Application of pear disease-resistant gene REA1 in prevention and control of pear fire blight

Abstract

The invention provides application of a pear disease-resistant gene REA1 in prevention and control of pear fire blight. The invention relates to a pear fire and blight resistance gene REA1 and application thereof in molecular identification and creation of pear disease-resistant varieties and pear fire and blight prevention and control. The REA1 gene provided by the invention is a novel pear fire and epidemic disease resistant gene resource, and the resistance to fire and epidemic diseases can be improved by overexpressing the gene in a susceptible variety. Through pear fruit gene silencing and overexpression analysis, the important role of the REA1 gene in pear fire and blight resistance is clarified for the first time, the REA1 gene provided by the invention is a new high-quality pear fire and blight resistance gene, and theoretically promotes the understanding of plants on dead body nutrition pathogen ETI immunity; in application, high-quality gene resources and a new direction are provided for follow-up creation of fire and blight resistant fruit tree varieties, a new strategy is provided for green prevention and control of pear fire and blight, and a new method for identifying fire and blight resistant molecules of pear varieties is provided.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of a pear disease-resistant gene REA1 in the prevention and control of fire blight of pear. Background Art

[0002] 1. Analysis of Pear Gene Function The function of plant genes can be elucidated by comparing the phenotypes or functional performances under the over-expression and normal-expression conditions of the genes. The over-expression of genes includes the expression higher than the normal level and the expression lower than the normal level. The expression higher than the normal level is mainly over-expression, which can be achieved by connecting a strong promoter to drive the expression of the target gene. The expression lower than the normal level can be achieved by RNA interference (RNAi), virus-induced gene silencing (VIGS), etc. RNAi is achieved by constructing and transforming a hairpin structure formed by inserting a same sequence fragment in the opposite direction with an intron or other non-expressing sequences inserted therein, and the result is the reduction of the expression of the target gene. VIGS is a technology developed based on the antiviral mechanism of plants and is applicable to the study of gene functions of species lacking a stable genetic transformation system. It can avoid complex transformation methods and effectively reduce the expression of endogenous genes in plants. Therefore, it is widely used in the research of multiple fields such as plant resistance and growth and development regulation. Pear is a woody plant, mainly propagated by grafting, with a high genomic heterozygosity and difficulties in tissue culture and stable genetic transformation. Therefore, the functions of pear genes can be analyzed and clarified by techniques such as transient gene over-expression and VIGS gene silencing.

[0003] 2. Immune Stimulation of Pear against Erwinia amylovora Plant immunity is a defense response activated by plants through the recognition of pathogen molecules by receptors. Plant immunity is divided into two levels. First, pattern recognition receptors (PRRs) located on the cell surface sense pathogen-associated molecular patterns (PAMPs) of invading pathogens or host-derived damage-associated molecular patterns (DAMPs), initiating pattern-triggered immunity (PTI). The other level is intracellular nucleotide-binding (NB)-leucine-rich repeat (LRR)-containing receptors (NLRs), which mediate the direct or indirect recognition of race-specific pathogen effector proteins delivered into plant cells, initiating effector-triggered immunity (ETI). RPS2 is an NLR protein in Arabidopsis thaliana with NB and LRR domains, located on the cytoplasmic membrane, binding to another membrane protein RIN4, and regulating the immunity triggered by the bacterial effector protein AvrRpt2. AvrRpt2 has cysteine protease activity, which activates RPS2 by cleaving the RIN4 protein, triggering resistance to Pseudomonas syringae. A similar immune mechanism has been found in apples. The wild apple Malus × robusta 5 contains a similar NLR protein FB_MR5 and its guarded MdRIN4, and the fire blight-causing Erwinia amylovora has AvrRpt2 EA . AvrRpt2 EA activates FB_MR5 after cleaving MdRIN4, triggering resistance to fire blight bacteria. This is currently the only example of ETI against necrotrophic pathogens. Pears and apples belong to the Rosaceae family. However, to date, there have been no reports of genes similar to RPS2 in pears. The identification and application of RPS2-like genes in pears will promote the understanding of plant ETI and provide new strategies and resources for the prevention and control of pear fire blight.

[0004] 3. Prevention and control of pear fire blight Pear fire blight is a devastating bacterial disease caused by Erwinia amylovora, and it is also a quarantine and class I crop disease in China, posing a great threat to the healthy development of the pear industry nationwide and globally. Due to the wide host range, strong virulence, fast migration speed of Erwinia amylovora, and the lack of pear disease-resistant resources, chemical control remains an important means. Due to problems such as ecological pollution, human and livestock poisoning, and the easy generation of pathogen resistance by some pesticides, the identification of pear fire blight-resistant genes, the creation, cultivation, and utilization of fire blight-resistant materials and varieties are crucial for the green prevention and control of pear fire blight. Summary of the invention

[0005] The object of the present invention is to provide an application of the pear disease-resistant gene REA1 in the prevention and control of fire blight, which is a pear fire blight-resistant gene REA1 and its application in the molecular identification and creation of disease-resistant pear varieties and the prevention and control of fire blight.

[0006] The pear disease-resistant gene REA1 provided by the present invention uses the cDNA of the white pear (Pyrus×bretschneideri) variety Yuluxiang as a template, and the Yuluxiang gene REA1 (Resistance to Erwinia amylovora 1) is obtained by PCR cloning. Its nucleotide sequence is shown in SEQ ID: 1. The open reading frame (ORF) of this gene is 2712 bp long and encodes a protein containing 903 amino acids, and its sequence is shown in SEQ ID: 2. The REA1 protein contains a nucleotide-binding domain and a leucine-rich repeat domain. The nucleotide sequence cloned in the present invention has 6 base differences from the nucleotide sequence of XP_009349127 of the white pear variety Hongxiangsu in the (Pyrus×bretschneideri) (taxid: 225117) database of NCBI, and these base changes result in 3 amino acid changes. Specifically: G2122 becomes C, resulting in the amino acid Glu708 becoming Gln; T2146 becomes C, resulting in the amino acid Ser716 becoming Pro; G2635 becomes C, resulting in the amino acid Glu879 becoming Gln.

