CN109652425B - Application of rice OsHIR3 gene and method for obtaining disease-resistant rice - Google Patents

Abstract

The present invention provides riceOsHIR3The application of the gene in preparing plants for defending against virus or bacterial infection hazards and a preparation method thereof, wherein the method comprises the following steps: rice (Oryza sativa L.) with improved resistance to stressOsHIR3The gene is constructed into a plant binary expression vector and is introduced into an agrobacterium strain through electric shock; overexpression obtained by rice mature embryo induction methodOsHIR3The transgenic rice of (1). Such plants have a basic antiviral or bacteriological capacity, mainly increasing the level of SA in the plant.

Description

Application of rice OsHIR3 gene and method for obtaining disease-resistant rice

Technical Field

The invention relates to the technical field of genetic engineering and the field of plant disease control, in particular to the application field of a plant OsHIR3s gene in plant basic resistance.

Background

Rice Stripe Virus (RSV) causes Rice stripe disease. Lapdelphax Striatellus (SBPH) is a mediator for RSV (respiratory syncytial virus) propagation, and rice stripe disease caused by the propagation of the Lapdelphax striatellus is an important virus disease in rice production in China and poses great threat to agricultural production safety.

Hypersensitive Response (HR) is a defense mechanism of plants against the spread of pathogen infestation and is primarily characterized by rapid death of local cells around the infestation, by inducing a local allergic response, i.e. blocking the site of injury, to defend against further infestation by the pathogen. The hypersensitive response gene (HIR) family is thought to be closely related to HR and is involved in the resistance response of plants to pathogens. Many members of the HIR family have been identified to be very similar to the common tobacco HR-inducing protein NG1, NG1 being an activator of HR and being able to induce HR-like necrotic lesions. HvHIR3 on the barley fast neutron mutant plant is up-regulated by 35 times, and the leaves show spontaneous HR, which indicates that the HIR gene can induce HR. Subsequently, HIRs in leguminous plants, cucumber, rice and wheat were identified as being associated with microspore development and bacterial infection reactions.

Previous studies have focused primarily on HIR1, with less research on other HIRs. Transgenic Arabidopsis overexpressing OsHIR1 of rice showed resistance to Pseudomonas syringae pst.DC3000. It is not clear whether HIR3s, like other HIR family members, can induce HR and participate in the defense response of plants against pathogenic bacteria.

Host plants are adapted to external environment stimulation, damage of biotic and abiotic stresses to the host plants is effectively reduced, and a series of complex and fine HR signal conduction mechanisms are formed by sensing and signal conduction of adversity signals and inducing expression of various defense genes in the long-term evolution process. Ca ²⁺ Ions, reactive oxygen species, various hormones, and other signaling molecules play important roles in the signaling process of HR. Three hormones, namely Salicylic Acid (SA), jasmonic Acid (JA) and Ethylene (ETH), participate in the HR signal transduction process, and the important role of the three hormones in plant defense reaction is widely researched and applied.

For the moment, research on HIR has focused primarily on HIR1, and the role of HIRs in plants to combat bacterial and fungal pathogen infestation has been studied more. There have been few functional studies on other HIRs, and it is not clear whether HIRs are involved in the process of viral infection in plants. It is necessary to discover or screen some other genes related to disease resistance, so that the genes can be used for manufacturing disease-resistant plants, thereby obtaining disease-resistant plants and reducing the harm of chemical pesticides.

Disclosure of Invention

The rice is used as a natural host of the rice stripe virus, and the OsHIR3 of the rice stripe virus is highly homologous with the NbHIR3s sequence of the experimental host Nisha NbHIR (the amino acid homology reaches 80%). We found that RSV infection induced upregulation of OsHIR3 expression in rice. We successfully cloned the OsHIR3 gene of the Nipponbare (Oryza sativa L.spp.japonica.cv.Nipponbare) to construct a transient expression vector. The gene is successfully overexpressed in the rice body by a rice mature embryo induction method, and compared with wild type Nipponbare rice, the conditions of seed germination, plant seedling growth, plant height and seed setting of the OsHIR3 gene-transferred rice have no obvious difference.

