CN117947094A - Method for improving rice blast resistance by Pi-Pprs42 gene and application - Google Patents
Method for improving rice blast resistance by Pi-Pprs42 gene and application Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, in particular to a method for improving rice blast resistance by Pi-Pprs gene and application thereof, wherein a novel broad-spectrum rice blast resistance gene Pi-Pprs is cloned through the research on the expression of miRNA target genes for negatively regulating and controlling rice disease resistance, pi-Pprs gene is induced to express by rice blast bacteria infection in rice plants, and the rice plants which are over expressed with Pi-Pprs42 gene are endowed with the broad-spectrum rice blast resistance by enhancing the accumulation of active oxygen H 2O2 and the expression of related genes PR1a and PBZ1, SA and JA pathway key genes OsNPR1 and OsCOI b. The research provides a new thought and a new method for identifying rice blast resistance genes, and provides new gene resources for improving rice variety resistance and breeding new varieties with broad-spectrum disease resistance.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for improving rice blast resistance by Pi-Pprs42 gene and application thereof.
Background
The rice blast is the most destructive disease in rice production, and the effective control of the rice blast is critical to the sustainable development of rice production and the world grain safety. The identification of novel rice blast resistance genes, especially broad-spectrum rice blast resistance genes and the application thereof in rice breeding are the most economical and effective measures for preventing and controlling rice blast.
To date, 146 rice blast-dominant resistance loci have been identified, but only 38 have been cloned. Some genes are widely used in rice disease-resistant breeding, and play an important role in rice blast prevention and control. The coiled CC-NBS-LRR protein Pb1 identified, for example, from indica 'Modan' has been introduced into several elite varieties commercially cultivated in Japan and shows no signs of resistance loss in the last 30 years. He Zuhua et al identified a broad-spectrum durable new site Pigm of pestilence resistance in breeding materials of agricultural varieties in China, which is a gene cluster containing a plurality of NBS-LRR disease-resistant genes. The bred variety improved by Pigm has broad-spectrum durable disease resistance and does not influence the final yield.
We identify a new miRNA for negative regulation of rice blast resistance in rice, miR9664, the predicted target gene is mainly a gene for encoding NBS-LRR protein, wherein the expression level of the predicted target gene LOC_Os12g13550 is obviously down-regulated in transgenic rice over-expressing miR9664, and is obviously up-regulated in transgenic rice knocking miR9664, and the predicted target gene is probably a main rice blast resistance gene under epigenetic regulation, so that the novel miRNA has important potential application value in rice blast prevention and control.
Disclosure of Invention
The invention aims to provide a method for improving rice blast resistance by Pi-Pprs gene and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme: the invention takes rice miR9664 predictive target gene LOC_Os12g13550 as an object, extracts total RNA from rice seed pre-44 and reversely transcribes the total RNA into cDNA, uses a PCR technology to amplify CDS region sequence 2883bp (shown as SEQ ID No. 1) of LOC_Os12g13550 gene, codes CC-NBS-LRR disease-resistant protein containing one protein phosphokinase 1 regulatory subunit 42 (protein phosphatase 1 regulatory subunit 42), which is the first discovered disease-resistant protein with phosphokinase domain (figure 1), and is named Pi-Pprs42.
The gene sequence SQE ID No. 1and the coded amino acid sequence are shown as SQE ID No. 2. The Pi-Pprs gene over-expression vector is constructed, and the agrobacterium-mediated genetic transformation method is adopted to introduce the over-expression vector into the infected rice TP309 to obtain the Pi-Pprs gene over-expression plant. The rice strain over-expressing Pi-Pprs gene obviously enhances the resistance to Pyricularia oryzae, the T1 generation positive transgenic strain has the advantages that the isolated leaves at the tillering stage hurt inoculated Pyricularia oryzae A7203-8, HN2, GUY11 and LP11 conidium suspensions (1.0X10 5/mL), and the average lesion length and the fungal spore quantity of the transgenic strain are obviously lower than those of a non-transgenic TP309 control. Pi-Pprs is shown to significantly enhance the resistance of rice to different pathogenic rice blast strains. DAB (3, 3' Diaminobenzodine-HCl) staining detection of rice leaf cell H 2O2 accumulation shows that the accumulation of H 2O2 in 48H leaf cells of rice plants over-expressing Pi-Pprs42 gene after infection by rice blast bacteria is significantly higher than that of non-transgenic TP309 control. The detection results of the qRT-PCR method on the expression levels of defense related genes OsPR1a and OsPBZ1, JA and SA pathway signal pathway key genes OsCOI b and OsNPR1 show that compared with a control, the expression of OsPR1a, osPBZ1, osNPR 1and OsCOI b in rice plants over-expressing Pi-Pprs42 genes is obviously up-regulated.
