CN116355067A - Rice OsGLP8-12 inhibiting sclerotinia and its application - Google Patents

Abstract

The invention belongs to the field of plant molecular biology, and particularly relates to an OsGLP8-12 inhibiting sclerotinia and application thereof. The rice OsGLP8-12cDNA sequence is shown as SEQ ID No.1, and the encoded protein amino acid sequence is shown as SEQ ID No. 2. The invention is different in arabidopsis thalianaThe source over-expressed OsGLP8-12 gene is cultivated to T after positive plants are identified by utilizing fluorescence quantitative means ₃ For the generation, the positive lines were then inoculated with leaf sclerotinia. The results show that the average plaque area (31.8 mm) of the Arabidopsis thaliana overexpression line OE_OsGLP8-12 ² ) Plaque area (47.0 mm compared to wild-type WT ² ) The reduction by 32.2%, extremely remarkable (p<0.01 Increased resistance of arabidopsis thaliana to sclerotinia sclerotiorum.

Description

Rice OsGLP8-12 inhibiting Sclerotinia and its application

technical field

The invention belongs to the field of plant molecular biology, and in particular relates to OsGLP8-12 for inhibiting sclerotinia and application thereof.

Background technique

Sclerotiorum (S. sclerotiorum) is a saprophytic filamentous fungus with a wide range of pathogenicity. S. sclerotiorum can infect more than 500 kinds of plants (Liang et al., 2018), including dicotyledonous rapeseed, cabbage, cabbage, etc., which has caused serious losses to our agricultural production (Boland et al., 2009). Due to the limitations of conventional breeding methods, disease resistance breeding has always been a major problem in agricultural production. For Sclerotinia, a variety of grasses, especially rice, were identified as non-host plants of S. sclerotiorum very early (Purdy, 1979), using the non-host resistance mechanism of rice-related genes, for rape, etc. The improvement of Sclerotinia resistance in host plants provides a new resource of disease resistance genes.

Germin, first discovered in wheat, is a water-soluble glycoprotein commonly found in exosomes or extracellular matrix. GLPs in plants generally have three functions: true germin (truegermin), plant immune response and regulation of plant growth and development (circadian rhythm, flowering) functions, etc. (Sawsan et al., 2001). OsGLPs in transgenic rice can enhance resistance to bacterial bacterial blight and fungal pathogens by affecting defense-related genes associated with JA-dependent pathways while increasing endogenous JA levels (Liu et al., 2016). Recently, researchers overexpressed soybean GmGLP7 into Arabidopsis thaliana, and observed the offspring phenotypes, and found that the positive line showed increased salt tolerance to salt, drought and oxidative stress, and was highly sensitive to exogenous ABA (Li et al., 2022) while GLP GmGLP9 significantly increased tolerance to salt stress in tobacco (Mo et al., 2010).

Contents of the invention

The object of the present invention is to provide rice OsGLP8-12 and its encoded protein and application.

First, the present invention provides rice OsGLP8-12 protein, which is:

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

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

The present invention also provides the gene encoding the protein. Preferably, the sequence of the gene is shown in SEQ ID No.1.

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

The present invention also provides the use of the gene in regulating the Sclerotinia host plant resistance to Sclerotinia sclerotiorum.

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

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

In this study, the candidate gene OsGLP8-12 was selected by analyzing the RNA-seq of rice inoculated with S. sclerotiorum and combining the transcriptome data of host plants such as cabbage. The OsGLP8-12 gene was heterologously overexpressed in Arabidopsis thaliana, and the positive plants were identified by fluorescence quantitative means, cultured to the T ₃ generation, and then the leaves of the positive lines were inoculated with Sclerotinia sclerotiorum. The results showed that the average plaque area (31.8mm ² ) of the Arabidopsis overexpression line OE_OsGLP8-12 was 32.2% lower than that of the wild-type WT (47.0mm ² ), which was significantly (p<0.01) increased. Resistance of Arabidopsis thaliana to Sclerotinia sclerotiorum.

Description of drawings

Figure 1 shows the phenotype observation of rice leaves after inoculation with wounded and non-wounded. -w: non-wounded treatment; +w: wounded treatment.

Figure 2 shows the differentially expressed genes of the samples.

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

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

Figure 5 shows the cloning of the OsGLP8-12 gene.

Figure 6 is a schematic diagram of the construction of the fusion vector.

Figure 7 shows the identification of the target fragment in Escherichia coli. (M=Marker 2000; lane1-4 are positive clones).

