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

Abstract

The present invention provides the use and preparation method of rice OsHIR3 gene in preparing plants to defend against virus or bacterial infection . Transgenic rice overexpressing OsHIR3 was obtained by induction of rice mature embryos. Such plants have basic ability to resist viral or bacterial diseases, mainly by increasing the level of SA in plants.

Description

Use 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 field of application of plant OsHIR3s gene in basic plant resistance.

Background technique

Rice stripe virus (RSV) can cause rice stripe blight. Lapdelphax striatellus (SBPH) is the mediator of RSV transmission. Rice stripe blight caused by SBPH is an important virus disease in rice production in my country, which poses a great threat to agricultural production safety.

Hypersensitivity response (HR) is a defense mechanism of plants to prevent the spread of pathogen infection, which is mainly characterized by the rapid death of local cells around the infection, by inducing a local allergic response, that is, sealing the injury site to resist further infection by pathogens. . The allergy-induced response (HIR) family is thought to be closely related to HR and involved in plant resistance to pathogens. Many members of the HIR family have been identified that are very similar to the common tobacco HR-inducing protein NG1, which acts as an activator of HR and can induce HR-like necrotic plaques. HvHIR3 was up-regulated 35-fold in barley fast neutron mutant plants, and leaves showed spontaneous HR, indicating that HIR gene can induce HR. Subsequently, HIRs in legumes, cucumbers, rice, and wheat were identified as associated with microspore development and bacterial infection responses.

Previous studies have mainly focused on HIR1, with less research on other HIRs. Transgenic Arabidopsis thaliana overexpressing rice OsHIR1 exhibited resistance to Pseudomonas syringae Pst.DC3000. It is unclear whether HIR3s, like other HIR family members, can induce HR and participate in plant defense responses against pathogens.

In order to adapt to external environmental stimuli and effectively reduce the damage of biotic and abiotic stresses to themselves, host plants have formed a series of complex and delicate structures through the perception of adversity signals, signal transduction, and induction of various defense gene expressions in the long-term evolution process. HR signaling mechanism. Ca ²⁺ ions, reactive oxygen species, various hormones and other signaling molecules play important functions in the signal transduction process of HR. Three types of hormones, Salicylic acid (SA), Jasmonic acid (JA), and Ethylene (ETH), are involved in the HR signal transduction process, and their important roles in plant defense responses have been widely studied and applied.

At present, the research on HIR mainly focuses on HIR1, and there are many studies on the role of HIRs in plant defense against bacterial and fungal pathogens. There are not many functional studies on other HIRs, and it is unclear whether HIRs are involved in the process of plant resistance to viral infection. It is necessary to find or screen some other genes related to disease resistance, so as to use these genes to manufacture disease-resistant plants, so as to obtain disease-resistant plants and reduce the harm of chemical pesticides.

SUMMARY OF THE INVENTION

Rice is the natural host of rice streak virus, and its OsHIR3 is highly homologous to the experimental host NbHIR3s of NbHIR3s (the amino acid homology reaches 80%). We found that RSV infection induced up-regulated expression of OsHIR3 in rice. We successfully cloned the OsHIR3 gene of Nipponbare rice (Oryza sativa L.spp.japonica.cv.Nipponbare) and constructed a transient expression vector. The above-mentioned genes were successfully overexpressed in rice by the rice mature embryo induction method. There was no significant difference in seed germination, plant seedling growth, plant height and seed setting between OsHIR3 transgenic rice and wild-type Nipponbare rice.

The wild-type and transgenic rice plants were treated in parallel with the virus-carrying SBPH. It was found that the OsHIR3 transgenic rice plants had reduced symptoms and dwarfing after RSV infection. The infected plants only showed a striped phenotype. RSV RNAs were significantly reduced in plant leaves. From this, we believe that OsHIR3 confers resistance to RSV in rice.

In addition, when the OsHIR3 transgenic rice plants were inoculated with an important bacterial pathogen Xanthomonas oryzae pv. Plants have enhanced resistance to Xoo. Taken together, OsHIR3 confers basal resistance to RSV and Xoo in rice.

Therefore, the present invention overexpresses rice OsHIR3 in vivo, and obtains RSV and Xoo-resistant transgenic rice with basic resistance through resistance identification. This is the first time that HIR3 gene is used as a basic resistance gene for transgenic resistance. Viral rice creation.