[0007] Before the present invention, there was no public report on the function of the REA1 gene. The present invention cloned this gene for the first time and clarified its role in the resistance to fire blight in pears. The gene was expressed in Nicotiana benthamiana using an Agrobacterium-mediated gene expression system, which could stimulate the hypersensitive cell necrosis reaction, indicating that it is a functional NLR gene. An overexpression vector and a TRV-mediated VIGS gene silencing vector of this gene were constructed, and the gene was overexpressed or silenced in pear fruits. The analysis results of the disease incidence area and pathogen biomass of the fruits after inoculation showed that overexpression of this gene significantly improved the resistance of the fruits of the susceptible variety Korla Fragrant Pear to Erwinia amylovora, while silencing the gene significantly reduced the resistance of the fruits of the disease-resistant variety Yuluxiang Pear to Erwinia amylovora, indicating that REA1 plays an important role in improving the resistance of pears to fire blight.

[0008] Based on the function of the REA1 gene clarified in the present invention above, the application object of the present invention is to use the REA1 gene to improve the resistance of pears to fire blight and to identify pear fire blight-resistant varieties based on this disease-resistant gene, including (1) functional identification of the Yuluxiang REA1 gene and its application in improving the disease resistance of susceptible varieties (such as Korla Fragrant Pear) to fire blight (Example 1); and (2) application in the molecular identification of pear fire blight-resistant varieties (such as Akizuki Pear) based on the REA1 gene (Example 2).

[0009] Functional identification of the Yuluxiang REA1 gene and its application in improving the resistance of susceptible varieties (such as Korla fragrant pear) to fire blight are specifically achieved through the following steps: (1) Construction and acquisition of the REA1 gene overexpression structure: Clone the open reading frame (ORF) of the Yuluxiang REA1 gene into a plant expression vector to drive its expression under the control of a strong promoter; (2) Acquisition of Agrobacterium tumefaciens transformed with the REA1 gene overexpression structure: Transform the constructed REA1 gene overexpression structure into an Agrobacterium tumefaciens strain by methods such as electroporation; (3) Identification and acquisition of the functional REA1 gene: Express the REA1 gene in Nicotiana benthamiana, observe the induction of the hypersensitive cell death response, and obtain the functional REA1 gene that can trigger hypersensitive necrosis; (4) Verification of the fire blight resistance function of the REA1 gene: Construct a TRV-mediated VIGS gene silencing vector for the REA1 gene, transform Agrobacterium tumefaciens, silence the REA1 gene on Yuluxiang fruits, and clarify that the resistance of Yuluxiang pear fruits to Erwinia amylovora is significantly reduced through inoculation analysis; (5) Acquisition of Korla fragrant pear fruits with improved fire blight resistance based on overexpression of REA1: Overexpress the REA1 gene in the susceptible variety Korla fragrant pear, inoculate and detect the resistance to Erwinia amylovora, and obtain Korla fragrant pear fruits with improved disease resistance.

[0010] Application in the molecular identification of fire blight resistant pear varieties (such as Akizuki pear) based on the REA1 gene. It is achieved through the following steps: (1) Cloning and sequence analysis of the Akizuki pear REA1 gene: Extract the RNA from the young leaves of Akizuki pear and reverse transcribe it into cDNA. Using it as a template, clone the Akizuki pear REA1 gene by PCR and sequence analysis to determine whether it is a full-length gene; (2) Construction and acquisition of the Akizuki pear REA1 gene overexpression structure: Clone the open reading frame (ORF) of the Akizuki pear REA1 gene into a plant expression vector to drive its expression under the control of a strong promoter; (3) Acquisition of Agrobacterium tumefaciens transformed with the Akizuki pear REA1 gene overexpression structure: Introduce the constructed Akizuki pear REA1 gene overexpression structure into an Agrobacterium tumefaciens strain by methods such as electroporation; (4) Functional identification of the Akizuki pear REA1 gene: Express the Akizuki pear REA1 gene in Nicotiana benthamiana, observe the induction of the hypersensitive cell death response, and the gene that can trigger the hypersensitive response is the functional REA1 gene; (5) Evaluation and verification of the resistance of Akizuki pear to Erwinia amylovora: Determine whether the variety is a fire blight resistant variety based on the presence or absence of the functional REA1 gene. Inoculate the young shoots of Akizuki pear with Erwinia amylovora and detect the disease incidence to verify the above resistance identification results based on the functional REA1 gene.

