Corn zmlecrk-g2 gene and application thereof

Abstract

The application discloses a corn ZmLecRK-G2 gene and application thereof, and belongs to the technical field of plant pathogen control. The corn ZmLecRK-G2 gene has a nucleotide sequence as shown in sequence 1. The extracellular Lectin domain of the ZmLecRK-G2 protein has the ability of binding beta-glucan; the corn ZmLecRK-G2 mutant is more sensitive to corn southern rust; and the transient expression of the ZmLecRK-G2 gene can improve the resistance of Nicotiana benthamiana to sclerotinia sclerotiorum.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant pathogen control, and particularly relates to a maize ZmlecRK-G2 gene and application thereof. BACKGROUND

[0002] Maize southern and common rusts are important diseases in maize production in China, which are caused by Puccinia polysora and P.sorghi, respectively. Maize southern rust is more harmful to China's maize industry, and can cause absolute yield loss in severe cases. China's maize varieties are generally less resistant to maize southern rust. In recent years, maize southern rust has broken out in many major maize producing areas in China, causing maize quality and yield reduction, posing a serious threat to maize production. If pesticides are used for a long time, it will have a significant impact on the environment. Therefore, mining new disease resistance genes and germplasm resources for disease resistance breeding and genetic improvement of varieties is an effective way to control the prevalence of maize southern rust.

[0003] In the long-term struggle between plants and pathogenic fungi, plants have evolved an immune system. Plants use two major types of immune receptors located on the cell surface and inside the cell to recognize viruses, bacteria, fungi, oomycetes, nematodes, insects and other invasive organisms, and activate the plant immune system. Cell surface immune receptors, also known as pattern recognition receptors (PRRs), are mainly composed of single transmembrane receptor-like kinases (RLKs) and receptor-like proteins (RLPs), which recognize molecular patterns from invasive organisms and plant-derived molecular patterns associated with infection damage on the cell surface. The activated immunity is called pattern-triggered immunity (PTI). For example, plant CEBiP and LYK5 can bind chitin in fungal cell walls to activate the immune response of plants. Lectin-like receptor kinases are a subfamily of receptor-like kinases. According to the different domains, lectin-like receptor kinases can be divided into L, G and C types. Among them, G-type lectin-like receptor protein kinases were once called B-type LecRLK, and their extracellular lectin domains have a beta-barre structure, which are alpha-D mannose-specific plant lectin receptor protein kinases. G-type lectin receptor kinases in plants mainly recognize lipopolysaccharides in bacteria to trigger downstream immune responses. In the interaction between plants and fungi, the role of G-type lectin receptor kinases is rarely reported. Therefore, it is urgent to explore new resistance sources and resistance genes, broaden the germplasm basis of plant disease resistance resources, and apply them to breeding practice to provide technical support for molecular marker-assisted selection breeding. SUMMARY

[0004] Therefore, the application aims to provide a corn ZmlecRK-G2 gene and application thereof, which can effectively improve the sensitivity of corn to southern rust and the resistance of tobacco to Sclerotinia sclerotiorum, and lay a foundation for widening the germplasm of corn or tobacco disease-resistant resources.

[0005] The application solves the above problems through the following technical scheme:

[0006] The corn ZmlecRK-G2 gene has a nucleotide sequence as shown in SEQ ID NO. 1.

[0007] The SNP variation of the ZmLecRK-G2 gene mutant of the application causes a G-type LecRLK receptor protein coding gene (Zm00001eb025540) to replace the Trp-102 codon (TGG) at the lectin domain with a stop codon (TGA).

[0008] Further, the expression vector or host cell containing the corn ZmlecRK-G2 gene also belongs to the protection scope of the application, and can be used for plant improvement, breeding or preparation of transgenic plants.

[0009] The extracellular lectin domain of the corn ZmlecRK-G2 protein binds to β-glucan in the cell wall of fungi, and β-glucan is an important component of all fungal cell walls, which indicates that the protein has broad-spectrum function in recognizing various fungal pathogens, and indicates that the gene can be applied to plant resistance to bacteria.

