Application of MdDRK1 and MdDRKIP1 genes in improvement of apple ring spot resistance

Abstract

The invention discloses application of MdDRK1 and MdDRKIP1 genes in improvement of apple ring spot resistance, and belongs to the technical field of gene engineering. According to the application of the MdDRK1 gene and the MdDRKIP1 gene in improving the resistance to the apple ring spot, a CDS sequence for coding the MdDRK1 gene is as shown in SEQ ID NO.7, and a CDS sequence for coding the MdDRKIP1 gene is as shown in SEQ ID NO.15. According to the application of the MdDRK1 gene and the MdDRKIP1 gene in improving the resistance to the apple ring spot. The contents of resistant substances, namely total phenols and total flavonoids, in the overexpression MdDRK1 apple callus and the overexpression MdDRKIP1 apple callus are obviously increased. The research shows that the MdDRK1 and MdDRKIP1 genes play an important role in improving the resistance of apples to the ring spot, have the potential to be applied to gene editing and cultivation of new varieties resistant to the ring spot, and have important significance on directed genetic improvement of the disease resistance of the apples.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and more particularly to the application of MdDRK1 and MdDRKIP1 genes in improving the resistance of apples to ring rot disease. Background Art

[0002] Apple (Malus domestica Borkh.) is one of the fruits with the largest planting area and the highest economic value in temperate regions. However, its yield and quality are easily affected by pests and diseases, seriously restricting the healthy development of the apple industry. Apple ring rot caused by Botryosphaeria dothidea results in fruit rot, weakens the tree vigor or causes the death of the whole plant, and is one of the most serious diseases endangering the main apple-producing areas in China. In production, fruit farmers often adopt control methods such as severely scraping the bark and spraying pesticides to control ring rot disease. These methods not only weaken the tree vigor and reduce the fruit quality, but also lead to an increase in the drug resistance of ring rot pathogens, causing serious environmental pollution and food safety problems. With the promotion of the concepts of green agriculture and sustainable development, exploring new strategies for environmentally friendly, efficient and long-lasting control of ring rot disease has become the focus of current research. Therefore, exploring the molecular mechanism of apple resistance to ring rot disease and using molecular biological breeding techniques to breed disease-resistant varieties are effective ways to solve the problem of apple ring rot disease control and are of great significance for promoting the high-quality development of the apple industry in China.

[0003] The mitogen-activated protein kinase (MAPK) cascade pathway is one of the earliest activated signal transduction pathways in plants after being infected by pathogenic bacteria and is a hub for the interaction between plants and microorganisms. A typical MAPK cascade pathway consists of three parts: MAPK kinase kinase (MKKK, or MEKK), MAPK kinase (MKK, or MEK), and MAPK. When stimulated by the outside world, the MAPK cascade pathway is phosphorylated and activated step by step, converting extracellular stimuli into intracellular responses in a cascading amplification manner and triggering various phosphorylation-mediated cellular responses. A large number of literature reports show that the activation of the MAPK cascade pathway is one of the earliest cellular responses in plants after being infected by pathogenic bacteria and regulates the immune response of plant cells. Bacterial leaf streak of rice is a devastating disease caused by the bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc). When Xoc infects rice, the OsMPKK10.2-OsMPK6 cascade pathway is activated, and this signaling pathway improves the resistance of rice to Xoc by regulating the expression of multiple disease resistance-related genes. The expression level of TaMKK5 in wheat varieties resistant to Rhizoctonia solani is significantly higher than that in susceptible varieties, and TaMKK5 participates in regulating the defense response of wheat to Rhizoctonia solani through the TaMKK5-TaMPK3-TaERF3 signaling pathway. The above research shows that the MAPK cascade pathway is a key node in the plant immune response regulation network. However, the molecular mechanism of the MAPK signaling pathway regulating the resistance of apples to ring rot disease is still unclear.

[0004] Therefore, exploring the function of the MAPK signaling pathway in apple resistance to ring rot disease, analyzing its molecular mechanism of regulating apple disease resistance and applying it are technical problems that need to be solved urgently by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention isolated and identified a MAPK cascade signaling pathway that improves apple resistance to ring rot disease, and this signaling pathway consists of MdDRK1 ( D isease R esistance K inase 1) and MdDRKIP1 (DRK I nteraction P rotein 1). And the applications of the MdDRK1 and MdDRKIP1 genes in improving apple resistance to ring rot disease are provided.

[0006] To achieve the above object, the present invention adopts the following technical solutions: Application of MdDRK1 and MdDRKIP1 genes in improving the resistance of apple to ring rot. The CDS sequence encoding the MdDRK1 gene is shown as SEQ ID NO.7, and the CDS sequence encoding the MdDRKIP1 gene is shown as SEQ ID NO.15.

[0007] Furthermore, the MdDRK1 protein sequence is shown as SEQ ID NO.8, and the MdDRKIP1 protein sequence is shown as SEQ ID NO.16.

[0008] Vectors for increasing the expression levels of the MdDRK1 and MdDRKIP1 genes, which are pCAMBIA1305-MdDRK1-3MYC and pPZP211-MdDRKIP1-3FLAG; The pCAMBIA1305-MdDRK1-3MYC is formed by ligating the pCAMBIA1305-3MYC vector and the MdDRK1 gene; The pPZP211-MdDRKIP1-3FLAG is formed by ligating the pPZP211-3FLAG vector and the MdDRKIP1 gene.

[0009] Yeast two-hybrid expression vectors prepared from the MdDRK1 and MdDRKIP1 genes, which are MdDRK1-AD and MdDRKIP1-BD; The MdDRK1-AD is formed by ligating the pGADT7-AD vector and the MdDRK1 gene; The MdDRKIP1-BD is formed by ligating the pGBKT7-BD vector and the MdDRKIP1 gene.