[0011] Advantages of the present invention: (1) The REA1 gene provided by the present invention is a novel gene resource for fire blight resistance in pears. Overexpression of this gene in susceptible varieties can improve resistance to fire blight. Fire blight resistance in pears is a quantitative trait resistance, and worldwide, materials resistant to fire blight are scarce. The REA1 gene is a typical NLR disease-resistant protein, which can endow pears with ETI immune resistance to Erwinia amylovora. It is the second (the first in pears) fire blight-resistant NLR gene in the world after apple FB_MR5, so it is a high-quality fire blight-resistant gene resource for pears, providing direction and reference for subsequent creation of fire blight-resistant fruit tree germplasms. (2) The REA1 gene provided by the present invention is an important fire blight-resistant gene, and varieties carrying the REA1 gene are an important type of disease-resistant varieties. Whether a functional REA1 gene is present can be used as one of the criteria for evaluating and identifying the resistance of pear varieties to Erwinia amylovora. A variety that can clone a functional REA1 gene can be identified as a fire blight-resistant variety. Therefore, the present invention provides a new molecular identification method for fire blight-resistant pear varieties, which is more convenient and accurate compared with traditional identification methods. Description of the Drawings

[0012] Figure 1 Provide evidence that Yuluxiang pear REA1 is a functional NLR gene, showing the ability of this gene to stimulate the hypersensitive necrosis response in plants after expression in Nicotiana benthamiana. Figure A shows that the corresponding gene of the Yuluxiang variety of the Pyrus bretschneideri sequence XP_009349127 with the highest homology to Arabidopsis AtRPS2 can stimulate the hypersensitive necrosis response, and it is named REA1 (Resistance to Erwinia amylovora 1). Figure B shows that Yuluxiang RIN4 YLX inhibits the hypersensitive necrosis response stimulated by REA1, and REA1, RIN4 YLX when co-expressed with the Erwinia amylovora AvrRpt2 EA in pears, the hypersensitive necrosis response is re-stimulated. The experiment was repeated three times with consistent results. The results show that Yuluxiang REA1 is an RPS2-like fire blight-resistant gene in pears that stimulates immunity and defends RIN4 EA by indirectly recognizing AvrRpt2. YLX

[0013] Figure 2Provide evidence for the gene silencing analysis that the REA1 gene plays an important role in the disease resistance of the disease-resistant pear cultivar Yuluxiang to fire blight. Five days after inoculating the fruits of Yuluxiang pears treated with REA1 gene silencing with the fluorescently labeled Erwinia amylovora strain Ea-Luc1, the fluorescence signals representing the pathogen amount on the surface and cross-section were significantly higher than those of the control group (A), indicating that the REA1 gene silencing treatment led to a significant decrease in disease resistance. The results of real-time fluorescence quantitative PCR showed that the REA1 gene silencing treatment led to a significant decrease in the expression level of this gene in the fruits of Yuluxiang pears (B). In the fruits of Yuluxiang pears with the REA1 gene silenced, the fluorescence signals representing the pathogen amount on the surface and cross-section were 6.71 times (C) and 3.24 times (D) that of the control group, respectively. The results of the pathogen amount determination analysis showed that the pathogen amount in the fruits with the REA1 gene silenced was extremely significantly higher than that of the control group after inoculation (E, F). These results indicate that the silencing of the REA1 gene significantly reduces the resistance of the fruits of the disease-resistant cultivar Yuluxiang pears to Erwinia amylovora. The above data were statistically analyzed by Student's t-test using GraphPad Prism, and the significance of the difference was indicated by different numbers of * (**, P < 0.01; ***, P < 0.001).

[0014] Figure 3 Provide evidence that the REA1 gene improves the resistance of the susceptible cultivar Korla Fragrant Pear to fire blight. Five days after inoculating the fruits of Korla Fragrant Pears overexpressing the REA1 gene with the fluorescently labeled Erwinia amylovora strain Ea-Luc1, the fluorescence signals representing the pathogen amount on the surface and cross-section were significantly lower than those of the control group (A), indicating that the overexpression treatment of the REA1 gene significantly improved disease resistance. The results of real-time fluorescence quantitative PCR showed that the overexpression treatments of both the REA1 gene and the homologous gene of Yuluxiang with high homology to RPS2 (XP_048439943) used as a control led to a significant increase in the expression levels of the corresponding genes in the fruits of Korla Fragrant Pears (B, C). In the fruits of Korla Fragrant Pears overexpressing the REA1 gene, the fluorescence signals representing the pathogen amount on the surface and cross-section were 7.94 times (D) and 7.69 times (E) that of the control gene treatment group, respectively. The results of the pathogen amount determination analysis showed that the pathogen amount in the fruits overexpressing the REA1 gene was extremely significantly lower than that of the control gene treatment group after inoculation (F, G). These results indicate that the transient overexpression of the REA1 gene significantly improves the resistance of the fruits of the susceptible cultivar Korla Fragrant Pears to Erwinia amylovora. The above data were statistically analyzed by Student's t-test using GraphPad Prism, and the significance of the difference was indicated by different numbers of * (**, P < 0.01; ***, P < 0.001, ****, P < 0.0001).

[0015] Figure 4The results provide evidence for the identification of fire blight resistant varieties (such as Qiuyue pear) based on the REA1 gene. Figure A shows the stimulation of hypersensitive cell necrosis response by expressing this gene in Nicotiana benthamiana. The results showed that the expression of Qiuyue pear REA1 (REA1 QY ) stimulates allergic cell necrosis; REA1 QY With RIN4 QY When co-expressed, allergic cell necrosis was inhibited; Qiuyue Pear REA1 QY 、RIN4 QY With AvrRpt2 EA When the three are co-expressed, allergic cell necrosis is re-stimulated. The experiment was repeated three times and the results were consistent. These results indicate that there is a protein in Qiuyue pear that recognizes AvrRpt2 EA The functional fire blight resistance gene REA1 stimulates immunity and identifies Qiuyue pear as a fire blight resistant variety. Figure BC shows the inoculation analysis evidence to verify the above identification results. The bacteria solution of fire blight pathogen was dipped into a sterilized toothpick and inoculated into the top tender branches of Yuluxiang and Qiuyue pear. After 7 days of cultivation in an artificial climate box at 28℃, 75% relative humidity and 12h of light, there was no significant difference in the disease situation of Qiuyue pear tender branches and the disease-resistant variety Yuluxiang (B). The statistical analysis results of the lesion length showed that the average lesion length of Yuluxiang and Qiuyue pear tender branches was 3.16mm and 4.66mm, respectively, with no significant difference between the two (C), which is consistent with the result of Figure A identifying it as a fire blight resistant variety by possessing a functional REA1 gene. The scale bar in Figure B is 3cm. The data in Figure C were statistically analyzed by Student's t-test using GraphPad Prism, ns: not significantly different. These results indicate that the fire blight resistance gene REA1 exists in Qiuyue pear, and whether or not a pear variety has a functional REA1 gene can be used as a molecular basis for identifying the resistance of pear varieties to fire blight. Varieties that can be preliminarily identified as fire blight resistant can be cloned with a functional REA1 gene. DETAILED DESCRIPTION

