CN116444636B

CN116444636B - Rice OsGLP3-6 for inhibiting sclerotinia and application thereof

Info

Publication number: CN116444636B
Application number: CN202310411370.3A
Authority: CN
Inventors: 梅家琴; 杨书贤; 左香君; 钱伟
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2023-04-17
Filing date: 2023-04-17
Publication date: 2024-02-20
Anticipated expiration: 2043-04-17
Also published as: CN116444636A

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 for inhibiting sclerotinia and application thereof

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to OsGLP3-6 for inhibiting sclerotinia and application thereof.

Background

Sclerotinia sclerotiorum (s.sclerotiorum) is a saprophytic filamentous fungus with a broad pathogenic range. Sclerotinia can infect more than 500 plants (Liang et al, 2018), including dicotyledonous plants rape, cabbage, etc., with severe losses for our agricultural production (Boland et al, 2009). The plant cell wall degrading enzyme secreted protein secreted by the sclerotinia sclerotiorum helps pathogenic bacteria to break through a barrier by degrading the plant cell wall and promotes the invasion of hyphae. For sclerotinia, a variety of gramineous plants, particularly rice, were identified as non-host plants of sclerotinia for a long time (Purdy, 1979), and a novel disease resistance gene resource is provided for improving sclerotinia resistance of host plants such as rape by utilizing a non-host resistance mechanism of rice-related genes.

In recent years, studies of plant germination proteins (GLPs) have been reported in plants such as arabidopsis thaliana (Arabidopsis thaliana), rice (Oryza sativa), and soybean (Glycine max). Studies have shown that part AtGLPs, osGLPs (Li et al 2016) and ZmGLPs (Fan et al 2005) are highly expressed in seed germination and young tissues, possibly involved in plant growth. More researches show that the plants are infected by pathogenic bacteria and bitten by herbivorous insects, the expression quantity of GLPs genes in the plants is increased, and the enzyme activities (SOD and OXO) of GLP family members are also increased. Play an important role in the management of pathogenic bacterial infections and some abiotic stresses (Zhang et al, 2017; pei et al, 2019;Banergee et al, 2017).

Disclosure of Invention

The invention aims to provide rice OsGLP3-6, and a coded protein and application thereof.

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

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

2) A protein derived from 1) which has equivalent activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.

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

The invention also provides a vector, a host cell and engineering bacteria containing the gene.

The invention also provides application of the gene in regulating and controlling sclerotinia sclerotiorum host plant sclerotinia sclerotiorum resistance.

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

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

The invention analyzes RNA-seq of rice inoculated sclerotinia and combines transcriptome data of host plants such as cabbage to select candidate gene OsGLP3-6. Heterogenous overexpression of OsGLP3-6 gene in Arabidopsis thaliana, identification of positive plants by fluorescence quantitative means, and cultivation 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.

Drawings

FIG. 1 shows phenotypic observations after wounded and non-wounded inoculation of rice leaves. -w: atraumatic treatment; +w: and (5) wound treatment.

FIG. 2 shows the differential expression of genes in samples.

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

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

FIG. 5 shows cloning of OsGLP3-6 gene.

FIG. 6 is a schematic diagram showing construction of a fusion vector.

FIG. 7 shows the identification of a fragment of interest in E.coli. (m=marker 2000; lane1-6 is a positive clone).

FIG. 8 shows the identification of a fragment of interest in Agrobacterium. (m=marker 2000; lane1-6 is a positive clone).

FIG. 9 shows the identification of the expression level of OsGLP3-6 in transgenic Arabidopsis thaliana.

FIG. 10 shows identification of resistance to transgenic Arabidopsis thaliana sclerotium disease.

FIG. 11 shows resistance identification of transiently transformed tobacco.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 2001), or in accordance with the manufacturer's instructions.