[0010] Luciferase complementation expression vectors prepared from the MdDRK1 and MdDRKIP1 genes, which are pCAMBIA1300-MdDRK1-Cluc and pCAMBIA1300-MdDRKIP1-NLuc; The pCAMBIA1300-MdDRK1-Cluc is formed by ligating the pCAMBIA1300-Cluc vector and the MdDRK1 gene; The pCAMBIA1300-MdDRKIP1-NLuc is formed by ligating the pCAMBIA1300-NLuc vector and the MdDRKIP1 gene.

[0011] Co-immunoprecipitation expression vectors prepared from the MdDRK1 and MdDRKIP1 genes, which are pCAMBIA1305-MdDRK1-3MYC and pPZP211-MdDRKIP1-3FLAG.

[0012] The total phenol (TP) detection kit was used to determine the total phenol content in apple calli overexpressing the MdDRK1 gene and apple calli overexpressing the MdDRKIP1 gene; the total flavonoid (TF) detection kit was used to determine the total flavonoid content in apple calli overexpressing the MdDRK1 gene and apple calli overexpressing the MdDRKIP1 gene.

[0013] As can be seen from the above technical solutions, compared with the prior art, the beneficial effects of the present invention are as follows: The present invention first designed primers using gene homology and successfully cloned a resistance gene in the apple scab-resistant cultivar 'Golden Delicious'. Through domain and database alignment, it was confirmed that this resistance gene is a gene of the MAPKK family in the MAPK cascade signaling pathway, so it was named MdDRK1 ( D isease R esistance K inase 1). The MdDRK1 gene was transferred into apple calli and apple fruits by genetic transformation method. After inoculating with Physalospora piricola, overexpression of MdDRK1 improved the resistance of apple calli and apple fruits to Physalospora piricola. The yeast two-hybrid screening library was used to screen out the possible interacting protein MdDRKIP1 (DRK I nteraction P rotein 1) of MdDRK1. By constructing an expression vector, yeast two-hybrid, luciferase reporter system and co-immunoprecipitation techniques were used to prove the interaction between MdDRK1 and MdDRKIP1. The MdDRKIP1 gene was transferred into apple calli and apple fruits by genetic transformation method. After inoculating with Physalospora piricola, overexpression of MdDRKIP1 improved the resistance of apple calli and apple fruits to Physalospora piricola. Further studies showed that the contents of the resistance substances total phenol and total flavonoid in apple calli overexpressing MdDRK1 and apple calli overexpressing MdDRKIP1 were significantly increased. The above studies indicate that the MdDRK1 and MdDRKIP1 genes can enhance the resistance of apples to Physalospora piricola by increasing the contents of total phenol and total flavonoid, and have important application value in resistance molecular breeding. Brief Description of the Drawings

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0015] Figure 1The figure is the 1% agarose gel electrophoresis map of the PCR product of the full-length CDS of the MdDRK1 gene of the present invention; M is the marker, and 1 and 2 are the CDS cloning fragments of the MdDRK1 gene; Figure 2 The figure is the qRT-PCR and Western positive detection and verification map of the overexpressed MdDRK1 transgenic apple callus of the present invention; among them, A is the protein level detection of the overexpressed MdDRK1 transgenic apple callus; B is the MdDRK1 gene expression level detection of the overexpressed MdDRK1 transgenic apple callus; WT is the wild-type control, and MdDRK1-OE is the overexpressed MdDRK1 transgenic apple callus line; Figure 3 The figure is the phenotype of the overexpressed MdDRK1 transgenic apple callus inoculated with Botryosphaeria berengeriana; among them, A is the phenotype of the overexpressed MdDRK1 transgenic apple callus after inoculation with Botryosphaeria berengeriana; B is the lesion diameter of the overexpressed MdDRK1 transgenic apple callus after inoculation with Botryosphaeria berengeriana; C is the pathogen biomass of the overexpressed MdDRK1 transgenic apple callus after inoculation with Botryosphaeria berengeriana; WT is the wild-type control, and MdDRK1-OE is the overexpressed MdDRK1 transgenic apple callus line; Figure 4 The figure is the phenotype of the transiently overexpressed MdDRK1 apple fruit inoculated with Botryosphaeria berengeriana; among them, A is the phenotype of the transiently overexpressed MdDRK1 apple fruit after inoculation with Botryosphaeria berengeriana; B is the lesion diameter of the transiently overexpressed MdDRK1 apple fruit after inoculation with Botryosphaeria berengeriana; C is the pathogen biomass of the transiently overexpressed MdDRK1 apple fruit after inoculation with Botryosphaeria berengeriana; Vec is the empty vector control, and MdDRK1-aOE is the transiently overexpressed MdDRK1 apple fruit; Figure 5 The figure is the 1% agarose gel electrophoresis map of the PCR product of the full-length CDS of the MdDRKIP1 gene of the present invention; M is the marker, and 1 and 2 are the CDS cloning fragments of the MdDRKIP1 gene; Figure 6 The figure is the qRT-PCR and Western positive detection and verification map of the overexpressed MdDRKIP1 transgenic apple callus of the present invention; among them, A is the protein level detection of the overexpressed MdDRKIP1 transgenic apple callus; B is the MdDRKIP1 gene expression level detection of the overexpressed MdDRKIP1 transgenic apple callus; WT is the wild-type control, and MdDRKIP1-OE is the overexpressed MdDRKIP1 transgenic apple callus line; Figure 7The accompanying drawings are for verifying the interaction relationship between MdDRK1 and MdDRKIP1 in the present invention; wherein, A is for verifying the interaction relationship between MdDRK1 and MdDRKIP1 using the yeast two-hybrid technique; positive control: pGBKT7-53 and pGADT7-T; negative control: pGBKT7-lam and pGADT7-T; DDO: SD / -Leu / -Trp solid medium; QDO: SD / -Ade / -His / -Leu / -Trp solid medium; QDO / X: SD / -Ade / -His / -Leu / -Trp solid medium plus X-α-gal; B is for verifying the interaction relationship between MdDRK1 and MdDRKIP1 through the luciferase reporter system; NLuc+Cluc: co-expression of pCAMBIA1300-Cluc vector and pCAMBIA1300-NLuc vector; NLuc+MdDRK1-Cluc: co-expression of pCAMBIA1300-NLuc vector and pCAMBIA1300-MdDRK1-Cluc vector; MdDRKIP1-NLuc+Cluc: co-expression of pCAMBIA1300-MdDRKIP1-NLuc vector and pCAMBIA1300-Cluc vector; MdDRKIP1-NLuc+MdDRK1-Cluc: co-expression of pCAMBIA1300-MdDRKIP1-NLuc vector and pCAMBIA1300-MdDRK1-Cluc vector; C is for verifying the interaction relationship between MdDRK1 and MdDRKIP1 using the co-immunoprecipitation technique; Figure 8 The accompanying drawings are for the phenotypes of apple calli overexpressing MdDRKIP1 transgenic inoculated with Physalospora piricola in the present invention; wherein, A is the phenotype of apple calli overexpressing MdDRKIP1 transgenic inoculated with Physalospora piricola; B is the lesion diameter of apple calli overexpressing MdDRKIP1 transgenic inoculated with Physalospora piricola; C is the pathogen biomass of apple calli overexpressing MdDRKIP1 transgenic inoculated with Physalospora piricola; WT is the wild-type control, and MdDRKIP1-OE is the apple calli line overexpressing MdDRKIP1 transgenic; Figure 9 The accompanying drawings are for the phenotypes of apple fruits transiently overexpressing MdDRKIP1 inoculated with Physalospora piricola in the present invention; wherein, A is the phenotype of apple fruits transiently overexpressing MdDRKIP1 inoculated with Physalospora piricola; B is the lesion diameter of apple fruits transiently overexpressing MdDRKIP1 inoculated with Physalospora piricola; C is the pathogen biomass of apple fruits transiently overexpressing MdDRKIP1 inoculated with Physalospora piricola; Vec is the empty vector control, and MdDRKIP1-aOE is the apple fruit transiently overexpressing MdDRKIP1; Figure 10The accompanying drawings show the total phenolic and total flavonoid contents of transgenic apple calli overexpressing MdDRK1 and overexpressing MdDRKIP1 of the present invention; among them, A is the total phenolic content of transgenic apple calli overexpressing MdDRK1; B is the total phenolic content of transgenic apple calli overexpressing MdDRKIP1; C is the total flavonoid content of transgenic apple calli overexpressing MdDRK1; D is the total flavonoid content of transgenic apple calli overexpressing MdDRKIP1; WT is the wild-type control, MdDRK1-OE is the transgenic apple calli line overexpressing MdDRK1, and MdDRKIP1-OE is the transgenic apple calli line overexpressing MdDRKIP1. Detailed implementation manners