[0016] The present invention is further described in conjunction with the accompanying drawings and embodiments.

[0017] Example 1 A REA1 gene was cloned and identified in Yuluxiang pears, and its role in resistance to Erwinia amylovora was elucidated for the first time. Overexpression of the REA1 gene significantly enhanced the resistance of fruits of the susceptible cultivar Korla fragrant pears to Erwinia amylovora, while silencing of the REA1 gene significantly reduced the resistance of fruits of the resistant cultivar Yuluxiang pears to Erwinia amylovora, indicating that REA1 plays an important role in improving the resistance of pears to fire blight. The REA1 gene is a high-quality fire blight resistance gene resource for pears. The identification of this gene provides a new idea for the study of the ETI immune mechanism of pears against fire blight and provides a direction and reference for the subsequent creation of fire blight-resistant fruit tree germplasms. The main steps for the cloning, identification of the REA1 gene in Yuluxiang pears and its application in improving the disease resistance of susceptible cultivars (such as Korla fragrant pears) to fire blight include:

[0018] 1) Cloning of the REA1 gene in Yuluxiang pears The REA1 gene in Yuluxiang pears provided by the present invention was cloned through the following steps. First, based on the white pear genome database (Pyrus×bretschneideri), the sequence XP_009349127 with the highest homology to RPS2 was analyzed, and primers Pbr9127-F (5’-aagtccggagctagctctaga atg ggg aac atg ttc tca gtc-3’, the italic part is the sequence containing the Xba I restriction site and consistent with the vector pCAMBIA1305) (the sequence is shown in SEQ ID: 3) and Pbr9127-R (5’-gcccttgctcaccatggatcc acc gcg tct gaa gca ggg-3’, the italic part is the sequence containing the Bam HI restriction site and consistent with the linearized vector pCAMBIA1305) (the sequence is shown in SEQ ID: 4) were designed according to this sequence. The total RNA of Yuluxiang pear leaves was extracted using the Tiangen RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit, and then the RNA was reverse transcribed into cDNA using the Novizan HiScript III RT SuperMix Reverse Transcription Kit. Using the cDNA of Yuluxiang leaves as a template, Pbr9127-F and Pbr9127-R as primers, the full-length sequence was amplified using a high-fidelity enzyme, and then the PCR product was recovered by gel cutting and purification after 1% agarose gel electrophoresis.

[0019] 2) Construction and acquisition of the overexpression structure of the REA1 gene in Yuluxiang pears The overexpression vector pCAMBIA1305 plasmid was digested with Xba I and Bam HI, and PCR One-Step Directional Cloning Kit was used to perform homologous recombination and ligation between the amplified target gene and the pCAMBIA1305 vector after digestion with restriction enzymes. The ligation product was transformed into Escherichia coli, screened on kanamycin plates, identified by PCR, and sent to a company for sequencing identification. The REA1 gene of Yuluxiang was successfully cloned, and the overexpression construct pCAMBIA1305-REA1 of the REA1 gene of Yuluxiang was obtained. The nucleotide sequence of the REA1 gene of Yuluxiang is shown in SEQ ID: 1. The open reading frame (ORF) of this gene is 2712 bp long and encodes a protein consisting of 903 amino acids, and its sequence is shown in SEQ ID: 2. The encoded product of this gene contains a nucleotide-binding domain and a leucine-rich repeat domain. The sequence of the REA1 gene of Yuluxiang cloned in this invention has 6 base differences from the nucleotide sequence of XP_009349127 of the red fragrant crisp pear variety in the NCBI (Pyrus×bretschneideri) database. These base changes result in 3 amino acid changes: G2122 changes to C, resulting in the amino acid Glu708 changing to Gln; T2146 changes to C, resulting in the amino acid Ser716 changing to Pro; G2635 changes to C, resulting in the amino acid Glu879 changing to Gln. Before this invention, there was no public report on the function of the REA1 gene of Yuluxiang.

[0020] 3) Obtaining Agrobacterium tumefaciens transformed with the overexpression construct pCAMBIA1305-REA1 of the REA1 gene of Yuluxiang The overexpression construct pCAMBIA1305-REA1 of the REA1 gene was transformed into the Agrobacterium tumefaciens strain GV3101 by methods such as electroporation. Transformants were screened on YEP medium containing kanamycin, and then identified by double digestion with Xba I and Bam HI and PCR. Agrobacterium tumefaciens carrying the overexpression construct pCAMBIA1305-REA1 of the REA1 gene of Yuluxiang was obtained and stored in a -80°C refrigerator for subsequent in vivo expression in tobacco.