Enzyme and kit: the gene cloning high-fidelity enzyme (ApexHF HS DNA Polymerase FS Master Mix) and the DNA gel recovery kit are purchased from Hunan Ai Kerui bioengineering Co., ltd; restriction enzymes were purchased from sameimers technology (china) limited; DNA markers (BM 5000/BM 2000) were purchased from Bomaide Biotechnology Co., ltd; plasmid extraction kits were purchased from Tiangen Biochemical technologies (Beijing); RNA extraction kit and total RNA reverse transcription kit were purchased from TIANGEN company. Nucleic acid dye Super GelRedTM was purchased from Suzhou Yuheng Co., ltd; other drugs: agarose is purchased from full gold biosystems, peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, etc. are commercially available as analytically pure, antibiotic powders such as kanamycin, rifampin, acetosyringone, etc. are purchased from Beijing Soy Bao technology Co.

Preparing a solution: the various reagents mentioned herein but not listed are formulated as described in third edition of the guidelines for molecular cloning experiments, with biochemical reagents being analytically pure or superior.

LB liquid medium: tryptone (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L; LB solid medium: 10g/L of Tryptone (Tryptone), 5g/L of Yeast extract (Yeast extract), 10g/L of sodium chloride (NaCl) and 15g/L of agar powder, and fixing the volume to 1L; LB selection Medium: before LB plate laying, adding antibiotics with corresponding concentration when the culture medium is sterilized under high pressure and cooled to 55 ℃, shaking uniformly, and then plate laying.

Test strain: the wild strain "1980" of sclerotinia sclerotiorum is preserved in laboratory, and the strain "TL" of sclerotinia sclerotiorum is cultured on PDA culture medium at 22 DEG C

The main instrument is as follows: PCR amplification apparatus (BIO-RAD), high-speed centrifuge (Hettich MIKRO 200R), electrophoresis apparatus (BIO-RAD), gel imaging system (BIO-RAD), fluorescent quantitative PCR apparatus (ABI 7500).

Example 1 disease resistance molecular network analysis of Rice against Sclerotinia

Test plant material:

the rice material is Hui 10, and is planted in a rice test base of southwest university, and the sclerotinia sclerotiorum wild type strain '1980' is cultured on a PDA culture medium at a temperature of 22 ℃.

The method for inoculating sclerotinia rice comprises the following steps:

seed-rice inoculation of sclerotinia: and cutting out two inverted rice leaves in a rice experimental field to inoculate sclerotinia. The rice leaf incision is covered with moist cotton, and when selecting the punching area, the sclerotinia sclerotiorum mycelium blocks with uniform and vigorous mycelium growth and diameter of 6mm are preferentially selected. During inoculation, the tweezers clamp the mycelium blocks to enable the mycelium surfaces to contact the front surfaces of rice leaves, and the veins are avoided for inoculation. The control group was inoculated with a blank PDA agar block without hyphae, as described above. 22 ℃ for 1-4 d (figure 1).

Transcriptome sequencing and analysis

Post-wounded rice leaves were inoculated with a blank PDA agar block (no control), two sclerotinia strains (1980 and TL), and after removal of the agar block and hyphal block 12 and 24 hours post-inoculation, a total of 8 samples were sent to Baimichael Biotechnology company for transcriptome sequencing (RNA-seq). The sequencing data were compared to a rice reference genome (reference gene source: oryza sativa; reference genome version: IRGSP-1.0; reference genome source: http:// plants. Ensembl. Org/Oryza_sativa/Info/Index; FIG. 2) for analysis of gene expression levels and Differentially Expressed Genes (DEGs) (FIG. 2), and finally KEGG enrichment analysis was performed on the DEGs. As shown in FIG. 3, inoculated rice was promoted in the biological processes of ndole-containing compound biosynthetic process, tryptophan biosynthetic process, etc., and in the pathways of Glutathione metabolism and Phenylalanine biosynthesis, etc., compared to the uninoculated control. Further, compared with transcriptome data before and after the cabbage is inoculated with sclerotinia, as shown in fig. 4, GLP family genes (plant germination proteins) in rice are subjected to bacteria-induced up-regulation expression, however, the family genes are not significantly up-regulated after the cabbage and other host plants are inoculated, and it is presumed that OsGLP has a certain important contribution to the resistance of sclerotinia, and OsGLP3-6 is used as a candidate gene for subsequent functional verification.

