ErLAC12 gene of pythium aphanidermatum and application thereof

Abstract

The application discloses a pythium curvatum ErLAC12 gene and application thereof. The cDNA sequence of the pythium curvatum ErLAC12 gene is shown as SEQ ID NO. 1. The ErLAC12 gene provided by the application can reduce the ROS level of weeds, and has a pathogenic effect on leafflower weeds, and can effectively prevent and control weeds, especially leafflower weeds.

Description

TECHNICAL FIELD

[0001] The present application relates to lactose permease gene and its application, and particularly to Conidiobolus brefeldianus ErLAC12 gene and its application. BACKGROUND

[0002] Lactose permease is a transmembrane protein with six transmembrane helices symmetrically distributed within the N- and C-terminal domains (shown by blue and red hemispheres in the model). Lactose is actively transported through the protein by six side chains, each of which is essential. Throughout the transport process, histidine 322, glutamate 325, and arginine 302 are important for proton transport or H+ proton transport. Lactose permease is a member of the "major facilitator" family and is a transmembrane protein that facilitates the transport of lactose across the phospholipid bilayer of the cell membrane. The active co-transport mechanism that drives this system is an inwardly directed H+ electrochemical gradient. Therefore, when lactose moves from the periplasm to the cytoplasm, a H+ proton moves with it. When lactose is consumed and absorbed by the cell, lactase breaks down the disaccharide into monosaccharide subunits. These are then further broken down into energy required by the cell during cellular respiration.

[0003] The LAC12 gene of lactose permease has specificity for lactose metabolism and is co-regulated by the same bidirectional promoter and four Gal4p binding sites upstream of the gene. In addition, the production and regulation of the enzyme also largely depends on the ability of the fungus to transport a variety of carbohydrates through transmembrane proteins called sugar transporters (STs). The LAC12 gene encodes a lactose permease that is involved in the intracellular hydrolysis of lactose into glucose and galactose, which is then metabolized through the glycolytic pathway. The LAC12 gene is a member of the major facilitator superfamily of proteins and functions through a proton symporter mechanism. It is saturable at high substrate concentrations and has also been reported to transport galactose. Therefore, it is considered to be a high-affinity lactose transporter in Klebsiella lactis. The LAC12 gene is transcribed from a promoter that contains key regulatory elements. This 2.8 kb intergenic region of LAC12 contains several binding sites for Lac9p, a well-studied homolog of the transcriptional activator Gal4p in S. cerevisiae. Therefore, the LAC12 gene is strongly induced by lactose in the growth medium, but can be inhibited by the presence of glucose. SUMMARY

[0004] One of the purposes of the present application is to provide Conidiobolus brefeldianus ErLAC12 gene.

[0005] Specifically, the cDNA sequence of the B. mucosa ErLAC12 gene provided in the present application is shown in SEQ ID NO. 1. The ErLAC12 gene provided in the present application can reduce the ROS level of weeds, and has a pathogenic effect on lespedeza, and can effectively prevent and control weeds, especially lespedeza.

[0006] The recombinant plasmid pCAMBIA2300-GFP-LAC12 containing the B. mucosa ErLAC12 gene.

[0007] The recombinant plasmid pCAMBIA-LAC12 containing the B. mucosa ErLAC12 gene.

[0008] The present application provides a B. mucosa ErLAC12 gene.

[0009] Specifically, the B. mucosa ErLAC12 gene is used in weed control, and is used in lawn weed control, and is used in lawn lespedeza control, and is used as a weed control agent, and is used as a lespedeza control agent. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 The LAC12 gene of B. mucosa is amplified.

[0011] Figure 2 The phylogenetic tree of the ErLAC12 family members is constructed by using MEGA 11 software for phylogenetic analysis of ExLAC12.

[0012] Figure 3 The LAC12 gene of strain Y9511-XO50 is amplified.

[0013] Figure 4 ptopo-blunt vector map.

[0014] Figure 5 LAC12 gene successfully transformed into ptopo-blunt end vector.

[0015] Figure 6 pCAMBIA restriction enzyme map for transforming LAC12 gene into E. coli

[0016] Note: LAC12 gene sequence was inserted between the promoter and OCS region. GFP was removed to create a GFP-free vector. This plasmid also contains a kanamycin resistance marker for selection in both fungi and bacteria. The plasmid structure was drawn using vector advance NTI 11.5 (2003) software.

[0017] Figure 7 Restriction enzyme map of pCAMBIA-GFP for transformation of LAC12 gene into E. coli.

[0018] Note: LAC12 gene sequence was inserted between the left and right T-border. This plasmid also contains a kanamycin resistance marker for selection in both fungi and bacteria. The plasmid structure was drawn using vector advance NTI 11.5 (2003) software.

[0019] Figure 8 Transformation of LAC12 gene into E. coli Dh5a.

[0020] Figure 9 Transformation of LAC12 gene into Agrobacterium tumefaciens GV3101,

[0021] Note: Band size 1720 bp shows successful transformation of LAC12 gene.

[0022] Figure 10 Differences in morphological characteristics of wild type Y9511, mutant Y9511-X050 and ErLAC12 gene overexpression strain

[0023] Note: Colony characteristics of wild type Y9511, mutant Y9511-X050 and ErLAC12 gene overexpression strain on PDA medium for 4 days and 7 days. *, ** and *** represent significant differences at P<0.05, P<0.01 and P<0.001 levels, respectively.

[0024] Figure 11 Subcellular localization of ErLAC12 gene ErLAC12-GFP and the original strain as a control transiently expressed in Exserohilum rostratum.

[0025] Figure 12 Subcellular localization of ErLAC12 gene ErLAC12-GFP and the original strain as a control transiently expressed in Exserohilum rostratum.

[0026] Figure 13 SEM analysis of fungal mycelium surface morphology,

[0027] Note: ErLAC12 showed high cell wall damage and prominent extracellular accumulation of growth. There were significant differences in the number of mycelial cells and cell damage among the three strains.

[0028] Figure 14 To determine the significant differences between the surface morphology of wild type Y9511, mutant Y9511-X050 and ErLAC12 overexpression strain

[0029] Note: (A) represents the surface morphology of S, (B) represents the diameter of D.

[0030] Figure 15 To determine the differences in pathogenicity of ErLAC12 overexpression strain, wild type Y9511 and mutant Y9511-X050 to Leptochloa chinensis

[0031] Note: (A). Control efficacy of 3 strains on Leptochloa chinensis at 3 leaf stage (D, E) Fresh weight control effect (B, C). All values indicate significant differences at P<0.01 and P<0.001 levels, respectively.

[0032] Figure 16 To determine the differences in pathogenicity of ErLAC12 overexpression strain, wild type and mutant to Leptochloa chinensis

[0033] Note: (A). Control efficacy of 3 strains on Leptochloa chinensis at 4 leaf stage (D, E) Fresh weight control effect (B, C). All values indicate significant differences at P<0.01 and P<0.001 levels, respectively.

[0034] Figure 17 Flow chart for DNS method to determine PG, PMG, CX, βG.

[0035] Figure 18 CWDEs activity of ErLAC12 overexpression strain, wild type Y9511 and mutant Y9511-X050

[0036] Note: (A) CWDEs activity of ErLAC12 overexpression strain, wild type Y9511 and mutant Y9511-X050 on PDA medium (B) Isolated L. chinensis leaves. PG, polygalacturonase; PMG, polymethylgalacturonase; CX, cellulase; βG, β-glucuronidase.