[0016] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0017] ‘Golden Delicious’ used in the examples was obtained from the Tianpinghu Experimental Base of the Shandong Institute of Pomology. The pericarp of mature apple fruits was collected in September 2023, frozen in liquid nitrogen, and stored at -80°C for later use. The callus is the callus of ‘Orin’ apple. The transiently infected apple fruit is ‘Yishui Red’ Fuji from the Tianpinghu Experimental Base of the Shandong Institute of Pomology.

[0018] Example 1 Cloning and expression vector construction of MdDRK1 gene

[0019] 1) Extraction of total RNA from apple pericarp Weigh 0.05 g of ‘Golden Delicious’ apple pericarp, quickly grind it into powder in liquid nitrogen, use the NobelPlant RNA extraction kit to extract total RNA according to the instructions, and detect it by 1% agarose gel electrophoresis, and store it at -80°C for later use.

[0020] 2) Primer design for MdDRK1 gene Using the apple genomic mRNA sequence as a template, primer sequences containing the CDS region of MdDRK1 were designed, and the primer sequences are as follows: MdDRK1-F1: 5’-CATATGAAGACGAAGACGCCATTG-3’, as shown in SEQ ID NO.1; MdDRK1-R1: 5’-GAATTCTCTTGGAAAATTTACAGG-3’, as shown in SEQ ID NO.2; Primer sequences designed for connecting the pCAMBIA1305-3MYC expression vector are as follows: MdDRK1-MYC-F: 5’-GGATCCATGAAGACGAAGACGCCA-3’, as shown in SEQ ID NO.3; MdDRK1-MYC-R: 5’-GTCGACTCTTGGAAAATTTACAGG-3’, as shown in SEQ ID NO.4; Specific primer sequences designed for real-time fluorescence quantitative PCR (qRT-PCR) are as follows: MdDRK1-qRT-F: 5’-AAACCAAGCAGCACAATGTCCAC-3’, as shown in SEQ ID NO.5; MdDRK1-qRT-R: 5’-AGCGAAGCACTCACGCCAAAATC-3’, as shown in SEQ ID NO.6.

[0021] 3) Cloning of the MdDRK1 gene The first strand of apple cDNA was synthesized using the Nobel reverse transcription kit, and the reaction system and reaction conditions were carried out according to the kit instructions. After the reaction, gene-specific PCR primers (MdDRK1-F1 and MdDRK1-R1) were used for amplification. The PCR amplification products were detected using 1% agarose, and a single band of about 1026 bp at the target position was obtained ( Figure 1 ).

[0022] The target fragment was recovered using the Tiangen gel recovery kit, and the recovered fragment was ligated into the T vector (pMD19-T vector) using T4 DNA ligase. 5 μL of the ligation product was transformed into 50 μL of DH5α Escherichia coli competent cells. After culturing on 1 mL of LB liquid medium, 100 μL was taken and centrifuged and spread on the LB solid medium for ampicillin resistance screening, and the resistant monoclonal colonies were verified by colony PCR using the universal M13 primers.