The first object of the present invention is to provide the rice OsHIR3 gene.

The second object of the present invention is to provide the use of the above-mentioned gene.

In order to realize the above-mentioned first purpose, the present invention adopts the following technical solutions:

There are 6 HIR family genes in Oryza sativa L.spp.japonica.cv.Nipponbare. Sequence analysis shows that except Os06g0136000, which belongs to HIR3 family, the nucleotide sequence of the gene is shown in SEQ ID NO: 1. Belongs to the HIR1 family. The above gene sequence was amplified by ordinary PCR using primers and rice cDNA as a template.

In order to realize the above-mentioned second purpose, the present invention adopts the following technical solutions:

The above OsHIR3 was constructed into a plant binary expression vector pCV1300, named pCV:OsHIR3, and electroporated into Agrobacterium strain EHA105. Transgenic rice overexpressing OsHIR3 was obtained by induction of rice mature embryos.

The above-mentioned transgenic plants were inoculated with RSV to obtain RSV-tolerant transgenic rice.

The above-mentioned transgenic plants were inoculated and identified by Xoo to obtain transgenic rice with basic resistance.

The OsHIR3 gene-transformed rice obtained by the invention is mainly used for RSV and Xoo infection of plants, so as to reduce the damage of virus and bacterial diseases. The invention has important theoretical and practical significance for cultivating transgenic plants with basic resistance, and also has a guiding role in other fields of plant disease control. Compared with the resistant strains obtained by other anti-virus strategies, the main advantage of the present invention is that the OsHIR3 gene is a gene existing in the plant itself, and has obvious safety compared to the resistance genes or fragments derived from other viruses; OsHIR3 transgenic rice has basic resistance to viral and bacterial diseases, and the resistance is wider.

Description of drawings

Figure 1.1: Map of the expression vector containing the OsHIR3 gene

Figure 1.2: Insertion plasmid pCV-eGFP-N1 vector map.

Figure 2: Molecular biological detection of OsHIR3 transgenic rice

Figure 3: Developmental phenotype of OsHIR3 transgenic rice

Figure 4: RSV resistance analysis of OsHIR3 transgenic rice

Figure 5: Anti-Xoo analysis of OsHIR3 transgenic rice

Figure 6: Exploration of the regulatory mechanism of OsHIR3-mediated basal resistance.

Figure 7 is a sequence diagram of the OsHIR3 gene.

Detailed ways

It should be noted that this embodiment is only an example to illustrate the new function of the gene we discovered. The validity of the present invention has been verified by the model plant Nicotiana benthamiana, which is not considered to be a limitation of the present invention.

Example 1: Cloning of the OsHIR3 gene

The plant transformed by the present invention is Nipponbare rice.

1. Acquisition of Recombinant Agrobacterium

1) Cloning of OsHIR3 gene

Using the primers OsHIR3-ORF-f and OsHIR3-ORF-r, and using Nipponbare rice cDNA as a template, it was amplified by ordinary PCR. The nucleotide sequence of the above gene is shown in SEQ ID NO:1.

The cloning primers are as follows:

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

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

Extraction of total plant RNA by Trizol method: To avoid RNA degradation, masks and gloves should be worn during RNA extraction.

1. Take an appropriate amount of sample into an imported 2mL Eppendorf (EP) tube containing steel balls, quickly freeze in liquid nitrogen, and quickly shake it on a mill at 18 rps (Revolutions Per Second, revolutions per second) for 30s-1min. After fully grinding, add an appropriate amount of Trizol. (1mL/100mg sample, to ensure that the sample is fully lysed), shake vigorously and mix, and place on ice for 5min (conducive to the dissociation of nucleic acid-protein complexes). Centrifuge at 13,000 rpm for 10 min at 4°C.

2. Take the supernatant into a new 2mL EP tube, add 1/5 volume of chloroform, shake vigorously for 30s, mix well, and let stand on ice for 2-3min. Centrifuge at 13,000 rpm for 30 min at 4°C.

3. The top layer of the EP tube is the colorless aqueous phase containing RNA, the middle white layer is the protein phase, and the bottom layer is the chloroform phase. Transfer the upper aqueous phase to a new 2mL EP tube (as gentle as possible to avoid aspirating the intermediate protein phase), and repeat step 2.