[0010] The application also discloses application of the corn ZmlecRK-G2 gene, and the corn ZmlecRK-G2 gene is applied to reducing the resistance of corn to southern rust, or the corn ZmLecRK-G2 mutant corn plant is more sensitive to southern rust of corn.

[0011] It is further found that the transient expression of the corn ZmlecRK-G2 gene can improve the resistance of tobacco to Sclerotinia sclerotiorum.

[0012] Further, the corn ZmlecRK-G2 gene is applied to preparation of transgenic plants.

[0013] Further, the corn ZmlecRK-G2 gene is applied to improvement and breeding of crops.

[0014] Beneficial effects:

[0015] The extracellular domain of the corn ZmlecRK-G2 gene can bind beta-glucan of fungi, which indicates that the gene has broad-spectrum antibacterial property, and experiments show that the transient expression of the gene can improve the resistance of N.benthamiana to Sclerotinia sclerotiorum, and provides a basis for the prevention and control technology of tobacco Sclerotinia sclerotiorum. Meanwhile, the mutant of the gene reduces the resistance of corn to southern rust, lays a foundation for widening the germplasm of corn disease-resistant resources, and has the development potential for further development of disease-resistant breeding. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 : SNP variation diagram of ZmLecRK-G2 gene mutant;

[0017] Figure 2 : Diagram of binding of extracellular structure of ZmLecRK-G2 gene to small molecules of fungi;

[0018] Figure 3 : Result diagram of ZmLecRK-G2 gene for improving the disease resistance of N.benthamiana;

[0019] Figure 4 : Comparison diagram of ZmLecRK-G2 gene mutant identification and resistance identification. DETAILED DESCRIPTION

[0020] The application will be described in detail below in combination with specific examples and drawings:

[0021] The SNP variation of the ZmLecRK-G2 gene mutant of the application causes the Trp-102 codon (TGG) of a G-type LecRLK receptor protein coding gene (Zm00001eb025540) at the lectin domain to be replaced by a stop codon (TGA), and the schematic diagram is as shown in Figure 1 .

[0022] Example 1:

[0023] The nucleotide sequence of the ZmLecRK-G2 gene disclosed in the application is shown in sequence 1:

[0024] Sequence 1:

[0025]

[0026] The amino acid sequence is as follows:

[0027] "MASVSLLLCLLLLVPPLAAAQQQMSSFSANNSTWSPTNSNRILVSINRE FAAGFVASTSARDRYHFAVWVVGSNSTDKAFIWYAHDTASYSPYEGNDTSKLAIDAAGRLTWTAGGNNNATIWSLPPPANTTTTPAVLQLNDTGSLVYGAAWSSFAEPTNTLMPGQAMPKGGNDTTLQSVNGHYRVVNSATLQFNNSMMYANISGGSALLNLTADGKLQFSGSQLIASDQGTTNRVRRLTLDDDGNLRLYSLVPKTRKWLVVWQVVQELCTIRGTCANGRICVPVGVDSTTCVCPPGYRNATPTDPCTPKKRYSGRGDDDTFVRMDFVSFSGAANSSASDPGPLMTKLTPQNLADCERLCRSNSTCVAFGYKFGGDRTCLQFTGLVDGYWSPATEMSTYLRVVASDNDSNPFTGMTTMIETVCPVQLALPVPPKESQTTIQNVAIITALFVVELLAGVLSFWAFLRKYSQYREMARTLGLEYLPAGGPRRFSHAELKQATKDFSNVVGRGAYGTVYRGELPDRRAVAVKQLQGVGGGEAEFWAEVTIIARMHHLNLVRMWGFCAEKEQRMLVYEYVPNGSLDKYLFAGGGGGGGSGEEDSSAEQQQQQPLLDLHTRYRIALGVARAIAYLHEECLEWVLHCDIKPENILLEDDFCPKVSDFGLSKLTSKKEKVTMSRIRGTRGYMAPEWVIHREPITAKADVYSFGMVLLEIVSGRRNYGFRQESVGSEDWYFPKWAYEKVYVERRIDDILDPRIAATYDDAASVATVERMVKTAMWCLQDRAEMRPSMGKVSKMLEGSVEITEPVKPTIFCVQDD".

