Application of pineapple calcium-dependent protein kinase gene in prevention and treatment of pineapple water core disease

Abstract

The application relates to the field of molecular biology technology, and particularly relates to application of a pineapple calcium-dependent protein kinase gene in prevention and treatment of pineapple water core disease; the pineapple calcium-dependent protein kinase gene comprises AcCPK4, AcCPK8 and / or AcCPK15 genes, the CDS sequence of which is shown as SEQ ID NO. 1-3; the gene is transiently expressed in pineapple fruits, can significantly inhibit the pineapple water core disease, and provides important theoretical support for analysis of a pineapple water core disease mechanism and cultivation of high-quality pineapple varieties resistant to the water core disease.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to the application of a pineapple calcium-dependent protein kinase gene in the prevention and treatment of pineapple watercore disease. Background Technology

[0002] Pineapple [Ananascomosus (L.) Merr] is the world's third most popular tropical fruit after bananas and citrus fruits, with extremely high production and economic value. Abnormal global climate fluctuations have led to a significant increase in the incidence of pineapple watercore disease, resulting in a substantial decline in the edible quality and commercial value of pineapple fruit. Pineapple watercore disease is a complex physiological and biochemical process, and its causes are not yet fully understood. Studies have shown that the incidence of watercore disease is closely related to external environmental factors, plant growth and development status, endogenous hormone levels, mineral nutrition in the fruit, and sugar content. Research has found that high-temperature treatment at 38℃ and low-temperature treatment at 10℃ 50 days after pineapple flowering can significantly induce pineapple watercore disease. Furthermore, the size of the pineapple crown bud is negatively correlated with the incidence of watercore disease; the larger the crown bud, the lower the incidence. Conversely, the size of the plant's leaves is positively correlated with the incidence of watercore disease; the more abundant the leaves, the higher the incidence.

[0003] Calcium is a crucial nutrient element in plants. It exists primarily as free and bound calcium, widely distributed in the cell nucleus, cytoplasm, vacuoles, cell membrane, and cell wall. Vacuoles, in particular, serve as calcium reservoirs within the cell and are vital for regulating osmotic pressure. Plants mainly acquire calcium from the soil through their roots. Calcium ions in the soil are transported from the xylem to more transpiratory tissues via apoplasts and symplasts under the influence of transpiration pull. Transpiration in leaves is greater than in fruits, resulting in more calcium being transferred to the leaves. This explains why plants with vigorous growth are more prone to calcium deficiency in their fruits. Calcium is not only essential for plants but also plays a vital role in the stability of cell membranes and cell walls, enzyme regulation, and osmotic regulation. For example, calcium... 2+ Calcium pectate plays a vital role in plant responses to biotic and abiotic stresses, acting as a second messenger. It can combine with the exposed carboxyl group of methyl galacturonate to form calcium pectate, stabilizing the cell wall structure. It can also couple with phospholipids and proteins on the cell membrane to stabilize the cell membrane structure. During the later stages of mitosis, the cell plate separating two daughter cells is primarily composed of calcium pectate. Calcium deficiency affects the formation of the cell plate and spindle fibers, leading to the inability of daughter cells to separate during cell division, resulting in binucleation and ultimately cell death.

[0004] Calcium-dependent protein kinases (CPKs) are a typical class of serine / threonine protein phosphatases, possessing four characteristic domains: a calcium-binding domain (CBD), a serine / threonine protein kinase domain (PKD), an autoinhibitory junction (AJ), and an N-terminal variable domain (NTD). During plant responses to environmental stress, intracytoplasmic calcium... 2+ Increased concentration promotes Ca 2+ It binds to CBD, thereby inducing a conformational change in the protein, relieving the inhibition of the kinase domain by the self-inhibition domain, and activating the CPK signal transduction function.

[0005] Studies have shown that CPKs are ubiquitous in plants, protozoa, oomycetes, and green algae, but have not yet been found in animals and fungi. CPKs constitute a large family of multi-gene kinases in plants, participating in the regulation of growth, development, and stress responses. Currently, 34, 31, and 17 members of the CPK family have been identified in Arabidopsis, rice, and pineapple, respectively, and the functions of some members have been thoroughly elucidated. For example, Qinet et al. (2020) identified 17 members of the AcoCPK family through a systematic analysis of the pineapple (Ananascomosus) genome and found that the expression of AcoCPK1, AcoCPK3, and AcoCPK6 was significantly inhibited by abiotic stresses (such as drought and salt stress), while these genes play a key regulatory role in plant disease resistance and immune responses.