EXAMPLE 2 cloning of OsGLP3-6 Gene in Rice

(1) Test material Hui 10 was planted in southwest university rice test base and managed according to the general field. The obtained part contains root, stem, leaf, flower and seeds of different development period, and the obtained material is rapidly put into liquid nitrogen for freezing, and stored in a refrigerator at-80 ℃ for standby. The plant DNA extraction adopts an improved CTAB method, and the total plant RNA extraction adopts a TIANGEN company kit.

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

The extracted total RNA of rice of each tissue is used as a template, and is reversely transcribed into cDNA by using a reverse transcription kit of TaKaRa, and the reaction system is shown in Table 1.

TABLE 1 reaction system

Remarks: the reaction process is carried out by incubating at 37deg.C for 15min, then preserving at 85deg.C for 5s and at-20deg.C

(3) Amplifying target gene by PCR reaction

Primers for OsGLP3-6 were designed using Oligo6 software. And carrying out PCR amplification on the rice OsGLP3-6 gene by taking the mixed cDNA obtained by reverse transcription as a template. The reaction system is shown in Table 2 and the target gene amplification system in Table 2

The conditions required for the PCR reaction were set as follows:

primer sequence:

OsGLP3-6-F:5′-ATGGAGCACAGCTTCAAAAC-3′

OsGLP3-6-R:5′-TTAGTACCCGCCGGTGAATT-3′

(4) After the completion of the reaction, the sample was stored at 4℃and detected by 1% agarose electrophoresis, and the band size was found to be effective when the expected design was satisfied (FIG. 5).

(5) And (5) performing gel cutting recovery on the target fragment by using a gel recovery kit.

(6) The products recovered from the above gel were ligated with pBin35SRed vector and transformed into E.coli.

(7) Overnight culture at 37℃from resistant LB medium was selected monoclonal to Kan-containing 600. Mu.L LB medium, and cultured at 37℃for 4 hours with shaking.

(8) And (3) performing PCR verification on bacterial liquid, and delivering bacterial liquid containing the size band of the target gene fragment for sequencing.

Homologous comparison and identification are carried out on the AtGLP1 (AT1G72610.1) gene of arabidopsis thaliana in a rice database, after a corresponding target sequence is found, primers are designed by adopting Oligo6 software, and a PCR (Polymerase Chain Reaction) technology is adopted to amplify a complete CDS sequence 690bp (SEQ ID No. 1) from R10, wherein an open reading frame ORF is 690bp, and 229 amino acid residues (SEQ ID No. 2) are encoded.

Example 3 construction of pBin35SRed: osGLP3-6 overexpression vector

1) Plasmid extraction and enzyme digestion

The plasmid extraction adopts TIANGEN company plasmid extraction kit, the plasmid concentration is detected to be 210 ng/. Mu.l, the agarose gel electrophoresis detection shows that the plasmid has no protein pollution, the test requirement is met, and the incision enzyme adopts Xba I-Sma I for double enzyme digestion.

2) Construction of pBin35SRed: osGLP3-6 overexpression vector

Amplification of ORF sequence of the target gene: xba I-Sma I is designed as an insertion site according to the map of the final vector pBin35SRed and a primer is synthesized, wherein the primer sequence is as follows:

In-OsGLP 3-6-F5'-ATTTGGAGAGGACACGAATTCATGGAGC ACAGCTTCAAAAC-3' (containing the cleavage site Xba I)

In-OsGLP 3-6-R5'-CCGCCTCGAGCCCGGGTCTAGATTAGTAC CCGCCGGTGAATT-3' (containing restriction enzyme cleavage site Sma I)

Amplifying by using high-fidelity enzyme to obtain the target fragment.

TABLE 3 target gene amplification System

(1) PCR reaction procedure:

pre-denaturation at 98 ℃ for 5min; the cycle is denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s and extension at 68 ℃ for 1min, and 30 cycles are taken; extending at 68℃for 5min.