The results show that Pi-Pprs42 can be used for endowing rice with broad-spectrum rice blast resistance by enhancing the accumulation of H 2O2 in rice plants and activating the expression of genes related to defense.
Therefore, the application claims the application of the Pi-Pprs gene shown in SEQ ID No.1 in improving the rice blast resistance of rice.
It is also within the scope of the present invention for a person skilled in the art to use the Pi-Pprs gene sequence shown as SEQ ID No.1 for cultivation of a new variety of rice.
The method over-expresses Pi-Pprs42 target genes in a rice variety TP309 to improve the resistance, so that the method can also be used for improving rice or breeding new varieties and also belongs to the protection scope of the invention.
A method for improving rice blast resistance of rice by using Pi-Pprs gene shown as SEQ ID No.1 or coded amino acid comprises the following steps:
(1) Extracting total RNA from wild rice seed pre-44, and then reversely transcribing the total RNA into cDNA;
(2) Amplifying a target gene Pi-Pprs by using a PCR technology, wherein the sequence of the target gene Pi-Pprs is shown as SEQ ID No. 1;
(3) Connecting the gene Pi-Pprs42 fragment to an expression vector to obtain a plasmid pCAMBIA 1300-Pi-Pprs 42 for over-expressing the Pi-Pprs target gene;
(4) Introducing the plasmid pCAMBIA 1300-Pi-Pprs into Agrobacterium;
(5) The agrobacterium containing the plasmid pCAMBIA 1300-Pi-Pprs is co-cultured with rice plant to obtain the rice plant with disease resistance.
Further, in the step (2), the primer sequences for amplifying the target gene Pi-Pprs are Pi-Pprs-42-F and Pi-Pprs-42-R:
Pi-Pprs42-F:5¢- ATTCGAGCTCGGTAATGTCGACGATGGTGGTGAG-3¢
Pi-Pprs42-R:5¢-AGGATCCCCGGGTACTACTTACATGCAACTGTTT-3¢。
The beneficial technical effects of the invention are as follows: the invention provides an expression study of miRNA target genes for negatively regulating rice disease resistance, which clones a novel broad-spectrum rice blast resistance gene Pi-Pprs, pi-Pprs gene is induced to be expressed by rice blast infection in rice plants, and the rice plants over-expressing Pi-Pprs42 gene endow the rice with broad-spectrum rice blast resistance by enhancing the accumulation of active oxygen H 2O2 and the expression of defense related genes PR1a and PBZ1 and SA and JA pathway key genes OsNPR1 and OsCOI b. The research provides a new thought and a new method for identifying rice blast resistance genes, and provides new gene resources for improving rice variety resistance and breeding new varieties with broad-spectrum disease resistance.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
Fig. 1: pi-Pprs42 protein structure prediction map.
Fig. 2: and (3) a graph of the expression level detection result of Pi-Pprs42 when rice is infected by rice blast bacteria.
Fig. 3: t0 generation over-expression Pi-Pprs42 transgenic rice line positive identification result diagram.
Fig. 4: the expression level detection result diagram of the T1 generation over-expression Pi-Pprs42 transgenic rice line Pi-Pprs.
Fig. 5: positive identification result diagram of T1 generation over-expression Pi-Pprs42 transgenic rice line: in the figure, A is a positive identification result diagram of OX-Pprs #14-T1, and B is a positive identification result diagram of OX-Pprs 42#16-T1.