[0037] Figure 19 ROS activity of ErLAC12 overexpression strain, wild type Y9511 and mutant Y9511-X050

[0038] Figure 20 Cellular observation of the effects of L. brevis on ErLAC12 gene overexpression strain, wild type Y9511 and mutant Y9511-X050;

[0039] Note: (A) control, (B) wild type Y9511, (C) mutant Y9511-X050 and (D) leaves treated by ErLAC12 gene overexpression strain. CW: cell wall, M: mitochondria, V: vacoule, Ch: chloroplast, PG: plasmagel, AS: airspace.

[0040] Figure 21 Effects of different treatments on the morphology of L. brevis;

[0041] Note: All values represent significant differences at P<0.05 and P<0.001 levels, respectively.

[0042] Figure 22 Effects of different treatments on the SPAD and antioxidant activity of L. brevis;

[0043] Note: All values represent significant differences at P<0.05 and P<0.001 levels. DETAILED DESCRIPTION

[0044] The application will be further described below in combination with specific examples.

[0045] 1. Plant and fungal strain sample collection

[0046] The mutagenic strain Exserohilum rostratum Y9511-X050 (Exserohilum rostratum Y9511-X050, hereinafter referred to as mutant Y9511-X050) was isolated and preserved by the Weed Laboratory of South China Agricultural University. The weed plant Leptochola chinensis was collected at the West China Teaching Practice Base of South China Agricultural University in Zengcheng District, Guangzhou, Guangdong Province. The seeds of the grass family plant were dried and stored in a cold environment. The indoor cultivation conditions were 26±1℃, relative humidity 50-75%, and light cycle 12L:12D.

[0047] The Exserohilum rostratum ErLAC12 gene overexpression strain (Exserohilum rostratum X50-ErLAC12-GFP) provided by the application has been preserved in the Guangdong Microbial Culture Collection Center on May 10, 2024, with the preservation number GDMCC No: 64602 and the preservation address being No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard.

[0048] (1) Preparation of culture medium

[0049] Modified Potato Dextrose Medium (Modified PDA): Potato Dextrose Agar 40.1 g, Agar 8 g, dissolved in 1 liter of water, autoclaved for 30 minutes.

[0050] (2) Preparation of fungal mycelium

[0051] The small mycelium plugs in the culture medium were transferred aseptically into Petri dishes containing the above modified PDA. The Petri dishes were incubated for 1-2 days (25°C, 12 hours / 12 hours light / dark, 35±5 μE / m 2 / s) until full growth of the colonies was observed. Wild type Y9511 and mutant Y9511-X050 were incubated on modified PDA for 14 days, respectively, washed from the Petri dishes with distilled water, and then filtered the mycelium with four layers of cheese cloth to obtain the mycelium. The bacteria were collected and the supernatant was discarded.

[0052] (3) Fungal RNA extraction

[0053] RNA isolation was performed by using Spin-column Fungal Total RNA Purification Kit (REF# B518659-0050) following the protocol below; the 14-day old fungal PDA plates were washed with sterile water and filtered through four layers of boletus cloth. The filtrate was discarded and the fungal mycelium was collected and RNA was extracted by the following steps. 20 mg of fungal mycelium was weighed and ground in liquid nitrogen and added to a 1.5 ml sterile centrifuge tube. 450 ul of Buffer Lysis FG shake vial was added, mixed well, and incubated at room temperature for 5 minutes. Centrifuged at 12000 rpm for 30 min at 4°C and the supernatant was transferred to a 1.5 ml RNase-free centrifuge tube. 1 / 2 volume of absolute ethanol was added and mixed well. The spin column was placed in a 1.5 RNase-free centrifuge tube, 40 ul of DEPC-treated H2O was added to the center of the adsorption membrane, incubated for 2 min, and centrifuged at 12000 rpm for 2 min. The RNA solution was stored at 80°C for further experiments. The concentration of RNA was measured by NanoDrop TM 2000.

[0054] 2. Preparation of first strand cDNA from RNA

[0055] 2.1 Method

[0056] First strand cDNA was prepared from RNA of wild type Y9511 and mutant Y9511-X050 (Hifairlll first strand cDNA synthesis CAT: 11141 ES60); Digestion solution was prepared in RNase-free centrifuge tube, RNase-free H2O 15ul + 5x gDNA digester mix 3ul, RNA 0.1 ud was introduced into RNase-free centrifuge tube, and it was mixed thoroughly by blowing with forceps, and then incubated at 40°C for 2 minutes. After blowing with a syringe and mixing, it was placed in a thermal cycler at 40°C for 2 minutes. Then 5ul 4x Hairpin III super mix plus was added directly into the tube, mixed gently with forceps, and then placed in a metal water bath. The reverse transcription system was as follows: 25°C for 5 minutes, 55°C for 15 minutes, and 85°C for 5 minutes. The cDNA was stored at -20°C for later use.

[0057] 2.2 Results

[0058] The concentrations of genomic RNA and cDNA extracted from wild type Y9511 and mutant Y9511-X050 were measured by NanoDrop. The concentration was 110 ng / μL, and the A260 / 280 ratio was 1.82. First strand cDNA was synthesized from RNA. Therefore, the quality and concentration of the purified cDNA met the requirements for PCR in gene amplification.

[0059] 3. Amplification and confirmation of lactase permease (LAC12)

[0060] 3.1 Method

[0061] RNA primers (Table 1) were synthesized by Snap gene (4.1.9) and Oligo.7 software for gene identification and amplification primer sequences. The PCR mixture was prepared with 2x Taq PCR mix 12.5ul, cDNA 2ul, primers F+R (1+1), ddH2O, and the total volume was 25ul. The primers for gene identification and amplification were sequenced according to the following PCR curve (Table 2). The PCR amplification products were electrophoresed in 1% agarose gel and photographed with an ultraviolet photographic system. Under ultraviolet irradiation, the corresponding band was cut according to the size of the gene of interest and placed in a 1.5 mL centrifuge tube. The universal DNA purification kit (Tangen Biotech (Beijing) Co., Ltd. Cat. # DP214-03) was used for recovery and purification. And it was sent to the company for sequencing.

[0062] Table 1 Primer List

[0063]

[0064] Table 2 PCR profile

[0065]

[0066]

[0067] Gel purification procedure:

[0068] Add 250BL activated silica gel membrane into the adsorption membrane, centrifuge at 12000xg for 1 minute. Discard the waste liquid. Cut the target DNA band from the gel under the ultraviolet lamp. Put the gel containing the target band into a 2ml sterile centrifuge tube, add 500ul buffer GL. Heat at 65℃ for 10 minutes to completely melt the gel. After cooling at room temperature, transfer the solution to the adsorption column, centrifuge at 12000xg for 1 minute, discard the waste liquid, and put the adsorption column back into the collection tube. Add 700ul buffer W2 to the adsorption column, stand at room temperature for 1 minute, repeat the operation. Centrifuge at 12000xg for 2 minutes. Put the adsorption column into a clean 1.5ml centrifuge tube, dry at room temperature for 2 minutes. Add 35-50 eluent to the middle of the adsorption column, incubate at room temperature for 2 minutes. Centrifuge at 12000xg for 2 minutes, then send to the company for sequencing to obtain the cDNA sequence of LAC12 of the target Pythium aphanidermatum of the application.

[0069] Sequencing alignment prediction:

[0070] The sequence alignment was performed using Snap gene (4.1.9) and BIOXM 2.7.1 software to compare the sequencing results of LAC12 prepared by mutant Y9511-X050 with the target LAC12 gene sequence.