[0023] The PCR products were sent to Sangon Biotech in Shanghai for sequencing. The sequencing results were compared using the NCBI database BALST and the GDDH13 Version 1.1 database (https: / / iris.angers.inra.fr / gddh13 / the-apple-genome-downloads.html). The target fragments were all predicted to be apple MAPKK family genes, named MdDRK1, and the gene CDS region sequence (1026 bp) was: The MdDRK1 protein sequence (341 residues) is as follows: MKTKTPLNLNLKQLKLNVPAQEANIKSFLTASGTFHDGDLLLNQKGLRLISEEKETPDQSSDSKELNFEITLEDLETIKVIGKGSGGVVQLVRHKWVGTLFALKVIQMNIQEEIRKQIVQELKINQAAQCPHVVVCHHSFYHNGAISLVLEYMDRGSLADVIRQVNTILEPYLAVVCKQVLQGLVYLHNERHVIHRDIKPSNLLVNHQGQVKITDFGVSASLASSMGQRDTFVGTYNYMSPERISGSTYDYSSDIWSLGLAVLECAIGRFPYMQSEDQQSWPSFYELLEAIVESPPPSAPPDQFSPESCSFVSSCIQKDPQHRSSCLDLLSHPFIKSRDKT, as shown in SEQ ID NO.8.

[0024] 4) Construction of the plant overexpression vector of the apple MdDRK1 gene Using the obtained MdDRK1 gene fragment as a template, amplification was performed using gene-specific PCR primers (MdDRK1-MYC-F and MdDRK1-MYC-R). PCR amplification was detected using 1% agarose, the target fragment was recovered, and the recovered fragment was ligated into a T-vector (pMD19-T vector) and transformed into competent DH5α Escherichia coli cells. The plasmid of the Escherichia coli bacterial liquid containing the MdDRK1 gene target fragment obtained above was extracted. The pCAMBIA1305-3MYC expression vector was selected, and restriction enzyme digestion reactions were performed on the vector and the plasmid using BamH I enzyme and Sal I enzyme. The restriction enzyme digestion products of the MdDRK1 fragment and the vector were recovered, and the vector and the MdDRK1 fragment were ligated using T4 DNA ligase. The ligation product was transformed into competent Escherichia coli DH5α cells. After culturing on 1 mL of LB liquid medium, 100 uL was taken and centrifuged and spread on LB solid medium containing kanamycin. Monoclonal colonies were picked and identified by colony PCR. Five positive monoclonal colonies were selected for sequencing and inoculated into 10 mL of LB liquid medium with kanamycin resistance for shaking culture. Finally, the plasmid of the bacterial liquid with correct sequencing was extracted and stored at -20°C for later use.

[0025] Example 2 Agrobacterium-mediated genetic transformation and identification of MdDRK1 apple callus 1) Prepare 'Wanglin' callus. The apple callus to be infected was cultured for about 12 - 14 days under dark conditions at 24°C; 2) Transformation of Agrobacterium. Add 1 μL of the positive plasmid of the pCAMBIA1305-MdDRK1-3MYC expression vector to 50 μL of LBA4404 Agrobacterium competent cells stored at -80°C. After transformation, add 1 mL of LB liquid medium and culture it on a shaker at 28°C and 200 rpm for 30 min for activation. The activated Agrobacterium is cultured on an LB solid medium containing kanamycin. Pick a single colony from the plate medium and inoculate it into an LB liquid medium containing kanamycin, and culture it on a shaker at 28°C and 200 rpm for 16 h. Use the MdDRK1-F1 and MdDRK1-R1 primers for colony PCR identification, and store the positive clone at -20°C for later use. Take 200 μL of the Agrobacterium liquid containing the target gene and add it to 10 mL of liquid LB medium containing rifampicin (50 mg / L) and kanamycin (100 mg / L), and culture it on a shaker at 28°C and 220 rpm for 12 - 24 h until the OD 600 = 0.6 - 0.8; 3) Take 2 mL of the activated Agrobacterium and add it to 50 mL of liquid LB medium without antibiotics, and culture it on a shaker at 28°C and 220 rpm for 4 - 6 h; 4) Transfer the bacterial liquid into a sterilized 50 mL centrifuge tube, centrifuge at 5000 rpm for 10 min at room temperature, discard the supernatant, add an appropriate amount of MS liquid medium to resuspend the bacterial liquid to make the OD600 value 0.4 - 0.6, pour it into a 50 mL conical flask, transfer the 'Wanglin' callus into the conical flask, and culture it on a shaker at 28°C and 200 rpm for 20 min; 5) Use a sterile filter paper to absorb the bacterial liquid on the surface of the callus and transfer it to an MS medium without antibiotics, and culture it in the dark at 24°C for 2 days; 6) Transfer the co-cultured callus to an MS selection medium containing kanamycin (100 mg / L) and carbenicillin (250 mg / L), and culture it in the dark at 24°C; 7) Screening of transgenic lines. The callus grown on the selection medium is transferred to a new selection medium until positive transgenic callus is screened out; 8) PCR identification of transgenic lines After the apple callus was transformed with pCAMBIA1305-MdDRK1-3MYC, the stably inherited transgenic apple callus was purified. Two methods were used in the present invention to identify the transgenic callus. First, using the MYC tag it contains, with the wild-type callus as a control, Anti-MYC antibody was applied, and Western-Blot technology was used to detect the expression of the MdDRK1-MYC fusion protein in 3 transgenic callus lines. Protein bands of the target length of MdDRK1 were amplified in all of them, while no such band was observed in the wild-type. The positive rate of the target transgene detection was 100% for all. Second, using qRT-PCR specific primers, with the wild-type callus as a control, qRT-PCR technology was used to detect the expression level of the MdDRK1 gene in 3 transgenic callus lines. The expression level of MdDRK1 in the transgenic lines was significantly higher than that in the wild-type, and the positive rate of the target transgene detection was 100% for all. The results showed that MdDRK1 was successfully transformed into apple callus and could be stably inherited. The results are shown respectively in Figure 2 .