(2) Electrophoresis detection and recovery:

the PCR product was electrophoresed on a 1.8% agarose gel, the voltage was adjusted to 90V, the electrophoresis was carried out for 1h, and the result was observed under an ultraviolet lamp, and the target band was cut off rapidly. The target fragment is recovered by using a gel recovery kit, and the specific method is carried out according to the kit instruction.

(3) Fusion expression vector construction:

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

Table 4 Gene of interest pBin35SRed recombinant plasmid ligation system

a. Sucking, beating, mixing, slightly centrifuging, and adding a drop of mineral oil;

b.16 ℃ for 2h;

c. after the connection, the mixture is put into a refrigerator at 4 ℃ for storage overnight.

Ligation product transformation of E.coli

a. Sterilizing with ultra-clean bench for 30min, taking out 100 μl of competent cells of Escherichia coli from-70deg.C ultra-low temperature refrigerator, placing on ice, and pre-cooling for 10min;

b. then adding 10 mu L of the connection product, sucking and beating the connection product by a pipetting gun, and then carrying out ice bath for 30min;

c. after ice bath is finished, placing the ice bath into a constant-temperature water bath kettle at 42 ℃ for heat shock for 90 seconds, and then rapidly placing the ice bath kettle into ice cubes for 2 minutes;

d. sucking 500 μl of LB liquid culture solution into Ep tube, mixing, placing into a shaking table at 160rpm, and shaking at 37deg.C for 1 hr;

e. taking out an Ep tube finished by a shaking table, centrifuging for 5min at 2000-3000 rmp, discarding 300 mu L of supernatant, gently sucking and beating the rest bacterial liquid, mixing uniformly, adding the bacterial liquid into an LB solid culture dish containing Kan, uniformly coating by using a glass coating rod, and drying;

f, culturing in a constant temperature incubator at 37 ℃ for 16-20 h.

g. The monoclonal was randomly picked for identification and the results are shown in FIG. 7.

The primer sequences used were:

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

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

h. The correct monoclonal was identified and propagated in LB medium containing Kan, on a 190rmp shaker at 37℃overnight, and the extracted plasmid was sequenced and stored at 4 ℃.

Ligation product transformation of Agrobacterium

a. Sterilizing with ultra-clean bench for 30min, taking out competent cells of 100 μm lGV3101 Agrobacterium from-70deg.C ultra-low temperature refrigerator, and placing on ice;

b. after waiting for competent slush, 100ng of recombinant plasmid was added to the Ep tube, followed by an ice bath for 10min;

c. immediately after liquid nitrogen for 5min, water bath is carried out for 5min at 37 ℃ and ice bath is carried out for 5min;

d. sucking 500 mu L of LB liquid culture solution into the Ep tube, uniformly mixing, and placing in a shaking table at 160rpm for 2h at 28 ℃;

e. taking out the Ep tube ending the shaking table, taking 200 mu L of bacterial liquid, sucking the bacterial liquid into an LB solid culture dish containing Kan/Rif, uniformly coating the bacterial liquid with a glass coating rod, and drying the bacterial liquid;

culturing in a constant temperature incubator at 28 ℃ for 24-36h.

g. The monoclonal was randomly picked for identification and the results are shown in FIG. 8.

EXAMPLE 4 acquisition of transgenic Arabidopsis thaliana

After the wild arabidopsis seeds (22 ℃ C., 16h light/8 h dark) are cultivated and enter the full bloom stage, the arabidopsis seeds are transformed by adopting a flower dipping method, and the arabidopsis seeds are appropriately 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 injection concentration was adjusted to OD600 approximately 0.8 by transformation medium, and left to stand in the dark for about 4 hours. The transformation of the wild arabidopsis adopts a dipping method, a gun head is used for sucking a little agrobacterium-containing transformation medium, the medium is dripped on the arabidopsis flower batting, and the medium is cultivated for 24 hours in dark. Taking out after light-shielding culture, culturing under normal culture conditions (22deg.C, 16h light/8 h dark), mixing and collecting T after pod maturation ₀ Seed of the generation. Then T is taken up ₀ After the generation seeds are screened on MS culture medium containing Kan, three strains are randomly selected and further subjected to qRT-PCR for identification, and the expression level of the Arabidopsis positive strain OsGLP3-6 is obviously improved (figure 9).