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

Figure 9 shows the identification of the expression level of OsGLP8-12 in transgenic Arabidopsis.

Figure 10 shows the identification of Sclerotinia resistance in transgenic Arabidopsis thaliana.

Figure 11 shows the identification of resistance in transiently transformed tobacco.

Detailed ways

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

Enzymes and kits: gene cloning high-fidelity enzyme (ApexHF HS DNA Polymerase FS Master Mix), 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 Biomed Biotechnology Co., Ltd.; plasmid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; RNA extraction kit, total RNA reverse transcription The kit was purchased from TIANGEN Company. Nucleic acid dye Super GelRedTM was purchased from Suzhou Yuheng Biological Co., Ltd.; other drugs: agarose was purchased from Quanshijin Biological Company, and peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, and sodium chloride were domestically produced Analytical pure, kanamycin, rifampicin, and acetosyringone and other antibiotic powders were purchased from Beijing Suolaibao Technology Co., Ltd.

Solution preparation: All reagents mentioned but not listed in this article were prepared according to the method in the third edition of "Molecular Cloning Experiment Guide", and the biochemical reagents were of analytical grade or above.

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

Tested strains: Sclerotinia wild-type strain "1980" was preserved in the laboratory, Sclerotinia "TL", the strain was cultured on PDA medium, and 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), fluorescent quantitative PCR instrument (ABI7500).

Example 1 Molecular Network Analysis of Rice Resistance to Sclerotinia

Test plant material:

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

Method of inoculating rice with Sclerotinia sclerotiorum:

Sclerotinia inoculation Rice inoculation: cut the second leaf of rice in the rice experimental field for Sclerotinia inoculation. Cover the incision of the rice leaf with moistened cotton, and when selecting the perforated area, preferentially select the Sclerotinia mycelium block with uniform and vigorous mycelium growth, with a diameter of 6 mm. When inoculating, clamp the mycelium block with tweezers so that the mycelium surface touches the front of the rice leaf, avoiding the leaf veins for inoculation. The control group was inoculated with blank PDA agar blocks without hyphae, and the method was the same as above. 22 ℃ moisture culture inoculation 1 ~ 4d (Figure 1).

Transcriptome sequencing and analysis

The wounded rice leaves were inoculated with blank PDA agar blocks (no inoculation control), two Sclerotinia strains (1980 and TL), and the agar blocks and mycelium blocks were removed 12 and 24 hours after inoculation, and a total of 8 samples were obtained. , sent to Baimic Biotechnology Company for transcriptome sequencing (RNA-seq). After quality assessment, the sequencing data was compared 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;) comparison analysis, Gene expression levels and differentially expressed genes (DEGs) were analyzed (Figure 2), and finally DEGs were subjected to KEGG enrichment analysis. As shown in Figure 3, biological processes such as ndole-containing compound biosynthetic process, tryptophan biosynthetic process, and pathways such as Glutathione metabolism and Phenylalanine biosynthesis were promoted in the inoculated rice compared with the uninoculated control. Further comparison with the transcriptome data before and after inoculation of cabbage with S. sclerotiorum, as shown in Figure 4, the expression of GLP family genes (plant germination-like proteins) in rice was induced by bacteria, but the family genes were not significantly expressed after inoculation of host plants such as cabbage. It is speculated that OsGLP has an important contribution to the resistance of S. sclerotiorum, and OsGLP8-12 is used as a candidate gene for subsequent functional verification.

Cloning of OsGLP8-12 gene in rice in embodiment 2

(1) The test material Jinhui 10 was planted in the rice experiment base of Southwest University and managed as a general field. The parts taken include roots, stems, leaves, flowers and seeds of different developmental 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 improved CTAB method, and plant total RNA was extracted using TIANGEN kits.

(2) 200ng RNA was reverse-transcribed into cDNA, and the cDNA solution of the reverse-transcribed product was diluted 4 times as a PCR reaction template.

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

Table 1 reaction system

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

(3) Perform PCR reaction to amplify the target gene

Oligo6 software was used to design the primers of OsGLP8-12. Using the mixed cDNA obtained by reverse transcription as a template, the rice OsGLP8-12 gene was amplified by PCR. The reaction system is shown in Table 2.

Table 2 Target gene amplification system

ingredients Volume/uL 2×PCR Buffer 25.0 2mM dNTPs 10.0 primers (10μM) 1.0 template 2.0 KOD FX Neo 1.0 ddH ₂ O up to 50

The conditions required for the PCR reaction were set as:

Primer sequence:

OsGLP8-12-F:5′-ATGGCCTCCTCTTCCTTATT-3′

OsGLP8-12-R:5′-TCAGTAGTTGTTCTCCCAGA-3′

(4) Store at 4° C. after the reaction, and detect with 1% agarose electrophoresis. If the band size meets the expected design, it is considered a valid result ( FIG. 5 ).