[0006] However, the application of AcoCPKs in pineapple watercore disease remains a blank in the research field. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention aims to provide an application of the pineapple calcium-dependent protein kinase gene in the prevention and control of pineapple watercore disease, thereby achieving the prevention and control of pineapple watercore disease and providing a way to cultivate watercore-resistant pineapple varieties.

[0008] To solve the above problems, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides the application of pineapple calcium-dependent protein kinase genes in the prevention and treatment of pineapple watercore disease or in the preparation of pineapple watercore disease prevention and treatment reagents, wherein the pineapple calcium-dependent protein kinase genes include AcoCPK4, AcoCPK8 and / or AcoCPK15 genes.

[0010] Secondly, the present invention provides the application of pineapple calcium-dependent protein kinase genes in the breeding of pineapple varieties resistant to watercore disease, wherein the pineapple calcium-dependent protein kinase genes include AcoCPK4, AcoCPK8 and / or AcoCPK15 genes.

[0011] Thirdly, the present invention provides a protein encoded by a pineapple calcium-dependent protein kinase gene for inhibiting pineapple watercore disease, wherein the pineapple calcium-dependent protein kinase gene for inhibiting pineapple watercore disease includes the AcoCPK4, AcoCPK8 and / or AcoCPK15 genes.

[0012] Furthermore, the CDS sequence of the AcoCPK4 gene is shown in SEQ ID NO.1; the CDS sequence of the AcoCPK8 gene is shown in SEQ ID NO.2; and the CDS sequence of the AcoCPK15 gene is shown in SEQ ID NO.3.

[0013] Furthermore, the amplification primers for the AcoCPK4 gene are shown in SEQ ID NO.7-SEQ ID NO.8; the amplification primers for the AcoCPK8 gene are shown in SEQ ID NO.9-SEQ ID NO.10; and the amplification primers for the AcoCPK15 gene are shown in SEQ ID NO.11-SEQ ID NO.12.

[0014] Fourthly, the present invention provides an expression vector for inhibiting pineapple watercore disease, comprising the aforementioned pineapple calcium-dependent protein kinase gene.

[0015] Fifthly, the present invention provides an engineered bacterium for inhibiting pineapple watercore disease, including the expression vector for inhibiting pineapple watercore disease.

[0016] Sixthly, the present invention provides a method for inhibiting pineapple watercore disease, comprising:

[0017] The expression vector for inhibiting pineapple watercore disease was transformed into pineapple fruit by infecting pineapple fruit with Agrobacterium.

[0018] In a seventh aspect, the present invention provides the expression vector for inhibiting pineapple watercore disease or the engineered bacteria for inhibiting pineapple watercore disease for use in the prevention and control of pineapple watercore disease or in the preparation of reagents for the prevention and control of pineapple watercore disease.

[0019] Eighthly, the present invention provides the application of the expression vector for inhibiting pineapple watercore disease or the engineered bacteria for inhibiting pineapple watercore disease in the breeding of watercore-resistant pineapple varieties.

[0020] The beneficial effects of this invention are as follows: Traditional hybridization breeding is time-consuming, labor-intensive, and has low breeding efficiency, requiring a long cycle for breeding new pineapple varieties resistant to watercore disease. In contrast, genetic engineering-based breeding is characterized by high efficiency, targeted approach, and short cycle. This invention discovers the important functions of calcium-dependent protein kinases AcoCPK4, AcoCPK8, and AcoCPK15 in pineapple during watercore disease. Later, molecular breeding techniques such as transgenics and site-directed gene editing can be used to cultivate high-quality pineapple germplasm resistant to watercore disease. This provides new ideas and research directions for breeding high-yielding, disease-resistant, and stress-resistant pineapple varieties, effectively solving the problems faced by the pineapple industry and promoting its economic benefits and rapid development. Attached Figure Description

[0021] Figure 1 Characterized by the AcoCPK4, AcoCPK8, and AcoCPK15 genes;

[0022] Figure 2 This is a gel electrophoresis image of the PCR amplification of the coding region sequences of the pineapple AcoCPK4, AcoCPK8, and AcoCPK15 genes in this invention;

[0023] Figure 3 This is a diagram showing the transcriptional levels of AcoCPK4, AcoCPK8, and AcoCPK15 induced by exogenous and endogenous CaCl2.

[0024] Figure 4 This is a tissue and subcellular localization map of the AcoCPK4, AcoCPK8, and AcoCPK15 genes of this invention;

[0025] Figure 5 This invention relates to the transcriptional levels of the AcoCPK4, AcoCPK8, and AcoCPK15 genes in pineapple fruits with different degrees of watercore disease.