[0026] Example 3 Phenotype of MdDRK1 transgenic apple callus after inoculation with Physalospora piricola The MdDRK1-OE1, MdDRK1-OE2 and MdDRK1-OE3 transgenic apple callus were propagated. 3 g of transgenic callus and wild-type callus were weighed respectively and placed in the same MS medium without any antibiotics for propagation culture. Each petri dish contained 25 mL of the medium, and each line had three replicates. After the apple callus was inoculated with Physalospora piricola, it was cultured in the dark at 24 °C for 12 days. The phenotypic differences between the positive transgenic callus and the wild-type were observed. As Figure 3 shown, after inoculation with Physalospora piricola, the lesion diameter of Physalospora piricola and the biomass of the pathogen in the MdDRK1-OE1, MdDRK1-OE2 and MdDRK1-OE3 transgenic apple callus were significantly smaller than those in the control group. If the function of this gene is applied to the breeding of new apple varieties, it has potential application value in improving the disease resistance of fruits.

[0027] Example 4 Phenotype observation of MdDRK1 transiently infecting apple fruits and after inoculation with Physalospora piricola 1) Prepare mature 'Yishuihong' Fuji apple fruits about 180 days after flowering for standby; 2) Agrobacterium transformation. Add 1 μL of the positive plasmid pCAMBIA1305-C-MdDRK1-3MYC to 50 μL of GV3101 Agrobacterium competent cells stored at -80°C. After transformation, add 1 mL of LB liquid medium and incubate on a shaker at 28°C and 200 rpm for 30 min for activation. The activated Agrobacterium is cultured on LB solid medium containing kanamycin. Pick a single colony from the plate medium and inoculate it into LB liquid medium containing kanamycin. Incubate on a shaker at 28°C and 200 rpm for 16 h. Perform bacterial liquid PCR identification using the MdDRK1-F1 and MdDRK1-R1 primers. The positive clone is stored at -20°C for later use. Take 200 μL of Agrobacterium liquid and add it to 10 mL of liquid LB medium containing rifampicin (50 mg / L) and kanamycin (100 mg / L). Incubate at 28°C and 220 rpm for 12 - 24 h until OD600 = 0.6 - 0.8; 3) Secondary activation. Take 200 μL of activated Agrobacterium and add it to 10 mL of liquid LB medium containing acetosyringone (200 μM), MES (10 mM), rifampicin (50 mg / L), and kanamycin (100 mg / L). Incubate at 28°C and 220 rpm for 12 - 24 h until OD600 = 0.6 - 0.8; 4) Collect Agrobacterium liquid or mixed plasmids and prepare the infection solution. The infection solution contains MgCl2 (10 mM), acetosyringone (200 μM), and MES (10 mM). Centrifuge the secondary-activated bacterial liquid to remove the supernatant and resuspend it with the infection solution; 5) Use a sterile syringe to inject the infection solution under the apple fruit epidermis respectively; 6) After dark incubation of the injected apple fruits for 24 h, inoculate Botryosphaeria berengeriana and observe; As Figure 4 shown, the lesion diameter and pathogen biomass of MdDRK1-aOE apple fruits are significantly smaller than those of the control.

[0028] Example 5 Cloning and construction of the MdDRKIP1 gene expression vector 1) Extraction of total RNA from apple peel Weigh 0.05 g of 'Golden Delicious' apple peel, quickly grind it into powder in liquid nitrogen, use the Novabio plant RNA extraction kit to extract total RNA according to the instructions, and detect it by 1% agarose gel electrophoresis. Store it at -80°C for later use.

[0029] 2) Primer design for the MdDRKIP1 gene Using the apple genomic mRNA sequence as a template, primer sequences containing the MdDRKIP1 CDS region were designed, and the primer sequences are as follows: MdDRKIP1-F1: 5'-CATATGTGGCAGACGCTGTTC-3', as shown in SEQ ID NO.9; MdDRKIP1-R1: 5'-GGATCCGGAAAACACTGCGGC-3', as shown in SEQ ID NO.10; Primer sequences designed for ligating to the pPZP211-3FLAG expression vector are as follows: MdDRKIP1-FLAG-F: 5'-GGATCCATGTGGCAGACGCTGTTC-3', as shown in SEQ ID NO.11; MdDRKIP1-FLAG-R: 5'-ACTAGTGGAAAACACTGCGGCATTG-3', as shown in SEQ ID NO.12; Specific primer sequences designed for real-time fluorescence quantitative PCR (qRT-PCR) are as follows: MdDRKIP1-qRT-F: 5'-GCCGATTCAGAGGAAGAGC-3', as shown in SEQ ID NO.13; MdDRKIP1-qRT-R: 5'-GTTTGCGTTGATAAGAAGGTTT-3', as shown in SEQ ID NO.14.

[0030] 3) Cloning of the MdDRKIP1 gene The first strand of apple cDNA was synthesized using the Nobel reverse transcription kit, and the reaction system and reaction conditions were carried out according to the kit instructions. After the reaction, amplification was performed using gene-specific PCR primers (MdDRKIP1, -F1 and MdDRKIP1-R1). The PCR amplification products were detected using 1% agarose, and a single band of about 1083 bp at the target position was obtained ( Figure 5 )

[0031] The target fragment was recovered using the Tiangen gel extraction kit, and the recovered fragment was ligated into the T vector (pMD19-T vector) using T4 DNA ligase. 5 μL of the ligation product was transformed into 50 μL of DH5α Escherichia coli competent cells. After culturing in 1 mL of LB liquid medium, 100 μL was taken and centrifuged and spread on the LB solid medium for ampicillin resistance screening, and the resistant monoclonal colonies were verified by colony PCR using the universal M13 primers.