The wild type and transgenic rice plants are subjected to parallel virus feeding treatment by utilizing the laodelphax striatellus with virus, and the results show that after the rice plants with the OsHIR3 gene are infected by RSV, symptoms are relieved, the plants are dwarfed and relieved, the infected plants only show a stripe phenotype, and RSV RNAs on leaves of the infected plants are obviously reduced. Thus, osHIR3 is thought to confer RSV tolerance to rice.

In addition, the OsHIR3 transgenic rice plant is inoculated with important bacterial pathogen Xanthomonas oryzae pv. Oryzae, xoo, and the length of the full lesion of the leaf of the OsHIR3 transgenic rice plant is obviously shorter than that of the wild type, which indicates that the OsHIR3 transgenic rice plant enhances the resistance to Xoo. Taken together, osHIR3 confers basal resistance to RSV and Xoo in rice.

Therefore, the invention obtains the transgenic rice with basic resistance of RSV and Xoo through the in vivo overexpression of the rice OsHIR3 plant and resistance identification, and the HIR3 gene is used for the creation of the transgenic virus-resistant rice as a gene with basic resistance for the first time.

The first purpose of the invention is to provide a rice OsHIR3 gene.

The second purpose of the present invention is to provide the use of the above gene.

In order to achieve the first object, the invention adopts the following technical scheme:

in the case of sunny rice japonica (Oryza sativa l. Spp. Japonica. Cv. Nipponbare), 6 HIR family genes exist, and sequence analysis shows that, except for Os06g0136000 belonging to the HIR3 family, the nucleotide sequence of the gene is SEQ ID NO:1, and others belong to the HIR1 family. The gene sequence is obtained by using a primer and rice cDNA as a template through common PCR amplification.

In order to achieve the second object, the invention adopts the following technical scheme:

the OsHIR3 is constructed into a plant binary expression vector pCV1300, named pCV: osHIR3, and is electrically shocked and introduced into an agrobacterium strain EHA105. Transgenic rice with over-expressed OsHIR3 is obtained by a rice mature embryo induction method.

And (3) performing virus inoculation identification of RSV on the transgenic plant to obtain RSV-tolerant transgenic rice.

And (3) carrying out Xoo inoculation identification on the transgenic plants to obtain the transgenic rice with basic resistance.

The OsHIR3 transgenic rice obtained by the invention is mainly applied to infection of RSV and Xoo of plants, and harm of virus and bacterial diseases is reduced. The invention has important theoretical and practical significance for cultivating transgenic plants with basic resistance and has guiding function in other fields of plant disease control. Compared with resistant strains obtained by other antiviral strategies, the invention has the main advantages that the OsHIR3 gene is a gene existing in plants, and has obvious safety compared with resistant genes or fragments from other viruses; the OsHIR3 transgenic rice has basic resistance to virus and bacterial diseases and wider resistance.

Drawings

FIG. 1.1: expression vector map containing OsHIR3 gene

FIG. 1.2: vector map of insert plasmid pCV-eGFP-N1.

FIG. 2 is a schematic diagram: molecular biological detection of OsHIR3 transgenic rice

FIG. 3: development phenotype of OsHIR3 transgenic rice

FIG. 4: transgenic rice with OsHIR3 gene for anti-RSV analysis

FIG. 5: transgenic rice with OsHIR3 gene for Xoo resistance analysis

FIG. 6: the control mechanism of OsHIR3 mediated basic resistance is explored.

FIG. 7 is a sequence diagram of OsHIR3 gene.

Detailed Description

It should be noted that this embodiment is merely an example to illustrate the novel functions of the genes that we have found. The effectiveness of the invention was demonstrated by verification on model plant Boehringer Mannheim and is not to be considered as limiting the invention

Example 1: cloning of OsHIR3 Gene

The plant of the invention is Nipponbare rice.

1. Obtaining of recombinant Agrobacterium

1) Cloning of OsHIR3 Gene

The primers OsHIR3-ORF-f and OsHIR3-ORF-r are used, and the cDNA of Nipponbare rice is taken as a template to be amplified by common PCR. The nucleotide sequence of the gene is SEQ ID NO:1 is shown.