[0021] 4) Cloning and transformation of other genes required for identifying the function of the REA1 gene of Yuluxiang Since the RIN4 gene in Yuluxiang is required YLX gene and AvrRpt2 in Erwinia amylovora EACo-expression of genes was used to verify the functionality of the REA1 gene. Therefore, cDNA from Yuluxiang leaves was used as a template, and RIN4-F (5’-gacgatgataagggcggtacc atg gca caa cgt tca cat gta cc-3’, the italic part is the sequence containing the Kpn I restriction site, which is the same as the sequence on the vector pCAMBIA1300) (the sequence is shown in SEQ ID: 5) and RIN4-R (5’-gtcctaggctacgtaggatcc tca ttt tct gct cca tgg aaa gc-3’, the italic part is the sequence containing the Bam HI restriction site, which is the same as the sequence on the linearized vector pCAMBIA1300) (the sequence is shown in SEQ ID: 6) were used as primers to clone RIN4 of Yuluxiang YLX gene; DNA from E. amylovora was used as a template, and AvrRpt2 EA -F (5’-ttacaattaccatggggcgcgccatg aaa gtc agt cat ctc aca tcc-3’, the italic part is the sequence containing the Asc I restriction site, which is the same as the sequence on the vector pFG1008) (the sequence is shown in SEQ ID: 7) and AvrRpt2 EA -R (5’-aacatcgtatgggtaggtacc attttc act gta taa cat ggc gtg t-3’, the italic part is the sequence containing the Kpn I restriction site, which is the same as the sequence on the vector pFG1008) (the sequence is shown in SEQ ID: 8) were used as primers to clone the AvrRpt2 EA gene. Using a one-step directional cloning kit for PCR, the amplified target genes were respectively ligated by homologous recombination with the digested pCAMBIA1300 and pFG1008 vectors. The ligation products were transformed into Escherichia coli, screened on corresponding resistance plates, identified by PCR, and sent to the company for sequencing identification to obtain the completely cloned RIN4 YLX gene and AvrRpt2 EA gene.

[0022] The overexpression construct pCAMBIA1300-RIN4 YLX of the RIN4 YLX gene and the overexpression construct pFG1008-AvrRpt2 EA of the AvrRpt2 EA gene were transformed into the Agrobacterium strain GV3101 by methods such as electroporation, and transformants were screened on YEP medium with corresponding antibiotics. Then, through double digestion and PCR identification, Yuluxiang RIN4 carrying the respectiveYLX Gene overexpression construct pCAMBIA1300 - RIN4 YLX and AvrRpt2 EA Gene overexpression construct pFG1008 - AvrRpt2 EA Agrobacterium was stored in an - 80°C refrigerator. It was used for the next step of in - vivo expression in tobacco.

[0023] 5) Functional identification of the Yuluxiang REA1 gene The stored GV3101 Agrobacterium was activated and cultured. Single colonies were picked for small - scale culture. It was cultured overnight at 28°C with a shaking speed of 220 rpm. Then, it was cultured with shaking at a ratio of 1:10 of the bacterial liquid to the liquid medium for 4 mL. It was cultured with shaking at 28°C and 220 rpm for 4 h. The OD 600 of the bacterial liquid was controlled to be between 0.8 and 0.9. It was centrifuged at 5000 rpm at room temperature for 8 min. The bacterial liquid was resuspended with the resuspension buffer (10 mM MgCl 2 , 10 mM MES, 200 mM acetosyringone) until the OD 600 was 0.5. It was restored at 28°C for 1.5 h. It was infiltrated into the back of the tobacco using a syringe without a needle, and the infiltration area was marked with a circle. After 48 h, it was observed whether hypersensitive cell necrosis could be induced to identify whether it was a functional NLR gene.

[0024] The results of in - vivo analysis of tobacco in this example showed that the Yuluxiang XP_009349127 homologous gene could induce hypersensitive cell necrosis ( Figure 1 A), indicating that it was a functional NLR gene, named REA1. Further analysis results of the anti - fire blight immune recognition function showed that when REA1 was co - expressed with RIN4 YLX , the hypersensitive cell necrosis reaction was inhibited; when REA1, RIN4 YLX and AvrRpt2 EA were co - expressed, the hypersensitive cell necrosis reaction was re - induced ( Figure 1 B), indicating that REA1 was an RPS2 - like disease - resistant gene in Yuluxiang that recognized AvrRpt2 EA of Erwinia amylovora to activate immunity and protect RIN4 YLX .

[0025] 6) Silencing analysis verification of the anti - fire blight function of the Yuluxiang REA1 gene By constructing a TRV - mediated VIGS gene silencing vector, a silencing analysis of the anti - fire blight function of the REA1 gene was carried out in the fruits of the disease - resistant variety Yuluxiang pear. The specific operation steps are as follows:

[0026] (i) Construction and acquisition of the VIGS silencing construct of the REA1 gene The CDS fragment of the Yuluxiang REA1 gene was constructed into the pTRV2 silencing vector. Using the pCAMBIA1305-REA1 plasmid as a template, REA1-VIGS-F (5’-cgtgagctcggtaccg gat ccg atg aac agg tgg gaa ca-3’, the italic part contains the Bam HI restriction site and is the same as the sequence on the vector pTRV2) (the sequence is as shown in SEQ ID: 9) and REA1-VIGS-R (5’-gtgagtaaggttaccgaattc tgt cag ctg gat cag-3’, the italic part contains the EcoR I restriction site and is the same as the sequence on the vector pTRV2) (the sequence is as shown in SEQ ID: 10) were used as primers to clone this fragment. Using the PCR One Step Directional Cloning Kit, it was ligated to the digested pTRV2 vector by homologous recombination. The ligation product was transformed into Escherichia coli, screened on a kanamycin plate, identified by PCR, and sent to the company for sequencing identification. The pTRV2-REA1 vector was successfully constructed.