Fig. 6: transgenic rice line seedling stage resistance test result diagram of over-expression Pi-Pprs42 gene: in the figure, A is a disease symptom graph after inoculation, and B is an average disease grade statistical graph.
Fig. 7: the identification result diagram of the resistance of the rice blast bacteria A7203-8 inoculated in the tillering stage of the transgenic rice strain which overexpresses the Pi-Pprs42 gene: in the graph, A is a disease symptom graph after inoculation, B is a rice blast disease spot length statistical graph, and C is a disease spot fungus spore amount statistical graph.
Fig. 8: the identification result diagram of the resistance of rice blast germ HN2 inoculated in the tillering stage of the transgenic rice strain with the over-expressed Pi-Pprs42 gene: in the graph, A is a disease symptom graph after inoculation, B is a rice blast disease spot length statistical graph, and C is a disease spot fungus spore amount statistical graph.
Fig. 9: transgenic rice strain over-expressing Pi-Pprs42 gene is inoculated with rice blast fungus GUY11 at tillering stage, and the result diagram is shown: in the graph, A is a disease symptom graph after inoculation, B is a rice blast disease spot length statistical graph, and C is a disease spot fungus spore amount statistical graph.
Fig. 10: transgenic rice strain over-expressing Pi-Pprs42 gene is inoculated with rice blast fungus LP11 resistance identification result diagram: in the graph, A is a disease symptom graph after inoculation, B is a rice blast disease spot length statistical graph, and C is a disease spot fungus spore amount statistical graph.
Fig. 11: the test result diagram of the accumulation of active oxygen in cells in leaves after the seedlings of transgenic rice lines of the control TP309 and the over-expressed Pi-Pprs42 are infected by rice blast bacteria: in the figure A, B, C is a DAB staining condition diagram, D is a relative H 2O2 content statistical diagram, and E is a OsRbohA gene expression level detection result diagram.
Fig. 12: expression level detection result graphs of OsPR1a, osPBZ1, osNPR1 and OsCOI b after transgenic rice line seedlings of control TP309 and over-expressed Pi-Pprs42 are infected by rice blast bacteria.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The construction of the over-expression Pi-Pprs gene vector comprises the following steps:
1. Total RNA was extracted from leaves of seedlings of oryza sativa at sub-44 four-leaf stage according to the kit instructions using MiniBEST plant RNA extraction kit (TaKaRa, dalian, china). RNA concentration and quality were determined using a NanoDrop 2000 uv-vis spectrophotometer (Thermo FISHER SCIENTIFC, waltham, ma, usa) and integrity distribution was checked by 2% agarose gel electrophoresis.
2. RNA was transcribed into cDNA by reverse transcription PCR.
3. The CDS sequence (shown as SEQ ID No.1, the coded amino acid series is shown as SEQ ID No. 2) of the Pi-Pprs gene with the enzyme cleavage site is amplified by PCR technology by using an amplification primer Pi-Pprs-F/R.
4. Electrophoresis was performed on 1% agarose gel at 120V for 23 minutes, the 2883bp electrophoresis fragment was excised under UV light, and the fragments were placed in a system for sol recovery, the recovery procedure was as described in the kit instructions of the particular manufacturer, and DNA was recovered by dissolution with water in a total volume of 30. Mu.L.
5. The vector was constructed using T4 ligation.
The plasmid used to construct the Pi-Pprs42 overexpression vector was pCAMBIA1300, containing two 35S strong promoters, one for the initiation of the gene of interest and the other for the initiation of the hygromycin gene (for the selection and identification of positive transgenic plants). The vector also contains the kanamycin (kan) resistance gene for screening positive bacterial clones during vector construction.
6. The pCAMBIA1300 plasmid was transferred into E.coli (DH 5 a) (see E.coli competent transformation standard method) and cultured overnight at 37℃and 200rpm, followed by plasmid extraction. Plasmid pCAMBIA1300 was then digested with restriction enzyme KpnI.