4. Extract the upper aqueous phase into a new 1.5 mL EP tube, add an equal volume (about 600 μL) of pre-cooled isopropanol, invert up and down to mix, and place at -70°C for 1 hour. Take it out and put it on ice until it is fully thawed, then centrifuge at 4°C and 13,000rpm for 30min.

5. Discard the supernatant, add 1 mL of pre-cooled 75% ethanol (prepared with RNase-free water), and wash the precipitate (sufficiently suspend the precipitate to ensure thorough washing). Centrifuge at 13,000 rpm for 5 min at 4°C.

6. Repeat step 5 to thoroughly wash off the residual salt.

7. Discard the supernatant, empty the tube at 4°C, centrifuge at 13,000 rpm for 2 minutes, carefully remove the residual liquid with a pipette, and air dry at room temperature. When the precipitate turns from white to transparent, add an appropriate amount of RNase-free water to dissolve.

8. The RNA concentration can be measured by UV spectrophotometer, and the RNA quality can also refer to the ratio of OD260/OD280 and _OD260 / _OD230 of RNA. RNA samples were placed at -80°C for later use.

cDNA was reversed from total RNA extracted by the Trizol method. The reverse system and conditions were as follows:

First, add the first four reagents to an RNase-free micro-EP tube, mix well, and denature at 70°C for 5 minutes; immediately put it on ice and leave it for 2 minutes. Then add the latter three reagents in turn, and invert them in the PCR machine according to the following conditions after mixing.

42℃, 2h→72℃, 10min

The PCR amplification system is as follows:

After mixing, PCR cycles were performed according to the following conditions:

Example 2: Vector Construction

Using primers OsHIR3-f and OsHIR3-r, taking the above-obtained full-length OsHIR3 gene as a template, amplifying the OsHIR3 sequence containing the corresponding restriction site under the same conditions as above, and connecting the two Multiple cloning site for meta expression vector pCV1300. The constructed vector map is shown in Figure 1.1, and the inserted plasmid vector is shown in Figure 1.2. Specifically, the position of the vector GFP (fluorescent protein) is replaced by the target gene OsHIR3, and finally a complete plasmid vector is formed. Such a plasmid vector is easily understood by those in the art, any plasmid vector is possible, and it is also a commonly used means in the art.

The primers are as follows (underlined TCTAGA and GGATCC indicate Xba I and Bam HI restriction sites, respectively):

OsHIR3-f: 5'-tgc TCTAGA ATGGTGAGCGCCTTCTTCCTGCT-3' (SEQ ID NO: 4)

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

Example 3: Agrobacterium transformation, identification and preservation of positive clones

The positive plasmid was transformed into Agrobacterium by electric shock, and the specific steps were as follows:

①Add 1 μL of purified plasmid DNA (obtained in Example 2) to the thawed Agrobacterium competent EHA105 (pre-removed and thawed on ice), gently pipetting and mixing, and then added to the electric shock cup;

②Put the above electric shock cup into the electric shock tank (the voltage of the electric shock meter is 2.2kV), press the electric shock button, and you can hear the beeping sound;

③ Extract the bacterial liquid into the EP tube, add 900 μL of non-resistant LB culture medium, 220 rpm, 28 ° C, and shake for 1 h.

④ Extract 200 μL of cultured bacterial liquid, spread evenly on LB plates (containing 50 μg/mL kanamycin (Kan) and 50 μg/mL rifampicin (Rif)), and culture at 28°C for 2 days.

Pick a single colony of transformed Agrobacterium and inoculate it in LB liquid medium containing 50 μg/mL kanamycin (Kan) and 50 μg/mL rifampicin (Rif), shake overnight at 28°C, 200 rpm, and take 1 μL of bacterial solution Perform PCR testing. The detection primers were the specific primer OsHIR3-detec-f and the carrier primer NOS-r (the schematic diagram of the binding direction of the primers is shown in Figure 2A). The bacterial liquid with positive test result was taken, mixed with 30% glycerol, placed in a glycerol tube, and stored in a -70°C ultra-low temperature refrigerator.

