Application of MdDRK1 and MdDRKIP1 genes in improving resistance to apple ring rot

Abstract

The present invention discloses the application of MdDRK1 and MdDRKIP1 genes in improving apple ring rot resistance, belonging to the field of genetic engineering technology. The application of MdDRK1 and MdDRKIP1 genes in improving apple ring rot resistance, 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. The contents of total phenols and total flavonoids, the resistance substances, in apple callus tissues overexpressing MdDRK1 and overexpressing MdDRKIP1 are significantly increased. The above studies show that MdDRK1 and MdDRKIP1 genes play an important role in improving apple resistance to ring rot, have the potential to be used in gene editing to cultivate new varieties resistant to ring rot, and are of great significance to the directed genetic improvement of apple disease resistance.

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 to apple ring rot. Background Art

[0002] Apple (Malus domestica Borkh.) is one of the most widely cultivated and economically valuable fruits in temperate regions. However, its yield and quality are susceptible to pests and diseases, severely hampering the healthy development of the apple industry. Apple ring rot, caused by the fungus Botryosphaeria dothidea, causes fruit rot, weakening the tree, or even the death of the entire plant, making it one of the most serious diseases affecting my country's major apple-producing areas. To control apple ring rot, fruit farmers often resort to harsh bark scraping and pesticide spraying. These methods not only weaken the tree and reduce fruit quality, but also lead to increased pesticide resistance in the pathogen, resulting in serious environmental pollution and food safety issues. With the advancement of green agriculture and sustainable development, exploring new, environmentally friendly, effective, and long-lasting strategies for controlling apple ring rot has become a research priority. Therefore, exploring the molecular mechanisms of apple ring rot resistance and using molecular biological breeding techniques to select resistant varieties are effective approaches to addressing apple ring rot and are of great significance for promoting the high-quality development of my country's apple industry.

[0003] The mitogen-activated protein kinase (MAPK) cascade is one of the earliest signal transduction pathways activated after plant pathogen infection and serves as a hub for plant-microbe interactions. A typical MAPK cascade consists of three components: MAPK kinase kinase (MKKK, or MEKK), MAPK kinase (MKK, or MEK), and MAPK. Upon exposure to external stimuli, the MAPK cascade is activated through sequential phosphorylation, transforming extracellular stimuli into intracellular responses through a cascade amplification process, triggering various phosphorylation-mediated cellular responses. Numerous reports indicate that activation of the MAPK cascade is one of the earliest cellular responses to pathogen infection in plants, regulating plant cell immune responses. Rice bacterial leaf streak is a devastating disease caused by the bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc). When Xoc infects rice, the OsMPKK10.2-OsMPK6 cascade is activated. This signaling pathway enhances rice resistance to Xoc by regulating the expression of multiple disease-resistance genes. TaMKK5 expression levels are significantly higher in wheat varieties resistant to R. solani than in susceptible varieties. TaMKK5 regulates wheat defense responses to R. solani through the TaMKK5-TaMPK3-TaERF3 signaling pathway. These studies demonstrate that the MAPK cascade is a key node in the plant immune response regulatory network. However, the molecular mechanisms by which the MAPK signaling pathway regulates apple resistance to ring rot remain largely unknown.

[0004] Therefore, exploring the function of the MAPK signaling pathway in apple ring rot resistance, analyzing its molecular mechanism of regulating apple disease resistance and applying it are technical problems that technicians in this field urgently need to solve. Summary of the Invention

[0005] In view of this, the present invention isolated and identified a MAPK cascade signaling pathway that improves apple's resistance to ring rot, which is mediated by MdDRK1 ( D isease R esistance K inase 1) and MdDRKIP1 (DRK I Interaction P The invention also provides the application of MdDRK1 and MdDRKIP1 genes in improving the resistance to apple ring rot.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The application of MdDRK1 and MdDRKIP1 genes in improving apple ring rot resistance, 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.

[0008] Furthermore, the MdDRK1 protein sequence is shown as SEQ ID NO.8, and the MdDRKIP1 protein sequence is shown as SEQ ID NO.16.

[0009] A vector for improving the expression levels of the MdDRK1 and MdDRKIP1 genes, wherein the vectors are pCAMBIA1305-MdDRK1-3MYC and pPZP211-MdDRKIP1-3FLAG;

[0010] The pCAMBIA1305-MdDRK1-3MYC is formed by connecting the pCAMBIA1305-3MYC vector and the MdDRK1 gene;

[0011] The pPZP211-MdDRKIP1-3FLAG is formed by connecting the pPZP211-3FLAG vector and the MdDRKIP1 gene.

[0012] Yeast two-hybrid expression vectors prepared from MdDRK1 and MdDRKIP1 genes, the vectors being MdDRK1-AD and MdDRKIP1-BD;

[0013] The MdDRK1-AD is formed by connecting the pGADT7-AD vector and the MdDRK1 gene;

[0014] The MdDRKIP1-BD is formed by connecting the pGBKT7-BD vector and the MdDRKIP1 gene.

[0015] Luciferase complementation expression vectors prepared from MdDRK1 and MdDRKIP1 genes, the vectors being pCAMBIA1300-MdDRK1-Cluc and pCAMBIA1300-MdDRKIP1-NLuc;

[0016] The pCAMBIA1300-MdDRK1-Cluc is formed by connecting the pCAMBIA1300-Cluc vector and the MdDRK1 gene;

[0017] The pCAMBIA1300-MdDRKIP1-NLuc is formed by connecting the pCAMBIA1300-NLuc vector and the MdDRKIP1 gene.

[0018] Immunoprecipitation expression vectors prepared from MdDRK1 and MdDRKIP1 genes, wherein the vectors are pCAMBIA1305-MdDRK1-3MYC and pPZP211-MdDRKIP1-3FLAG.

