the corresponding protein was predicted, including amino acid composition, physicochemical properties, etc. The online websites NetPhos3.1 Server, SOPMA, ProtScale, and ProtParam were used to predict protein phosphorylation sites, secondary structure, hydrophilicity, and physicochemical properties. The results showed that the RNA of the wild strain QWC was extracted and reverse transcribed into cDNA. After PCR amplification, ligation to the pMD19-TVector vector, and transformation into E. coli competent cells, sequencing was obtained. Pg00778 The cDNA sequence of the gene was successfully cloned into Pg00778 Gene. Bioinformatics analysis results showed that: according to ORFfinder software analysis Pg00778 The full length of the gene is 573 bp, and the ExPASy software translated Pg00778 Encoding 190 amino acids, the predicted physicochemical properties are as follows: Pg00778 The isoelectric point of the encoded protein is approximately 9.02, the protein molecular weight Mw is 21482.36 Da, the total number of atoms is 5544, and the molecular formula is C 950 H 1498 N 264 O 290 S7, the aliphatic index (Aliphatic index) is 77.58, the instability index (Instability index, II) is 33.70, and the protein is stable. Among the amino acids, alanine (Ala) and threonine (Thr) account for the largest proportion, both 8.9%, followed by asparagine (Asn) and leucine (Leu) accounting for 7.4%; the total number of positively charged residues (Arg + Lys) is 22, and the total number of negatively charged residues (Asp + Glu) is 18; the average hydrophilicity (GRAVY) is -0.466. The secondary structure of the protein shows that random coil (Cc) accounts for the largest proportion of 53.68%, followed by alpha helix (Hh), accounting for 42.63%, and the least extended chain (Ee), accounting for 3.68% ( Figure 2 A); Hydrophilicity analysis showed that the protein encoded by Pg00778 is a hydrophilic protein ( Figure 2 B); The phosphorylation sites showed that the protein encoded by the Pg00778 gene had serine (Ser), threonine (Thr) and tyrosine (Tyr) phosphorylation sites (see Appendix Figure 2 C).

[0027] Example 3: This example provides a barley stripe disease pathogenicity gene Pg00778 A vector construction method comprising: The CDS sequence of the gene was obtained through RNA-seq sequencing results, and the interference vector pSilent-1 was used according to the multiple cloning site on the vector map: Pg00778 Construction selection Kpn I and Bgl II. Xho I and Hin d III restriction site, pBARGPE1-Hyg: Pg00778 Construction and selection of overexpression vectors Bam HI and Eco RI restriction enzyme cutting site (Table 1). The target sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis.

[0028] The results showed that the target gene was amplified using the positive clone vector plasmid as a template using specific primers. The amplified products were recovered using the Tiangen Universal DNA Agarose Gel Extraction Kit (DP214, Tiangen, China). The target gene and expression vector were double-digested at 37°C. The target gene was then ligated to the expression vector using T4 DNA ligase at 16°C, completing the construction of the vector. Figure 3 ).

[0029] Table 1 Primer sequences

[0030] Example 4: This example provides a barley stripe disease pathogenicity gene Pg00778 Genetic transformation methods include: (1) Preparation of protoplasts Collect wild-type QWC mycelia by filtration using a sterile funnel, wash three times with 0.9 mol / L NaCl, and blot dry with sterile filter paper. Weigh 0.1 g of mycelia and place them in 10 mL of enzymatic hydrolysis solution (10 mg / mL lytic enzyme + 20 mg / mL cellulase R-10, prepared with 1.2 mol / L NaCl as a stabilizer). Incubate at 30°C and 80 rpm for 3-4 hours. Filter through sterile glass paper and collect the filtrate into a centrifuge tube. Centrifuge at 1996 rpm for 5 minutes and discard the filtrate. Wash twice with 300 μL of 1.2 mol / L NaCl, centrifuge, and discard the waste solution. Finally, resuspend the mycelia in NTC solution for storage.

[0031] (2) PEG-CaCl2-mediated genetic transformation Absorb pSilent-1: Pg00778, pBARGPE1-Hyg: Pg00778The plasmid (1-10 μg) was mixed with 100 μL NTC resuspended pellet and immediately cultured on ice for 20 min. 100 μL PEG4000 solution was slowly added, mixed and cultured on ice for another 20 min. 800 μL PEG4000 was added and cultured on ice for another 20 min. 500 μL of the mixture was added and evenly spread on the surface of the rPDA culture medium. After the liquid dried naturally, the culture dish was sealed with sealing film and then cultured in a 25°C constant temperature incubator until independent single colonies were formed. Single colonies were selected and cultured on Hyg B-PDA plates for three consecutive subcultures.

[0032] The results show that: through Hyg The transformants were verified by PCR using specific primers of B, and two interfering strains were screened out ( Pg00778-RNAi-3 and Pg00778-RNAi-7 )( Figure 4 A) and three overexpression strains ( Pg00778-OE-D 、 Pg00778- OE-E and Pg00778-OE-I )( Figure 4 B). q RT-PCR showed that the relative expression levels of the two interfering strains were 58.36% and 46.03%, respectively, which were reduced by 41.64% and 53.97% compared with the control strain ( P <0.05), which indicates that the expression of the interference strain was effectively inhibited ( Figure 5 A). The relative expression levels of the three overexpression transformants were 389.98%, 497.39% and 363.65%, respectively, which were increased by 289.98%, 397.39% and 263.65%, respectively ( P <0.05) ( Figure 5 B).