[0032] The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were aligned using the NCBI database BLAST and the GDDH13 Version 1.1 database (https: / / iris.angers.inra.fr / gddh13 / the-apple-genome-downloads.html). The target fragments were all predicted to be apple MAPK family genes, named MdDRKIP1. The gene CDS region sequence (1083 bp) is as follows: The MdDRKIP1 protein sequence (360 residues) is as follows: MWQTLFEIDTKYVPIKPIGRGAYGIVCSSVNRETNEKVAIKKINNAFENRVDALRTLREMKLLQHLRHENVIALKDVMMPIQRKSFKDVYLVYELMDTDLHQIIKSSQPLSNDHCQYFLFQLLRGLKYLHSANILHRDLKPGNLLVNANCDLKICDFGLARTSTGKDQFMTEYVVTRWYRAPELLLCCDNYGTSIDVWSVGCIFAELLGRKPIFPGTECLNQLKLIINILGSQREEDLQFIDNPKATKYIKSLPFSLGTPLTRLYPDAHPLAIDLLQKMLVFDPSKRISVTEALQHPYMAPLYDPSNNPPAEVPIDLDIDEDLETDDKGDDVEGNASLPSRSCYRQCRSVFRIRRPAAAA, as shown in SEQ ID NO.16.

[0033] 4) Construction of the plant overexpression vector of the apple MdDRKIP1 gene Using the obtained MdDRKIP1 gene fragment as a template, gene-specific PCR primers (MdDRKIP1-FLAG-F and MdDRKIP1-FLAG-R) were used for amplification. PCR amplification was detected using 1% agarose, and the target fragment was recovered. The recovered fragment was ligated to the T-vector (pMD19-T vector) and transformed into DH5α Escherichia coli competent cells. The plasmid of the Escherichia coli broth containing the MdDRKIP1 gene target fragment obtained above was extracted. The pPZP211-3FLAG expression vector was selected, and the vector and plasmid were digested with BamH Ⅰ enzyme and Bcu Ⅰ enzyme. The digested products of the MdDRKIP1 fragment and the vector were recovered, and the T4 DNA ligase was used to ligate the vector and the MdDRKIP1 fragment. The ligation product was transformed into Escherichia coli DH5α competent cells. After culturing in 1 mL of LB liquid medium, 100 μL was taken and centrifuged and spread on the LB solid medium containing spectinomycin. Monoclonal colonies were picked and identified by colony PCR. Five positive monoclonal colonies were selected for sequencing and inoculated into 10 mL of LB liquid medium with spectinomycin resistance for shaking culture. Finally, the plasmid of the broth with correct sequencing was extracted and stored at -20 °C for later use.

[0034] Example 6 Agrobacterium-mediated genetic transformation and identification of MdDRKIP1 apple callus 1) Prepare the 'Wanglin' callus. Incubate the apple callus to be infected under dark conditions at 24 °C for about 12 - 14 days; 2) Transform Agrobacterium tumefaciens. Add 1 μL of the positive plasmid of the pPZP211-MdDRKIP1-3FLAG expression vector to 50 μL of LBA4404 Agrobacterium competent cells stored at -80 °C. After transformation, add 1 mL of LB liquid medium, and culture it on a shaker at 28 °C and 200 rpm for 30 min for activation. The activated Agrobacterium is cultured on an LB solid medium containing spectinomycin. Pick a single colony from the plate medium and inoculate it into an LB liquid medium containing spectinomycin, and culture it on a shaker at 28 °C and 200 rpm for 16 h. Use the MdDRKIP1-F1 and MdDRKIP1-R1 primers for colony PCR identification, and store the positive clones at -20 °C for later use. Take 200 μL of the Agrobacterium liquid containing the target gene and add it to 10 mL of liquid LB medium containing rifampicin (50 mg / L) and spectinomycin (50 mg / L), and culture it on a shaker at 28 °C and 220 rpm for 12 - 24 h until OD 600 = 0.6 - 0.8; 3) Add 2 mL of the activated Agrobacterium to 50 mL of antibiotic-free liquid LB medium, and culture it on a shaker at 28 °C and 220 rpm for 4 - 6 h; 4) Transfer the bacterial liquid into a sterilized 50 mL centrifuge tube, centrifuge at 5000 rpm for 10 min at room temperature, discard the supernatant, add an appropriate amount of MS liquid medium to resuspend the bacterial liquid to make the OD600 value 0.4 - 0.6, pour it into a 50 mL conical flask, transfer the 'Wanglin' callus into the conical flask, and culture it on a shaker at 28 °C and 200 rpm for 20 min; 5) Use sterile filter paper to absorb the bacterial liquid on the surface of the callus and transfer it to an MS medium without antibiotics, and culture it in the dark at 24 °C for 2 days; 6) Transfer the co-cultured callus to an MS selection medium supplemented with hygromycin (50 mg / L) and carbenicillin (250 mg / L), and culture it in the dark at 24 °C; 7) Screening of transgenic lines. Transfer the callus grown on the selection medium to a new selection medium until positive transgenic callus is screened out; 8) PCR identification of transgenic lines After the apple callus was transformed with pPZP211-MdDRKIP1-3FLAG respectively, the stably inherited transgenic apple callus was purified. The present invention uses two methods to identify the transgenic callus. First, using its FLAG tag and the wild-type callus as a control, applying Anti-FLAG antibody and using Western-Blot technology to detect the expression of MdDRKIP1-FLAG fusion protein in 3 transgenic callus lines, and amplifying the protein band of the target length of MdDRKIP1. There is no such band in the wild-type, and the positive rate of the target transgene detected is 100%. Secondly, using qRT-PCR specific primers and the wild-type callus as a control, applying qRT-PCR technology to detect the expression level of MdDRKIP1 gene in 3 transgenic callus lines. The expression level of MdMdDRKIP1 in the MdDRKIP1 transgenic lines is significantly higher than that of the wild-type, and the positive rate of the target transgene detected is 100%. The results show that MdDRKIP1 has been successfully transformed into apple callus and can be stably inherited. The results are shown in Figure 6 。

