CN116444636B - Rice OsGLP3-6 that inhibits Sclerotinia sclerotiorum and its application - Google Patents

Abstract

The invention belongs to the field of plant molecular biology, and particularly relates to an OsGLP3-6 for inhibiting sclerotinia and application thereof. The rice OsGLP3-6cDNA sequence is shown as SEQ ID No.1, and the encoded protein amino acid sequence is shown as SEQ ID No. 2. The invention uses fluorescence quantitative means to identify positive plants by heterogenous over-expression OsGLP3-6 gene in Arabidopsis thaliana, and cultures the positive plants to T ₃ For the generation, the positive lines were then inoculated with leaf sclerotinia. The results showed that the average plaque area of the Arabidopsis over-expression line OE_OsGLP3-6 (65.2 mm ² ) Plaque area (79.9 mm compared to wild-type WT ² ) The reduction by 22.4%, extremely remarkable (p<0.01 Increased resistance of arabidopsis thaliana to sclerotinia sclerotiorum.

Description

Rice OsGLP3-6 that inhibits Sclerotinia sclerotiorum and its application

Technical field

The invention belongs to the field of plant molecular biology, and specifically relates to OsGLP3-6 that inhibits Sclerotinia sclerotiorum and its application.

Background technique

S. sclerotiorum is a saprophytic filamentous fungus that causes a wide range of diseases. Sclerotinia sclerotiorum can infect more than 500 types of plants (Liang et al., 2018), including dicotyledonous plants such as rape, cabbage, cabbage, etc., causing serious losses to our agricultural production (Boland et al., 2009). The plant cell wall-degrading enzyme secreted protein secreted by Sclerotinia sclerotiorum helps pathogenic bacteria break through the barrier and promote the invasion of hyphae by degrading plant cell walls. For Sclerotinia sclerotiorum, a variety of grass plants, especially rice, were identified as non-host plants of Sclerotinia sclerotiorum very early (Purdy, 1979). Using the non-host resistance mechanism of rice-related genes, it has been used for rapeseed, etc. Improvement of S. sclerotiorum resistance in host plants provides a new resource of disease resistance genes.

In recent years, research on plant germin-like proteins (GLPs) has been reported in Arabidopsis thaliana, rice (Oryza sativa), soybean (Glycine max) and other plants. Studies have shown that some AtGLPs, OsGLPs (Li et al., 2016) and ZmGLPs (Fan et al., 2005) are highly expressed in seed germination and young tissues and may be involved in plant growth and development. More studies have shown that when plants are infected by pathogenic bacteria and bitten by herbivorous insects, the expression of GLPs genes in plants increases, and the enzyme activities of GLP family members (SOD and OXO) also increase. It plays an important role in responding to pathogenic bacterial infections and some abiotic stresses (Zhang et al., 2017; Pei et al., 2019; Banergee et al., 2017).

Contents of the invention

The purpose of the present invention is to provide rice OsGLP3-6 and its encoded proteins and applications.

First, the present invention provides rice OsGLP3-6 protein, which is:

1) A protein composed of the amino acids shown in SEQ ID No. 2; or

2) A protein derived from 1) in which one or several amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID No. 2 and has equivalent activity.

The invention also provides genes encoding said proteins. Preferably, the sequence of the gene is shown in SEQ ID No. 1.

The invention also provides vectors, host cells and engineering bacteria containing the genes.

The present invention also provides the use of the gene in regulating sclerotinia resistance of S. sclerotiorum host plants.

In one embodiment of the present invention, the gene is transferred into the host plant gene and overexpressed in the transgenic plant to improve plant sclerotinia resistance.

The invention also provides a method for improving the resistance of Sclerotinia sclerotiorum host plants to Sclerotinia sclerotiorum, which is characterized in that the vector containing the gene is transferred into the plant genome and overexpressed in the transgenic plant.