The cloning primers were as follows:

OsHIR3-ORF-f:5'-ATGGTGAGCGCCTTCTTCCTGCT-3'(SEQ ID NO：2)

OsHIR3-ORF-r:5'-TTACACGTTGCTGCAGGACGCTT-3'(SEQ ID NO：3)

extracting plant total RNA by a Trizol method: to avoid RNA degradation, the RNA extraction process requires wearing a mask and gloves.

1. Taking a proper amount of sample in an imported 2mL Eppendorf (EP) tube containing steel beads, quickly freezing the sample by liquid nitrogen, then quickly oscillating the sample on a mill for 30s-1min at 18rps (Revolations Per Second), fully grinding the sample, adding a proper amount of Trizol (1 mL/100mg of sample to ensure that the sample is fully cracked), violently oscillating and uniformly mixing the sample, and placing the sample on ice for 5min (which is beneficial to the dissociation of a nucleic acid-protein complex). Centrifuge at 13,000rpm for 10min at 4 ℃.

2. And (3) taking the supernatant into a new 2mL EP tube, adding 1/5 volume of chloroform, violently shaking for 30s, fully mixing uniformly, and standing for 2-3 min on ice. Centrifuge at 13,000rpm for 30min at 4 ℃.

The top layer of the EP tube is colorless aqueous phase containing RNA, the middle white layer is protein phase, and the bottom layer is chloroform phase. The upper aqueous phase was transferred to a new 2mL EP tube (action was gentle enough to avoid aspiration of the intermediate protein phase) and step 2 was repeated.

4. The upper aqueous phase was drawn off into a new 1.5mL EP tube, an equal volume (about 600. Mu.L) of pre-cooled isopropanol was added and mixed by inversion from top to bottom and left at-70 ℃ for 1h. Taking out and placing on ice, and centrifuging at 13,000rpm for 30min at 4 ℃ after fully thawing.

5. The supernatant was discarded, 1mL of pre-cooled 75% ethanol (prepared in RNase-free water) was added, and the pellet was washed (to allow the pellet to be sufficiently suspended to ensure thorough and thorough washing). Centrifuge at 13,000rpm for 5min at 4 ℃.

6. And (5) repeating the step (5) and completely washing the residual salt.

7. Discarding the supernatant, centrifuging at 13,000rpm for 2min at 4 ℃ in an empty tube, carefully removing the residual liquid by using a pipette gun, drying at room temperature until the precipitate is white to transparent, and adding a proper amount of RNase-free water for dissolving.

The RNA concentration can be measured by ultraviolet spectrophotometer, and the RNA quality can be determined by reference to OD260/OD280, OD of RNA ₂₆₀ /OD ₂₃₀ A ratio. The RNA sample was placed at-80 ℃ until use.

The cDNA was inverted from total RNA extracted by Trizol method, the inversion system and conditions were as follows:

firstly, adding the first four reagents into a trace EP tube without RNase, uniformly mixing, and denaturing at 70 ℃ for 5min; immediately put on ice and left for 2min. Then the latter three reagents are added in sequence, and after mixing uniformly, inversion is carried out in a PCR instrument according to the following conditions.

42℃，2h→72℃，10min

The PCR amplification system is as follows:

after mixing uniformly, PCR circulation is carried out according to the following conditions:

example 2 of implementation: vector construction

And (3) amplifying under the same condition by using the obtained OsHIR3 gene full length as a template and using primers OsHIR3-f and OsHIR3-r to obtain an OsHIR3 sequence containing corresponding enzyme cutting sites, and connecting into the multiple cloning sites of the binary expression vector pCV1300 through conventional enzyme cutting and connection. The map of the constructed vector is shown in FIG. 1.1, the map of the inserted plasmid vector is shown in FIG. 1.2, specifically, the position of the vector GFP (fluorescent protein) is replaced by the target gene OsHIR3, and finally, the complete plasmid vector is formed. Such plasmid vectors are readily understood by those skilled in the art, and any plasmid vector is possible and is a means commonly used in the art.