(5) Use the gel recovery kit to recover the target fragments by cutting the gel.

(6) The product recovered from the above gel was connected to the pBin35SRed vector and transformed into Escherichia coli.

(7) Overnight culture at 37°C Pick a single clone from the resistant LB medium and place it in 600 μL of Kan-containing LB medium for shaking at 37°C for 4 hours.

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

The AtGLP1 (AT1G72610.1) gene of Arabidopsis thaliana was homologously compared and identified in the rice database, and after finding the corresponding target sequence, primers were designed using Oligo 6 software, and PCR (Polymerase Chain Reaction) technology was used to extract from Jinhui 10 A complete CDS sequence of 675bp (SEQ ID No.1) was amplified, wherein the open reading frame ORF was 675bp, encoding 224 amino acid residues (SEQ ID No.2).

Example 3 Construction of pBin35SRed::OsGLP8-12 overexpression vector

1) Plasmid extraction and digestion

The plasmid was extracted using the plasmid extraction kit from TIANGEN Company. The concentration of the plasmid was detected to be 210ng/μl. It was detected by agarose gel electrophoresis, and there was no protein contamination, which met the test requirements. Xba I-Sma I was used as the endonuclease for double digestion.

2) Construction of pBin35SRed::OsGLP8-12 overexpression vector

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

In-OsGLP8-12-F: 5′-ATTTGGAGAGGACACGAATTCATGGCCT CCTCTTCCTTATT-3′ (contains restriction site Xba I)

In-OsGLP8-12-R: 5′-CCGCCTCGAGCCCGGGTCTAGATCAGT AGTTGTTCTCCCAGA-3′ (with enzyme cutting site Sma I)

Use high-fidelity enzymes to amplify the target fragment.

Table 3 Target gene amplification system

(1) PCR reaction procedure:

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

(2) Electrophoresis detection and recovery:

The PCR product was electrophoresed on 1.8% agarose gel, and the voltage was adjusted to 90V, and the electrophoresis was performed for 1 hour. The result was observed under ultraviolet light, and the target band was cut off quickly. The target fragment was recovered with a gel recovery kit, and the specific method was carried out according to the kit instructions.

(3) Fusion expression vector construction:

The schematic diagram of the construction of the fusion expression vector by connecting the plasmid pBin35SRed digested with Xba I-Sma I to the target gene OsGLP8-12 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. Suction and beat to mix, then add a drop of mineral oil after a little centrifugation;

b. Connect at 16°C for 2 hours;

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

Ligation product transformed into Escherichia coli

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

b. Then add 10 μL of the ligation product, mix with a pipette gun, and then ice-bath for 30 minutes;

c. After the ice bath is over, 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 LB liquid culture solution into the Ep tube, mix well, place on a shaker at 160 rpm, and shake at 37°C for 1 hour;

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

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

g. Randomly pick 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. Propagate the identified correct single clone in LB medium containing Kan, overnight at 37°C on a shaker at 190rmp, extract the plasmid and send it to Qingke Company for sequencing and store it at 4°C.

Ligation product transformed into Agrobacterium

a. Sterilize the ultra-clean workbench for 30 minutes, take out 100 μlGV3101 Agrobacterium competent cells from the -70 ℃ ultra-low temperature refrigerator, and put them on ice;

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

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

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 the bacterial solution, pipette it into the LB solid culture dish containing Kan/Rif, spread it evenly with a glass stick, and dry it;

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

g. Randomly pick single clones for identification, the results are shown in Figure 8.

Embodiment 4 Acquisition of transgenic Arabidopsis

After waiting for the wild-type Arabidopsis seed (22°C, 16h light/8h dark) to enter the full flowering stage, the flower soaking method was used for transformation, and the Arabidopsis was properly watered one day before the transformation. The positive Agrobacterium solution prepared in Example 3 was shaken overnight in Kan and Rif double-resistant liquid LB 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 method of dipping flowers. Use a pipette tip to absorb a little transformation medium containing Agrobacterium, drop it on Arabidopsis flocs, and incubate in the dark for 24 hours. After cultivating in the dark, take them out and culture them under normal culture conditions (22°C, 16h light/8h dark), and harvest the seeds of the T ₀ generation after the fruit pods mature. Then, the T0 generation seeds were screened on the MS medium containing Kan, and further identified by qRT-PCR. It was found that the expression level of the Arabidopsis positive line OsGLP8-12 was significantly increased ( FIG. 9 ).