The present invention selects the candidate gene OsGLP3-6 by analyzing the RNA-seq of rice inoculated with Sclerotinia sclerotiorum and combining it with the transcriptome data of host plants such as cabbage. The OsGLP3-6 gene was heterologously overexpressed in Arabidopsis. After identifying positive plants using fluorescence quantitative methods, they were cultured to _{the third} T generation, and then the leaves of the positive plants were inoculated with S. sclerotiorum. The results showed that the average plaque area of the Arabidopsis overexpression line OE_OsGLP3-6 (65.2mm ² ) was reduced by 22.4% compared to the plaque area of the wild type WT (79.9mm ² ), which was a very significant (p<0.01) increase. Resistance of Arabidopsis thaliana to Sclerotinia sclerotiorum.

Description of the drawings

Figure 1 shows the phenotypic observation of rice leaves after wounding and unwounded inoculation. -w: No trauma treatment; +w: Trauma treatment.

Figure 2 shows the differentially expressed genes in the samples.

Figure 3 shows the GO enrichment (A) and KEGG enrichment results (B) of rice inoculated for 12 h and the GO enrichment (C) and KEGG enrichment results (D) of rice inoculated for 24 h.

Figure 4 shows the expression of GLP family genes before and after inoculation of rice and cabbage.

Figure 5 shows the cloning of OsGLP3-6 gene.

Figure 6 shows a schematic diagram of fusion vector construction.

Figure 7 shows the identification of target fragments in E. coli. (M=Marker 2000; lane1-6 is a positive clone).

Figure 8 shows the identification of target fragments in Agrobacterium. (M=Marker 2000; lane1-6 is a positive clone).

Figure 9 shows the identification of OsGLP3-6 expression in transgenic Arabidopsis.

Figure 10 shows the identification of sclerotinia resistance in transgenic Arabidopsis.

Figure 11 shows the resistance identification of transiently transformed tobacco.

Detailed ways

The following examples are used to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular cloning experimental manual (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or in accordance with the conditions recommended by the manufacturer's instructions.

Enzymes and kits: Gene cloning high-fidelity enzyme (ApexHF HS DNA Polymerase FS Master Mix) and DNA gel recovery kit were purchased from Hunan Aikerui Bioengineering Co., Ltd.; restriction endonucleases were purchased from Thermo Fisher Technology (China) Co., Ltd.; DNA marker (BM5000/BM2000) was purchased from Bomed Biotechnology Co., Ltd.; plasmid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; RNA extraction kit, total RNA reverse transcription The test kit was purchased from TIANGEN Company. The nucleic acid dye Super GelRedTM was purchased from Suzhou Yuheng Biological Co., Ltd.; other drugs: agarose was purchased from Quanshijin Biological Co., Ltd., and peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropyl alcohol, sodium chloride, etc. were domestically produced. Analytically pure antibiotic powders such as kanamycin, rifampin, and acetosyringone were purchased from Beijing Solebao Technology Co., Ltd.

Solution preparation: Various reagents mentioned but not listed in this article are prepared according to the methods in the third edition of "Molecular Cloning Experiment Guide". Biochemical reagents are of analytical grade or above.

LB liquid medium: tryptone 10g/L, yeast extract 5g/L, sodium chloride (NaCl) 10g/L; LB solid medium: tryptone 10g/L, yeast Extract (Yeastextract) 5g/L, sodium chloride (NaCl) 10g/L, agar powder 15g/L, adjust the volume to 1L; LB selection medium: Before LB plating, wait for the medium to be autoclaved and cooled to 55 Add corresponding concentration of antibiotics at ℃, shake well and spread the plate.

Test strains: Sclerotinia sclerotiorum wild-type strain "1980" was preserved in the laboratory, Sclerotinia sclerotiorum "TL", the strain was cultured on PDA medium, the culture temperature was 22°C

Main instruments: PCR amplification instrument (BIO-RAD), high-speed centrifuge (Hettich MIKRO 200R), electrophoresis equipment (BIO-RAD), gel imaging system (BIO-RAD), fluorescence quantitative PCR instrument (ABI7500).