[0071] 3.2 Results

[0072] The amplification of LAC12 was completed by using specific gene primers under the PCR conditions optimized in the above point 2. The DNA bands in the gel were observed by gel electrophoresis under ultraviolet light, and the specific band size of 1720bp showed the amplification and confirmation of LAC12 gene Figure 1 ).

[0073] The amplification and confirmation of LAC12 gene of the first strand cDNA synthesized by RNA of mutant Y9511-X050 are shown in Figure 3 , which is consistent with Figure 1 results, with a specific band of 1720bp, and the gene amplification is confirmed by PCR and sequencing results.

[0074] The cDNA sequence of the target LAC12 of the application:

[0075]

[0076] 4. Construction of phylogenetic tree and protein sequence alignment

[0077] Evolutionary history was inferred using the neighbor-joining method. The percentages of replicate trees for which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances were computed using the maximum composite likelihood method, and are in the units of base substitutions per site. The analysis involved 60 nucleotide sequences. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). A total of 1603 positions were in the final dataset. Evolutionary analyses were conducted in MEGA11.

[0078] Based on the whole genome sequence of P. oryzae, the inventors performed transcriptome analysis to identify functional DEGs. The cDNA sequence of P. oryzae consists of 34322-bp and 12782 bp protein-coding genes. To identify the subfamily of LAC12 enzyme subunits, a phylogenetic analysis was constructed using the molecular evolutionary genetics analysis (MEGA) software version 11 using the neighbor-joining method (NJ) based on 1000 replicates. Figure 2 ) Evolutionary distances were computed using the maximum composite likelihood method, and are in the units of base substitutions per site. The analysis involved 60 nucleotide sequences. All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). A total of 1603 positions were in the final dataset.

[0079] 5. Cloning and transformation of LAC12 gene

[0080] The mutant Y9511-X050 cDNA was stored at -20°C. The EZ pTOPOII cloning kit was purchased from www.weidibio.com. pCAMBIA2300-35S-OCS was constructed and recombined. Commonly used enzymes and biochemical reagents (Table 3).

[0081] Table 3 Commonly used enzymes and biochemical reagents

[0082]

[0083] Strains and vectors: *E. coli* strain Dh5α was obtained from the laboratory of Joerg Kudla, University of Münster, Germany. *Agrobacterium* strain GV3101 (CAT#AC 1001S) was purchased from www.weidibio.com. The binary expression vector pCambia2300-35S-OCS was stored in our laboratory. A list of the culture media used in this experiment is shown in Tables 4 and 5. The pH was adjusted to 7.0. The culture was autoclaved at 121°C for 15 minutes; solid culture medium containing 15g of agar powder was added before sterilization.

[0084] Table 4 Preparation of standard culture medium and LB medium (1L)

[0085]

[0086] Table 5 YEP medium (1L)

[0087]

[0088] Autoclave at 121°C for 15 minutes; add solid culture medium containing 15g AGAR powder before sterilization.

[0089] IM Solid Culture Media: The stock solutions for each induction medium are formulated as follows: (a) 1.25M K₂HPO₄ (pH 4.8): First, prepare 1.25M K₂HPO₄ and 1.25M KH₂PO₄ separately, then adjust the pH of KH₂PO₄ to 4.8 with K₂HPO₄. 28.5g K₂HPO₄, 100ml; 17g KH₂PO₄, 100ml. (b) MN Buffer (100ml): MgSO₄·7H₂O 3g, NaCl 1.5g, volume 100ml. (c) 0.01% FeSO₄ (100ml): MgSO₄·7H₂O 0.01g, add water to 100ml. (d) 1% CaCl₂… l250ml, CaCL2.2H2O 0.5g add water to 50ml. (e) Spore element (100ml), ZnSO4.7H2O: 0.01g, CuSO4.5H2O 0.01g, H3BO3 0.01g, MnSO4.H2O: 0.01g, Na2MoO4.2H2O: 0.01g, add the above reagents one by one and dissolve, then add water to 100ml. (f) 20% NH4NO3 (100ml): NH4NO3 20g, add water to 100ml. (g) 20% glucose: 20g glucose dissolved in water, add water to 100ml. (h) 1M MES (pH 5.5), MES (MW: 213.25): 21.32g dissolved in water to 100ml, adjust pH to 5.5 with NaOH. Add water to the corresponding volume, add 15g / L agar powder, autoclave. Add acetosyringone (AS) before plating, final concentration 200μmol / L. Add water to the corresponding volume and autoclave. Add AS before use, final concentration 200μM.

[0090] 5.1 Transformation of LAC12 target gene by EZ pTOPOII cloning kit

[0091] The lactase permease LAC12 gene was transformed into the EZ pTOPOII blunt vector by the following steps to obtain the ligation product. The competent cells were quickly removed and placed on ice until thawed. The reaction system was prepared according to (Table 6) at room temperature, stirred with a centrifuge tube finger, and then incubated at 28-37°C for 5 minutes.

[0092] Table 6 Ligation mixture ratio

[0093]

[0094] 5ul of the ligation product was added to 50μl of competent cells. Mix evenly with a finger. Ice bath for 10-30 minutes. Heat at 42°C for 60 seconds. Ice bath for 2 minutes. Add 800μl of LB medium and incubate at 37°C, 225rpm on a shaker for 1 hour. Centrifuge at 5000rpm for 2 minutes, discard the supernatant, and leave 100ul of bacterial solution. Resuspend the particles with the bacterial solution and evenly spread on YLB solid medium containing kanamycin. Invert and incubate in a 28°C constant temperature incubator for 12-16 hours.

[0095] Screen and identify the transformants. Colony PCR identification was performed using the following PCR mixture: ex-taq green mixture 5ul, M13 primer mixture 2ul, ddH2O 3ul. Use a toothpick to pick a colony from the LB medium plate and mix it into the above mixture. Positive colonies were placed in LB liquid medium and incubated in a 37°C, 225rpm shaking incubator for 12-16 hours.

[0096] LAC12 gene was cloned and transformed into universal topo blunt vector Figure 4 ). 10 colonies were randomly picked from LB (kanaR) culture plate. Positive colonies were determined by colony PCR using M13 universal primers according to optimized PCR conditions. PCR and sequencing results showed that LAC12 gene was successfully transformed and cloned into topo vector Figure 5

[0097] 5.2 Gene transformation using the desire vector pCAMBIA2300-35S-OCS

[0098] To transform Exserohilum rostratum fungus, a vector was designed to insert the gene of interest between the left and right borders of T-DNA, and A. tumefaciens was used as a vector to integrate the gene of interest into the fungal genome. The design and synthesis of primers were completed according to the nucleic acid sequence of the template, and the primers were designed using Snapgene software (Table 7). The synthesized primers were prepared into a 100 pmol / μl stock solution with sterilized ddH2O and stored in a -20°C refrigerator, and the working solution concentration was 10 pmol / μl.

[0099] The pCAMBIA2300-35S-OCS vector was stored in glycerol bacteria and was in an inactive state. First, it was activated by the following method.