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 cycles were performed according to the following conditions:

Example 4: Transgenic plants obtained by induction of callus from mature embryos of rice

1) Preparation of bacterial solution

Take the positive transformed strain (Example 3) stored at -70 °C, streak on the LB plate containing 50 μg/mL Kan and 50 μg/mL Rif, cultivate at 28 °C until a single colony is formed, pick a single colony containing 50 μg/mL. Culture in LB liquid medium of Kan, 100 μg/mL Rif, 28 ° C, 220 rpm shaking culture overnight; Dilute the bacterial liquid at 1:100 with fresh LB medium, continue to shake and culture until the OD ₆₀₀ is about 1.0.

2) Transgenic plants are obtained by inducing callus from mature embryos of rice, and the specific steps are as follows:

1. Disinfection:

①Take the young ears of Nippon Qingyang flower for about two weeks (filling period), thresh and shell them manually or mechanically, select the seeds with full, smooth and sterile spots, rinse the seeds with sterile water, and remove the floating shriveled grains;

②Put the seeds into a sterile glass tube, and rinse the seeds with sterile water 2 to 3 times;

③ Add 70% alcohol to disinfect for 1min, pour off the alcohol, and rinse with sterile water for 2 to 3 times;

④Add 30% sodium hypochlorite (NaClO, 5.2% available chlorine, containing a few drops of Tween-20) solution, let it stand for 30min soaking; pour off the sodium hypochlorite solution, rinse with sterile water 2 to 3 times, and finally soak the seeds with sterile water , let stand for 30 to 45 minutes.

2. Induction culture:

Spread the seeds on sterile filter paper, absorb the excess water, place 5-10 seeds per dish in the mature embryo induction medium; seal the petri dish with parafilm, and place it in a 28°C light incubator for about 10 minutes. 20d.

3. Subculture:

When the seeds grow pale yellow, dense and spherical embryogenic callus, open the petri dish in the ultra-clean workbench, pick the intact embryogenic callus that divides naturally with tweezers, and place it in the subculture medium at 28°C. In a lighted incubator, subculture for 1 week (if not used immediately, it can be moved to a dark place and cultured at 22°C for 1 week).

4. Co-cultivation:

①Pick Agrobacterium monoclonal and shake until the OD ₆₀₀ of the bacterial solution is about 1.0; collect the bacterial cells, resuspend the bacterial cells with AAM infective solution (containing 200 μM As), and adjust the concentration of the bacterial solution to an OD ₆₀₀ of about 0.1;

② Pick the callus of suitable size, put it into the Agrobacterium suspension prepared above, soak it fully for 5 minutes; take out the callus and dry it on sterile filter paper for 0.5-1 hour; spread the callus on the co-culture medium cultured at 25°C in the dark for 2-2.5 days.

5. Screening culture:

① Take out the callus, wash it with sterile water, and shake it continuously; wash and soak it in sterile water containing 500 mg/L cefradine for 30 minutes, wash it three times, and spread the callus on sterile filter paper for 2 hours;

②Transfer the air-dried callus to the selection medium (containing 500mg/L cefradine and 50mg/L hygromycin) for the first round of screening, and cultivate in a light incubator at 28°C for 14 days;

③Pick out the initial callus of the newly emerged resistant callus and place it in a new selection medium (containing 500mg/L cefradine and 50mg/L hygromycin) for the second round of selection, in a 28 ℃ light incubator, culture About 10d, until the granular resistant callus grows.

6. Differentiation culture:

Pick bright yellow resistant callus, transfer it to a plastic jar containing differentiation medium (4-5 per bottle), place it in a constant temperature incubator, and differentiate into seedlings (15-30 d).

7. Rooting and strong seedlings and transplanting:

When the seedlings differentiated from the callus grow to about 2-3cm, take out the seedlings, remove the root callus, move them to the rooting medium, and cultivate for 1-2 weeks; add an appropriate amount of sterile water to the well-growing seedlings (when the seedlings grow To the top of the test tube, open the cover in time), and harden the seedlings for 3-7 days; wash off the root medium, and transplant the seedlings into the soil.

Fourteen transgenic rice plants with OsHIR3 gene were obtained by inducing callus from mature embryos of rice.