Example 1 Molecular network analysis of rice resistance to Sclerotinia sclerotiorum

Plant materials for testing:

The rice material was Jinhui 10, which was planted at the Rice Experimental Base of Southwest University. The S. sclerotiorum wild-type strain "1980" was cultured on PDA medium at a culture temperature of 22°C.

Method for inoculating rice with Sclerotinia sclerotiorum:

Sclerotinia sclerotiorum inoculation Rice inoculation: Cut the first two leaves of rice in the rice experimental field to inoculate Sclerotinia sclerotiorum. Cover the incisions of the rice leaves with moist cotton. When selecting the punching area, give priority to the S. sclerotiorum mycelium pieces with a diameter of 6 mm that grow evenly and vigorously. During inoculation, use tweezers to pick up the mycelial pieces so that the mycelial surface contacts the front of the rice leaves and avoids the leaf veins for inoculation. In the control group, blank PDA agar blocks without hyphae were used for inoculation, and the method was the same as above. Culture and inoculate in a humidified environment at 22°C for 1 to 4 days (Figure 1).

Transcriptome sequencing and analysis

The injured rice leaves were inoculated with blank PDA agar blocks (no inoculation control) and two S. sclerotiorum strains (1980 and TL). After removing the agar blocks and mycelium blocks 12 and 24 hours after inoculation, a total of 8 samples were collected. , sent to Biomic Biotechnology Company for transcriptome sequencing (RNA-seq). After quality assessment, the sequencing data were compared and analyzed with the rice reference genome (reference gene source: Oryzasativa; reference genome version: IRGSP-1.0; reference genome source: http://plants.ensembl.org/Oryza_sativa/Info/Index;). Gene expression levels and differentially expressed genes (DEGs) were analyzed (Figure 2), and finally KEGG enrichment analysis was performed on DEGs. As shown in Figure 3, compared with the uninoculated control, inoculated rice was promoted in biological processes such as ndole-containing compound biosynthetic process, tryptophan biosynthetic process, and pathways such as Glutathione metabolism and Phenylalanine biosynthesis. Further comparison with the transcriptome data of cabbage before and after inoculation with Sclerotinia sclerotiorum, as shown in Figure 4, the expression of GLP family genes (plant-like germination proteins) in rice was induced by the bacteria. However, the expression of this family gene was not significant after inoculation with host plants such as cabbage. Up-regulated, it is speculated that OsGLP has an important contribution to the resistance of Sclerotinia sclerotiorum, and OsGLP3-6 is used as a candidate gene for subsequent functional verification.

Example 2 Cloning of OsGLP3-6 gene in rice

(1) The test material Jinhui 10 was planted in the rice experimental base of Southwest University and was managed as a general field. The parts taken included roots, stems, leaves, flowers and seeds at different development stages. The taken materials were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for later use. Plant DNA was extracted using the modified CTAB method, and plant total RNA was extracted using the TIANGEN company kit.

(2) Reverse-transcribe 200ng RNA into cDNA, and dilute the reverse-transcription product cDNA solution 4 times as a PCR reaction template.

The extracted total rice RNA from each tissue was used as a template, and the TaKaRa reverse transcription kit was used to reverse-transcribe it into cDNA. The reaction system is shown in Table 1.

Table 1 Reaction system

Note: The reaction process is first incubated in the PCR instrument at 37°C for 15 minutes, then 85°C for 5 seconds, and finally stored at -20°C for later use.

(3) Perform PCR reaction to amplify the target gene

Oligo6 software was used to design primers for OsGLP3-6. The rice OsGLP3-6 gene was PCR amplified using the mixed cDNA obtained by reverse transcription as a template. The reaction system is shown in Table 2. Table 2 Target gene amplification system.