[0100] Table 7 Vector construction and primer information used

[0101]

[0102]

[0103] (I) Plasmid DNA extraction

[0104] ​LB (kanamycin) plate stripe to activate pCAMBIA-2300. Pick single colony and put into liquid LB (kanamycin) medium, shake at 37°C, 220 rpm overnight. Use plasmid extraction kit to extract plasmid according to the following steps. Put the adsorption column into the collection tube and add 250ul buffer BL to activate silica gel membrane. Centrifuge at 12000 rpm for 1 minute, discard the waste liquid. Take 1-4ml bacterial culture overnight, centrifuge at 12000 rpm for 1 minute, collect bacteria and try to remove the supernatant. Add 250ul buffer PA to resuspend the bacterial pellet, vortex until there is no obvious bacterial mass; add 250ul buffer PB, gently up and down 6-8 times. Add 350ul buffer PC, gently up and down 6-8 times, mix well, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, put the adsorption column back into the empty collection tube. Add 500ul buffer PWA to the adsorption column, centrifuge at 12000 rpm for 1 minute, discard the waste liquid. Add 600ul buffer PWB to the adsorption column, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, repeat the operation. Put the adsorption column back into the collection tube, centrifuge at 12000 rpm for 2 minutes. Take out the adsorption column, stand at 20-25 degrees Celsius for 2 minutes to evaporate the residual ethanol. Add 35-50ul elution buffer (65°C preheated) to the middle of the adsorption column. Incubate at room temperature for 2 minutes. Centrifuge at 12000 rpm for 2 minutes and store at -20 degrees Celsius for later use. Double digestion of LAC12 gene and pCAMBIA vector. The operation steps of the enzyme system are as follows (Table 8).

[0105] Table 8 Double digestion mixture optimization concentration

[0106]

[0107] The above enzyme system was reacted overnight in a metal bath at 37°C, and the enzyme digestion product was recovered and purified by gel purification kit.

[0108] (II) Ligation of target gene and vector, as shown in the following Table 9.

[0109] Table 9 Ligation product ratio

[0110]

[0111] After agarose gel electrophoresis of the corresponding size bands, the recovered product was added according to the above system and reacted in a metal bath at 16°C overnight.

[0112] Positive colonies were confirmed by colony PCR. Single positive colony was picked from LB plate and put into LB liquid medium, shaken at 37°C, 225 rpm overnight, plasmid was extracted and then sent to the company for sequencing. Positive colonies were confirmed by PCR and sequencing.

[0113] Two vectors were successfully constructed, 1) pCAMBIA2300-35S-OCS. The MCS region of pCAMBIA2300-35S-OCS was digested with BamHI and speI, and the 1720 bp lactose permease gene was inserted. The LAC12 gene was inserted between the promoter and OCS region of the plasmid, creating the recombinant plasmid pCAMBIA2300-GFP-LAC12 Figure 6 ) to determine the subcellular localization of ErLAC12 in Exserohilum rostratum. 2) To determine the functional analysis of the ErLAC12 gene, a pCAMBIA vector without GFP was constructed. The pCAMBIA2300-35S-OCS vector was digested with BamHI and PstI to remove the GFP nucleotide sequence first, and then the LAC12 gene was inserted between the promoter and OCS region Figure 7 ). The recombinant vector without GFP was used to determine the functional analysis of the LAC12 gene in L. chinensis.

[0114] 5.3 Gene transformation of DH5a E. coli using heat shock method.

[0115] The transformation steps of DH5a E. coli are as follows: Thaw the qualified cells on ice, source: Invitrogen MAXEfficiency DH5a, cat no. 18258012. Take 20 μl of cells per 1.5 mL tube as negative control, positive control, and experiment, respectively.

[0116] 1) Set up control tubes. Negative control: no DNA. Positive control: 1 μL pUC19 DNA (0.01 μg / ml stock). Add 1 μL of new ligation to each DH5a cell tube (experimental tubes). Flick the bottom of the tube with your finger to mix. Freeze for 30 minutes. Heat shock in a 42°C water bath for 45 seconds. Freeze for 2 minutes. Add 900 μL of Luria-Bertani (LB) medium without ampicillin. Incubate at 37°C with 225 rpm shaker for 1 hour. Positive and negative controls: use 100 μL of undiluted sample. Each experimental tube / ligation reaction, experiment uses 100 μL of 10-fold diluted sample, i.e. 100 μL of undiluted sample + 900 μL of LB medium. Plate all tubes containing DH5a under all conditions on LB + Amp (50 μg / mL) plates. Invert the plates (agar side up) and incubate at 37°C overnight. Randomly pick 10 colonies from the LB (kanamycin) plates.

[0117] Two recombinant vectors pCAMBIA2300-GFP-LAC12 and pCAMBIA-LAC12 were successfully transformed into E. coli by heat shock method. In addition, kanamycin selection marker simplified the screening process of positive transformants. The most colonies were cultured in Luria-Bertani (LB) (kanamycin) plate at 37°C for 16 hours. Ten colonies were randomly picked from LB (kanaR) colony plate. Colony PCR was performed using specific primers of gene and sequence combination to confirm positive colonies. Figure 8

[0118] 6. Agrobacterium tumefaciens GV3101 recipient cell transformation (freeze-thaw method)

[0119] GV3101 recipient cells were quickly removed and placed on ice until thawed. 5 μl of the above ligation product was added to 100 μl of qualified cells, mixed well with the fingertips of the centrifuge tube, and then ice-bathed for 30 minutes. Liquid nitrogen was frozen for 1 minute and 37°C metal bath for 5 minutes. Add 500 μl of liquid medium without any antibiotic YEP, and place it on a shaking table at 28°C, 160 rpm for 3 hours. Centrifuge at 5000 rpm for 2 minutes, discard the supernatant, add 50 μL of sterile ddH2O to suspend the bacteria, and evenly spread on YEB solid medium containing the corresponding antibiotic. Inverted culture in a 28°C constant temperature incubator for 2-3 days. The colony PCR identification method is the same as that of E. coli in point 5 above.

[0120] LAC12 gene was successfully transformed into Agrobacterium (GV3101) by freeze-thaw method. The maximum expression of colonies was observed at 24h. Positive colonies were confirmed by colony PCR using specific primers in point 3 above. Figure 9

[0121] 7. Establishment of Agrobacterium tumefaciens-mediated genetic transformation system of Pythium delavense

[0122] ​​Fresh colonies of Agrobacterium tumefaciens GV3101 containing T-DNA binary vectors pCBAMBIA-LAC12 and pCBAMBIA-GFP-LAC12 were grown at 28°C for 24 hours with 220 rpm shaking in YEB liquid medium supplemented with 100 lg / ml kanamycin. Agrobacterium tumefaciens cells were collected by centrifugation and diluted to an optical density of 660 nm (OD660). 1 ml YEB, 1 ul (50 mg / L) was added to 1 ml of liquid induction medium (IM) containing 200 uM. The cells were then induced at 28°C for 6 hours with 220 rpm shaking. After 5 days of growth on potato dextrose agar (PDA) and YEB (agar) at 28°C, the conidia of Agrobacterium tumefaciens GV3101 were collected using sterilized physiological saline and resuspended to 10 5 Conidia / ml in IM liquid medium. The conidial suspension was germinated at 28°C for 0-5 hours. For co-incubation, 100 ul of mutant Y9511-X050 conidial suspension was mixed with an equal volume of Agrobacterium tumefaciens GV3101 cells, spread on the surface of a circular nitrocellulose filter, and placed horizontally on an IM plate containing the liquid IM medium plate component. These cells were co-cultured in the dark at 28°C for 3-5 days, the nitrocellulose membrane was removed, and the transgenic cubes were excised from the IM medium plate and transferred to solid PDA medium supplemented with 100 lg / ml kanamycin to inhibit the growth of Agrobacterium tumefaciens GV3101 cells. Putative transformants were formed within 2-4 days.