[0035] Example 7 Proving the Interaction between MdDRK1 and MdDRKIP1 by Yeast Two-Hybrid Technology 1) Perform digestion reactions on the plasmid containing the full-length CDS of MdDRK1 and the pGADT7-AD expression vector obtained above using Nde Ⅰ enzyme and EcoR Ⅰ enzyme; perform digestion reactions on the plasmid containing the full-length CDS of MdDRKIP1 and the pGBKT7-BD expression vector obtained above using Nde Ⅰ enzyme and BamH Ⅰ enzyme; 2) Recover the digestion products of the MdDRK1 fragment and the pGADT7-AD vector, and use T4 DNA ligase to ligate the vector with the MdDRK1 fragment to obtain the recombinant expression vector MdDRK1-AD; recover the digestion products of the MdDRKIP1 fragment and the pGBKT7-BD vector, and use T4 DNA ligase to ligate the vector with the MdDRKIP1 fragment to obtain the recombinant expression vector MdDRKIP1-BD; 3) After mixing the two ligation products and Carrier DNA, co-transform the competent cells of yeast Y2HGold. Incubate in a 30 °C water bath for 30 min; add 20 μL of dimethyl sulfoxide and incubate in a 42 °C water bath for 15 min; centrifuge at 12000 rpm for 30 sec to remove the supernatant, add 1 mL of YPDA and resuscitate at 30 °C for 1 h; centrifuge at 12000 rpm for 30 sec to remove the supernatant, add 100 μL of 0.9% NaCl solution to resuspend the cells, and spot them on different media; The positive control and the yeast strain co-transformed with MdDRK1-AD and MdDRKIP1-BD recombinant vectors can grow normally on SD / -Ade / -His / -Leu / -Trp solid medium, the negative control cannot grow on SD / -Ade / -His / -Leu / -Trp solid medium, all three can grow on SD / -Leu / -Trp solid medium, and the positive control and the yeast strain co-transformed with MdDRK1-AD and MdDRKIP1-BD recombinant vectors turn blue after adding X-ɑ-gal, indicating that there is an interaction between MdDRK1 and MdDRKIP1 (see Figure 7 A in ).

[0036] Example 8 Using the luciferase reporter system to demonstrate the interaction between MdDRK1 and MdDRKIP1 1) The full-length CDS of MdDRK1 obtained above was connected to the pCAMBIA1300-CLuc vector to obtain the recombinant expression vector MdDRK1-CLUC; the full-length CDS of MdDRKIP1 was connected to the pCAMBIA1300-NLuc vector to obtain the recombinant expression vector MdDRKIP1-NLUC; 2) The two recombinant expression vectors are transformed into Agrobacterium GV3101 respectively, and the successfully transformed Agrobacterium is used to prepare the infection solution; 3) Prepare tobacco in good growth condition for future use; 4) Primary activation: Take 200 μL of the preserved Agrobacterium culture medium and inoculate it into 10 mL of LB liquid medium, add the corresponding antibiotics, and culture at 28°C, 180 rpm with shaking overnight; 5) Secondary activation: Take 200 μL of the first activated bacterial solution and inoculate it into 10 mL of LB liquid medium, add the corresponding antibiotics, and culture overnight at 28°C and 180 rpm; 6) Prepare infection solution, collect bacteria by centrifugation, resuspend bacteria in infection solution, and place in the dark for 4 hours; 7) Use a 1 mL syringe to inject the infection solution into the back of tobacco leaves and culture under normal light conditions for 2 days; 8) After 2 days, use an in vivo fluorescence imager to observe the fluorescence of the injection site in the tobacco leaves and take photos.

[0037] The sites of tobacco leaves co-expressing MdDRK1-CLUC and MdDRKIP1-NLUC emitted obvious fluorescence, indicating that there is an interaction between MdDRK1 and MdDRKIP1 (see Figure 7 B in the figure).

[0038] Example 9 Using co-immunoprecipitation technology to prove the interaction between MdDRK1 and MdDRKIP1 1) Extract the proteins from wild-type callus, MdDRK1-OE, and MdDRKIP1-OE transgenic apple callus respectively, and store them at -80 °C for later use; 2) Pretreat anti-MYC Beads: Vortex the anti-MYC Beads to mix well, pipette 20 μL of the Beads suspension into a new 1.5 mL centrifuge tube, add 500 μL of PBS buffer, resuspend and wash the Beads. After standing on a magnetic stand for 10 sec, discard the supernatant, and repeat the washing twice; 3) Add 500 μL of protein extraction solution to the above Beads precipitate (Group 1: 500 μL of WT; Group 2: 250 μL of WT + 250 μL of MdDRKIP1; Group 3: 250 μL of WT + 250 μL of MdDRK1; Group 4: 250 μL of MdDRKIP1 + 250 μL of MdDRK1). Gently pipette to resuspend the Beads, and incubate with shaking overnight at 4 °C; 4) Transfer 60 μL of the protein extraction solution from different callus that has not been incubated with Beads to a 1.5 mL centrifuge tube containing 20 μL of 4× protein loading buffer, mix well, denature, and store at -20 °C for later use; 5) After overnight incubation, let the Beads stand on the magnetic stand for 10 sec, and discard the supernatant; 6) Add 500 μL of PBS buffer to the precipitate, pipette to resuspend and wash the Beads. After standing on the magnetic stand for 10 sec, discard the supernatant, and repeat the washing twice; 7) Add 50 μL of 1× protein loading buffer to the precipitate obtained after three washes, perform protein denaturation, and store at -20 °C for later use; 8) According to the above Western blot experimental method, detect the interaction relationship between the two proteins.

[0039] Figure 7 The Co-IP experimental results of C in... showed that MdDRK1-MYC could specifically immunoprecipitate MdDRKIP1-FLAG protein. This result was consistent with the results of yeast two-hybrid and luciferase reporter system experiments, further confirming the interaction relationship between MdDRK1 and MdDRKIP1.