The conditions required for the PCR reaction are set to:

Primer sequence:

OsGLP3-6-F:5′-ATGGAGCACAGCTTCAAAAC-3′

OsGLP3-6-R:5′-TTAGTACCCGCCGGTGAATT-3′

(4) After the reaction, store it at 4°C and use 1% agarose electrophoresis for detection. If the band size is in line with the expected design, it is considered a valid result (Figure 5).

(5) Use a gel recovery kit to cut and recover the target fragment.

(6) Connect the product recovered from the above gel to the pBin35SRed vector and transform into E. coli.

(7) Overnight culture at 37°C. Pick single clones from the resistant LB medium and culture them in 600 μL LB medium containing Kan for 4 hours at 37°C with shaking.

(8) Bacterial liquid PCR verification, send bacterial liquid containing the size band of the target gene fragment for sequencing.

The AtGLP1 (AT1G72610.1) gene of Arabidopsis thaliana was homologously compared and identified in the rice database. After finding the corresponding target sequence, Oligo 6 software was used to design primers, and PCR (Polymerase Chain Reaction) technology was used to extract the gene from Jinhui 10. The complete CDS sequence of 690 bp (SEQ ID No. 1) was amplified, in which the open reading frame ORF was 690 bp, encoding 229 amino acid residues (SEQ ID No. 2).

Example 3 Construction of pBin35SRed::OsGLP3-6 overexpression vector

1) Plasmid extraction and enzyme digestion

The plasmid was extracted using a plasmid extraction kit from TIANGEN. The plasmid concentration was tested to be 210ng/μl. The agarose gel electrophoresis showed that there was no protein contamination and met the test requirements. Xba I-Sma I was used as the endonuclease for double enzyme digestion.

2) Construction of pBin35SRed::OsGLP3-6 overexpression vector

Amplification of target gene ORF sequence: Design Xba I-Sma I as insertion sites according to the map of the final vector pBin35SRed and synthesize primers. The primer sequences are as follows:

In-OsGLP3-6-F:5′-ATTTGGAGAGGACACGAATTCATGGAGC ACAGCTTCAAAAC-3′ (contains restriction site Xba I)

In-OsGLP3-6-R:5′-CCGCCTCGAGCCCGGGTCTAGATTAGTAC CCGCCGGTGAATT-3′ (containing restriction site Sma I)

Use high-fidelity enzymes to amplify the target fragments.

Table 3 Target gene amplification system

(1)PCR reaction procedure:

Pre-denaturation at 98°C for 5 minutes; cycle of denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, extension at 68°C for 1 minute, a total of 30 cycles; extension at 68°C for 5 minutes.

(2) Electrophoresis detection and recovery:

The PCR product was electrophoresed in a 1.8% agarose gel, the voltage was adjusted to 90V, electrophoresed for 1 hour, the results were observed under UV light, and the target band was quickly cut out. Use a gel recovery kit to recover the target fragment. The specific method is carried out according to the kit instructions.

(3) Fusion expression vector construction:

The Xba I-Sma I digested plasmid pBin35SRed was connected to the target gene OsGLP3-6, and the schematic diagram of the construction of the fusion expression vector is shown in Figure 6. The connection system is shown in Table 4.

Table 4 Connection system between target gene and pBin35SRed recombinant plasmid

a. Mix well by pipetting, centrifuge slightly and add a drop of mineral oil;

b. Connect at 16°C for 2 hours;

c. After the connection is completed, store it in a 4°C refrigerator overnight.

Transformation of E. coli with the ligation product

a. Sterilize the ultra-clean workbench for 30 minutes, take out 100 μL of E. coli competent cells from the -70°C ultra-low temperature refrigerator, place it on ice, and pre-cool for 10 minutes;

b. Then add 10 μL of ligation product, mix well with a pipette, and then incubate on ice for 30 minutes;

c. After the ice bath, place it in a constant temperature water bath at 42°C for 90 seconds, then quickly put it into ice cubes and bathe in ice for 2 minutes;

d. Aspirate 500 μL of LB liquid culture medium into the Ep tube, mix well, and place in a shaker at 160 rpm and shake at 37°C for 1 hour;

e. Take out the Ep tube at the end of the shaker, centrifuge at 2000-3000 rpm for 5 minutes, discard 300 μL of the supernatant, and gently pipette the remaining bacterial liquid to mix evenly, then add it to the LB solid culture dish containing Kan, and spread evenly with a glass applicator stick. Dry;

f. Cultivate in a constant temperature incubator at 37°C for 16 to 20 hours.

g. Randomly select single clones for identification. The results are shown in Figure 7.