[0123] Transgenic strain target gene detection: Fungal genomic DNA was extracted using a fungal DNA extraction kit. The presence of the LAC12 gene in transgenic E. rostratum DNA was determined by PCR using LAC12-specific primers.

[0124] Screening of E. rostratum transformants: Putative transformants were screened on nitrocellulose membranes containing kanamycin (50 mg / L) at 28°C in the dark for 2-6 days, and the surviving / resistant transformants were subcultured for 2 weeks, the peripherally grown mycelium was excised and subcultured under the same conditions. Selective subculturing was repeated twice until the screened transformant culture, i.e., the ErLAC12 gene overexpression strain: strain ErLAC12-GV3101, was obtained.

[0125] 8. Determination of colony growth rate

[0126] A 7 mm diameter punch was used to take a colony cake from the edge of the colony to be tested and placed in the center of a PDA medium, which was then placed in a biochemical incubator at 28°C in the dark. The colony diameter was measured every 24 hours with a vernier caliper. A mycelial block of the same size and age was taken from the edge of the plate and inoculated on a fresh PDA plate with a diameter of 9 cm, which was then incubated at 28°C in the dark. The growth of the colony was observed every day under a microscope and photographed. The colony diameter was measured every day. The plate was slowly rinsed with 10 mL of sterile water using a pipette, then filtered with two layers of gauze and placed in a conical flask. A few drops of spore suspension were taken from the filtrate and placed on a hemocytometer, covered with a cover glass, and counted under a Carl Zeiss microscope (37081) for the number of conidia, repeating the process three times to obtain an average value.

[0127] Microscopic morphological observation results showed that the colony color, growth characteristics, mycelium, spore production, conidial structure, septum, and growth rate of wild-type Y9511, mutant strain Y9511-X050, and strain ErLAC12-GV3101 (hereinafter referred to as strain ErLAC12) were different Figure 10 ) The colony color of strain ErLAC12 was darker than that of the mutant strain on the 4th day after inoculation, and was black, while the wild-type Y9511 strain was brown. Similarly, on the 7th day of culture, the colony of strain ErLAC12 was dense and dark, and the aerial mycelium on the surface of the colony was more than that of the mutant strain, while the number of mycelium of wild-type Y9511 was relatively small. Figure 10 A describes the obvious differences in colony color of wild-type Y9511, strain Y9511-X050, and strain ErLAC12 on PDA medium for 3 days and 7 days. Within 2-4 days after inoculation, the growth rate of strain ErLAC12 was significantly faster than that of strain Y9511-X050, and the growth rate of wild-type Y9511 was slower, but the difference in growth rate between the two strains was small after 5 days. However, after 7 days of inoculation, the sporulation rate of strain ErLAC12 was almost the same as that of mutant strain Y9511-X050 and wild-type Y9511 Figure 10 B), and the colony plate was covered with mycelium. The conidial structure of Exserohilum rostratum strain showed greater variability. The conidial and mycelial septum of wild-type Y9511 was obvious, but the conidial mycelium of mutant strain Y9511-X050 and strain ErLAC12 did not have this structure Figure 10 C, D).

[0128] 9. Determine the GFP for gene subcellular localization

[0129] To determine the subcellular localization of ErLAC12, the pCAMBIA-LA12-GFP fusion vector was constructed, and pCAMBIA-LAC12-GFP was used as a GFP positive control localization vector. LAC12 gene was overexpressed in E. rostratum by ATMT. Microscopic observation found that ErLAC12-GFP was located in the hyphae and sheath of E. rostratum Figures 11-12 ). The fluorescence signal was located in the cell wall, hyphae and conidia of P. parasitica. These results show and confirm that ErLAC12 is located in the hyphae and conidia.

[0130] The subcellular localization of ErLAC12 gene in E. rostratum was verified by overexpression using the pCAMBIA-2300-LAC12-GFP fusion vector. The recombinant vector pCAMBIA-2300-LAC12-GFP and empty vector were transformed into Agrobacterium GV3101 strain by freeze-thaw method. The combined inoculation of fungal cells and bacterial cells was achieved by transgenic. The green fluorescent protein (GFP) signal was detected by ZEISS LSM800 (Germany) microscope.

[0131] 10. Scanning electron microscope detection of morphological differences between wild type Y9511, mutant strain Y9511-X050 and strain ErLAC12

[0132] Sample preparation: The fungal suspension was directly cultured under low speed centrifugation (1500g-2000g), the cells were precipitated, the supernatant was removed, washed with PBS (room temperature) for 1-2 times, and dehydrated by fractional ethanol series. Then stored in a fixed solution (2.5% glutaraldehyde solution) at 4 degrees, suspended by gentle shaking, transferred to 1.5 milliliter decanter for subsequent processing. The sample was loaded into an aluminum sample holder, freeze-dried with Scan-vac cs110-4 freeze dryer (Labogene, Bjarkesvej, Denmark), and coated with a layer of 10 nanometer gold / palladium (60:40). Observed under Hitachi S-3000N scanning electron microscope (Hitachi, Tokyo, Japan). Each treatment has at least three independent biological replicates.

[0133] Morphology quantification was performed using scanning electron microscope. Parameters such as particle distribution, cell damage, exposed area, surface roughness, surface irregularity and surface area were detected. The number of hyphal cells of ErLAC12 strain was the most, and arranged in large quantities. Compared with the mutant strain, the cell damage rate of ErLAC12 was the highest, while that of the wild strain was the lowest. Similarly, strain ErLAC12 showed the accumulation of extracellular growth, while wild type Y9511 and mutant strain Y9511-X050 did not have this structure Figure 13). The cell surface and surface area of strain ErLAC12 also had significant differences. The diameter of strain ErLAC12 was larger than that of the wild strain, and the mycelial cell diameter of wild type Y9511 was the smallest Figure 18 A). Similarly, the cell surface morphology of the three strains also had differences. Compared with mutant strain Y9511-X050, the cell surface of strain ErLAC12 was rougher, while the cell surface roughness of wild type Y9511 was lower Figure 14 B).

[0134] 11. Differences in pathogenicity of wild type mutagenized strain (strain X050) and overexpression of ErLAC12 gene on Lactobacillus (L.)

[0135] 1) Preparation of fungal conidial suspension or inoculum

[0136] After the two strains were cultured on PDA for 14 days, the culture dishes were washed with 0.5‰ (v / v) Tween 80 aqueous solution, and then the mycelium was filtered out with four layers of gauze to obtain a spore suspension. The concentration of the spore suspension was 1.0 x 105 spores / mL (counted with a hemocytometer).

[0137] 2) Measurement of fresh weight and plant control efficiency

[0138] The stems and leaves at the three-leaf to four-leaf stage were sprayed with 20 mL of fungal filtrate using 0.5‰ (v / v) Tween 80 aqueous solution as a control. After the treated P. hysterophorus was moistened for 48 h, it was moved to a greenhouse for growth (28°C, 12L:12D), and each treatment was repeated three times. After 14 d, the plant control efficiency and fresh weight control were investigated. At the time of harvest, the yield parameters fresh weight and the number of surviving plants were recorded. The fresh weight control efficiency (%) and plant control efficiency (%) of each treatment were calculated according to the following formulas (1) and (2), respectively:

[0139]

[0140]

[0141] Where Ew and En represent the fresh weight control effect and the plant control effect, respectively; W0 represents the fresh weight of the control weeds; W1 represents the fresh weight of the treated weeds; N0 represents the number of control weeds; and N1 represents the number of treated weeds.