[0019] The total phenolic (TP) detection kit was used to determine the total phenolic content in the apple calli overexpressing MdDRK1 gene and MdDRKIP1 gene. The total flavonoid (TF) detection kit was used to determine the total flavonoid content in the apple calli overexpressing MdDRK1 gene and MdDRKIP1 gene.

[0020] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are:

[0021] The present invention firstly utilized gene homology to design primers and successfully cloned a resistance gene in the apple variety 'Golden Crown' that was resistant to ring rot. By comparing the domain structure with the database, it was confirmed that the resistance gene belonged to the MAPKK family gene in the MAPK cascade signaling pathway, and was therefore named MdDRK1 ( D isease R esistance K Inase 1). The MdDRK1 gene was transferred into apple callus and apple fruit by genetic transformation. After inoculation with the ring rot pathogen, the overexpression of MdDRK1 improved the resistance of apple callus and apple fruit to the ring rot. The possible interacting protein of MdDRK1, MdDRKIP1 (DRK I Interaction P rotein 1). By constructing an expression vector, the interaction between MdDRK1 and MdDRKIP1 was demonstrated using yeast two-hybrid, luciferase reporter, and immunoprecipitation techniques. The MdDRKIP1 gene was introduced into apple callus and fruit using genetic transformation. After inoculation with the ring rot pathogen, overexpression of MdDRKIP1 enhanced the resistance of apple callus and fruit to the disease. Further studies revealed that the levels of total phenolics and flavonoids, the resistance-enhancing substances, were significantly increased in apple callus overexpressing MdDRK1 and MdDRKIP1. These studies demonstrate that the MdDRK1 and MdDRKIP1 genes can enhance apple resistance to ring rot by increasing their levels of total phenolics and flavonoids, and thus have important application value in molecular breeding for resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0023] Figure 1 The accompanying figure is a 1% agarose gel electrophoresis diagram of the PCR product of the full-length CDS of the MdDRK1 gene of the present invention; M is a marker, and 1 and 2 are cloned fragments of the CDS of the MdDRK1 gene;

[0024] Figure 2 The accompanying drawings are diagrams showing the validation of qRT-PCR and Western blot analysis of transgenic apple callus overexpressing MdDRK1 according to the present invention; wherein A is the protein level detection of transgenic apple callus overexpressing MdDRK1; B is the MdDRK1 gene expression level detection of transgenic apple callus overexpressing MdDRK1; WT is the wild-type control, and MdDRK1-OE is the transgenic apple callus line overexpressing MdDRK1;

[0025] Figure 3 The accompanying drawings show the phenotypes of transgenic apple callus overexpressing MdDRK1 and inoculated with ring rot pathogens of the present invention; wherein A is the phenotype of transgenic apple callus overexpressing MdDRK1 after inoculation with ring rot pathogens; B is the lesion diameter of transgenic apple callus overexpressing MdDRK1 after inoculation with ring rot pathogens; C is the biomass of pathogens after inoculation with ring rot pathogens of transgenic apple callus overexpressing MdDRK1; WT is the wild-type control, and MdDRK1-OE is a transgenic apple callus strain overexpressing MdDRK1;

[0026] Figure 4 The accompanying drawings show the phenotypes of apple fruits transiently overexpressing MdDRK1 and inoculated with ring rot pathogens according to the present invention; wherein A is the phenotype of apple fruits transiently overexpressing MdDRK1 and inoculated with ring rot pathogens; B is the lesion diameter of apple fruits transiently overexpressing MdDRK1 and inoculated with ring rot pathogens; C is the biomass of the pathogen in apple fruits transiently overexpressing MdDRK1 and inoculated with ring rot pathogens; Vec is an empty vector control, and MdDRK1-aOE is an apple fruit transiently overexpressing MdDRK1;

[0027] Figure 5 The accompanying figure is a 1% agarose gel electrophoresis diagram of the PCR product of the full-length CDS of the MdDRKIP1 gene of the present invention; M is a marker, and 1 and 2 are cloned fragments of the CDS of the MdDRKIP1 gene;

[0028] Figure 6The accompanying drawings are diagrams showing the validation of qRT-PCR and Western PCR for the transgenic apple callus overexpressing MdDRKIP1 of the present invention; wherein A is the protein level detection of the transgenic apple callus overexpressing MdDRKIP1; B is the MdDRKIP1 gene expression level detection of the transgenic apple callus overexpressing MdDRKIP1; WT is the wild-type control, and MdDRKIP1-OE is the transgenic apple callus line overexpressing MdDRKIP1;

[0029] Figure 7 The accompanying drawings show the verification of the interaction between MdDRK1 and MdDRKIP1 by the present invention; wherein, A is the verification of the interaction 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 culture medium; QDO: SD / -Ade / -His / -Leu / -Trp solid culture medium; QDO / X: SD / -Ade / -His / -Leu / -Trp solid culture medium plus X-α-gal; B is the verification of the interaction between MdDRK1 and MdDRKIP1 by the luciferase reporter system; NLuc+Cluc: pCAMBIA1300-Cluc Vector and pCAMBIA1300-NLuc vector were co-expressed; NLuc+MdDRK1-Cluc: pCAMBIA1300-NLuc vector and pCAMBIA1300-MdDRK1-Cluc vector were co-expressed; MdDRKIP1-NLuc+Cluc: pCAMBIA1300-MdDRKIP1-NLuc vector and pCAMBIA1300-Cluc vector were co-expressed; MdDRKIP1-NLuc+MdDRK1-Cluc: pCAMBIA1300-MdDRKIP1-NLuc vector and pCAMBIA1300-MdDRK1-Cluc vector were co-expressed; C is the interaction between MdDRK1 and MdDRKIP1 verified by immunoprecipitation technology;