[0028] If not otherwise indicated, the technical means used in the examples are those conventional to the person skilled in the art; if not otherwise indicated, the reagents used in the examples are commercially available.

[0029] Example 2: Construction of ZmLecRK-G2 gene expression vector

[0030] (1) Total RNA was extracted from corn leaves by TRNzol method, and then RNA was reverse transcribed into cDNA by reverse transcription kit (TaKaRa, 2641A), and the specific operation was as follows.

[0031] Corn total RNA extraction:

[0032] 1) About 0.1 g of leaves was ground into powder in liquid nitrogen, and the sample powder was quickly transferred into a pre-cooled 2 mL nuclease-free special centrifuge tube, 800 μL of TRNzol Reagent was added, and it was mixed well by upside-down, and then it was placed in ice for 3 min, and the subsequent steps were completed in a clean bench;

[0033] 2) 800 μL of phenol:chloroform:isopropyl alcohol (25:24:1, pH=4.5) was added, and it was mixed well by upside-down, and then it was placed in ice for 5 min, and then it was placed in a pre-cooled 4°C centrifuge at 12000 rpm for 10 min;

[0034] 3) The water phase was absorbed into a new 1.5 mL nuclease-free centrifuge tube, about 450 μL of pre-cooled chloroform was added, and it was shaken vigorously for 15 seconds, and then it was placed in ice for 3 min, and then it was placed in a 4°C centrifuge at 12000 rpm for 10 min;

[0035] 4) The water phase in the centrifuge tube was reserved, and an equal volume of pre-cooled isopropyl alcohol was added, and an equal volume of pre-cooled isopropyl alcohol was added, and it was placed in 20°C for 3 h, and then it was placed in a 4°C centrifuge at 12000 rpm for 10 min;

[0036] 5) The supernatant was poured out, 1 mL of 75% ethanol was added again, and the white precipitate at the bottom of the tube was suspended by shaking, and then it was placed in a 4°C centrifuge at 12000 rpm for 2 min;

[0037] 6) The step (5) was repeated;

[0038] 7) The supernatant was poured out, and it was dried in a clean bench; an appropriate amount of nuclease-free ddH2O was added to dissolve the precipitate, and the total RNA was obtained;

[0039] 8) 1 μL of the obtained total RNA was diluted to 10 μL with 9 μL of nuclease-free ddH2O, and 1 μL was taken for concentration measurement, and the remaining was detected by electrophoresis, and the unused RNA was placed at -80°C.

[0040] First strand cDNA synthesis:

[0041] 1) Take 1 μg total RNA, add 1 μL Oligo(dT)12-18 Primer (50 μM), 0.5 μL dNTP Mixture, RNase free H2O to 6 μL;

[0042] 2) Incubate at 65°C for 5 minutes, then quickly cool on ice for more than 2 minutes;

[0043] 3) Centrifuge for several seconds to make the denatured template RNA / primer solution gather at the bottom of the tube;

[0044] 4) The above denatured template RNA / primer solution 6 μL; 5X Reverse Transcription Buffer 2 μL; RNase Inhibitor (40 U / μl) 0.25 μL; Reverse Transcriptase M-MLV (RNase H-) (200 U / μl) 0.5 μL; RNase free H2O to 10 μL;

[0045] 5) Incubate at 42°C for 1 hour, then incubate at 70°C for 15 minutes, and cool on ice to obtain cDNA.

[0046] (2) The obtained corn cDNA is used as a template to clone ZmLecRK-G2, and the primer sequence is (F: ATGGCCTCCGTCTCCCTC R: GTCGTCCTGAACGCAGAAGA). The PCR procedure (98°C 1 min, (98°C 10 s, 55°C 10 s, 72°C 1 min) x 35, 72°C 10 min, 16°C 5 min) is used to obtain the ZmLecRK-G2 gene, and the sequence is shown in SEQ ID NO: 1.