[0142] Different leaf ages of plants have different resistances to fungi. The fresh weight control effects of strain ErLAC12 and mutant strain Y9511-X050 and wild type Y9511 on P. hysterophorus at the 3-leaf and 4-leaf stages were determined Figure 15 ) The results showed that at an inoculation concentration of 1.0 x 10 5The fresh weight control effect of the strain ErLAC12 fungus on the Chinese cabbage was significantly higher than that of the wild type (P<0.001), reaching 96% at the three-leaf stage, while the mutant strain was 83%, and the wild type Y9511 was relatively reduced to 67%. At the same time, at the four-leaf stage, the control effect of the strain ErLAC12 on the fresh weight of the plant remained at 88%, while the control effect of the fresh weight of the mutant and the wild type was lower Figure 16 ). Therefore, the E. rostratum transposon fungus strain ErLAC12 is considered to be more pathogenic to L. chinensis Nees (L.).

[0143] 12. Mechanism of action of the ErLAC12 gene on Chinese cabbage

[0144] About 6-7 pieces of P. oryzae were cut from the edge of a growing PDA plate and placed in 100 mL of Richard liquid medium, which was shaken at 30°C, 160 rpm, and in the dark for 48 hours. The filtrate was centrifuged, and the supernatant was stored in a -20°C refrigerator for later use. The mycelial dry weight of each sample was calculated, and the experiment was repeated three times.

[0145] Substrate preparation: After the substrate was prepared, it was stored in a 4°C refrigerator, and the buffer and substrate solution were freshly prepared. The substrate preparation method is as follows:

[0146] (1) Polygalacturonase (PG) substrate: Prepare a 10 g / L polygalacturonase solution, add 1.00 g of polygalacturonase, dissolve in 50 mol / L, pH 5.5 sodium acetate buffer, and reduce the volume to 100 mL.

[0147] (2) Pectin methylgalacturonase (PMG) substrate: Prepare a 10 g / L pectin solution, weigh 1.0 g of pectin, dissolve in 50 mol / L, pH 5.5 sodium acetate buffer, and dilute to 100 mL.

[0148] (3) Cellulase (Cx) substrate: Prepare a 10 g / L carboxymethyl cellulose solution, weigh 1.00 g of carboxymethyl cellulose, dissolve in 50 mol / L, pH 5.0 citric acid-sodium citrate buffer, heat to dissolve, and dilute to 100 mL.

[0149] (4) β-glucosidase (βG) substrate: 10 g / L salicylic acid solution 1.00 g salicylic acid (CMC) was dissolved in 50 mol / L, pH 4.5 citric acid-sodium citrate buffer, and made up to 100 mL. The activities of polygalacturonase (PG) and pectin methylgalacturonase (PMG) were determined using the 3,5 dinitrosalicylic acid method (DNS). Two 25 mL stoppered test tubes were taken, and 0.5 mL of the substrate solution and 1.0 mL of acetic acid-sodium acetate buffer (PMG substrate: pectin, PG substrate: polygalacturonic acid) were added to tubes a and b, respectively. After 5 minutes in a 37°C water bath, 0.5 mL of enzyme solution was added to tube A, and an equal amount of inactivated enzyme solution (heated in boiling water for 5 minutes) was added to tube B as a control. 1.5 mL of DNS reagent was added to each tube. After the solutions in the two tubes were quickly cooled to room temperature in a boiling water bath for 5 minutes, distilled water was added to dilute to the 25 mL mark and mixed. The absorbance value was measured at 540 nm using a spectrophotometer, and the corresponding reducing sugar content was obtained from the standard curve, and the activities of PG and PMG were calculated. One unit of enzyme activity refers to the amount of enzyme required to catalyze the formation of 1 μmol of galacturonic acid per minute at 37°C. The above process was repeated 3 times.

[0150] Cellulase and β-glucosidase activity determination: 1.5 mL of 10 g.L-1 substrate solution (βG substrate: salicylic acid, CX substrate: carboxymethyl cellulose) was first added, heated in a 37°C water bath for 5 minutes, 0.5 mL of enzyme solution was added to tube A, and 0.5 mL of inactivated enzyme solution was added as a control, 1.5 mL of DNS was added, incubated in a 37°C water bath for 1 hour, heated in a boiling water bath for 5 minutes, quickly cooled to room temperature, diluted with distilled water to the 25 mL mark, and mixed. The absorbance was measured at 540 nm, and the reducing sugar content was calculated from the standard curve. The enzyme activity unit of cellulase is the amount of enzyme required to catalyze the formation of 1 μmol of glucose from the substrate per minute at 37°C. The above process was repeated 3 times. The in vitro activities of PG, PMG, βG, and CX can be calculated as follows:

[0151]

[0152] m: mass of galacturonic acid in the standard curve (unit: milligrams);

[0153] 5.14: micromoles of 1 milligram of galacturonic acid (1000 / 194.14);

[0154] V': total volume of the enzyme solution after filtration of the mycelium (unit: milliliters);

[0155] V: total volume of the sample taken for determination (unit: mL);

[0156] t: enzyme reaction time (unit: min); m': dry mycelium mass (unit: mg).

[0157] Effect of ErLAC12 on cell wall-degrading enzyme activity: According to the transcriptome sequence of B. emerssonii, cell wall-degrading enzymes play a key role in improving the virulence of B. emerssonii. The main cell wall-degrading enzymes belonging to the Cazymes family are PG, CMC, CX, and βG, which are significantly involved in the pathogenicity of B. emerssonii to P. chinensis. Therefore, the present experiment determined the effect of cell wall-degrading enzymes of ErLAC12 on L. chinensis, paving the way for determining the mechanism of ErLAC12 involved in controlling L. chinensis. The results showed that the activity of cell wall-degrading enzymes was significantly improved after applying ErLAC12. Compared with the mutant (0.624 U / mg), the activity of polygalacturonase (PG) was significantly increased (0.786 U / mg) after applying ErLAC12. Similarly, compared with the mutant Y9511-X050 (1.078 U / mg), the PMG activity of strain ErLAC12 was significantly increased (1.363 U / mg), while the wild-type strain had the lowest activity of poly-methylgalacturonase (0.53 U / mg) on L. chinensis leaves after applying ErLAC12 Figure 18A). The strain ErLAC12 showed the highest beta-glucosidase activity (0.9571 U / mg) compared to the mutant strain Y9511-X050 (0.49 U / mg) and the wild type Y9511 (0.125 U / mg). The cellulase activity of the ErLAC12 treated leaves (0.342 U / mg) was also significantly increased compared to the wild type strain (0.133 U / mg) and the mutant strain (0.210 U / mg). Likewise, we also determined the difference in cell wall degrading enzyme activity of the strain ErLAC12. The results showed that the PG, PMG, CX and βG activities of the strain ErLAC12 were drastically increased, with more significant effects on PG, PMG and βG activities. The cellulase activity of the strain ErLAC12 was increased (0.00427 U / mg) compared to the wild type Y9511 (0.00085 U / mg) and the mutant strain Y9511-X050 (0.0036 U / mg). Likewise, its PG activity (0.0088 U / mg) was also significantly increased compared to the wild type Y9511 (0.00103 U / mg) and the mutant strain Y9511-X050 ((0.00677 U / mg). The PMG activity of ErLAC12 (0.00786 U / mg) was also higher than the wild type Y9511 (0.00192 U / mg) and the mutant strain Y9511-X050 (0.0062 U / mg). Likewise, the βG activity of ErLAC12 (0.00538 U / mg) was also significantly increased compared to the wild type strain (0.00097 U / mg) and the mutant strain Y9511-X050 (0.0033 U / mg). Based on the current results, it can be concluded that the overexpression of the lactose permease gene in ErLAC12 has a significant effect on the cell wall degrading enzyme activity of the fungus Figure 18 B). Since the lactose permease gene LAC12 is a functional key gene of the fungus R. oryzae, overexpression of the LAC12 gene (ErLAC12) can increase the pathogenicity enzyme characteristics of the specific fungus. It can be seen that the overexpression of the required functional gene can affect the activities of these major cell wall degrading enzymes.