The primer sequences used are:

pBin35sRed-F primer: 5’-CGCACAATCCCACTATCCTT-3’

pBin35sRed-R primer: 5'-AAAAGACAAAAGTGGGGTAG-3'

h. Expand the identified correct single clone in LB medium containing Kan, shake it at 190 rpm at 37°C overnight, extract the plasmid and send it to Qingke Company for sequencing and store it at 4°C.

Transformation of ligation products into Agrobacterium

a. Sterilize the ultra-clean workbench for 30 minutes, take out 100 µl GV3101 Agrobacterium competent cells from the -70°C ultra-low temperature refrigerator, and place them on ice;

b. After the competent state is half melted, add 100ng of recombinant plasmid to the Ep tube, and then incubate on ice for 10 minutes;

c. Followed by 5 minutes of liquid nitrogen, 5 minutes of 37°C water bath, and 5 minutes of ice bath;

d. Pipette 500 μL of LB liquid culture solution into the Ep tube, mix well, place in a shaker at 160 rpm, and incubate at 28°C for 2 hours;

e. Take out the Ep tube at the end of the shaker, take 200 μL of bacterial liquid, pipet it into an LB solid petri dish containing Kan/Rif, spread it evenly with a glass coating rod, and apply it dry;

f. Cultivate in a constant temperature incubator at 28°C for 24-36 hours.

g. Randomly select single clones for identification. The results are shown in Figure 8.

Example 4 Obtaining transgenic Arabidopsis thaliana

Wait for the wild-type Arabidopsis seeds (22°C, 16h light/8h dark) to enter the full flowering stage, then use the flower dipping method for transformation, and water the Arabidopsis appropriately the day before transformation. The positive Agrobacterium solution prepared in Example 3 was shaken overnight in Kan and Rif double-resistant liquid LB culture medium, and the concentration of the injection solution was adjusted to about OD600≈0.8 through the transformation medium, and left to stand in the dark for about 4 hours. The transformation of wild-type Arabidopsis thaliana adopts the flower dipping method. Use a pipette tip to absorb a little transformation medium containing Agrobacterium, drop it on the Arabidopsis thaliana flowers, and cultivate it in the dark and away from light for 24 hours. After culturing in the dark, take them out and cultivate according to normal culture conditions (22°C, 16h light/8h dark). After the fruit pods mature, the seeds of the T ₀ generation are mixed and harvested. Then the T ₀ generation seeds were screened on MS medium containing Kan, and three lines were randomly selected for further identification by qRT-PCR. It was found that the expression level of OsGLP3-6 in the Arabidopsis positive line was significantly increased (Figure 9 ).

Primer sequence:

qRT-GLP3-6-F:GAGCACAGCTTCAAAACCATAG

qRT-GLP3-6-R: TGGCAATCTTGGAGGAGAAG

At-Actin-F:AGAAACCCTCGTAGATTGGCAC

At-Actin-R:ACTCTCCCGCTATGTATGTCGC

Example 5 Resistance identification of transgenic Arabidopsis thaliana

Arabidopsis thaliana was sown on 1/2MS medium and placed in a greenhouse with a temperature of 22°C, an air humidity of 70%, a light intensity of 50 μmlo·m ^-2 ·s ^-1 , and a photoperiod of light:dark=16h:8:h. After germination, the seedlings were transplanted to nutrient soil for culture, and 30 days later, the seedlings were inoculated with S. sclerotiorum.