[0026] Figure 6 The present invention provides that the instantaneous transformation of pineapple fruit by AcoCPK4, AcoCPK8, and AcoCPK15 can significantly inhibit pineapple watercore disease. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments.

[0028] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0029] Example

[0030] Construction of pineapple cDNA library

[0031] This invention involves extracting total RNA from mature pineapple pulp tissue using an RNA extraction kit (catalog number: 0416-100GK) from Beijing Huayueyang Biotechnology Co., Ltd. Using the obtained total RNA as a template, double-stranded cDNA is synthesized using a reverse transcription kit from Beijing TransGen Biotech Co., Ltd., under the action of reverse transcriptase and polymerase.

[0032] AcoCPK4, AcoCPK8, and AcoCPK15 gene amplification

[0033] This invention designs six specific primers, and amplifies the AcoCPK4, AcoCPK8, and AcoCPK15 genes based on the primer sequences. See also... Figure 1 AcoCPK4, AcoCPK8, and AcoCPK15 are located on different chromosomes, encoding 522, 546, and 550 amino acids, respectively. Their relative molecular masses range from 58.878 kDa to 61.302 kDa, and their isoelectric points are 6.10, 5.37, and 8.25, respectively. The CDS sequences of the three genes are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, and their encoded amino acid sequences are shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.

[0034] SEQ ID NO.1:

[0035] >CPK4

[0036]

[0037] SEQ ID NO.2:

[0038] >CPK8

[0039]

[0040] SEQ ID NO.3:

[0041] >CPK15

[0042]

[0043] SEQ ID NO.4:

[0044] >CPK4

[0045] MGSRTSRHRSDPDRRAPPQPQPQPQPWPHRAPKPYVPAAAAPPQASASAAAAAAPVGRVLGRPMEDVRASYVFGRELGRGQFGVTYLVTHRATGERFACKSIATRKLVHRDDVEDVRREVQIMHHLTGHRNIVELRGAYEDRHSVNLVMELCEGGELFDRIIARGHYTERAAAALCREIVAVVHSCHSMGVMHRDLKPENFLFLNKSEDSPLKATDFGLSVFFKPGEVYKDLVGSAYYVAPEVLKRHYGPEADIWSAGVILYILLSGVPPFWAENEDGIFNEIRRGHVDFLSDPWPSISSGAKDLVKKMLRQDPKERLTAAEILNHPWIREDGEAPDKPLDNTVISRMKQFRAMNKLKKVALKVVAENLSEEEIVGLKEMFKSIDTDNSGTITLEELRTGLPKLGTKISESELMQLMEAADVDGNGSIDYLEFISATMHMNRLEKEGHIYKAFEYFDKDNSGYITMEELEQALKKYDMGDEKTIKEIIAEVDADHDGRINYEEFVAMMKNNSQDIAPIRRRK。

[0046] SEQ ID NO.5:

[0047] >CPK8

[0048] MGNTCRGSLASTSNYFSRYSSSSASSSSSLGGDAAAEADAADDRPPRPLSAPMRASSSAAASVAAEAASVLGHKTRNLHELYALGRKLGQGQFGTTYLCTELATGARFACKSIAKRKLISKEDVEDVRREIQIMHHLSGHRNVVAIKGAYEDQVHVHIVMELCEGGELFDRIIERGHFTERKAADLARIVVGVVEACHSLGVMHRDLKPENFLLANKDDDSSLKAIDFGLSVFFKPGQVFTDVVGSPYYVAPEVLCKHYGPEADVWTAGVIIYILLSGVPPFWAETQQGIFDAVLKGVVDFEAEPWPMISESAKDLIRKMLCPRPSERLTAHEVLCHPWICENGVAPDRALDPAVLSRLKNFSAMNKLKKMALRVIADSLSEEEIAGLREMFRAMDTDNSGAITFDELKEGLRRYGSNLKESEIRALMEAADVDNSGTIDYGEFIAATVHLNKLDREEHLVAAFSYFDKDGSGYITVDELQQACKEHNMTDVIIDDIIKEADQDNDGRIDYNEFVAMMLKGNMGLGRRTMTNSLNVSMWGAPRPSC。

[0049] SEQ ID NO.6:

[0050] >CPK15

[0051] MGNCCASPAAAGAGGGGGGGGGKQRGRKKNKNRDKAKGKKPNPFSIDYSRGPAAAAGVPRITVLKDPTGRDIAGRYELGRELGRGEFGVTYLCTDKVSGEGYACKSISKKKLRTAVDVEDVRREVDIMRQLPPHPNIVSLKDTYEDDYAVHLVMELCEGGELFDRIVARGHYTERAAAEVTRTIVEVVQVCHKHGVMHRDLKPENFLFASKKENSPLKAIDFGLSVFFKPGERFTEIVGSPYYMAPEVLKRDYGPEVDVWSAGVILYILLCGVPPFWAETEQGVAQAIIRSVIDFKREPWPRVSDNAKDLVKRMLDPDPKRRLTAQEVLDHPWLQNAKKAPNVSLGETVRARLQQFAVMNKFKKRALRVVAEHLSVEEVADIKNMFQKMDIHNNGTITFEELKYGLRKLGHQISDADAQILMNAADVDGSGTLDYGEFVAVSIHLKKLGNDEHLRKAFAYFDQNRSGYIEIEELRDSLADDLGPNHEEVIDAIIRDVDTDKDGKISYEEFATMMKAGTDWRKASRQYSRERFSSLSLKLQKDGSLQVAAN。

[0052] Using cDNA as a template, amplification was carried out using the designed primers and Phusion high-fidelity enzyme. The primers are as follows:

[0053] AcoCPK4-SmaI-F (SEQ ID NO.7):

[0054] CCCGGGATGGGTAGCCGCACCTCTCGC.

[0055] AcoCPK4-XbaI-R (SEQ ID NO.8):

[0056] TCTAGACTTCCGTCGCCTTATGGGAGC.

[0057] AcoCPK8-SmaI-F (SEQ ID NO.9):

[0058] CCCGGGATGGGAAACACGTGCCGC.

[0059] AcoCPK8-XbaI-R (SEQ ID NO.10):

[0060] TCTAGAGCAACTAGGGCGCGGGGC.

[0061] AcoCPK15-SmaI-F (SEQ ID NO.11):

[0062] CCCGGGATGGGCAATTGCTGTGCGTC.

[0063] AcoCPK15-XbaI-R(SEQ ID NO.12):

[0064] TCTAGAGTTTGCTGCTACCTGCAATGAT.

[0065] The amplification system is shown in Table 1.

[0066] Table 1. Phusion enzyme amplification system

[0067] Phusion enzyme amplification system 30μL 5×HFbuffer 6μL 10mMdNTPs 0.6μL 20μMF primers 0.6μL 20μMR primers 0.6μL Phusion enzyme 0.3μL cDNA 1μL <![CDATA[ddH2O]]> 21.2μL

[0068] The amplification procedure is shown in Table 2.

[0069] Table 2. Phusion enzyme amplification program

[0070] step ℃ time goto Cycle number 1 98 1min - - 2 98 10s - - 3 annealing 30s - - 4 72 15-30s / kb 2 34 5 72 10min - - 6 4 30min - -

[0071] Annealing temperature depends on the primers and the characteristics of the Phusion enzyme, and can be predicted on the TmCalculator website (https: / / www.thermofisher.cn / cn / ); extension time (Phusion enzyme amplification rate 15–30 s / kb) depends on gene length. After amplification, the specificity and integrity of the PCR product are detected by agarose gel electrophoresis, specifically observing whether the bands are monochromatic and their brightness. If the bands are monochromatic, the PCR product is recovered; if the bands are not monochromatic, the correct-sized band is cut and fragments are recovered using a gel recovery kit from Shanghai Sangon Biotech Co., Ltd. See [link to relevant documentation]. Figure 2 The target fragments AcoCPK4, AcoCPK8, and AcoCPK15 were obtained by PCR amplification.

[0072] CaCl2-induced changes in transcriptional levels of AcoCPK4, AcoCPK8, and AcoCPK15

[0073] Under sunny conditions, collect 'Bali' suckers with a stem base diameter of 0.5 cm or more and normal development. Bring them back to the laboratory, completely remove senescent leaves from the base, rinse the suckers with tap water, and cut off all leaves about 0.5 cm above the sucker's growth point. Pre-sterilize the suckers with 2% NaClO for 10 min, rinse three times with sterile water, then sterilize with 0.1% HgCl for 8 min, and rinse three times with sterile water. Cut off each white leaf base attached to the sucker, including a portion of the sucker tissue, and sterilize the leaf base with 0.1% HgCl for 5 minutes, rinse three times with sterile water. Inoculate the leaf base onto callus induction medium (MS + 2.0 mg / L BA + 2.5 mg / L NAA) for callus induction and culture under light.