[0030] Figure 8 The accompanying drawings show the phenotypes of transgenic apple callus overexpressing MdDRKIP1 and inoculated with ring rot pathogens of the present invention; wherein A is the phenotype of transgenic apple callus overexpressing MdDRKIP1 after inoculation with ring rot pathogens; B is the lesion diameter of transgenic apple callus overexpressing MdDRKIP1 after inoculation with ring rot pathogens; C is the biomass of pathogens after inoculation with ring rot pathogens of transgenic apple callus overexpressing MdDRKIP1; WT is the wild-type control, and MdDRKIP1-OE is a transgenic apple callus strain overexpressing MdDRKIP1;

[0031] Figure 9 The accompanying drawings show the phenotypes of apple fruits transiently overexpressing MdDRKIP1 and inoculated with ring rot pathogens according to the present invention; wherein A is the phenotype of apple fruits transiently overexpressing MdDRKIP1 after inoculation with ring rot pathogens; B is the lesion diameter of apple fruits transiently overexpressing MdDRKIP1 after inoculation with ring rot pathogens; C is the biomass of the pathogen in apple fruits transiently overexpressing MdDRKIP1 after inoculation with ring rot pathogens; Vec is an empty vector control, and MdDRKIP1-aOE is an apple fruit transiently overexpressing MdDRKIP1;

[0032] Figure 10 The accompanying drawings show the total phenolic and total flavonoid contents of the callus tissues of the transgenic apples overexpressing MdDRK1 and overexpressing MdDRKIP1 of the present invention; wherein, A is the total phenolic content of the callus tissues of the transgenic apples overexpressing MdDRK1; B is the total phenolic content of the callus tissues of the transgenic apples overexpressing MdDRKIP1; C is the total flavonoid content of the callus tissues of the transgenic apples overexpressing MdDRK1; D is the total flavonoid content of the callus tissues of the transgenic apples overexpressing MdDRKIP1; WT is the wild-type control, MdDRK1-OE is the transgenic apple callus tissue line overexpressing MdDRK1, and MdDRKIP1-OE is the transgenic apple callus tissue line overexpressing MdDRKIP1. DETAILED DESCRIPTION

[0033] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] The 'Golden Crown' used in the examples was obtained from the Tianping Lake Experimental Base of the Shandong Fruit Research Institute. The peels of mature apple fruits were collected in September 2023, frozen in liquid nitrogen, and stored at -80°C for later use.

[0035] The callus tissue is the callus tissue of 'Wanglin' ​​apple;

[0036] The apple fruits used for instant infection were Fuji 'Yishuihong' grown at the Tianping Lake Experimental Base of Shandong Fruit Research Institute.

[0037] Example 1 Cloning of MdDRK1 gene and construction of expression vector

[0038] 1) Total RNA extraction from apple peel

[0039] 0.05 g of 'Golden Crown' apple peel was weighed and quickly ground into powder in liquid nitrogen. Total RNA was extracted using the Nobel Plant RNA Extraction Kit according to the manufacturer's instructions and detected by 1% agarose gel electrophoresis. The RNA was stored at -80°C until use.

[0040] 2) MdDRK1 gene primer design

[0041] Using the apple genomic mRNA sequence as a template, primer sequences encompassing the MdDRK1 CDS region were designed. The primer sequences are as follows:

[0042] MdDRK1-F1: 5′-CATATGAAGACGAAGACGCCATTG-3′, as shown in SEQ ID NO. 1;

[0043] MdDRK1-R1: 5′-GAATTCTCTTGGAAAATTTACAGG-3′, as shown in SEQ ID NO. 2;

[0044] The primer sequences designed for ligation to the pCAMBIA1305-3MYC expression vector are as follows:

[0045] MdDRK1-MYC-F: 5′-GGATCCATGAAGACGAAGACGCCA-3′, as shown in SEQ ID NO. 3;

[0046] MdDRK1-MYC-R: 5′-GTCGACTCTTGGAAAATTTACAGG-3′, as shown in SEQ ID NO. 4;

[0047] Design specific primer sequences for real-time fluorescence quantitative PCR (qRT-PCR). The primer sequences are as follows:

[0048] MdDRK1-qRT-F: 5′-AAACCAAGCAGCACAATGTCCAC-3′, as shown in SEQ ID NO. 5;

[0049] MdDRK1-qRT-R: 5′-AGCGAAGCACTCACGCCAAAATC-3′, as shown in SEQ ID NO. 6.

[0050] 3) Cloning of the MdDRK1 gene

[0051] The first strand of apple cDNA was synthesized using the Nobel Reverse Transcription Kit. The reaction system and 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. PCR amplification products were detected using 1% agarose gel, and a single band around 1026 bp was obtained at the target position ( Figure 1 ).

[0052] Recover the target fragment using a Tiangen Gum Extraction Kit and ligate it into a T-vector (pMD19-T vector) using T4 DNA ligase. Transform 5 μL of the ligation product into 50 μL of DH5α competent E. coli cells. After culturing in 1 mL of LB liquid medium, 100 μL of the aliquot was centrifuged and plated onto LB solid medium for ampicillin resistance screening. Resistant clones were verified by colony PCR using the universal M13 primer.