[0047] (3) Primer sequence:

[0048] F: cggGGTACCATGGCCTCCGTCTCCCTC

[0049] R: tgcTCTAGACTGGATCGTCGTCTGCGA

[0050] The primer is used for amplification to obtain the extracellular domain fragment of ZmLecRK-G2. The target fragment and the pCold vector (TaKaRa) are double-digested with Kpn1 and Xba1, and the recovered vector fragment and the target fragment are ligated by T4 DNA Ligase (TaKaRa, 2011A) at 16°C for 2 hours. The reaction product is transformed into E. coli competent TOP10, and incubated at 37°C overnight.

[0051] (4) Using carrier universal primer for colony PCR detection, the PCR product of the purpose band was sent to GenScript Biotech Corporation for sequencing and comparing the sequencing results, and the recombinant vector pCold-ZmLecRK-G2 extracellular domain was successfully constructed;

[0052] (5) The constructed recombinant vector was transformed into competent BL21, and cultured at 37°C overnight, and a single colony was picked into LB liquid medium containing ampicillin (50 μg / ml) for culture.

[0053] 1. Prokaryotic expression and protein purification analysis

[0054] The bacteria liquid was inoculated into liquid LB medium containing 50 μg / ml ampicillin at a ratio of 1:100, and when the A600 value of the bacteria liquid reached 0.6-0.8, isopropyl thiogalactoside (IPTG) was added to the culture medium at a final concentration gradient of 0.6 mmol / L, and the expression was induced at 18°C, 200 rpm / min overnight. The bacteria were collected by centrifugation at 8000 rpm / min and resuspended with 1xPBS buffer, and ultrasonic broken treatment (broken 3s, stop 5s, 25min) for half an hour, and added one thousandth PMSF. The supernatant was collected by centrifugation, and the His protein purification kit (Shanghai Shenguo, C600332-0001) was used for protein purification according to the instructions.

[0055] 2. Microscale thermophoresis (MST) analysis

[0056] The ZmLecRK-G2 extracellular domain protein with 6xHis tag was labeled according to the manufacturer's (NanoTemper) labeling kit (Monolith His-Tag Labeling Kit RED-tris-NTA, MO-L018) instructions. 16 different concentrations of fungal small molecule compounds (maximum concentration 50 mg / ml, gradient dilution) were incubated with the labeled protein (1:1, v / v) at room temperature. MST analysis was performed using a capillary on a NanoTemper instrument (Monolith NT.115). The data were analyzed using NanoTemper analysis software MO.Affinity Analysis (v.2.3).

[0057] The results are shown in Figure 2 , and the results of the analysis Figure 2 are as follows:

[0058] From Figure 2As can be seen, the extracellular domain of ZmLecRK-G2 gene can bind β-glucan and mannose, and does not bind chitin and ergosterol. These four substances are small molecules of fungi, and it is known that mannose can bind to G-type agglutinin receptor kinase. Our results show that the binding ability of ZmLecRK-G2 to β-glucan is stronger than that to mannose, and the β-glucan of fungi is a potential ligand of ZmLecRK-G2, which may have a certain broad-spectrum antibacterial property. The transient expression of the gene may improve the resistance of N. benthamiana to S. sclerotiorum, and the gene reduces the resistance of corn to southern rust. Therefore, the following examples are verified.