[0027] (ii) Obtaining Agrobacterium tumefaciens transformed with the pTRV2-REA1 silencing construct The pTRV2-REA1 vector was introduced into Agrobacterium tumefaciens GV3101, and transformants were screened on a YEP medium with kanamycin resistance. Then, through double digestion and PCR identification, the transformants carrying pTRV2-REA1 were obtained and stored in a -80 °C refrigerator.

[0028] (iii) Obtaining Yuluxiang pears with silenced REA1 The stored GV3101 strain was activated, a single colony was picked for small-scale culture, and cultured overnight at 28 °C and 220 rpm. Then, it was cultured in 10 mL according to the ratio of the bacterial liquid to the YEB liquid medium of 1:10. Shaken at 28 °C and 220 rpm for 4 h, and the OD 600 of the bacterial liquid was controlled at 0.8 - 1.2, centrifuged at 5000 rpm at room temperature for 8 min, and the bacterial liquid was resuspended with the resuspension buffer (10 mM MgCl 2 , 10 mM MES, 200 mM acetosyringone). The OD 600 was adjusted to 0.8 - 1.0, and recovered at 28 °C for 1.5 h. The bacterial liquid was injected into Yuluxiang pear fruits with a sterile syringe, cultured in the dark for 24 h, and then cultured under light for 5 days.

[0029] (iv) Detection and analysis of the silencing efficiency of the REA1 gene in Yuluxiang pear fruits RNA was extracted from Yuluxiang pear fruits 6 days after the TRV-mediated VIGS gene silencing treatment, and the expression level of REA1 in pear fruits was detected by real-time fluorescence quantitative PCR to clarify the silencing efficiency of the REA1 gene.

[0030] (v)Inoculation analysis and evaluation of the resistance of Yuluxiang pear fruits to fire blight The Erwinia amylovora strain Ea1-luc with fluorescence signal stored at -80 °C was streaked on NA solid medium and cultured at 28 °C for 1 day. Single colonies were picked and transferred into a 2 mL EP tube containing 400 μL of NA liquid medium, and cultured in a shaker at 28 °C. The next day, the ratio of the bacterial liquid to the NA liquid medium was 1:10 for medium-scale culture, and the bacteria were shaken until the OD 600 was 0.6 - 0.8. After centrifugation at 5000 rpm for 6 min, it was resuspended to an OD 600 of 0.8. After recovery for 1 hour, 10 μL was taken and inoculated into the pulp of Yuluxiang pear after REA1 gene silencing, and cultured under moisturized conditions at 27 °C. After 5 days, the fluorescence signal intensity was detected by a single-photon counting imaging system to characterize the disease incidence. In addition, the diseased fruits were disinfected twice with 75% ethanol and rinsed three times with sterile water, and then 1 cm 3 of the diseased fruits was cut and placed into a 2 mL EP tube, 100 μL of ddH 2 O and several grinding beads were added, and ground with a grinder for 2 min. 10 μL of this liquid was taken and diluted in gradient, and 10 μL was pipetted onto the NA solid medium and cultured at 28 °C for 2 days. Photos were taken and the amount of pathogenic bacteria was statistically analyzed and recorded.

[0031] In this example, the results of REA1 gene silencing and inoculation of the fruits of the disease-resistant variety Yuluxiang pear showed that the expression level of the REA1 gene in pear fruits decreased significantly, indicating effective silencing ( Figure 2 B). Five days after inoculation of the fruits with silenced REA1 gene, the fluorescence signals representing the amount of pathogenic bacteria on the surface and cross-section were significantly higher than those of the control group ( Figure 2 A); the fluorescence signals representing the amount of pathogenic bacteria on the surface and cross-section of the fruits with silenced REA1 gene were 6.71 times ( Figure 2 C) and 3.24 times ( Figure 2 D) of the control group; the amount of pathogenic bacteria in the fruits with silenced REA1 gene after inoculation was extremely significantly higher than that of the control group ( Figure 2 E, F). These results indicate that silencing the REA1 gene significantly reduces the resistance of the fruits of the disease-resistant variety Yuluxiang pear to Erwinia amylovora; the REA1 gene plays an important role in the resistance to fire blight.

[0032] 7) Verification and application of the REA1 gene in improving the resistance of susceptible varieties (such as Korla fragrant pear) to fire blight The stored Agrobacterium tumefaciens strain pCAMBIA1305-REA1 (REA1-OE) was activated, and single colonies were picked for small-scale culture, cultured overnight at 28 °C and 220 rpm, and cultured 10 mL according to the ratio of the bacterial liquid to the YEB liquid medium of 1:10. Shaken at 28 °C and 220 rpm for 4 h, the OD of the bacterial liquid600 Control at 0.8 - 1.2, centrifuge at 5000 rpm at room temperature for 8 min, resuspend the bacterial solution with resuspension buffer (10 mM MgCl 2 , 10 mM MES, 200 mM acetosyringone), and adjust the OD 600 to 0.8 - 1.0, and recover at 28 °C for 1.5 h. Inject the bacterial solution into Korla fragrant pear fruits with a sterile syringe. After placing them in the dark for 12 h, culture them in an incubator for 3 days. Extract the RNA of Korla fragrant pear fruits 4 days after the overexpression treatment, and detect the expression of the REA1 gene by real-time fluorescence quantitative PCR. The culture, inoculation, and statistical analysis of the pathogen infection situation are the same as described in 6)(v).