[0053] The PCR products were sent to Shanghai Bioengineering for sequencing. The sequencing results were aligned 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. The gene CDS region sequence (1026 bp) is:

[0054]

[0055] The MdDRK1 protein sequence (341 residues) is:

[0056] MKTKTPLNLNLKQLKLNVPAQEANIKSFLTASGTFHDGDLLLNQKGLRLISEEKETPDQSSDSKELNFEITLEDLETIKVIGKGSGGVVQLVRHKWVGTLFALKVIQMNIQEEIRKQIVQELKINQAAQCPHVVVCHHSFYHNGAISLVLEYMDRGSLADVIRQVNTILEPYL AVVCKQVLQGLVYLHNERHVIHRDIKPSNLLVNHQGQVKITDFGVSASLASSMGQRDTFVGTYNYMSPERISGSTYDYSSDIWSLGLAVLECAIGRFPYMQSEDQQSWPSFYELLEAIVESPPPSAPPDQFSPESCSFVSSCIQKDPQHRSSCLDLLSHPFIKSRDKT, such as SEQ Shown as ID NO.8.

[0057] 4) Construction of plant overexpression vector for the apple MdDRK1 gene

[0058] The obtained MdDRK1 gene fragment was used as a template for amplification using gene-specific PCR primers (MdDRK1-MYC-F and MdDRK1-MYC-R). PCR amplification was performed on 1% agarose gel. The target fragment was recovered and ligated into a T-vector (pMD19-T vector) for transformation into DH5α competent E. coli cells. The plasmid containing the target MdDRK1 gene fragment was extracted from the E. coli bacterial culture. The pCAMBIA1305-3MYC expression vector was selected and digested with BamH I and Sal I enzymes. The digested products of the MdDRK1 fragment and vector were recovered and ligated with T4 DNA ligase. The ligation product was then transformed into DH5α competent E. coli cells. After culturing in 1 mL of LB liquid medium, 100 μL of the aliquot was centrifuged and applied to LB solid medium containing kanamycin. Single colonies were isolated and identified by colony PCR. Five positive clones were selected for sequencing and inoculated into 10 mL of LB liquid medium containing kanamycin resistance. Finally, the plasmids from the correctly sequenced bacterial suspension were extracted and stored at -20°C for future use.

[0059] Example 2 Agrobacterium-mediated genetic transformation and identification of MdDRK1 apple callus

[0060] 1) Prepare 'Wanglin' ​​callus. Incubate the infected apple callus at 24°C in the dark for 12-14 days.

[0061] 2) Transform Agrobacterium. Add 1 µL of the positive plasmid from the pCAMBIA1305-MdDRK1-3MYC expression vector to 50 µL of competent Agrobacterium LBA4404 cells stored at -80°C. After transformation, add 1 mL of LB liquid medium and incubate the cells at 28°C at 200 rpm for 30 minutes for activation. Activated Agrobacterium should be cultured on LB solid medium supplemented with kanamycin. Single colonies should be picked from the plate and inoculated into LB liquid medium supplemented with kanamycin. Incubate the cells at 28°C at 200 rpm for 16 hours. Identify the cells by PCR using primers MdDRK1-F1 and MdDRK1-R1. Positive clones should be stored at -20°C for future use. Take 200 μL of Agrobacterium containing the target gene 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 the OD reaches 0. 600 =0.6~0.8;

[0062] 3) Add 2 mL of activated Agrobacterium to 50 mL of liquid LB medium without antibiotics and culture at 28°C with shaking at 220 rpm for 4-6 hours.

[0063] 4) Place the bacterial suspension into a sterilized 50 mL centrifuge tube and centrifuge at 5000 rpm for 10 min at room temperature. Discard the supernatant and resuspend the suspension in an appropriate amount of MS liquid medium to an OD600 value of 0.4-0.6. Pour the suspension into a 50 mL conical flask. Transfer the 'Wanglin' ​​callus to the conical flask and culture at 28°C with shaking at 200 rpm for 20 min.

[0064] 5) Use sterile filter paper to remove the bacterial liquid on the surface of the callus tissue and transfer it to MS medium without antibiotics. Incubate in the dark at 24°C for 2 days.

[0065] 6) Transfer the co-cultured callus to MS selection medium containing kanamycin (100 mg / L) and carbenicillin (250 mg / L) and culture in the dark at 24°C.

[0066] 7) Screening of transgenic lines. Calli grown on the screening medium are transferred to new screening medium until positive transgenic calli are screened out.

[0067] 8) PCR identification of transgenic lines

[0068] After pCAMBIA1305-MdDRK1-3MYC was used to transform apple callus tissue, stably inherited transgenic apple callus tissue was purified. The present invention uses two methods to identify transgenic callus tissue. First, the MYC tag contained therein was used, with wild-type callus tissue as the control, Anti-MYC antibody was applied, and Western-Blot technology was used to detect the expression of MdDRK1-MYC fusion protein in three transgenic callus tissue lines. Protein bands of the target length of MdDRK1 were amplified, while the wild type had no such band. The positive rate of detecting target transgenics was 100%. Secondly, qRT-PCR specific primers were used, with wild-type callus tissue as the control, and qRT-PCR technology was applied to detect the expression level of the MdDRK1 gene in the three transgenic callus tissue lines. The expression level of MdDRK1 in the transgenic line was significantly higher than that of the wild type, and the positive rate of detecting target transgenics was 100%. The results showed that MdDRK1 successfully transformed apple callus tissue and could be stably inherited. The results are shown in Table 1. Figure 2 .