[0059] Example 3: Overexpression of ZmLecRK-G2 in N. benthamiana

[0060] (1) The obtained corn cDNA was used as a template, and the primers were as follows:

[0061] F: gcTCTAGAATGGCCTCCGTCTCCCTC

[0062] ZmLecRK-G2-R: ggACTAGTGTCGTCCTGAACGCAGAAGA

[0063] ZmLecRK-G2ΔKinase-R: ggACTAGTGGGGAGGTACTCCAGTCC

[0064] ZmLecRK-G2ΔTM-R: ggACTAGTCTGGATCGTCGTCTGCGA

[0065] PCR amplification was performed, and the procedure was the same as in Example 2, to obtain ZmLecRK-G2, ZmLecRK-G2ΔKinase, and ZmLecRK-G2ΔTM fragments. Then, using a homologous recombination kit (Tiangen, VI202), the pCNF3 expression vector fragment digested with Xba1 and Spe1 was combined with the ZmLecRK-G2, ZmLecRK-G2ΔKinase, and ZmLecRK-G2ΔTM fragments, respectively, and 5 μL of 2×EasyGenoSingle Assembly Mix was added, ddH2O was added to 10 μL, and 50°C incubation for 15 min was performed for homologous recombination. The reaction product was transformed into E. coli competent TOP10, and 37°C overnight culture was performed.

[0066] (2) Colony PCR was performed using a vector universal primer, and the PCR product of the target band was sent to GenScript Biotech Corporation for sequencing and comparison of the sequencing results. The recombinant vectors pCNF3-ZmLecRK-G2, pCNF3-ZmLecRK-G2ΔKinase, and pCNF3-ZmLecRK-G2ΔTM were successfully constructed.

[0067] (3) Transform the constructed recombinant vector into Agrobacterium competent GV3101, cultivate at 28°C for 48h, pick single colonies into LB liquid medium with kanamycin (50μg / ml) and rifampicin (25μg / ml) for cultivation.

[0068] (4) After the bacterial solution is turbid, centrifuge at 5000rpm / min for 10 minutes, resuspend the precipitate with resuspension solution, adjust the OD600 to 0.6-0.8 with a spectrophotometer, shield from light for 2-3h, inject the resuspension into N. benthamiana with a 1ml syringe, and use the area injected with pCNF3-eGFP (1ml) as a control.

[0069] Take samples after 48h for Western blot analysis, and inoculate the activated S. sclerotiorum (S. sclerotiorum activation: after sterilizing a 0.5cm puncher, punch the S. sclerotiorum culture dish stored at 4°C. Inoculate on PDA medium and cultivate at 20°C, and use after 48h) in the injection area, punch the S. sclerotiorum with a 0.5cm puncher, and measure the lesion diameter after 24h of inoculation.

[0070] Perform trypan blue staining and Western blot analysis on the above experiment, with the following specific operations:

[0071] 1. Trypan blue staining: place the N. benthamiana leaves of this experiment in 0.4% trypan blue dye, use a vacuum freeze dryer to vacuum for 5min, then boil in a water bath for 5-10min, and decolorize to transparency with 95% alcohol.

[0072] 2. Western blot: take 0.1g of N. benthamiana leaves in the injection area, grind and add 200μl of Western and IP cell lysis solution (Bi Yun Tian, P0013), lyse on ice for 30min, centrifuge at 10000rpm / min, 4°C for 5min. Take the supernatant into a clean centrifuge tube, add 5× protein loading buffer, boil in a water bath for 5min, centrifuge at 10000rpm / min, 4°C for 5min. Take the supernatant for SDS-PAGE protein electrophoresis, after electrophoresis, transfer the membrane, 15V, 20min, put the membrane into blocking solution (50% skim milk powder) for overnight blocking at 4°C. The next day, put into 50% skim milk powder with GFP antibody (Shanghai Shenguo, D191040) in a horizontal shaker for slow shaking for 2 hours, elute with TBST for 3-5 times, 10min each time. Put the NC membrane into 50% skim milk powder with HRP antibody for slow shaking for 1 hour, elute with TBST for 3-5 times, 5min each time, drop high-sensitivity ECL luminescent reagent on the membrane, mix the luminescent reagent and color developing reagent 1:1 (Shanghai Shenguo, C500044), treat in the dark for 3min, and observe under the nucleic acid marker system.

[0073] The results are shown in Figure 6A and 6B. Figure 3 The results are shown in Figure 6A and 6B. Figure 3 The results are shown in Figure 6A and 6B.