[0033] Example 5: This example provides a barley stripe disease pathogenicity gene Pg00778 Applications include: (1) Identification of growth and differentiation of mutant strains Two interference transformed strains and three overexpression transformed strains were inoculated on PDA medium and grown for 7 days. Their growth status was counted, and the wild strain QWC was used as the control. Three replicates were set for each strain.

[0034] The results showed that the strains were inoculated on PDA plates and found that both the wild strain and the transformed strain of barley streak pathogen grew as white, flat and compact fuzzy colonies on PDA medium ( Figure 6A). The colony diameters of each strain were measured on the 7th day, and the colony diameters of the interfering strains were found to be 4.80 cm and 5.40 cm, respectively, which were 30.13% and 21.40% lower than those of the control (6.87 cm). P <0.05). The colony diameters of the overexpression strains were significantly larger than those of the wild-type strain, at 7.73 cm, 7.74 cm, and 7.37 cm, respectively, which were 12.52%, 12.66%, and 7.28% higher than those of the control (6.87 cm). P <0.05) ( Figure 6 B). The above results show that Pg00778 Genes related to hyphal growth of barley streak pathogen.

[0035] (2) Identification of pathogenicity of mutant strains Two interference-transformed strains, three overexpression-transformed strains, and the wild-type strain QWC were inoculated onto PDA medium for 7 days before artificial inoculation pathogenicity testing using the "sandwich method." Plump barley cultivar Alexis seeds were placed in a sterilized Petri dish (9 cm diameter). An appropriate amount of sterile water was added, shaken for 30 seconds, and then the sterile water was discarded. 70% anhydrous ethanol was added, shaken for 30 seconds, and then the ethanol was discarded. The dish was rinsed 3-5 times with sterile water, then soaked in 5% sodium hypochlorite for 5 minutes. After discarding the sodium hypochlorite, the seeds were rinsed 3-5 times with sterile water. The seeds were then spread flat on sterile filter paper and allowed to dry. Three replicates were set up, using blank PDA medium as a control. Seeds were inoculated in a dark incubator at 6°C for 20 days and then planted in pots of the same size under a 12 h light / 12 h dark cycle at 20°C light / 18°C dark conditions. After 20 days of growth, the incidence of the disease was counted and photographed. Three biological replicates were performed for each strain, and the wild-type strain QWC was used as a control.

[0036] The results showed that after the wild strain QWC, two interference strains and three overexpression strains infected the highly susceptible barley variety Alexis, the incidence of the two interference strains was 35.33% and 37.50%, respectively, which was 52.91% and 50.01% lower than that of the wild strain QWC (75.02%). P <0.05); the incidence rates of the three overexpression strains were 89.26%, 90.24%, and 90.91%, respectively, which were 18.98%, 20.29%, and 21.18% higher than those of the wild-type QWC (75.02%). P <0.05) ( Figure 7 D).

[0037] When the strains infected the barley variety Alexis, the QWC strain infected barley leaves shrank significantly, with large areas of lesions on the leaves, and the disease was more serious. The barley infected with the interference strain grew better overall, with reduced lesion areas on the leaves and significantly weakened symptoms. However, the lesion area on the leaves of the overexpression transformant increased, and the symptoms were significantly enhanced ( Figure 7 A). Trypan blue and DAB staining revealed that the degree of cell death and reactive oxygen species (ROS) content in the leaves of the overexpression transformant were significantly stronger than those of the QWC and interference transformants, while the degree of cell death and ROS content in the leaves of the interference transformant were lower than those of the QWC and overexpression transformants ( Figure 7 B); Chlorophyll content analysis found that the relative chlorophyll content of the interference transformant was higher than that of the wild strain QWC, and the relative chlorophyll content of the overexpression transformant was lower than that of the wild strain QWC ( Figure 7 C). In summary, barley streak pathogen strains Pg00778 The gene is related to pathogenicity and is involved in the pathogenicity of Pseudomonas aeruginosa strains.

[0038] The above is only a specific implementation method of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

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Claims

1. Barley stripe disease pathogenicity gene Pg00778 , characterized in that, described Pg00778 The full-length sequence of the transcript is shown in SEQ ID NO: 1, wherein the CDS region is 51-623 bp.

2. Barley stripe disease pathogenicity gene Pg00778 The acquisition method is characterized in that The method comprises: (1) culturing a wild strain of barley stripe disease QWC; (2) Pg00778 Gene cloning; (3) Pg00778 Bioinformatics analysis of genes.

3. Barley stripe disease pathogenicity gene Pg00778 The vector construction method is characterized in that: The method comprises: obtaining by RNA-seq sequencing results Pg00778 The CDS sequence of the gene is based on the multiple cloning site on the vector map and interferes with the vector pSilent-1: Pg00778 Construction selection Kpn I and Bgl II. Xho I and Hin d III restriction site, pBARGPE1-Hyg: Pg00778 Construction and selection of overexpression vectors Bam HI and Eco RI restriction site, and the target sequence was synthesized.

4. Barley stripe disease pathogenicity gene Pg00778 The genetic transformation method is characterized in that The method comprises: (1) preparing wild strain QWC protoplasts; (2) PEG-CaCl2-mediated genetic transformation; and (3) screening and identification of transformed strains.

5. Interference with the pathogenicity gene of barley stripe disease Pg00778 The application of expression in regulating the nutritional growth of a strain is characterized in that described Pg00778 The full-length sequence of the transcript is shown in SEQ ID NO: 1, wherein the CDS region is 51-623 bp.

6. Interference with the pathogenicity gene of barley stripe disease Pg00778 The use of expression in reducing the pathogenicity of the fungus Pyricularia truncatula is characterized in that, described Pg00778 The full-length sequence of the transcript is shown in SEQ ID NO: 1, wherein the CDS region is 51-623 bp.

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