[0069] Example 3 Phenotype of MdDRK1 transgenic apple callus after inoculation with ring rot pathogen

[0070] Transgenic apple calli of MdDRK1-OE1, MdDRK1-OE2, and MdDRK1-OE3 were propagated. 3 g of transgenic calli and wild-type calli were weighed and cultured in the same MS medium without any antibiotics. Each culture dish contained 25 mL of medium, and each strain was replicated three times. After inoculation with ring rot pathogen, the apple calli were cultured in the dark at 24°C for 12 days. The phenotypic differences between the positive transgenic calli and the wild-type were observed. Figure 3 As shown in the results, after inoculation with the ring rot pathogen, the diameter of the ring rot lesions and the biomass of the pathogen in the calli of transgenic apples MdDRK1-OE1, MdDRK1-OE2, and MdDRK1-OE3 were significantly smaller than those in the control group. This gene function has potential application value in improving the disease resistance of apple fruits if applied to the breeding of new apple varieties.

[0071] Example 4 Phenotypic observation after transient infection of apple fruit with MdDRK1 and inoculation with ring rot pathogen

[0072] 1) Prepare mature 'Yishuihong' Fuji apple fruits about 180 days after flowering;

[0073] 2) Transform Agrobacterium. Add 1 µL of the pCAMBIA1305-C-MdDRK1-3MYC positive plasmid to 50 µL of GV3101 competent Agrobacterium cells stored at -80°C. After transformation, add 1 mL of LB liquid medium and incubate the cells at 28°C at 200 rpm for 30 minutes for activation. Activated Agrobacterium should be cultured on LB solid medium supplemented with kanamycin. Single colonies should be picked from the plate and inoculated into LB liquid medium supplemented with kanamycin. Incubate the cells at 28°C at 200 rpm for 16 hours. Identify the cells by PCR using primers MdDRK1-F1 and MdDRK1-R1. Positive clones should be stored at -20°C for future use. Take 200 μL of Agrobacterium culture 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 with shaking at 220 rpm for 12-24 h until OD600 = 0.6-0.8.

[0074] 3) Secondary activation: Add 200 μL of activated Agrobacterium to 10 mL of liquid LB medium containing 200 μM acetosyringone, 10 mM MES, 50 mg / L rifampicin, and 100 mg / L kanamycin. Incubate at 28°C with shaking at 220 rpm for 12–24 h until the OD600 reaches 0.6–0.8.

[0075] 4) Collect the Agrobacterium culture or plasmid mixture and prepare an infection solution containing 10 mM MgCl₂, 200 μM acetosyringone, and 10 mM MES. Centrifuge the secondary activated culture, remove the supernatant, and resuspend in the infection solution.

[0076] 5) Use a sterile syringe to inject the infection solution under the skin of the apple fruit;

[0077] 6) After the injected apple fruit was incubated in the dark for 24 hours, it was inoculated with the ring rot pathogen and observed;

[0078] like Figure 4 As shown in the figure, the lesion diameter and pathogen biomass of MdDRK1-aOE apple fruits were significantly smaller than those of the control.

[0079] Example 5 Cloning of the MdDRKIP1 gene and construction of an expression vector

[0080] 1) Total RNA extraction from apple peel

[0081] 0.05 g of 'Golden Crown' apple peel was weighed and quickly ground into powder in liquid nitrogen. Total RNA was extracted using the Nobel Plant RNA Extraction Kit according to the manufacturer's instructions and detected by 1% agarose gel electrophoresis. The RNA was stored at -80°C until use.

[0082] 2) MdDRKIP1 gene primer design

[0083] Using the apple genomic mRNA sequence as a template, primer sequences encompassing the MdDRKIP1 CDS region were designed. The primer sequences are as follows:

[0084] MdDRKIP1-F1: 5′-CATATGTGGCAGACGCTGTTC-3′, as shown in SEQ ID NO. 9;

[0085] MdDRKIP1-R1: 5′-GGATCCGGAAAACACTGCGGC-3′, as shown in SEQ ID NO. 10;

[0086] The primer sequences designed for ligation to the pPZP211-3FLAG expression vector are as follows:

[0087] MdDRKIP1-FLAG-F: 5′-GGATCCATGTGGCAGACGCTGTTC-3′, as shown in SEQ ID NO. 11;

[0088] MdDRKIP1-FLAG-R: 5′-ACTAGTGGAAAACACTGCGGCATTG-3′, as shown in SEQ ID NO. 12;

[0089] Design specific primer sequences for real-time fluorescence quantitative PCR (qRT-PCR). The primer sequences are as follows:

[0090] MdDRKIP1-qRT-F: 5′-GCCGATTCAGAGGAAGAGC-3′, as shown in SEQ ID NO. 13;

[0091] MdDRKIP1-qRT-R: 5′-GTTTGCGTTGATAAGAAGGTTT-3′, as shown in SEQ ID NO.14.

[0092] 3) Cloning of the MdDRKIP1 gene

[0093] The first strand of apple cDNA was synthesized using the Nobel Reverse Transcription Kit. The reaction system and conditions were carried out according to the kit instructions. After the reaction, gene-specific PCR primers (MdDRKIP1, -F1 and MdDRKIP1-R1) were used for amplification. PCR amplification products were detected using 1% agarose gel, and a single band around 1083 bp was obtained at the target position ( Figure 5 ).

[0094] Recover the target fragment using a Tiangen Gum Extraction Kit and ligate it into a T-vector (pMD19-T vector) using T4 DNA ligase. Transform 5 μL of the ligation product into 50 μL of DH5α competent E. coli cells. After culturing in 1 mL of LB liquid medium, 100 μL of the aliquot was centrifuged and plated onto LB solid medium for ampicillin resistance screening. Resistant clones were verified by colony PCR using the universal M13 primer.