The primers are as follows (underlined)TCTAGAAndGGATCCxba I and Bam HI cleavage sites, respectively):

OsHIR3-f:5'-tgcTCTAGAATGGTGAGCGCCTTCTTCCTGCT-3'(SEQ ID NO：4)

OsHIR3-r:5'-cgcGGATCCTTACACGTTGCTGCAGGACGCTT-3(SEQ ID NO：5)'

example 3: agrobacterium transformation, positive cloning identification and preservation

The positive plasmid is transformed into agrobacterium by an electric shock method, and the specific steps are as follows:

(1) adding 1 μ L of the purified plasmid DNA (obtained in example 2) to thawed Agrobacterium-infected EHA105 (taken out in advance and thawed on ice), gently whipping and mixing, and adding into an electric beaker;

(2) putting the electric shock cup into an electric shock groove (the voltage of an electric shock instrument is 2.2 kV), pressing an electric shock button, and hearing the dropping sound;

(3) the culture broth was transferred to an EP tube, and 900. Mu.L of non-resistant LB medium was added thereto, and the mixture was subjected to shaking culture at 220rpm,28 ℃ for 1 hour.

(4) 200. Mu.L of the cultured broth was collected and spread evenly on LB plates (containing 50. Mu.g/mL kanamycin (Kan) and 50. Mu.g/mL rifampicin (Rif)), and cultured at 28 ℃ for 2 days.

A single colony of transformed Agrobacterium was picked and inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin (Kan) and 50. Mu.g/mL rifampicin (Rif), incubated overnight at 28 ℃ and 200rpm, and 1. Mu.L of the resulting culture was taken for PCR detection. The detection primers are a specific primer OsHIR3-detec-f and a carrier primer NOS-r (the schematic diagram of the primer binding direction is shown in FIG. 2A). And (3) taking the bacterium liquid with a positive detection result, mixing with 30% glycerol, placing in a glycerol tube, and storing in an ultra-low temperature refrigerator at-70 ℃.

The detection primers are as follows:

OsHIR3-detec-f:5'-AAGGTGATGGGAGATTATGGTTAC-3'(SEQ ID NO：6)

NOS-r：5’-GATAATCATCGCAAGACCGG-3’(SEQ ID NO：7)

the PCR detection system is as follows:

after mixing, PCR circulation is carried out according to the following conditions:

example 4: transgenic plant obtained by rice mature embryo induction callus method

1) Preparation of bacterial liquid

Taking a positive transformant (example 3) stored at-70 ℃, streaking the positive transformant on an LB plate containing 50. Mu.g/mL Kan and 50. Mu.g/mL Rif, culturing at 28 ℃ until a single colony is formed, picking the single colony to culture in an LB liquid medium containing 50. Mu.g/mL Kan and 100. Mu.g/mL Rif, and performing shaking culture at 28 ℃ and 220rpm overnight; diluting the bacterial liquid with a fresh LB culture medium according to the proportion of 1 ₆₀₀ Is about 1.0.

2) A transgenic plant is obtained by a rice mature embryo induced callus method, and the specific steps are as follows:

1. and (3) disinfection:

(1) taking young ears of around two weeks (filling period) of the Japan fine flower, manually or mechanically threshing and shelling, selecting full, smooth and non-bacterial plaque seeds, washing the seeds with sterile water, and removing floated shrunken grains;

(2) putting the seeds into a sterile glass tube, and washing the seeds for 2-3 times by using sterile water;

(3) adding 70% alcohol for disinfection for 1min, pouring out the alcohol, and washing with sterile water for 2-3 times;

(4) adding 30% sodium hypochlorite (NaClO, available chlorine 5.2%, containing several drops of Tween-20) solution, standing and soaking for 30min; pouring out the sodium hypochlorite solution, washing with sterile water for 2-3 times, finally soaking the seeds with sterile water, and standing for 30-45 min.

2. And (3) induction culture:

spreading the seeds on sterile filter paper, sucking off excessive water, and placing 5-10 seeds in each dish in a mature embryo induction culture medium; plastic-packaging culture dish with sealing film, and culturing in 28 deg.C illumination incubator for about 20 days.

3. Subculturing:

after the seeds grow light yellow, compact and spherical embryogenic callus, the culture dish is opened in an ultraclean workbench, the naturally-divided whole embryogenic callus is picked by forceps and placed in a subculture medium, and the subculture medium is placed in an illumination incubator at 28 ℃ for 1 week (if the tissue is not used immediately, the tissue can be moved to a dark place, and the tissue is cultured for 1 week at 22 ℃).