[0040] Example 10 Observation of the phenotype of MdDRKIP1 transgenic apple callus The MdDRKIP1 - OE1, MdDRKIP1 - OE2, and MdDRKIP1 - OE3 transgenic apple calli were subcultured. Weigh 3 g of transgenic apple calli and wild - type apple calli respectively and place them in the same MS medium without any antibiotics for propagation culture. Each petri dish contains 25 mL of the medium, and each strain is replicated three times. After inoculating the apple calli with Botryosphaeria berengeriana, they were cultured in the dark at 24°C for 12 days. Observe the phenotypic differences between wild - type and positive transgenic apple calli. As Figure 8 shown, after inoculation with Botryosphaeria berengeriana, the lesion diameters of Botryosphaeria berengeriana and the fungal biomass of MdDRKIP1 - OE1, MdDRKIP1 - OE2, and MdDRKIP1 - OE3 transgenic apple calli were significantly smaller than those of the control group. If the function of this gene is applied to the breeding of new apple varieties, it has potential application value in improving the disease resistance of fruits.

[0041] Example 11 Transient infection of apple fruits with MdDRKIP1 1) Prepare mature 'Yishuihong' Fuji apple fruits about 180 days after flowering for use; 2) Transform Agrobacterium tumefaciens. Add 1 μL of the positive plasmid pPZP211 - MdDRKIP1 - 3FLAG to 50 μL of Agrobacterium tumefaciens competent cells stored at - 80°C respectively. After transformation, add 1 mL of LB liquid medium and culture it on a shaker at 28°C and 200 rpm for 30 min for activation. The activated Agrobacterium tumefaciens was cultured on LB solid medium containing spectinomycin. Pick a single colony from the plate medium and inoculate it into LB liquid medium containing spectinomycin, and culture it on a shaker at 28°C and 200 rpm for 16 h. Use the MdDRKIP1 - F1 and MdDRKIP1 - R1 primers for colony - PCR identification, and store the positive clones at - 20°C for later use. Take 200 μL of the Agrobacterium tumefaciens liquid containing the MdDRKIP1 gene and add it to 10 mL of liquid LB medium containing rifampicin (50 mg / L) and spectinomycin (100 mg / L), and culture it on a shaker at 28°C and 220 rpm for 12 - 24 h until OD600 = 0.6 - 0.8; 3) Secondary activation. Take 200 μL of the activated Agrobacterium tumefaciens containing the MdDRKIP1 gene and add it to 10 mL of liquid LB medium containing acetosyringone (200 μM), MES (10 mM), rifampicin (50 mg / L), and spectinomycin (100 mg / L), and culture it on a shaker at 28°C and 220 rpm for 12 - 24 h until OD600 = 0.6 - 0.8; 4) Collect the Agrobacterium liquid or mixed plasmids and prepare an infection solution. The infection solution contains MgCl2 (10 mM), acetosyringone (200 μM), and MES (10 mM). Take the secondarily activated bacterial liquid, centrifuge to remove the supernatant, and resuspend it with the infection solution; 5) Use a sterile syringe to inject the infection solution under the epidermis of apple fruits; 6) After the injected apple fruits are incubated in the dark for 24 h, inoculate with Botryosphaeria berengeriana f. sp. piricola for observation.

[0042] As Figure 9 shown, the lesion diameters and pathogen biomass of MdDRKIP1-aOE apple fruits are significantly smaller than those of the control.

[0043] Example 12 Detection of resistance substances in MdDRK1- and MdDRKIP1-transgenic apple calli To further explore the mechanism of MdDRK1-MdDRKIP1 against Botryosphaeria berengeriana f. sp. piricola, the present invention uses a total phenol (TP) detection kit to determine the total phenol content in apple calli overexpressing the MdDRK1 gene and the MdDRKIP1 gene; a total flavonoid (TF) detection kit to determine the total flavonoid content in apple calli overexpressing the MdDRK1 gene and the MdDRKIP1 gene, and compare the differences in total phenol and total flavonoid contents in different apple calli. The results are as Figure 10 shown, the total phenol and total flavonoid contents in both MdDRK1-OE transgenic apple calli and MdDRKIP1-OE transgenic apple calli are higher than those of the control.

[0044] The above research shows that MdDRK1-MdDRKIP1 improves the resistance of apples to Botryosphaeria berengeriana f. sp. piricola by regulating the total phenol and total flavonoid contents in apples, and has potential application value in improving the disease resistance of apples.

[0045] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference can be made to each other.

[0046] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of MdDRK1 and MdDRKIP1 genes in improving the resistance of apples to ring rot, characterized in that, The CDS sequence encoding the MdDRK1 gene is shown in SEQ ID NO.7, and the CDS sequence encoding the MdDRKIP1 gene is shown in SEQ ID NO.

15.

2. The application according to claim 1, characterized in that, The MdDRK1 protein sequence is shown in SEQ ID NO.8, and the MdDRKIP1 protein sequence is shown in SEQ ID NO.

16.

3. A vector for increasing the expression levels of MdDRK1 and MdDRKIP1 genes, characterized in that, The vectors are pCAMBIA1305-MdDRK1-3MYC and pPZP211-MdDRKIP1-3FLAG; The pCAMBIA1305-MdDRK1-3MYC is formed by ligating the pCAMBIA1305-3MYC vector and the MdDRK1 gene; The pPZP211-MdDRKIP1-3FLAG is formed by ligating the pPZP211-3FLAG vector and the MdDRKIP1 gene.

4. A yeast two-hybrid expression vector prepared from the MdDRK1 and MdDRKIP1 genes, characterized in that the vector is MdDRK1-AD and MdDRKIP1-BD; The MdDRK1-AD is formed by ligating the pGADT7-AD vector and the MdDRK1 gene; The MdDRKIP1-BD is formed by ligating the pGBKT7-BD vector and the MdDRKIP1 gene.

5. A luciferase complementation expression vector prepared from the MdDRK1 and MdDRKIP1 genes, characterized in that the vector is pCAMBIA1300-MdDRK1-Cluc and pCAMBIA1300-MdDRKIP1-NLuc; The pCAMBIA1300-MdDRK1-Cluc is formed by ligating the pCAMBIA1300-Cluc vector and the MdDRK1 gene; The pCAMBIA1300-MdDRKIP1-NLuc is formed by ligating the pCAMBIA1300-NLuc vector and the MdDRKIP1 gene.

Images