[0158] 13. Measurement of reactive oxygen species (ROS) levels in weed leaves

[0159] To further evaluate the mechanism of the effect of ErLAC12 on the pathogenicity of P. catenatum to Euphorbia lathyrys, the role of the ErLAC12 gene in the ROS level of the weed leaves was also determined.

[0160] (1) Measurement of malondialdehyde (MDA) and hydrogen peroxide (H2O2) activity

[0161] MDA extraction about 1 g of plant material was taken, chopped, added 2 mL of 5% trichloroacetic acid and a small amount of quartz sand, ground to homogenate, added 8 mL of trichloroacetic acid further grinding, the homogenate was centrifuged at 4000 r / min for 10 min, the supernatant was the sample extract. Add 2 mL of centrifugal supernatant (add 2 mL of distilled water to the control group), then add 2 mL of 0.6% TBA solution, shake well. The test tube was placed in a boiling water bath for 10 minutes (from the test tube solution appeared small bubbles), the test tube was taken out and cooled, centrifuged at 3000 r / min for 15 minutes, the supernatant was taken and the volume was measured. With 0.6% TBA solution as blank, the absorbance values at 532 nm, 600 nm and 450 nm were measured. Hydrogen peroxide activity was measured by grinding the leaves (0.25 g) with 5 mL of TCA solution, determining the content of hydrogen peroxide (H2O2), and then measuring the absorbance of the supernatant at 390 nm with a spectrophotometer.

[0162] (2) Relative electrolyte leakage (REL)

[0163] In the test tube, 200 mg of leaf tissue was added, soaked in sterile distilled water at 25°C for 2 hours, and then the conductivity (E1) was measured. The test tube containing leaf material was boiled for 30 minutes, and the conductivity (E2) was measured again. The relative electrolyte leakage rate (%) was calculated using the formula:

[0164] REL (%) = E1 / E2 x 100 (2.4)

[0165] The accumulation of reactive oxygen species in Euphorbia esula leaves treated with ErLAC12 cell filtrate was significantly increased. From the increase of malondialdehyde (MDA), hydrogen peroxide (H2O2) and exosmosis (EL%) levels in weeds, it can be seen that the overexpression of ErLAC12 obviously triggered the oxidative stress of Euphorbia esula Figure 19) compared to mutant Y9511-X050 and wild type Y9511, but not obvious on the accumulation of H2O2 content. Compared to mutant Y9511-X050 and wild type Y9511 of P. catenatum, strain ErLAC12 increased the accumulation of superoxide anion and the level of ROS toxicity in weeds. Strain ErLAC12 caused a significant increase in MDA content and electrolyte leakage in weeds. Compared to wild type strain (45.74%) and mutant strain (41.53%), strain ErLAC12 caused a sharp increase in EL% content of weed leaves (55.14%). Compared to the control group, all these fungal strains significantly increased the accumulation of EL%. Similarly, compared to wild type Y9511 (3.021 μmol / g) and mutant Y9511-X050 (3.59 μmol / g), strain ErLAC12 (4.5 μmol / g) also increased the MDA content. All these fungal strains significantly increased the accumulation of MDA content and the level of EL% in weeds, resulting in a significant decrease in the growth of weeds Figure 19 ) in Euphorbia maculata leaves. This indicates that ErLAC12 has a significant systemic effect on the physiology of Rhus chinensis. An important fact is that, unlike superoxide anions, H2O2 can easily cross biological membranes and be transported away from its source. Among ROS, H2O2 and O2 are generally reactive, but both can generate the most reactive ROS species, OH. ROS concentrations above optimal levels can cause damage to plant tissues, often leading to premature cell death. Our study shows that the amount of ROS produced by plants increases significantly after being subjected to biotic stress.

[0166] 14. Cytological observation of Euphorbia maculata leaves

[0167] Transmission electron microscopy used JEM1200; resin was a SPI company package kit; anhydrous ethanol and acetone reagents were analytical pure AR from National Pharmaceutical Group Chemical Reagents Co., Ltd.; glutaraldehyde was Solarbio; osmium acid, uranyl acetate, and lead citrate were analytical pure AR from Xie Xin Experimental Supplies Co., Ltd.

[0168] The experimental process was to fix L. chinensis seedling leaf samples in a 2.5% glutaraldehyde solution overnight at 4°C, and then follow these steps: pour out the fixative, rinse the sample with 0.1M, pH 7.0 phosphate buffer three times for 15 minutes each time. Fix the sample with 1% osmium acid solution for 1-2 hours. Carefully remove the osmium acid waste liquid, rinse the sample with 0.1M, pH 7.0 phosphate buffer three times for 15 minutes each time, and use five gradient concentrations (including 30%, 50%, 70%, 80%, 90%, and 100%).

[0169] Dehydration was performed using ethanol solutions of different concentrations, with each concentration treated for 15 minutes. The samples were then transferred to pure acetone for 20 minutes, treated with a 3:1 mixture of acetone and resin for 2 hours, and dehydrated with a 1:1 mixture of acetone and resin. Alternatively, samples were treated for 3 hours with a 1:3 mixture of acetone and resin, and finally treated overnight with pure resin. The soaked samples were placed in a mold and subjected to gradient heating (35℃-60℃-80℃) for 5 hours at each step, resulting in embedded samples. After rough trimming, the samples were sectioned using a LEICA EMUC7 ultramicrotome to obtain sections with a resolution of 70-90 nm. The sections were stained with acetic acid (15 minutes) and lead citrate (5 minutes), then dried and photographed.

[0170] Plant cell walls are highly complex, composed of polysaccharides and protein polymers, and are crucial in monitoring plant-pathogen interactions. The plant cell wall is the first barrier against pathogens attempting to infect plant tissues, thus functioning as a passive defense barrier. Transmission microscopy confirmed the damage to *Echinochloa crus-galli* leaves. These abnormalities, such as cell malformation, significant deformation of cell vacuoles, disruption of cell walls and plasma membranes, dispersion of the intercellular matrix, abnormalities of microcysts and endoplasmic reticulum, and destruction and malformation of chloroplasts, were observed in leaves of two plant species under fungal stress, with the most severe effects observed in leaves of strain ErLAC12. After application of strain ErLAC12, chloroplasts showed the highest plasmoglobin accumulation and a lack of cellular peroxisomes due to lipid metabolism disorders. Furthermore, strain ErLAC12 exhibited the most severe cell and protoplast damage compared to wild-type Y9511 and the mutant Y9511-X050. All treatments significantly damaged the cell walls and organelles of *Echinochloa crus-galli* leaves. Figure 20 ).

[0171] 15. Assessment of the safety management of *Helicobacter buergerianum* for rice crops.

[0172] The safety of *Helicobacter ostreatus* to rice crops was evaluated by measuring growth parameters, physiological properties, and biochemical parameters.

[0173] (1) Measurement of growth parameters

[0174] The safety of fungi to rice plants was determined through in vitro pot experiments. At harvest, the rice plants were removed from the potting soil, and standard measurement techniques were used with slight modifications. (et al., 2020) evaluated various growth parameters such as plant height, fresh weight, and dry weight.