4. Co-culturing:

(1) selecting agrobacterium tumefaciens to shake bacteria to a bacterial liquid OD ₆₀₀ About 1.0; collecting the cells, resuspending the cells with AAM infected cell suspension (containing 200. Mu.M As), and adjusting the OD of the cell suspension concentration ₆₀₀ About 0.1;

(2) selecting callus with appropriate size, placing into the prepared Agrobacterium tumefaciens suspension, and soaking for 5min; taking out the callus and drying the callus on sterile filter paper for 0.5 to 1 hour; the callus is spread on a co-culture medium and cultured in dark at 25 ℃ for 2-2.5 days.

5. Screening and culturing:

(1) taking out the callus, cleaning the callus by sterile water, and continuously oscillating the callus; washing with sterile water containing 500mg/L cefradine for 30min, washing for 3 times, spreading the callus on sterile filter paper, and air drying for 2 hr;

(2) transferring the air-dried callus to a selection culture medium (containing 500mg/L cefradine and 50mg/L hygromycin) for first screening, and culturing in a light incubator at 28 ℃ for 14 days;

(3) the initial callus from which the resistant callus was newly generated was picked and placed in a new selection medium (containing 500mg/L cephradine and 50mg/L hygromycin) for a second selection, and cultured in a light incubator at 28 ℃ for about 10 days until the granular resistant callus was grown.

6. And (3) differentiation culture:

the resistant callus with the fresh yellow color is picked up and transferred to a plastic wide-mouth bottle containing a differentiation culture medium (4-5 pieces are placed in each bottle), and the bottle is placed in a constant temperature incubator to be differentiated into seedlings (15-30 days).

7. Rooting, strengthening seedlings and transplanting:

when the seedlings differentiated from the callus grow to about 2-3 cm, taking out the seedlings, removing the root callus, transferring the seedlings to a rooting culture medium, and culturing for 1-2 weeks; adding a proper amount of sterile water into the well-grown seedlings (when the seedlings grow to the tops of the test tubes, opening the caps in time), and hardening the seedlings for 3-7 d; washing off the culture medium at the root, transplanting the seedlings into the soil, and culturing in normal greenhouse growth environment, wherein the water surface is suitable for not submerging the seedlings.

14 rice plants of OsHIR3 transgenic rice are obtained by a rice mature embryo induced callus method.

3) Molecular biological detection of transgenic plants

The DNA of the OsHIR3 gene-transferred rice is extracted by a CTAB method, and the specific steps are as follows:

(1) putting a proper amount of plant material into a 2mL EP tube, quickly and completely grinding the plant material by liquid nitrogen, adding 500 mu L of 2 xCTAB, and violently shaking;

(2) reacting in a water bath at 65 ℃ for 30min, and uniformly mixing the mixture by turning upside down every 10min;

(3) adding 500mL of chloroform, shaking and mixing uniformly, centrifuging at room temperature of 12,000rpm for 10min, and taking the supernatant to a new EP tube;

(4) repeating the step (3) once;

(5) adding isopropyl alcohol with the same volume and NaAc (3M, pH 5.2) with the volume of 1/10, shaking and uniformly mixing, and standing at-20 ℃ for 15min;

(6) centrifuging at room temperature of 12,000rpm for 10min;

(7) discarding the supernatant, washing the precipitate with 75% ethanol, and centrifuging at 12,000rpm for 5min at room temperature;

(8) repeating the step (7) once;

(9) discarding supernatant, opening cover, standing at room temperature for 15min to dry precipitate, and precipitating with 40 μ L ddH ₂ And dissolving the O.

As OsHIR3 is a rice endogenous gene, PCR detection of a specific primer (OsHIR 3-detec-f: 5-.