3) Molecular biological detection of transgenic plants

The above-mentioned OsHIR3 gene transgenic rice DNA was extracted by CTAB method, and the specific steps were as follows:

①Put an appropriate amount of plant material into a 2mL EP tube, grind it completely with liquid nitrogen, add 500μL of 2×CTAB, and shake vigorously;

②Reaction in a water bath at 65°C for 30min, invert and mix once every 10min;

③ Add 500 mL of chloroform, shake and mix, centrifuge at 12,000 rpm for 10 min at room temperature, and take the supernatant into a new EP tube;

④ Repeat step ③ once;

⑤ Add equal volume of isopropanol and 1/10 volume of NaAc (3M, pH 5.2), shake and mix, and place at -20°C for 15min;

⑥ Centrifuge at 12,000rpm for 10min at room temperature;

⑦ Discard the supernatant, wash the precipitate with 75% ethanol, and centrifuge at 12,000 rpm for 5 min at room temperature;

⑧ Repeat step ⑦ once;

⑨ Discard the supernatant, open the lid and place at room temperature for 15 min to dry the precipitate, and dissolve the precipitate with 40 μL ddH ₂ O.

Given that OsHIR3 is an endogenous gene in rice, a specific primer (OsHIR3-detec-f: 5'-AAGGTGATGGGAGATTATGGTTAC-3' (SEQ ID NO: 8)) and a vector primer (NOS-r: 5'-GATAATCATCGCAAGACCGG-3' were selected (SEQ ID NO: 9)) PCR detection was carried out, and the results showed that there were 12 positive plants and 2 negative plants in transgenic OsHIR3 rice, with a positive rate of 86% (Fig. 2B).

In order to test whether OsHIR3 was successfully integrated into the genome and highly expressed, we extracted the total protein and total RNA of the leaves of the T2 generation plants of the positive line, and performed Western blot and qRT-PCR detection on them. The OsHIR3 protein level and OsHIR3 mRNA expression of the OsHIR3 transgenic rice lines OE6, OE8 and OE12 were significantly higher than those of the wild type (Figure 2C and Figure 2D). The phenotypic observation showed that there was no significant difference in seed germination, plant seedling growth, and seed setting between the T2 generation OsHIR3 transgenic rice and wild-type Nipponbare rice (Figure 3), indicating that the up-regulation of OsHIR3 did not affect the growth and development of rice plants. Significantly affected.

Example 5: RSV inoculation identification of OsHIR3 transgenic rice

The present invention selects T2 generation positive transgenic OsHIR3 gene rice for RSV inoculation identification. The specific operations are as follows:

1) Purification and identification of the high virulence rate of S. striatellus

The SBPH colonies were reared with RSV-infected rice plants for 5 to 7 days (day, d) to ensure that the parasites could fully acquire the virus. Then, 5th instar females were individually captured into test tubes (2-3 rice seedlings were planted in the test tubes for their feeding) for single-worm feeding. Single worms were raised for 2 to 3 weeks (when the second generation of larvae grew to about 2 to 3 instars), 3 young larvae were captured in each tube for single worm RT-PCR detection of the virus carrying rate, and then the positive worm lines were transferred to large beakers. Propagation in expansion. The breeding offspring were sampled and tested, and after the virus-carrying rate was stable, the poison-carrying S. lugens was collectively raised.

2) OsHIR3 transgenic rice inoculated with RSV

The OsHIR3 transgenic rice T2 generation line was selected as the test material, and the wild-type rice (Nipponbare) was used as the control, and were sown at the same time. Sterile water treatment, soaking and germination in a 37°C constant temperature incubator, and selecting 20 to 40 healthy seeds for each strain. After about 2 days, the seeds were sown, and each line was planted in a nutrient pot (10cm×10cm) and cultivated in a greenhouse environment.

When the rice grows to the 3-leaf stage, it is moved to the inoculating cage, and according to the effective inoculation amount of 3 to 5 heads per plant, the 2-4 instar larvae of the above-mentioned purified high-carriage-carrying (RSV) rate S. striatellus Into the inoculation cage, the wild and transgenic plants were fed with poison in parallel for 2 to 3 days. During the infestation period, ensure that the inoculated rice seedlings are evenly poisoned, and the insects are removed twice a day. After the feeding of the poison was completed, all the planthoppers were removed, and after the rice seedlings were relieved in the greenhouse for 2-3 days, the plants were transplanted to the field for disease investigation and analysis.