[0033] In this example, the overexpression of the REA1 gene and the inoculation results in the susceptible cultivar Korla fragrant pear showed that the expression level of the REA1 gene and the overexpression of the Yuluxiang homologous gene of another sequence XP_048439943 with high homology to RPS2 as a control were significantly increased after the overexpression treatment, and overexpression was obtained ( Figure 3 B, C). Five days after inoculation of the fruits with overexpressed REA1 gene, the fluorescence signals representing the pathogen amount on the fruit surface and cross-section were significantly lower than those of the control group ( Figure 3 A), and the fluorescence signals on the fruit surface and cross-section of the control group were 7.94 times ( Figure 3 D) and 7.69 times ( Figure 3 E) that of the overexpressed REA1 gene group; the pathogen amount in the overexpressed REA1 gene group was extremely significantly lower than that of the control group after inoculation ( Figure 3 F, G). These results indicate that overexpressing the REA1 gene significantly improves the resistance of the fruits of the susceptible cultivar Korla fragrant pear to Erwinia amylovora; the REA1 gene can be applied to improve the resistance of susceptible cultivars to Erwinia amylovora.

[0034] These results of this example indicate that expressing the REA1 gene in Nicotiana benthamiana using an Agrobacterium-mediated gene expression system can stimulate the hypersensitive cell necrosis reaction, indicating that it is a functional NLR gene. Silencing the REA1 gene in fruits significantly reduces the resistance of the fruits of the disease-resistant cultivar Yuluxiang pear to Erwinia amylovora, and overexpressing the REA1 gene in fruits significantly improves the resistance of the fruits of the susceptible cultivar Korla fragrant pear to Erwinia amylovora. It shows that REA1 plays an important role in improving the resistance of pears to fire blight, and it is a high-quality fire blight-resistant gene resource for pears, providing directions and references for the subsequent creation of fire blight-resistant fruit tree germplasms.

[0035] Example 2 As described above, the present invention identifies a pear fire blight resistance gene REA1. This embodiment establishes a set of evaluation and identification criteria for pear varieties' resistance to fire blight based on whether they have a functional REA1 gene, and provides a new molecular identification method for pear varieties' resistance to fire blight. Taking Qiuyue pear as an example, the main implementation steps include:

[0036] 1) Cloning of Qiuyue Pear REA1 gene and construction of overexpression structure Total RNA from Qiuyue Pear leaves was extracted using Tiangen RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit, and then RNA was reverse transcribed into cDNA using Novizan HiScript III RT SuperMix Reverse Transcription Kit. The cDNA from Qiuyue Pear leaves was used as a template, Pbr9127-F and Pbr9127-R were used as primers (sequences are shown in SEQ ID: 3 and 4), and the full-length sequence was amplified using high-fidelity enzymes. The PCR product was then purified and recovered by 1% agarose gel electrophoresis. The PCR one-step directional cloning kit was used to connect the amplified target gene with the pCAMBIA1305 vector after enzyme digestion by homologous recombination. The ligation product was transformed into E. coli, screened on kanamycin plates, identified by PCR, and sent to the company for sequencing identification. The Qiuyue Pear REA1 gene was successfully cloned. Its nucleic acid and protein sequences are shown in SEQ ID: 11 and 12, respectively. The gene overexpression structure pCAMBIA1305-REA1 was constructed by referring to the steps described in Example 1 2). QY .

[0037] 2) Obtaining Agrobacterium transformed with the Qiuyue Pear REA1 gene overexpression structure The Qiuyue pear REA1 gene overexpression construct pCAMBIA1305-REA1 QY The Agrobacterium strain GV3101 was transformed by electroporation and other methods, and the transformants were screened on YEP medium containing kanamycin. Then, double restriction digestion and PCR identification were performed to obtain the overexpression construct pCAMBIA1305-REA1 carrying the Qiuyue Pear REA1 gene. QY Agrobacterium was stored in a -80℃ refrigerator for subsequent in vivo expression in tobacco.

[0038] 3) Functional identification of the Qiuyue pear REA1 gene The stored GV3101 strain was activated, and a single colony was selected for small-scale culture. The culture was shaken at 28°C and 220 rpm overnight, and 4 mL of the culture medium was shaken at a ratio of 1:10. The culture medium OD was 0.04. 600 The concentration was controlled at 0.8-0.9, centrifuged at 5000 rpm for 8 min at room temperature, and resuspended in a resuspension buffer (10 mM MgCl 2, 10 mM MES, 200 mM acetosyringone) resuspend the bacterial suspension, OD 600 is 0.5, recover at 28 °C for 1.5 h, infiltrate the back of the tobacco with a sterile syringe needle without a needle, and mark the infiltration range by drawing a circle. After 48 h, observe whether it can trigger the hypersensitive cell necrosis reaction to identify whether it is a functional NLR gene.

[0039] The results of this example show that the single expression of REA1 QY can trigger hypersensitive cell necrosis, and REA1 QY co-expressed with RIN4 QY when co-expressed, the hypersensitive cell necrosis reaction is inhibited. REA1 QY , RIN4 QY co-expressed with AvrRpt2 EA when the three are co-expressed, the hypersensitive cell necrosis is re-triggered ( Figure 4 A), indicating that the REA1 gene of autumn pear is a functional gene for recognizing the fire blight pathogen AvrRpt2 EA to stimulate immunity and defend RIN4 QY of the RPS2-like disease-resistant gene.