In order to detect whether OsHIR3 is successfully integrated into a genome and is highly expressed, total leaf protein and total RNA of a positive strain T2 generation plant are extracted and subjected to Western blot and qRT-PCR detection, and the result shows that the expression quantity of OsHIR3 in the positive strain is different, wherein the OsHIR3 protein level and the OsHIR3mRNA expression quantity of OsHIR3 transgenic rice strains OE6, OE8 and OE12 are obviously higher than those of a wild type (fig. 2C and fig. 2D). Phenotypic observation shows that the seed germination, plant seedling growth and fructification conditions of the T2 generation OsHIR3 transgenic rice are not obviously different from those of wild type Nipponbare rice (figure 3), and the fact that the OsHIR3 up-regulation expression does not have a significant influence on the growth and development conditions of rice plants is shown.

Example 5: RSV inoculation identification of OsHIR3 transgenic rice

The invention selects the T2 generation positive OsHIR3 transgenic rice to carry out RSV inoculation identification. The specific operation content is as follows:

1) Purification and identification of laodelphax striatellus with high toxicity rate

The rice plants infected with RSV are used for feeding Laodelphax striatellus groups for 5-7 days (day, d) to ensure that the insect bodies can fully obtain the virus. Then, female insects with 5 ages are separately caught and put into a test tube (2-3 rice seedlings are planted in the test tube for feeding) for single insect breeding. Breeding the single insects for 2-3 weeks (when the second generation larvae grow to about 2-3 years old), capturing 3 low-age larvae per tube, detecting the toxicity rate of the single insects by RT-PCR, and transferring the positive insect lines into a large beaker for expanding propagation. And sampling and detecting the propagated offspring, and gathering and feeding the laodelphax striatellus after the toxicity rate is stable.

2) OsHIR3 transgenic rice inoculated RSV

The OsHIR3 transgenic rice T2 generation line was selected as a test material and wild type rice (Nipponbare) was selected as a control, and simultaneously sowed. Sterile water treatment, seed soaking and germination acceleration in a constant-temperature incubator at 37 ℃, and 20-40 healthy seeds are selected for each strain. After about 2 days, the seeds are sown after white emergence, and each line is planted in a nutrition pot (10 cm multiplied by 10 cm) and cultured in a greenhouse environment.

When the rice grows to 3-leaf stage, the rice is moved to an insect receiving cage, the purified 2-4-year-old larvae with high virus (RSV) rate are transferred to the insect receiving cage according to the effective insect receiving amount of 3-5 heads of each plant, and wild plants and transgenic plants are simultaneously subjected to virus feeding for 2-3 d in parallel. During the period of inoculation, the inoculated rice seedlings are ensured to be evenly poisoned, and the insects are repelled twice every day. And (3) after the feeding of the virus is finished, removing all the laodelphax striatellus, transplanting the rice seedlings to a field after the rice seedlings are relieved for 2-3 days in a greenhouse environment, and carrying out disease investigation and analysis.

3) RSV inoculation identification of OsHIR3 transgenic rice

After inoculation of RSV for about 4-6 days, rice seedlings begin to curl, seedlings with serious infection begin to die after inoculation for about 8-12 days, newborn leaves with serious diseases after inoculation for 20 days have obvious disease spots and curl, then some plants with serious diseases die gradually, plant leaves with strong resistance capacity have disease spots, and some plants gradually become cryptogamic along with the growth of the plants.

In order to analyze whether the overexpression of OsHIR3 endows rice plants with RSV resistance, T2 generation seedlings of three independent rice lines OE6, OE8 and OE12 with OsHIR3 gene transfer are selected for RSV resistance identification, 30 rice lines are planted in each rice line, and wild type Nipponbare is used as a control for carrying out the same treatment. Transplanting the rice plants after the inoculation into the field, and continuously observing and counting the mortality rate of the rice plants. After 20d of inoculation, the mortality statistics show that the mortality of all three tested independent lines OE6, OE8 and OE12 is significantly lower than that of the control plants (FIG. 4A). After 30 days of virus inoculation, the growth condition of the transgenic rice plant is obviously better than that of the control plant. Of the control plants, most severely affected plants had died; among the three transgenic lines tested, the plants with severe infection only show weak growth. The survival contrast susceptible plants are short and small in growth and severely curled; while the surviving transgenic rice susceptible lines exhibited a streak phenotype only in the leaves (FIG. 4B).