[0074] Figure 3 As shown in Figure 6A and 6B, compared with the control, the area of the lesion of transient expression of ZmLecRK-G2 region was significantly reduced, followed by transient expression of ZmLecRK-G2AKinase region, and transient expression of ZmLecRK-G2ATM region had no significant difference with the control. The lesion was stained by trypan blue staining method, which was consistent with the above results. And Western blotting proved that GFP, ZmLecRK-G2, ZmLecRK-G2AKinase, ZmLecRK-G2ATM were successfully expressed in N. benthamiana. At the same time, these results proved that transient expression of ZmLecRK-G2 could improve the resistance of N. benthamiana to S. sclerotiorum.

[0075] Example 4: Identification of ZmLecRK-G2 mutants of corn and identification of disease resistance

[0076] According to the website information (http: / / elabcaas.cn / memd / public / index.html# / ), a ZmLecRK-G2 mutant with B73 as the background was obtained, and ZmLecRK-G2 homozygous mutant was screened by PCR detection and sequencing.

[0077] The corn was cultured in a greenhouse at 25±2℃, 16h light and 8h dark, and when the corn grew to two-leaf-one-heart stage, fresh corn southern rust spores were added to ultrapure water and mixed (10mg / ml), and the spore suspension was evenly smeared on the front of the leaf with a cotton swab, and placed in a humidity box for dark and humid treatment for 24h, and then the culture condition was restored to 16h light and 8h dark. After the disease occurred, the corn southern rust disease grade division standard was used:

[0078] 1: No lesion or only hypersensitive reaction without spore pile on the leaf;

[0079] 3: A small amount of spore pile on the leaf, accounting for less than 25% of the leaf area;

[0080] 5: Medium amount of spore pile on the leaf, accounting for 26%-50% of the leaf area;

[0081] 7: A large amount of spore pile on the leaf, accounting for 51%-75% of the leaf area;

[0082] 9: A large amount of spore pile on the leaf, accounting for 76%-100% of the leaf area;

[0083] The disease grades of wild type and mutant were counted, and the change of biomass was detected by real-time fluorescent quantitative PCR, and the results are shown in Figure 6A and 6B.Figure 4 As shown in the analysis Figure 4 It can be seen that:

[0084] Compared with B73, the mutation of ZmLecRK-G2 gene caused the corn resistance to decrease, the ZmLecRK-G2 mutant showed more sensitive to corn southern rust, the number of spore piles on the leaf increased and no necrosis symptom, the disease grade increased, and the biomass accumulation increased. In addition, the ZmLecRK-G2 mutant plant growth was relatively short. This indicates that ZmLecRK-G2 is a new corn southern rust resistance gene, which has further development potential for disease breeding.

[0085] The above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the present application, and they should be covered in the scope of the claims of the present application. The technical, shape and structure parts not described in detail in the present application are well-known technologies.

Claims

1. The use of overexpressing the ZmlecRK-G2 gene of Zea mays in improving the resistance of tobacco to Sclerotinia sclerotiorum, characterized in that, The nucleotide sequence of the corn ZmlecRK-G2 gene is shown as SEQ ID NO.

1.

2. Use of the maize ZmlecRK-G2 gene in the preparation of transgenic tobacco, characterized in that, Overexpression of the corn ZmlecRK-G2 gene improves the resistance of tobacco to Sclerotinia sclerotiorum, and the nucleotide sequence of the corn ZmlecRK-G2 gene is shown as SEQ ID NO.

1.

3. The application of the maize ZmlecRK-G2 gene in tobacco breeding and seed production, characterized in that, Overexpression of the corn ZmlecRK-G2 gene improves the resistance of tobacco to Sclerotinia sclerotiorum, and the nucleotide sequence of the corn ZmlecRK-G2 gene is shown as SEQ ID NO.

1. Overexpression of the corn ZmlecRK-G2 gene improves the resistance of tobacco to Sclerotinia sclerotiorum, and the nucleotide sequence of the corn ZmlecRK-G2 gene is shown as SEQ ID NO.1.

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