Primer sequence:

qRT-GLP8-12-F: CCTCCTCTTCCTTATTTCTCCTG

qRT-GLP8-12-R: CAAATCCATTCACTCGCACAG

At-Actin-F: AGAAACCCTCGTAGATTGGCAC

At-Actin-R: ACTCTCCCGCTATGTATGTCGC

Example 5 Resistance Identification of Transgenic Arabidopsis

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

Take a square petri dish with a length and width of 90 mm, place filter paper of similar size on the bottom, add an appropriate amount of water to soak the filter paper, select Arabidopsis leaves with similar size and flat surface, and spread them on the filter paper. Place a sterilized cotton sliver on the leaf veins, soak the sliver with appropriate amount of water with a pipette gun to keep the leaves moist, take a sclerotinia block with a diameter of about 2.5 mm, place it on the leaves of Arabidopsis thaliana, and place it in the greenhouse After 36 hours of medium treatment, the lesion area was measured and recorded by photographing.

Live leaf inoculation was carried out on Arabidopsis thaliana at the seedling stage, and the results were shown in Figure 10 through the statistics of the plaque area. The average plaque area (38.1mm ² ) of the OE_OsGLP8-12 transgenic overexpression line was 32.2% smaller than that of the WT (47.0mm ² ). sex.

Example 6 Resistance Identification of Transiently Transformed Tobacco

A 500 ml tobacco injection suspension was prepared including 2-morpholineethanesulfonic acid (MES, 1.02 g), magnesium chloride hexahydrate (0.98 g) and acetosyringone (As, 50 mM). Add an appropriate amount of tobacco infection suspension to the centrifuge tube, suspend the Agrobacterium cell precipitate carrying the pBin35SRed::OsGLP8-12 overexpression vector prepared in Example 3, adjust the OD600 of the bacterial solution to 1.0, and keep the incubator at 28°C away from light Incubate for 2h. Use a disposable sterile syringe to suck up the infection liquid and inject it from the back of the tobacco leaves. Lightly touch the front of the leaf at the injection site with your index finger and inject slowly. The water-soaked round spots can be seen spreading evenly from the injection site to the leaves with the naked eye. Use a marker pen on the back of the leaves Mark the location of bacterial infection. After injection, the tobacco was sealed in a humid environment and cultured for 36-48 hours in the dark.

Take a square Petri dish with a length and a width of 90 mm, place filter paper of similar size on the bottom, add an appropriate amount of water to soak the filter paper, select 4-5 weeks of tobacco leaves with similar size and flat surface, and spread it on the On the filter paper, place a sterilized cotton sliver at the leaf vein, soak the sliver with appropriate water with a pipette gun to keep the leaves moist, take a sclerotinia block with a diameter of about 2.5mm, and place it in the area where the bacterial solution is injected After 36 hours, the plaque size was counted, samples were taken and photographed for record.

The results are shown in Figure 11 through the statistical discovery of the bacterial plaque area of the tobacco leaves. The plaque area of the four leaves transiently expressing 35S::OsGLP8-12 was 35.42% smaller than that of CK. The above results all indicated that the overexpression of OsGLP8-12 could 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 on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (9)

1. Rice OsGLP8-12 protein, which is: 1) A protein consisting of amino acids shown in SEQ ID No.2; or 2) A protein derived from 1) that is substituted, deleted or added with one or several amino acids in the amino acid sequence shown in SEQ ID No. 2 and has equivalent activity. 2. The gene encoding the rice OsGLP8-12 protein according to claim 1. 3. The gene according to claim 2, wherein the sequence is as shown in SEQ ID No.1. 4. A vector comprising the gene of claim 2 or 3. 5. A host cell comprising the vector of claim 4. 6. The engineering bacterium containing the gene described in claim 2 or 3. 7. The use of the gene according to claim 2 or 3 in regulating Sclerotinia host plant Sclerotinia resistance. 8. The use according to claim 7, characterized in that the gene is transferred into a host plant gene and overexpressed in the transgenic plant to improve plant Sclerotinia resistance. 9. A method for improving Sclerotinia host plant resistance to Sclerotinia sclerotiorum, characterized in that, the carrier containing the gene described in claim 2 or 3 is transferred into the plant genome, and overexpressed in the transgenic plant .

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