7. And recombining the target gene with the plasmid. The ligation product was added to E.coli (DH 5 a) and incubated overnight at 37 ℃. The monoclonal is picked up and placed on LB liquid medium containing kan resistance on a constant temperature shaking table (200 rpm/h at 37 ℃) for 12 hours, then bacterial plaque PCR identification is carried out by using a specific primer (Pi-Pprs-F/R), and plasmid miniextraction is carried out on positive colonies with correct sequencing to obtain purified plasmids.
8. The plasmid was then transferred to Agrobacterium tumefaciens EHA105, and then placed on Kan+ hygromycin antibiotic plates and incubated at 28℃overnight with inversion. The following day single colonies were picked for PCR colony identification.
9. Rice callus induction
(1) The seeds were washed with 75% alcohol for 10s and then three times with sterile water.
(2) Adding 1.5% sodium hypochlorite to soak the seeds, shaking in an ultra-clean workbench for 10min, pouring out the sodium hypochlorite, and then washing with sterile water until no peculiar smell exists.
(3) The liquid on the seed surface was blotted dry with sterilized filter paper. The dried seeds were placed evenly on N6 medium with sterile forceps. Sealing the flat plate with sealing film, and culturing in a 30 deg.c incubator for 7-10 d until tender yellow callus grows.
10. Infection and co-cultivation
(1) Agrobacterium strains with transformation vectors were streaked onto LB (plus Kan and Rif antibiotics) plates and activated in a 28℃incubator.
(2) Single colonies were picked up in 2mL of liquid LB (Kan and Rif antibiotics were added), at 28℃and 200rpm were shaken overnight, 1mL of the bacterial liquid was aspirated the next day to 50 mL TY medium, the concentration was measured after shaking at 200rpm for 1h at 28℃and the OD600 value was adjusted to 0.05-0.1.
(3) In an ultra-clean workbench, rice callus stripped in advance is soaked in bacterial liquid and gently shaken for 25min (pollution is avoided).
(4) Sucking the bacterial liquid on the surface of the callus with sterile filter paper, and blowing for 30min in an ultra-clean bench until the surface of the callus is dried.
(5) The dried calli were transferred to co-culture medium and dark cultured at 22℃for 3 days.
11. Screening, differentiation and rooting culture
(1) The calli were placed on the selection medium from the co-culture medium using sterile forceps, and the selection medium was changed every 10 days during the culture with light at 30℃for about one month.
(2) The selected surviving calli were transferred to primary differentiation medium at loose intervals and incubated at 30℃for about 14 days with light.
(3) Transferring the callus subjected to primary differentiation to a redifferentiation medium until the callus is differentiated into green buds.
(4) And (5) transferring the whole tissue to a rooting culture medium after the green buds grow strong.
12. When the tissue in the rooting culture medium grows into a strong root and leaves grow to about 10cm (the seedling is formed), taking the seedling out of the culture medium, washing the culture medium of the root, soaking the culture medium in tap water for 3-5 d, and transplanting the healthy and survival seedling into soil.
Example 2
Expression levels of Pi-Pprs42 gene 0, 3, 12 and 24 hours after the disease resistance Shui Daozi pre 44 (ZY 44) and the disease-susceptible rice Jiangnan Oryza Glutinosa (JNXN) were inoculated with Pyricularia oryzae LP 11.
To determine the expression pattern of Pi-Pprs42 between the anti-sensory materials, rice pre-44 (ZY 44) and rice Jiangnan fragrant glutinous rice (JNXN) three leaves-heart stage seedlings were inoculated with a suspension of conidia of rice blast bacteria LP11 (1.5x10 5/mL), 0,3, 12, 24h leaves after inoculation were taken to extract RNA, and qRT-PCR was used to detect expression of Pi-Pprs42 at different time points in the two materials. Pi-Pprs42 had similar expression patterns in disease-resistant rice ZY44 and disease-sensitive rice JNXN, with 3hpi significantly upregulated expression, 12hpi at the highest level and 24hpi significantly downregulated expression, but its level of rice blast-induced expression in disease-resistant rice ZY44 was significantly higher than that in disease-sensitive rice JNXN (FIG. 2). Pi-Pprs.sup.42 was shown to play an important role in early stage of rice-Pyricularia oryzae interaction.