Take a square petri dish with a length and width of 90mm, place filter paper of similar size at the bottom, add an appropriate amount of water to soak the filter paper, select Arabidopsis leaves of similar size and flat surface, and lay them flat on the filter paper. Place a sterilized cotton swab at the leaf veins, use a pipette gun to absorb appropriate water to soak the cotton swab, keep the leaves moist, take a S. sclerotiorum bacteria block with a diameter of about 2.5mm, place it on the Arabidopsis leaves, and place it in the greenhouse After 36 hours of treatment, the area of the lesions was measured and photographed for recording.

Arabidopsis thaliana leaves were inoculated at the seedling stage, and the plaque area was statistically found. The results are shown in Figure 10. The plaque areas of the three OE_OsGLP3-6 transgenic lines were 65.9mm ² , 64.6mm ² and 65.1mm ² respectively. The average plaque area of the overexpression line (65.2mm ² ) was 22.4% smaller than that of the WT (79.9mm ² ). The above results indicate that overexpression of OsGLP3-6 can significantly improve the sclerotinia resistance of Arabidopsis thaliana.

Example 6 Resistance identification of transiently transformed tobacco

Prepare 500 ml of tobacco injection suspension, including 2-morpholinoethanesulfonic acid (MES, 1.02g), magnesium chloride hexahydrate (0.98g), and acetosyringone (As, 50mM). Add an appropriate amount of tobacco infection suspension to the centrifuge tube, suspend the Agrobacterium cell pellet carrying the pBin35SRed::OsGLP3-6 overexpression vector prepared in Example 3, adjust the OD600 of the bacterial solution to 1.0, and keep it in a 28°C incubator away from light. Incubate for 2h. Use a disposable sterile syringe to absorb the infection fluid and inject it from the back of the tobacco leaf. Place your index finger lightly on the front of the leaf at the injection site and slowly inject. The water-stained round spots will be visible to the naked eye and spread evenly from the injection site to the leaf. Use a marker pen to mark the back of the leaf. Mark the location of bacterial infection. After injection, the tobacco was sealed in a humid environment to avoid light and cultured for 36 to 48 hours.

Take a square petri dish with a length and width of 90mm, place filter paper of similar size at the bottom, add an appropriate amount of water to soak the filter paper, select 4-5 weeks old tobacco leaves of similar size and flat surface, and lay them flat on the On the filter paper, place a sterilized cotton swab on the leaf veins. Use a pipette gun to absorb appropriate water to soak the cotton swab to keep the leaves moist. Take a S. sclerotiorum bacteria block with a diameter of about 2.5mm and place it in the injection area. , count the plaque size after 36 hours, take samples and take photos and records.

Through statistics on the plaque area of tobacco leaves, the results are shown in Figure 11. The plaque area of the three leaves transiently expressing 35S::OsGLP3-6 was 35.45% smaller than that of CK. The above results showed that overexpression of OsGLP3-6 can significantly improve the resistance to Sclerotinia sclerotiorum.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims (5)

1. The application of the gene for encoding the rice OsGLP3-6 protein in regulating and controlling sclerotinia sclerotiorum resistance of sclerotinia sclerotiorum host plants is provided, wherein the amino acid sequence of the rice OsGLP3-6 protein is shown as SEQ ID No. 2.

2. The use according to claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 1.

3. Use according to claim 1 or 2, wherein the gene is transferred into a host plant gene and overexpressed in a transgenic plant to increase sclerotinia sclerotiorum resistance.

4. A method for improving sclerotinia sclerotiorum resistance of sclerotinia sclerotiorum host plants is characterized by transferring a vector containing a gene encoding rice OsGLP3-6 protein into a plant genome and over-expressing the vector in a transgenic plant, wherein the amino acid sequence of the rice OsGLP3-6 protein is shown as SEQ ID No. 2.

5. The method of claim 4, wherein the nucleotide sequence of the gene is set forth in SEQ ID No. 1.