[0095] The PCR products were sent to Shanghai Bioengineering for sequencing. The sequencing results were aligned 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 MAPK family genes, named MdDRKIP1. The gene CDS region sequence (1083 bp) is:

[0096]

[0097] The protein sequence of MdDRKIP1 (360 residues) is:

[0098] MWQTLFEIDTKYVPIKPIGRGAYGIVCSSVNRETNEKVAIKKINNAFENRVDALRTLREMKLLQHLRHENVIALKDVMMPIQRKSFKDVYLVYELMDTDLHQIIKSSQPLSNDHCQYFLFQLLRGLKYLHSANILHRDLKPGNLLVNANCDLKICDFGLARTSTGKDQFMTEYVVTRWYRAP ELLLCCDNYGTSIDVWSVGCIFAELLGRKPIFPGTECLNQLKLIINILGSQREEDLQFIDNPKATKYIKSLPFSLGTPLTRLYPDAHPLAIDLLQKMLVFDPSKRISVTEALQHPYMAPLYDPSNNPPAEVPIDLDIDEDLETDDKGDDVEGNASLPSRSCYRQCRSVFRIRRPAAAA, as in SEQ Shown as IDNO.16.

[0099] 4) Construction of plant overexpression vector for the apple MdDRKIP1 gene

[0100] The obtained MdDRKIP1 gene fragment was used as a template for amplification using gene-specific PCR primers (MdDRKIP1-FLAG-F and MdDRKIP1-FLAG-R). PCR amplification was performed on 1% agarose gel, and the target fragment was recovered. The recovered fragment was ligated into a T vector (pMD19-T vector) and transformed into DH5α competent E. coli cells. The plasmid containing the target MdDRKIP1 gene fragment was extracted from the E. coli bacterial culture. The pPZP211-3FLAG expression vector was selected and digested with BamHI and BcuI enzymes. The digested product of the MdDRKIP1 fragment and vector was recovered and ligated with T4 DNA ligase. The ligation product was then transformed into DH5α competent E. coli cells. After culturing in 1 mL of LB liquid medium, 100 μL of the aliquot was centrifuged and plated onto LB solid medium containing spectinomycin. Single colonies were picked and identified by colony PCR. Five positive clones were selected for sequencing and inoculated into 10 mL of LB liquid medium containing spectinomycin resistance. Finally, the plasmids from the correctly sequenced bacterial suspension were extracted and stored at -20°C for future use.

[0101] Example 6 Agrobacterium-mediated genetic transformation and identification of MdDRKIP1 apple callus

[0102] 1) Prepare 'Wanglin' ​​callus. Incubate the infected apple callus at 24°C in the dark for 12-14 days.

[0103] 2) Transform Agrobacterium. Add 1 µL of the positive plasmid from the pPZP211-MdDRKIP1-3FLAG expression vector to 50 µL of competent Agrobacterium LBA4404 cells stored at -80°C. After transformation, add 1 mL of LB liquid medium and incubate the cells at 28°C at 200 rpm for 30 minutes for activation. Activated Agrobacterium should be cultured on LB solid medium supplemented with spectinomycin. Single colonies should be picked from the plate and inoculated into LB liquid medium supplemented with spectinomycin. Incubate the cells at 28°C at 200 rpm for 16 hours. Identify the cells by PCR using primers MdDRKIP1-F1 and MdDRKIP1-R1. Positive clones should be stored at -20°C for future use. Take 200 μL of Agrobacterium containing the target gene and add it to 10 mL of liquid LB medium containing rifampicin (50 mg / L) and spectinomycin (50 mg / L). Incubate at 28°C and 220 rpm for 12-24 h until the OD 600 =0.6~0.8;

[0104] 3) Add 2 mL of activated Agrobacterium to 50 mL of liquid LB medium without antibiotics and culture at 28°C with shaking at 220 rpm for 4-6 hours.

[0105] 4) Place the bacterial suspension into a sterilized 50 mL centrifuge tube and centrifuge at 5000 rpm for 10 min at room temperature. Discard the supernatant and resuspend the suspension in an appropriate amount of MS liquid medium to an OD600 value of 0.4-0.6. Pour the suspension into a 50 mL conical flask. Transfer the 'Wanglin' ​​callus to the conical flask and culture at 28°C with shaking at 200 rpm for 20 min.

[0106] 5) Use sterile filter paper to remove the bacterial liquid on the surface of the callus tissue and transfer it to MS medium without antibiotics. Incubate in the dark at 24°C for 2 days.

[0107] 6) Transfer the co-cultured callus to MS selection medium supplemented with hygromycin (50 mg / L) and carbenicillin (250 mg / L) and culture in the dark at 24°C.

[0108] 7) Screening of transgenic lines. Calli grown on the screening medium are transferred to new screening medium until positive transgenic calli are screened out.

[0109] 8) PCR identification of transgenic lines

[0110] After apple callus was transformed with pPZP211-MdDRKIP1-3FLAG, stably inherited transgenic apple callus was purified. The present invention uses two methods to identify transgenic callus. First, using the FLAG tag, the wild-type callus was used as a control. Anti-FLAG antibodies were used to detect the expression of the MdDRKIP1-FLAG fusion protein in three transgenic callus lines using Western Blot technology. A protein band of the target length for MdDRKIP1 was amplified, while the wild-type callus did not have this band. The positive rate for the target transgene was 100%. Second, using qRT-PCR-specific primers, the wild-type callus was used as a control. The expression level of the MdDRKIP1 gene in the three transgenic callus lines was detected using qRT-PCR technology. The expression level of MdDRKIP1 in the MdDRKIP1 transgenic lines was significantly higher than that in the wild-type, and the positive rate for the target transgene was 100%. The results showed that MdDRKIP1 was successfully transformed into apple callus and stably inherited. See the results Figure 6 .