Example 3
And (5) identifying a positive rice strain over-expressing the Pi-Pprs gene.
Firstly, a hygromycin soaking method is utilized to carry out preliminary identification on transgenic plants. The specific method comprises the following steps: to 1L of hygromycin soak solution, 650. Mu.L of 50 mg/mL hygromycin solution was added, and 1mL of 1mg/mL 6-BA mother liquor was added to adjust the pH to 7. And marking a serial number on the back of the rice leaf to be detected, cutting rice leaves with the length of 2cm containing the serial number (the other identical serial number is left on the plant), placing the rice leaf into hygromycin soaking liquid, placing the rice leaf into a climatic chamber for 3-5 days, observing whether a brown mark exists at the incision of the leaf, if so, indicating that the rice leaf is a negative plant, and if the whole leaf is green, indicating that the rice leaf is a positive plant.
The positive transgenic plants identified above were further PCR-identified using hygromycin specific primers Pprs-PI.
Pprs42-PI-F: 5¢- TCCTTCGCAAGACCCTTCCTC-3¢
Pprs42-PI-R: 5¢- TACGCCGCTCGTTTATCTCCTG-3¢。
The specific method comprises the following steps: the DNA of the transgenic rice is extracted by CTAB method, and hygromycin gene is amplified to identify whether the transformed seedlings are positive. Finally 8 positive T0 transgenic plants were identified (FIG. 3). The expression level of Pi-Pprs42 of the T1-generation transgenic plants was then detected using specific primers Pprs-RT-F/R (FIG. 4).
Pprs42-RT-F: 5¢-TCAACAGCTGTTGCTTCTGCAGG-3¢
Pprs42-RT-R: 5¢-TCCTTGGGAGCACGGTAAATGC-3¢。
T1 generation plants of OX-Pprs #14 and OX-Pprs #16 lines, which had expression levels 16.6-fold and 9.0-fold higher than the control, were positively identified using specific primers Pprs-PI-F/R, to obtain 8 (FIG. 5A) and 15 positive T1 generation transgenic plants, respectively (FIG. 5B).
Example 4
And (5) identifying the seedling stage resistance of the transgenic rice line over-expressing the Pi-Pprs gene.
By using non-transgenic TP309 as a control, T1 generation positive transgenic plants, three-leaf-first-heart seedlings were sprayed with a suspension of Magnaporthe grisea LP11 conidium (1.5X10 5/mL), and 7. 7 d were used for investigation of disease conditions. The results showed that transgenic T1 lines #14 and #16 overexpressing Pi-Pprs42 had significantly increased blast resistance (FIG. 6A), with a 2.2 and 1.6 level reduction in the incidence level, respectively, compared to the control (FIG. 6B).
Example 5
And (5) identifying the resistance of the transgenic rice line over-expressing the Pi-Pprs gene in the tillering stage.
To further observe the resistance contribution of Pi-Pprs4 to tillering stage rice, tillering stage T1 generation positive transgenic plants were inoculated with Pyricularia oryzae A7203-8 (FIG. 7), HN2 (FIG. 8), GUY11 (FIG. 9) and LP11 (FIG. 10) conidium suspensions (1.0X10: 10 5/mL) ex vivo, and 5-7 d were subjected to resistance identification, and after 9 d, the leaf wounds were examined for fungal spore amounts. The results showed that the OX-Pprs42 transgenic plants developed less frequently than the controls, and that the average lesion length of OX-Pprs42 transgenic plants was significantly reduced after inoculation with different Pyricularia oryzae (figures 7B, 8B, 9B, 10B). The spore amount detection results showed that the fungal spore amount on the lesions of the OX-Pprs42 transgenic plants was significantly reduced (FIGS. 7C, 8C, 9C, 10C) compared to the control, consistent with the resistance identification results (FIGS. 7A, 8A, 9A, 10A), indicating that over-expression of Pi-Pprs4 enhanced the resistance of the tillering stage rice to multiple rice blast strains. Taken together, pi-Pprs4 was shown to confer broad-spectrum rice blast resistance to rice.