The accumulation amount of RSV RNAs in OsHIR3 transgenic rice and wild type susceptible plants is analyzed by Northern blot, and the result shows that the accumulation amount of RSV RNA3 of leaves of three tested transgenic independent plants OE6, OE8 and OE12 susceptible plants is lower than that of a control plant (figure 4C). The results show that the OsHIR3 overexpression effectively inhibits the accumulation of RSV RNAs, and rice plants with transferred OsHIR3 genes show RSV disease tolerance.

Example 6: transgenic rice with OsHIR3 gene for Xoo resistance analysis

1) Xoo inoculation

Bacterial strain P10 (Xoo)Inoculating to the liquid culture medium of Chachishi, culturing at 28 deg.C in a shaker at 200r/min for 1d, collecting thallus, and culturing with ddH ₂ O was formulated to have an OD600 of about 0.5. The OsHIR3 transgenic rice T2 generation strain is selected as a test material, wild type rice (Nipponbare) is selected as a control, and the rice is simultaneously sown and planted in a greenhouse. After the rice grows for about 2 months (before heading), inoculating the bacterial leaf blight strain P10 to the rice by adopting an artificial leaf-cutting inoculation method, observing the disease occurrence condition of rice leaves after inoculating for 1-2 weeks, measuring the length of disease spots of the diseased leaves of different rice materials, and carrying out statistical comparison to evaluate the resistance of the rice.

2) Determination of the Total lesion Length

Two weeks after inoculation with Xoo P10 races, the total lesion length of infected leaves was measured. Statistical results show that compared with wild type, lesion length of three independent lines OE6, OE8 and OE12 of OsHIR3 transgenic rice is shorter (FIG. 5A). The length of disease spot of wild type Nipponbare is (10.5 +/-0.6) cm; the lesion length of three independent lines OE6, OE8 and OE12 of OsHIR3 transgenic rice is (5.2 +/-0.5) cm, (2.1 +/-0.3) cm and (6.1 +/-0.2) cm respectively, and statistical results show that the three independent lines OE6, OE8 and OE12 have significant difference in pairs compared with wild type (FIG. 5B). The results show that the OsHIR3 transgenic rice plant enhances the disease tolerance to Xoo. Taken together, osHIR 3-mediated basal resistance is not only against RSV, but also targets other pathogens, such as Xoo.

Example 7 nbhir3s basic resistance was obtained by forward regulation of the SA pathway

Positive plants with high OsHIR3 expression level are screened out through the detection, and SA content determination and SA pathway key gene real-time fluorescence quantitative qRT-PCR analysis are carried out. With reference to the qRT-PCR specification, the specific operation is as follows:

sequentially adding the reagents into an RNase-free EP tube, fully and uniformly mixing, spotting into a 384-hole quantitative plate according to 10 mu L/hole, covering a membrane, instantaneously centrifuging, and placing in a qRT-PCR instrument for reaction: 5min at 95 ℃; 40 cycles were performed: 95 ℃ 20s → 58 ℃ 20s → 72 ℃ 20s; 10min at 72 ℃.

The specific quantitative primers used for the real-time fluorescent quantitative PCR analysis are as follows:

compared with wild type, the SA content in leaf cells of three independent lines OE6, OE8 and OE12 of OsHIR3 transgenic rice is remarkably increased (figure 6A), the key genes PBZ1 (also named NPR 1) of the SA pathway, and PR1 and PR5 genes are remarkably up-regulated and expressed (figure 6B). This fully suggests that the OsHIR3 gene positively regulates the SA pathway to gain basal resistance.

This is because Systemic Resistance, which results in Resistance to pathogenic bacteria at remote uninfected sites after the plant is infected with pathogenic bacteria, is called Systemic Acquired Resistance (SAR), a phenomenon that has been demonstrated in many plant-pathogen interactions. SAR is typically manifested by limiting the growth of pathogenic bacteria and inhibiting their symptomatic development. In plants, the role of SA in SAR has been reported, and the mainstream view is that SA is an important signal molecule in SAR process, and the accumulation of SA can stimulate SAR response. The large expression of the disease course related protein (PR protein) is an important mark of SAR reaction, and a plurality of PR proteins coordinate with each other rather than a specific PR protein acts alone to cause SAR reaction. In the body of the Tobacco treated by SA or aspirin, a large amount of PR protein is accumulated, and the Tobacco is resistant to infection of Tobacco mosaic virus (TMW); TMV infection can induce the content of endogenous SA of the tobacco to be increased sharply, and the SA content of resistant varieties is obviously higher than that of susceptible varieties; the mutant plants of sid1 and sid2 can not accumulate SA in vivo, can not start SAR, show sensitivity to pseudomonas syringae, and further prove that SA is a key signal molecule in the SAR process.