[0111] Example 7: Using yeast two-hybrid technology to prove the interaction between MdDRK1 and MdDRKIP1

[0112] 1) The plasmid containing the full-length CDS of MdDRK1 and the pGADT7-AD expression vector obtained above were digested with Nde Ⅰ and EcoR Ⅰ enzymes; the plasmid containing the full-length CDS of MdDRKIP1 and the pGBKT7-BD expression vector obtained above were digested with Nde Ⅰ and BamH Ⅰ enzymes;

[0113] 2) Recovering the digestion products of the MdDRK1 fragment and the pGADT7-AD vector, ligating the vector and the MdDRK1 fragment using T4 DNA ligase to obtain the recombinant expression vector MdDRK1-AD; recovering the digestion products of the MdDRKIP1 fragment and the pGBKT7-BD vector, ligating the vector and the MdDRKIP1 fragment using T4 DNA ligase to obtain the recombinant expression vector MdDRKIP1-BD;

[0114] 3) Mix the two ligation products and the carrier DNA and co-transform yeast Y2HGold competent cells. Incubate at 30°C in a water bath for 30 min. Add 20 μL of dimethyl sulfoxide and incubate at 42°C in a water bath for 15 min. Centrifuge at 12,000 rpm for 30 sec, remove the supernatant, add 1 mL of YPDA and resuspend at 30°C for 1 h. Centrifuge at 12,000 rpm for 30 sec, remove the supernatant, and resuspend the cells in 100 μL of 0.9% NaCl solution. Spot the cells onto different culture media.

[0115] The positive control and the yeast strain co-transformed with the MdDRK1-AD and MdDRKIP1-BD recombinant vectors were able to grow normally on SD / -Ade / -His / -Leu / -Trp solid medium, while the negative control could not grow on SD / -Ade / -His / -Leu / -Trp solid medium. All three strains were able to grow on SD / -Leu / -Trp solid medium, and the positive control and the yeast strain co-transformed with the MdDRK1-AD and MdDRKIP1-BD recombinant vectors turned blue after the addition of X-ɑ-gal, indicating that there is an interaction between MdDRK1 and MdDRKIP1 (see Figure 7 A in ).

[0116] Example 8 Using the luciferase reporter system to demonstrate the interaction between MdDRK1 and MdDRKIP1

[0117] 1) The full-length CDS of MdDRK1 obtained above was ligated into the pCAMBIA1300-CLuc vector to obtain the recombinant expression vector MdDRK1-CLUC; the full-length CDS of MdDRKIP1 was ligated into the pCAMBIA1300-NLuc vector to obtain the recombinant expression vector MdDRKIP1-NLUC;

[0118] 2) The two recombinant expression vectors were transformed into Agrobacterium GV3101 respectively, and the successfully transformed Agrobacterium was used to prepare the infection solution;

[0119] 3) Prepare tobacco in good growth condition for future use;

[0120] 4) Primary activation: Take 200 μL of the preserved Agrobacterium culture solution and inoculate it into 10 mL of LB liquid medium. Add the corresponding antibiotics and shake at 28°C and 180 rpm overnight.

[0121] 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 with shaking at 28°C and 180 rpm overnight.

[0122] 6) Prepare the infection solution, collect the bacteria by centrifugation, resuspend the bacteria in the infection solution, and place them in the dark for 4 hours;

[0123] 7) Use a 1 mL syringe to inject the infection solution onto the back of tobacco leaves and incubate under normal light conditions for 2 days;

[0124] 8) After 2 days, use an in vivo fluorescence imaging device to observe the fluorescence of the injection site in the tobacco leaves and take photos.

[0125] The sites of tobacco leaves co-expressing MdDRK1-CLUC and MdDRKIP1-NLUC emitted significant fluorescence, indicating that there is an interaction between MdDRK1 and MdDRKIP1 (see Figure 7 B in ).

[0126] Example 9: Using co-immunoprecipitation technology to prove the interaction between MdDRK1 and MdDRKIP1

[0127] 1) Proteins were extracted from wild-type callus, MdDRK1-OE, and MdDRKIP1-OE transgenic apple callus and stored at -80°C until use;

[0128] 2) Pretreatment of anti-MYC beads: Mix the anti-MYC beads by pipetting with a pipette tip. 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 rack for 10 seconds, discard the supernatant and repeat the wash process twice.

[0129] 3) Add 500 μL of protein extract to the above beads pellet (500 μL WT for group 1; 250 μL WT + 250 μL MdDRKIP1 for group 2; 250 μL WT + 250 μL MdDRK1 for group 3; 250 μL MdDRKIP1 + 250 μL MdDRK1 for group 4). Resuspend the beads by gently pipetting with a pipette tip and incubate with shaking at 4°C overnight.

[0130] 4) Transfer 60 μL of protein extracts from different callus tissues that have not been incubated with beads to 1.5 mL centrifuge tubes containing 20 μL of 4× protein loading buffer. Mix well, denature, and store at -20°C until use.

[0131] 5) Place the beads incubated overnight on a magnetic rack for 10 seconds and discard the supernatant.

[0132] 6) Add 500 μL of PBS buffer to the pellet, pipette to resuspend and wash the beads, let them stand on a magnetic rack for 10 seconds, discard the supernatant, and repeat the wash process twice;

[0133] 7) Add 50 μL of 1× protein loading buffer to the precipitate obtained after three washes, denature the protein, and store at -20°C until use.

[0134] 8) Detect the interaction between the two proteins using the Western blot assay described above.

[0135] Figure 7The Co-IP experimental results of C in Figure 3 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 between MdDRK1 and MdDRKIP1.