Primer sequence:

qRT-GLP3-6-F：GAGCACAGCTTCAAAACCATAG

qRT-GLP3-6-R：TGGCAATCTTGGAGGAGAAG

At-Actin-F：AGAAACCCTCGTAGATTGGCAC

At-Actin-R：ACTCTCCCGCTATGTATGTCGC

example 5 identification of resistance of transgenic Arabidopsis thaliana

Sowing Arabidopsis thaliana on 1/2MS culture medium, placing at temperature 22 deg.C, air humidity 70% and illumination intensity 50 mu mlo.m ^-2 ·s ^-1 Photoperiod is illumination: dark = 16h:8:h, transplanting the seedlings to nutrient soil for culturing after germination, and inoculating sclerotinia after 30 d.

Square petri dishes with the length and the width of 90mm are taken, filter paper with similar size is placed at the bottom of the square petri dishes, a proper amount of water is added to soak the filter paper, arabidopsis thaliana leaves with similar size and flat surface are selected and laid on the filter paper, a sterilizing cotton sliver is placed at the leaf veins, a liquid-transfering gun is used for sucking proper water to soak the cotton sliver, the leaves are kept moist, sclerotinia sclerotiorum bacterial block with the diameter of about 2.5mm is taken, the nuclear sclerotinia sclerotiorum bacterial block is placed on the Arabidopsis thaliana leaves, and after the treatment in a greenhouse for 36 hours, the area of the disease spots is measured and photographed and recorded.

Leaf inoculation of arabidopsis was performed during the seedling stage, and the results were found by counting the area of plaque, and are shown in fig. 10. Plaque areas of three OE_OsGLP3-6 transgenic lines are 65.9mm respectively ² 、64.6mm ² And 65.1mm ² Average plaque area of the over-expression system (65.2 mm ² ) Specific WT (79.9 mm) ² ) The reduction of 22.4 percent shows that the overexpression of OsGLP3-6 can obviously improve the sclerotinia resistance of arabidopsis thaliana.

Example 6 identification of resistance to transient transformed tobacco

500ml of tobacco injection suspension was prepared, comprising 2-morpholinoethanesulfonic acid (MES, 1.02 g), magnesium chloride hexahydrate (0.98 g) and acetosyringone (As, 50 mM). An appropriate amount of tobacco infection suspension is added into a centrifuge tube, agrobacterium tumefaciens bacterial pellet carrying pBin35SRed:: osGLP3-6 over-expression vector prepared in example 3 is suspended, bacterial liquid OD600 is adjusted to 1.0, and an incubator at 28 ℃ is incubated for 2 hours in a dark place. Sucking the invasion solution by a disposable sterile injector to inject from the back of the tobacco leaf, lightly propping the front of the leaf at the injection position by an index finger, slowly injecting, uniformly diffusing the water stain-shaped circular spots from the injection position to the leaf by naked eyes, and marking the bacterial solution invasion position on the back of the leaf by a Mark pen. After injection, the tobacco is sealed in a moist environment and cultivated for 36 to 48 hours in a dark place.

Taking square culture dishes with the length and the width of 90mm, placing filter papers with similar sizes at the bottoms of the square culture dishes, adding a proper amount of water to soak the filter papers, selecting tobacco leaves with similar sizes and flat surfaces for 4-5 weeks, spreading the tobacco leaves on the filter papers, placing a sterilizing cotton sliver at the veins, sucking proper water by a pipette to soak the cotton sliver, keeping the leaves moist, taking sclerotinia bacteria blocks with the diameter of about 2.5mm, placing the sclerotinia bacteria blocks in an injection bacteria liquid area, counting the size of bacterial plaques after 36 hours, sampling and taking pictures for recording.

The results of statistical findings on the plaque area of tobacco leaves are shown in fig. 11. The three transient expression 35S are that the bacterial plaque areas of the OsGLP3-6 leaves are respectively reduced by 35.45 percent compared with CK, and the results show that the overexpression of the OsGLP3-6 can obviously improve the resistance to sclerotinia.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

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.