The regulatory protein NPR1 is a key component in an SA-mediated signal transduction pathway, and the NPR1 can induce the expression of PR-1 and other resistance genes, thereby enhancing the disease resistance of plants. When the pathogen is infected, the affected mutant nim1 of NPR1 gene expression has normal SA level but can not induce SAR, which indicates that NPR1 acts on the downstream of SA and is a key regulatory factor in SAR signal transduction pathway. Desspres et al found that NPR1 was able to interact with members of the TGA family of leucine-rich (bZIP) transcription factors in Arabidopsis, while NPR1 mutants lost their interaction with TGA2, suggesting that NPR 1-mediated TGA2 binding is critical for activation of defense genes. When SAR is induced, NPR1 activates PR-1 gene through interaction with transcription factor in PR gene promoter region, which shows that NPR1 activity is closely related to PR gene expression regulation. It was found that four-point mutants of NPR1 blocked the SA signal and lost the interaction with TGA2 and TGA3, that TGA2 and TGA3 were able to bind the SA response element of the Arabidopsis PR-1 promoter, and that NPR1 was linked to SA-induced PR-1 gene expression by TGA transcription factors.

Sequence listing

<110> Ningbo university

<120> application of rice OsHIR3 gene and method for obtaining disease-resistant rice

<130> 19-100070-00011864

<141> 2019-01-08

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agagcaatga atgaaataaa tgcagcacaa aggcttcagc ttgcaagtgt ctacaaagga 540

gaggcggaga agattcttct ggtgaagaaa gcagaagcag aggcagaggc aaaacacctt 600

tccggtgtcg gcattgctag acagcggcag gcgataactg atggcctgag agagaacatc 660

ctgaacttct cgcactcggt ttcaggcacc tcagcaaaag aagtcatgga tctcatcatg 720

gtcacgcagt acttcgacac catcaaagaa cttggggatg gctcgaagaa caccacggtg 780

ttcatacctc atggcccagg ccatgtcagg gatatcagcg agcaaatccg gaatggtatg 840

atggaagcgt cctgcagcaa cgtgtaa 867

<210> 2

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggtgagcg ccttcttcct gct 23

<210> 3

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ttacacgttg ctgcaggacg ctt 23

<210> 4

<211> 32

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgctctagaa tggtgagcgc cttcttcctg ct 32

<210> 5

<211> 32

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cgcggatcct tacacgttgc tgcaggacgc tt 32

<210> 6

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aaggtgatgg gagattatgg ttac 24

<210> 7

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gataatcatc gcaagaccgg 20

<210> 8

<211> 24

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aaggtgatgg gagattatgg ttac 24

<210> 9

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gataatcatc gcaagaccgg 20

<210> 10

<211> 25

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggtatccatg agactacata caact 25

<210> 11

<211> 23

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tactcagcct tggcaatcca cat 23

<210> 12

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cacactcgac ggagacgaag 20

<210> 13

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gccatagtag ccatccacga t 21

<210> 14

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tatccaagct ggccattgct 20

<210> 15

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ttctctggct ggcgtagttc 20

<210> 16

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cgcaacaact gcacctacac 20

<210> 17

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ggctaggaac gagacgttgg 20

Claims (3)

1. Rice (Oryza sativa L.) with improved resistance to stressOsHIR3Gene production with defense against RSV andXooapplication to rice infected with harm; saidOsHIR3The gene is SEQ ID NO: 1.

2. The use according to claim 1, ofOsHIR3The gene is from Nipponbare rice.

3. Use according to claim 1, whereinOsHIR3The gene has the ability to confer basal resistance by upregulating PBZ1 (also known as NPR 1), PR1 and PR5, leading to upregulation of SA.

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