[0136] Example 10 Phenotypic Observation of MdDRKIP1 Transgenic Apple Callus

[0137] The transgenic apple calli of MdDRKIP1-OE1, MdDRKIP1-OE2 and MdDRKIP1-OE3 were propagated. 3 g of transgenic apple calli and wild-type apple calli were weighed and placed in the same MS medium without any antibiotics for propagation and culture. Each culture dish contained 25 mL of medium, and each strain was replicated three times. After the apple calli were inoculated with ring rot pathogens, they were cultured in the dark at 24°C for 12 days. The phenotypic differences between wild-type and positive transgenic apple calli were observed. Figure 8 As shown in the results, after inoculation with the ring rot pathogen, the diameter of the ring rot lesions and the biomass of the pathogen in the calli of transgenic apples MdDRKIP1-OE1, MdDRKIP1-OE2, and MdDRKIP1-OE3 were significantly smaller than those in the control group. This gene function has potential application in improving the disease resistance of apple fruits if applied to the breeding of new apple varieties.

[0138] Example 11 MdDRKIP1 transiently infects apple fruit

[0139] 1) Prepare mature 'Yishuihong' Fuji apple fruits about 180 days after flowering;

[0140] 2) Transform Agrobacterium. Add 1 µL of the pPZP211-MdDRKIP1-3FLAG positive plasmid to 50 µL of GV3101 competent Agrobacterium cells stored at -80°C. After transformation, add 1 mL of LB liquid medium and incubate the cells at 28°C at 200 rpm for 30 minutes for activation. Activated Agrobacterium should be cultured on LB solid medium supplemented with spectinomycin. Single colonies should be picked from the plate and inoculated into LB liquid medium supplemented with spectinomycin. Incubate the cells at 28°C at 200 rpm for 16 hours. Identify the cells by PCR using primers MdDRKIP1-F1 and MdDRKIP1-R1. Positive clones should be stored at -20°C for future use. Take 200 μL of Agrobacterium containing the MdDRKIP1 gene and add it to 10 mL of liquid LB medium containing rifampicin (50 mg / L) and spectinomycin (100 mg / L). Incubate with shaking at 220 rpm at 28°C for 12-24 h until the OD600 reaches 0.6-0.8.

[0141] 3) Secondary activation: Add 200 μL of activated Agrobacterium containing the MdDRKIP1 gene to 10 mL of liquid LB medium containing 200 μM acetosyringone, 10 mM MES, 50 mg / L rifampicin, and 100 mg / L spectinomycin. Incubate at 28°C with shaking at 220 rpm for 12–24 h until the OD600 reaches 0.6–0.8.

[0142] 4) Collect the Agrobacterium culture or plasmid mixture and prepare an infection solution containing 10 mM MgCl₂, 200 μM acetosyringone, and 10 mM MES. Centrifuge the secondary activated culture, remove the supernatant, and resuspend in the infection solution.

[0143] 5) Use a sterile syringe to inject the infection solution under the skin of the apple fruit;

[0144] 6) After the injection, the apple fruits were incubated in the dark for 24 hours and then inoculated with the ring rot pathogen for observation.

[0145] like Figure 9 As shown, the lesion diameter and pathogen biomass of MdDRKIP1-aOE apple fruits were significantly smaller than those of the control.

[0146] Example 12 Detection of resistance substances in MdDRK1 and MdDRKIP1 transgenic apple callus

[0147] To further explore the anti-ring rot mechanism of MdDRK1-MdDRKIP1, the present invention used a total phenol (TP) detection kit to determine the total phenol content in apple calli overexpressing the MdDRK1 gene and the MdDRKIP1 gene; and a total flavonoid (TF) detection kit to determine the total flavonoid content in apple calli overexpressing the MdDRK1 gene and the MdDRKIP1 gene. The differences in total phenol and total flavonoid contents in different apple calli were compared. Figure 10 As shown in the figure, the total phenolic and total flavonoid contents in the MdDRK1-OE transgenic apple callus and the MdDRKIP1-OE transgenic apple callus were higher than those in the control.

[0148] The above studies show that MdDRK1-MdDRKIP1 improves apple's resistance to ring rot by regulating the content of total phenols and total flavonoids in apple, and has potential application value in improving apple's disease resistance.

[0149] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0150] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of MdDRK1 and MdDRKIP1 genes in improving resistance to apple ring rot, characterized in that: The method for improving resistance is to overexpress MdDRK1 and MdDRKIP1 genes. 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. The use according to claim 1, characterized in that The vectors for improving the expression levels of the MdDRK1 and MdDRKIP1 genes are pCAMBIA1305-MdDRK1-3MYC and pPZP211-MdDRKIP1-3FLAG; The pCAMBIA1305-MdDRK1-3MYC is formed by connecting the pCAMBIA1305-3MYC vector and the MdDRK1 gene; The pPZP211-MdDRKIP1-3FLAG is formed by connecting the pPZP211-3FLAG vector and the MdDRKIP1 gene.

4. The use according to claim 1, characterized in that The yeast two-hybrid expression vectors prepared by the MdDRK1 and MdDRKIP1 genes are MdDRK1-AD and MdDRKIP1-BD; The MdDRK1-AD is formed by connecting the pGADT7-AD vector and the MdDRK1 gene; The MdDRKIP1-BD is formed by connecting the pGBKT7-BD vector and the MdDRKIP1 gene.

5. The use according to claim 1, characterized in that The luciferase complementary expression vectors prepared from the MdDRK1 and MdDRKIP1 genes are pCAMBIA1300-MdDRK1-Cluc and pCAMBIA1300-MdDRKIP1-NLuc; The pCAMBIA1300-MdDRK1-Cluc is formed by connecting the pCAMBIA1300-Cluc vector and the MdDRK1 gene; The pCAMBIA1300-MdDRKIP1-NLuc is formed by connecting the pCAMBIA1300-NLuc vector and the MdDRKIP1 gene.

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