3) Identification of RSV inoculation of OsHIR3 transgenic rice

About 4 to 6 days after inoculation with RSV, rice seedlings began to curl, and the severely susceptible seedlings began to die about 8 to 12 days after inoculation. The newly infected leaves showed obvious lesions and curls after 20 days of inoculation. Gradually die, and the leaves of plants with strong resistance will appear diseased spots, and some will gradually disappear as the plants grow.

To analyze whether OsHIR3 overexpression confers resistance to RSV in rice plants, we selected the T2 generation seedlings of three independent lines of OsHIR3 transgenic rice, OE6, OE8, and OE12, for RSV resistance identification. Nippon Fine was used as a control, and the same treatment was performed. After the inoculated rice plants were transplanted into the field, the mortality was continued to be observed and counted. After 20 days of inoculation, the mortality statistics showed that the mortality of the three independent lines OE6, OE8 and OE12 was significantly lower than that of the control plants (Fig. 4A). After 30 days of inoculation, the growth of transgenic rice plants was significantly better than that of control plants. In the control plants, most of the severely susceptible plants died; while among the three transgenic lines examined, the severely susceptible plants only showed weak growth. The surviving control susceptible plants had dwarf growth and severely curled leaves, while the surviving transgenic rice susceptible lines only showed a striped phenotype on the leaves (Fig. 4B).

The accumulation of RSV RNAs in OsHIR3 transgenic rice and wild-type susceptible plants was analyzed by Northern blot. The results showed that the RSV RNA3 accumulation in leaves of susceptible plants of the three independent transgenic lines OE6, OE8 and OE12 were all lower than those of the control. plants (Fig. 4C). The above results indicated that OsHIR3 overexpression effectively inhibited the accumulation of RSV RNAs, and OsHIR3 transgenic rice plants showed resistance to RSV.

Example 6: Anti-Xoo analysis of OsHIR3 transgenic rice

1) Xoo vaccination

Bacterial blight strain P10 ( Xoo) was transferred to Xiebenzhe's liquid medium, cultured for 1 d at 28°C on a shaker at 200 r/min, and the cells were collected and prepared with ddH ₂ O to an OD600 of about 0.5. The OsHIR3 gene transgenic rice T2 line was selected as the test material, and the wild-type rice (Nipponbare) was selected as the control, which was sown at the same time and planted in the greenhouse. After the rice was grown for about 2 months (before heading), the rice was inoculated with the bacterial blight strain P10 by artificial leaf cutting and inoculation. After 1 to 2 weeks of inoculation, the diseased leaves of the rice were observed, and the disease spots of the diseased leaves of different rice materials were measured. Length, statistical comparison, and evaluation of rice resistance.

2) Determination of whole lesion length

Two weeks after inoculation of Xoo P10 races, the length of full lesions on infected leaves was measured. The statistical results showed that, compared with the wild type, the three independent lines of OsHIR3 transgenic rice, OE6, OE8, and OE12, had shorter lesion lengths (Fig. 5A). The lesion length of wild-type Nipponbare was (10.5±0.6) cm; while the lesion lengths of three independent lines of OsHIR3 transgenic rice, OE6, OE8, and OE12, were (5.2±0.5) cm, (2.1±0.3) cm, respectively. (6.1±0.2) cm, the statistical results showed that there were significant differences between the three independent lines OE6, OE8, OE12 compared with the wild type (Fig. 5B). The above results indicated that the OsHIR3 transgenic rice plants had enhanced disease resistance to Xoo. Taken together, OsHIR3-mediated basal resistance not only targets RSV, but also targets other pathogens, such as Xoo.

Example 7: NbHIR3s gain basal resistance by positively regulating the SA pathway

The positive plants with higher OsHIR3 expression were screened out through the above detection, and the SA content was determined and the key genes of SA pathway were analyzed by real-time fluorescence quantitative qRT-PCR. Refer to the qRT-PCR instruction manual, the specific operations are as follows:

Add the above reagents to RNase-free EP tubes in turn, mix well, spot 10 μL/well into a 384-well quantitative plate, cover with membrane, centrifuge briefly, and place in a qRT-PCR instrument for reaction: 95°C for 5 min ; Carry out 40 cycles: 95°C for 20s→58°C for 20s→72°C for 20s; 72°C for 10min.