[0040] 4) Resistance evaluation and verification of autumn pear to fire blight The above results indicate that autumn pear carries the functional REA1 gene, so this variety is determined to be a fire blight-resistant variety. To verify this result, inoculation analysis was carried out. Collect healthy young branches of Yuluxiang pear and autumn pear from the orchard, wrap the base with sterile absorbent cotton soaked in water, and then wrap it with plastic wrap for moisture preservation and bring it back to the laboratory. Cut the pear branches into 30 cm segments, disinfect the surface with 75% alcohol, dip a sterilized toothpick into the bacterial suspension of Erwinia amylovora, and inoculate by pricking the tender branches at the top of the branches. Culture in an artificial climate chamber at 28 °C, relative humidity 75%, and light 12 h. Record the lesion length, the proportion of the lesion length to the inoculated branch length, and the disease grade 7 days after inoculation, calculate the incidence rate and disease index, and formulate the pear disease resistance classification standard according to the classification index.

[0041] The inoculation results of this example show that the lesion length of the disease-resistant variety Yuluxiang is 3.16 mm, and the lesion length of autumn pear is 4.66 mm ( Figure 4 B), and there is no significant difference between the two ( Figure 4 C), indicating that autumn pear is a fire blight-resistant variety with a resistance level similar to that of Yuluxiang. The results of this inoculation analysis confirm the molecular identification results of the disease resistance of pear varieties based on the REA1 gene, indicating the feasibility of the molecular identification of the disease resistance of pear varieties based on the REA1 gene, that is, whether having a functional REA1 gene can be used as one of the resistance evaluation and identification criteria for pear varieties to Erwinia amylovora, and a pear variety with a functional REA1 gene cloned can be initially identified as a fire blight-resistant variety.

[0042] In summary, the present invention combines Figures 1 - 4 the results of, and for the first time clarifies the important role of the REA1 gene in resistance to Erwinia amylovora, and provides application strategies and examples for this gene in improving the resistance of susceptible pear varieties to Erwinia amylovora and in the molecular identification of the resistance of pear varieties to Erwinia amylovora. The present invention provides a new high-quality gene resistant to Erwinia amylovora, provides resources and directions for the subsequent creation of fruit tree germplasms resistant to Erwinia amylovora, and proposes a new method for the molecular identification of the resistance of pear varieties to Erwinia amylovora.

Claims

1. Application of a pear disease resistance gene REA1 in the prevention and control of apricot fire blight, characterized in that: The nucleotide sequence of the gene REA1 is shown in SEQ ID: 1, and the protein sequence encoded by the gene REA1 is shown in SEQ ID:

2.

2. The use according to claim 1, characterized in that: The application is to use the REA1 gene to improve the resistance to amylopectin and to identify amylopectin resistance varieties based on the disease resistance gene.

3. The use according to claim 2, characterized in that: The function of the REA1 gene is identified and its application in improving the disease resistance of susceptible varieties to fire blight.

4. The use according to claim 2, characterized in that: Application of REA1 gene in molecular identification of amylopectin resistance varieties.

5. The use according to claim 3, characterized in that: The functional identification of REA1 gene and its application in obtaining pear materials with enhanced fire blight resistance were achieved through the following steps: (1) Construction and acquisition of REA1 gene overexpression structure The open reading frame of the REA1 gene of Gypsum frutescens was cloned into a plant expression vector, so that it was expressed under the drive of a strong promoter; (2) Obtaining Agrobacterium Transformed with REA1 Gene Overexpression Structure The constructed REA1 gene overexpression structure is transformed into the Agrobacterium strain by electroporation or other methods; (3) Identification and acquisition of functional REA1 gene Express REA1 gene in Nicotiana benthamiana, observe the stimulation of allergic cell necrosis reaction, and obtain the functional REA1 gene that can induce allergic cell necrosis; (4) Verification of the REA1 gene's function in resistance to fire blight The TRV-mediated VIGS gene silencing vector of REA1 gene was constructed and transformed with Agrobacterium tumefaciens to silence REA1 gene in Yuluxiang pear fruit. Inoculation analysis detected that the resistance of Yuluxiang pear fruit to Erwinia amylovora was significantly reduced. (5) Obtaining Korla pear fruit with improved fire blight resistance based on overexpression of REA1 The REA1 gene was overexpressed in the susceptible Korla pear variety, and the resistance to the amygdalitis pathogen was tested by inoculation to obtain Korla pear fruits with improved disease resistance.

6. The use according to claim 4, characterized in that: The application of REA1 gene in molecular identification of amylopectin resistance varieties is achieved through the following steps: (1) Cloning and sequence analysis of Qiuyue pear REA1 gene: RNA was extracted from young leaves of Qiuyue Pear and reverse transcribed into cDNA. Using it as a template, PCR was used to clone the Qiuyue Pear REA1 gene, and sequencing was performed to determine whether it was a full-length gene. (2) Construction and acquisition of Qiuyue pear REA1 gene overexpression structure The open reading frame of the Qiuyue pear REA1 gene was cloned into a plant expression vector, so that it was expressed under the drive of a strong promoter; (3) Obtaining Agrobacterium Transformed with the Akizuki Pear REA1 Gene Overexpression Structure The constructed Qiuyue pear REA1 gene overexpression structure was introduced into the Agrobacterium strain by electroporation or other methods; (4) Functional identification of Qiuyue pear REA1 gene The REA1 gene of Akizuki pear was expressed in Nicotiana benthamiana, and the stimulation of allergic cell necrosis was observed. The one that could stimulate allergic reaction was a functional REA1 gene. (5) Evaluation and verification of Qiuyue pear's resistance to fire blight pathogen Whether a variety is fire blight resistant is determined by whether it has a functional REA1 gene. The tender branches of Qiuyue pear are inoculated with amylovora amylovora to detect the disease and verify the above resistance identification results based on the functional REA1 gene.

Images