[0175] (2) Physiological attribute determination

[0176] Leaf chlorophyll content was estimated using a SPAD-502plus (Konica Minolta Sensing Americas, Inc.) instrument following the procedure described in detail in (Ling et al., 2011). For SPAD estimation, fully expanded uppermost leaves were used. The average of SPAD values was calculated from ten readings following the method of (León et al., 2007).

[0177] (3) Determination of antioxidant activity in rice

[0178] Preparation of enzyme solution: 0.5 g of rice leaves were weighed into a 10 mL centrifuge tube, 5 mL of pre-cooled phosphate buffer was added, and a small amount of steel ball was added. The mixture was ground into a homogenate in a low-temperature grinder (-35°C), and then 10 mL of buffer was added to make up the volume. The mixture was centrifuged at 6000 rpm for 15 min, and the supernatant was used as the crude enzyme extract for various enzyme activity assays.

[0179] Preparation of 0.05 mol / L phosphate buffer (PBS, pH 7.8): Take 228.75 mL of solution A and 21.25 mL of solution B, respectively, and make up to 1 L with pure water. Add 10 grams of polyvinylpyrrolidone (PVP) and 0.029 grams of ethylenediaminetetraacetic acid disodium salt (EDTA-Na2). The subsequent experiments all use this buffer. Solution (A): 0.2 mol / L sodium phosphate dibasic solution. Na2HPO4-12H2O (molecular weight 358.14) 71.7 grams; Solution (B): 0.2 mol / L sodium phosphate dibasic solution. Take 31.2 g of NaH2PO4-2H2O (molecular weight 156.01). Prepare 1 L of volume with pure water for later use.

[0180] 1) Superoxide dismutase (SOD) activity determination

[0181] The method of (Jalali-e-Emam et al., 2011) was used with slight modifications, and the specific operation steps are as follows:

[0182] Reagent preparation: 130 mmol / L methionine (Met) solution: 1.399 g of Met was weighed into 100 mL of phosphate buffer. 100 μmol / L EDTA-Na2 solution: 750 μmol / L nitrogen blue tetrazolium (NBT) solution: 0.06133 g of NBT was added to 100 mL of phosphate buffer. 20 μmol / L riboflavin solution: 0.0075 g of riboflavin was weighed and made up to 1 L with pure water. Color development: 5 mL centrifuge tubes were used, 3 for sample tubes, 1 for control tube, and 1 for blank tube. The reagents were added quickly according to the table. After mixing, the blank tube was placed in the dark, and the other tubes were exposed to sunlight in the morning for 20 min (the direction was adjusted from time to time to ensure that all tubes received the same amount of light) (Table 10).

[0183] Table 10 List of reagents and volumes

[0184]

[0185] After the reaction, the absorbance of the sample and control tubes was measured at 560 nm wavelength under the UV spectrophotometer with a blank tube set to zero. The SOD activity unit was taken as 50% inhibition of NBT photochemical reduction as the enzyme activity unit.

[0186] 2) Peroxidase (POD) activity assay (guaiacol method)

[0187] The dehydrogenation of guaiacol was monitored to quantify the activity of peroxidase. To prepare the crude enzyme extract, a fine powder of frozen leaf (100 mg) was added to 1 ml of phosphate buffer solution (PBS) (pH 7.0). The mixture was centrifuged at 5°C and 8,064 x g for 5 minutes. The enzyme was extracted in 3 ml of 100 mM PBS (pH 7.0) and stirred at 5°C and 8064 x g for 15 minutes. In 0.1 ml of plant extract, 3 ml of PBS (100 mmol), 0.05 ml of guaiacol (20 mmol) and 0.03 ml of H2O2 (12.3 mmol or 0.04%) were added, then vortexed. The optical density was monitored at a wavelength of 436 nm, and the absorbance was recorded over time.

[0188] 3) Catalase (CAT) activity assay

[0189] A 0.5 g sample of frozen leaf was added to 25 mmol of potassium dihydrogen phosphate buffer (pH 7.8), 1 mmol of ascorbic acid, 0.4 mmol of ethylenediaminetetraacetic acid and 2% (w / v) polyvinylpyrrolidone to prepare a crude enzyme extract. The mixture was shaken at a speed of 15,000 rpm for 1 minute to homogenize its contents, then centrifuged at a speed of 15,000 g for 20 minutes to remove debris. In the crude enzyme extract, 50 mM potassium dihydrogen phosphate buffer and 10 mM H2O2 were added. The activity of catalase was detected by monitoring the rate of decomposition of H2O2 at a wavelength of 240 nm.

[0190] The effects of ErLAC12 on plant morphological, physiological and biochemical characteristics were studied using standard measurement techniques. Under the biological stress of Pythium aphanidermatum, the plant height, fresh weight of tender shoots and dry weight of tender shoots of rice did not decrease significantly compared to healthy plants. Therefore, according to our research results, we can conclude that overexpression of lactose permease ErLAC12 gene does not have a significant impact on plant morphological characteristics and is a safe treatment method for rice plants.

[0191] (1) Effect of ErLAC12 on the plant, physiology and biochemical properties of rice

[0192] The present experiment recorded the safety of ErLAC12 on rice, and the results showed that ErLAC12 did not significantly reduce the morphological characteristics of the plant Figure 21 ). The application of ErLAC12 can maintain the SPAD value of rice leaves. Under normal environmental conditions, antioxidant activity can reduce the proportion of autoxidation processes by reducing the initial event or interacting with chain free radicals. Compared with wild type Y9511 (190.8 μ / g.min), mutant Y9511-X050 (290.8 μ / g.min) and control plants (173.66 μ / g.min), ErLAC12 significantly increased the activity of POD (321.68 μ / g.min). Compared with the control plants (52.86 μg-1.FW), the SOD activity of ErLAC12 (150.01 μg-1.FW) was improved, and the wild type Y9511 (153.98 μg-1.FW) and mutant Y9511-X050 (166.78 μg-1.FW) had a greater impact. Compared with mutant Y9511-X050 (55 μg-1.FW) and wild type Y9511 (50 μg-1.FW), the CAT activity of ErLAC12 (57 μg-1.FW) was also improved, thereby enhancing the defense mechanism of rice by catalyzing the decomposition of two H2O2 molecules into water and O2. The results also confirmed that under biological stress, Pythium protrudens can significantly improve the antioxidant activity of rice plants, and is therefore considered a safe method of treating rice plants. Enzyme antioxidants include superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activity. After the biological stress signal is sent, the plant will start the antioxidant defense mechanism to handle ROS and restore cell balance. Enzymes such as CAT can directly or indirectly neutralize ROS. In plants under biological stress, excess H2O2 will be degraded by CAT. ErLAC12 reduces hydrogen peroxide activity by increasing POD activity, which provides a safe mechanism for rice crops. In the present experimental group, the activity of the three scavenging enzymes increased significantly, indicating that the balance between ROS generation and clearance was out of control Figure 22 ).

Claims

1. A gene of Erwinia herbicola ErLAC12, the nucleotide sequence of which is shown in SEQ ID NO.

1. 2.A recombinant plasmid comprising the gene of Erwinia herbicola ErLAC12 according to claim 1. 3.The gene of Erwinia herbicola ErLAC12 according to claim 1 is used in the prevention and control of Leptinella fimbriata. 4.The gene of Erwinia herbicola ErLAC12 according to claim 1 is used as a Leptinella fimbriata prevention and control agent.

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