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

Compared with the wild type, SA content was significantly increased in leaf cells of three independent lines of OsHIR3 transgenic rice, OE6, OE8, and OE12 (Fig. 6A), and the SA pathway key genes PBZ1 (also known as NPR1), PR1 and PR5 genes were significantly increased. up-regulated expression (Fig. 6B). This fully indicates that OsHIR3 gene obtains basal resistance by positively regulating SA pathway.

This is because after the plant is infected by pathogenic bacteria, systemic resistance leads to resistance to pathogenic bacteria in the remote uninfected parts. This induced resistance is called Systemic Acquired Resistance (SAR). It has been confirmed in many plant-pathogen interactions. The typical manifestation of SAR is to limit the growth of pathogenic bacteria and inhibit the development of their symptoms. In plants, there have been many reports on the role of SA in SAR. The mainstream view is that SA is an important signaling molecule in the SAR process, and the accumulation of SA will stimulate the SAR response. The massive expression of pathogenesis related proteins (PR proteins) is an important marker of SAR response. Multiple PR proteins work together in coordination rather than a specific PR protein alone to cause SAR response. In tobacco treated with SA or aspirin, a large amount of PR protein accumulates, and resistance to Tobacco mosaic virus (TMW) infection occurs; TMV infection can induce a sharp increase in endogenous SA content in tobacco, and resistant varieties The SA content of sid1 and sid2 mutants was significantly higher than that of susceptible varieties; sid1 and sid2 mutant plants could not accumulate SA in vivo and could not initiate SAR, and showed sensitivity to Pseudomonas syringae, further proving that SA is a key signaling molecule in the process of SAR.

The regulatory protein NPR1 is a key component in the SA-mediated signal transduction pathway, and NPR1 can induce the expression of resistance genes such as PR-1, thereby enhancing plant disease resistance. Upon pathogen infection, NPR1 gene expresses the affected mutant nim1, and SA levels are normal, but SAR cannot be induced, indicating that NPR1 acts downstream of SA and is a key regulator in the SAR signal transduction pathway. Despres et al. found that NPR1 can interact with members of the TGA family of Arabidopsis leucine-rich (bZIP) transcription factors, while npr1 mutants lost the interaction with TGA2, indicating that NPR1-mediated TGA2 binding Activation of defense genes is critical. When SAR is induced, NPR1 activates the PR-1 gene through the interaction with transcription factors in the promoter region of the PR gene, indicating that NPR1 activity is closely related to the regulation of PR gene expression. The study found that four point mutants of NPR1 blocked SA signaling and lost the interaction with TGA2 and TGA3, which were able to bind the SA response element of the Arabidopsis PR-1 promoter. NPR1 is linked to SA-induced PR-1 gene expression.

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<213> Artificial Sequence

<400> 5

cgcggatcct tacacgttgc tgcaggacgc tt 32

<210> 6

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<400> 6

aaggtgatgg gagattatgg ttac 24

<210> 7

<211> 20

<212> DNA/RNA

<213> Artificial Sequence

<400> 7

gataatcatc gcaagaccgg 20

<210> 8

<211> 24

<212> DNA/RNA

<213> Artificial Sequence

<400> 8

aaggtgatgg gagattatgg ttac 24

<210> 9

<211> 20

<212> DNA/RNA

<213> Artificial Sequence

<400> 9

gataatcatc gcaagaccgg 20

<210> 10

<211> 25

<212> DNA/RNA

<213> Artificial Sequence

<400> 10

ggtatccatg agactacata caact 25

<210> 11

<211> 23

<212> DNA/RNA

<213> Artificial Sequence

<400> 11

tactcagcct tggcaatcca cat 23

<210> 12

<211> 20

<212> DNA/RNA

<213> Artificial Sequence

<400> 12

cacactcgac ggagacgaag 20

<210> 13

<211> 21

<212> DNA/RNA

<213> Artificial Sequence

<400> 13

gccatagtag ccatccacga t 21

<210> 14

<211> 20

<212> DNA/RNA

<213> Artificial Sequence

<400> 14

tatccaagct ggccattgct 20

<210> 15

<211> 20

<212> DNA/RNA

<213> Artificial Sequence

<400> 15

ttctctggct ggcgtagttc 20

<210> 16

<211> 20

<212> DNA/RNA

<213> Artificial Sequence

<400> 16

cgcaacaact gcacctacac 20

<210> 17

<211> 20

<212> DNA/RNA

<213> 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.

Images