Example 6
Response of active oxygen in Pi-Pprs4 over-expressed transgenic seedling stage rice leaves to Pyricularia oryzae infection
DAB (3, 3' diaminobenzodine-HCl) staining was performed on three-leaf-heart rice leaves after inoculation with Pyricularia oryzae 48H, and H 2O2 outbreak was detected. The results showed that after 48H infestations with Pyricularia oryzae, the brown area was greater in Pi-Pprs4 overexpressing transgenic plants (FIG. 11B, C) and the accumulation of H 2O2 was significantly increased, with 1.75 and 1.13 fold increase in lines #14 and #16, respectively, compared to control TP309 (FIG. 11A). 48 The expression level of the hpi active oxo associated gene OSRbohA was significantly higher than that of TP309 in the OX-Pprs rice line (FIG. 11E). The outbreak enhancement of H 2O2 is shown to be one of the ways in which Pi-Pprs4 enhances the resistance of rice plants to rice blast.
Example 7
Expression level detection of OsPR1a, osPBZ1, osNPR1 and OsCOI b after infection of transgenic rice line seedlings of control TP309 and over-expressed Pi-Pprs by Pyricularia oryzae
Expression of OsPR1a, osPBZ1, osCOI1b and OsNPR1 in TP309 and OX-Pprs #14 was examined at 0.0 h and 24. 24h, respectively, after inoculation of the suspension of Pyricularia oryzae LP11 conidia (1.0X10 5/mL). The results show that OsPR1a (FIG. 12A), osPBZ1 (FIG. 12B), osNPR1 (FIG. 12C) and OsCOI B (FIG. 12D) are all induced to be expressed after inoculation, and the expression level of 24 hpi in an OX-Pprs42 plant is significantly higher than that of TP309. It is shown that Pi-Pprs42 overexpression enhances the expression of defense-related genes and enhances the rice blast resistance of rice plants through SA and JA signaling pathways.
Finally, what should be said is: the above embodiments are only for illustrating the technical aspects of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention, which is intended to be encompassed by the claims.
Claims (5)
1. The Pi-Pprs gene shown as SEQ ID No.1 is applied to improving the rice blast resistance of rice.
2. The application of the amino acid sequence coded by the Pi-Pprs gene shown as SEQ ID No.2 in improving rice blast resistance of rice.
3. The Pi-Pprs gene shown as SEQ ID No.1 is contained in the application of rice breeding.
4. A method for improving rice blast resistance of rice by using Pi-Pprs gene shown in SEQ ID No.1 or coded amino acid is characterized by comprising the following steps:
(1) Extracting total RNA from wild rice seed pre-44, and then reversely transcribing the total RNA into cDNA;
(2) Amplifying a target gene Pi-Pprs by using a PCR technology, wherein the sequence of the target gene Pi-Pprs is shown as SEQ ID No. 1;
(3) Connecting the gene Pi-Pprs42 fragment to an expression vector to obtain a plasmid pCAMBIA 1300-Pi-Pprs 42 for over-expressing the Pi-Pprs target gene;
(4) Introducing the plasmid pCAMBIA 1300-Pi-Pprs into Agrobacterium;
(5) The agrobacterium containing the plasmid pCAMBIA 1300-Pi-Pprs is co-cultured with rice plant to obtain the rice plant with disease resistance.
5. The method according to claim 4, wherein in the step (2), the primer sequences for amplifying the target gene Pi-Pprs are Pi-Pprs-42-F and Pi-Pprs-42-R:
Pi-Pprs42-F: 5¢-ATTCGAGCTCGGTAATGTCGACGATGGTGGTGAG-3¢
Pi-Pprs42-R:5¢-AGGATCCCCGGGTACTACTTACATGCAACTGTTT-3¢。
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