[0074] After four weeks of culture, callus tissue was excised and transferred to a growth medium containing Mock and 200 Mm CaCl2. After 24 hours of treatment, the callus material was harvested. The callus material was ground in liquid nitrogen, and total RNA was extracted using an RNA extraction kit (catalog number: 0416-100GK) from Beijing Huayueyang Biotechnology Co., Ltd. Using the obtained total RNA as a template, double-stranded cDNA was synthesized using a reverse transcription kit (catalog number: M20105) from Beijing TransGen Biotech Co., Ltd., under the action of reverse transcriptase and polymerase.

[0075] Using Perfect Start Green Qpcr SuperMix (catalog number: TG-AQ601-02-V2) from Beijing TransGen Biotech Co., Ltd., and the above-mentioned primers, quantitative PCR was performed with cDNA as a template to detect the transcription levels of AcoCPK4, AcoCPK8, and AcoCPK15.

[0076] The transcriptional levels of AcoCPK4, AcoCPK8, and AcoCPK15 are induced by exogenous and endogenous CaCl2, as follows: Figure 3 As shown, the results indicate that as the pineapple fruit matures (endogenous calcium content gradually decreases), the transcriptional levels of AcoCPK4, AcoCPK8, and AcoCPK15 gradually decrease.

[0077] Tissue and subcellular localization of AcoCPK4, AcoCPK8, and AcoCPK15 genes

[0078] Pulp from mature pineapple fruits, stems, old leaves, young leaves, and inflorescences from plants before flowering were collected and flash-frozen in liquid nitrogen. The collected samples were then ground in liquid nitrogen, and total RNA was extracted using an RNA extraction kit (catalog number: 0416-100GK) from Beijing Huayueyang Biotechnology Co., Ltd. Using the obtained total RNA as a template, double-stranded cDNA was synthesized using a reverse transcription kit (catalog number: M20105) from Beijing TransGen Biotech Co., Ltd., under the action of reverse transcriptase and polymerase. Quantitative PCR was performed using the cDNA as a template with a PerfectStart GreenQpcrSuperMix (catalog number: TG-AQ601-02-V2) from Beijing TransGen Biotech Co., Ltd., to detect the transcription levels of AcoCPK4, AcoCPK8, and AcoCPK15. The results are as follows: Figure 4 As shown, AcoCPK4, AcoCPK8, and AcoCPK15 are widely expressed in plants, and subcellular localization results show that AcoCPK4, AcoCPK8, and AcoCPK15 are all localized to the plasma membrane.

[0079] In addition, using the successfully sequenced AcoCPK4, AcoCPK8, and AcoCPK15 as templates, amplification was performed using primers and Phusion high-fidelity enzyme. The amplified PCR products were recovered using the SanPrep Column PCR Product Purification Kit (Catalog No.: B518141-0050). The recovered PCR products (2 μg) and pCOMBIA2300 vector (2 μg) were digested with SmaI (0.5 μL) and XbaI (0.5 μL) at 37°C for 30 minutes. The digested products were subjected to gel electrophoresis, and the target band was excised and recovered using the SanPrep Column DNA Gel Extraction Kit (Catalog No.: B518131-0100). The recovered AcoCPK4, AcoCPK8, AcoCPK15 products and pCOMBIA2300 vector were ligated using Thermo Fisher Scientific T4 DNA ligase (Catalog No.: EL0011).

[0080] Add 5 μL of the ligation product to 50 μL of LDH5α competent cells and gently mix. Incubate on ice for 30 min, then incubate at 42°C for 60 s, then on ice for 2 min. Add 1 mL of LB liquid medium and incubate at 37°C with a shaker at 150 rpm for 1 h. Spread 200 μL of the bacterial culture onto solid medium containing Kana and incubate upside down at 37°C overnight. Pick a single colony and incubate in 1 mL of liquid medium containing Kana antibiotic. Incubate at 37°C with 150 rpm for 12 h and send to Genewiz for sequencing. The expression vectors P2300-AcoCPK4, P2300-AcoCPK8, and P2300-AcoCPK15 were finally obtained.

[0081] Add 100 ng of the above-mentioned plasmid to 50 μL of GV3101 competent cells (Angyu Biotechnology, catalog number: G6039), and gently mix. Incubate sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 28°C water bath for 5 min, and in an ice bath for 5 min. Then add 700 μL of antibiotic-free LB or YEB liquid medium and incubate at 28°C with shaking for 2–3 hours. Centrifuge at 6000 rpm for one minute to collect the bacteria. Retain approximately 100 μL of supernatant, gently resuspend the bacterial cells, and spread them onto LB or YEB plates containing the appropriate antibiotic. Invert the plates and incubate at 28°C for 2–3 days. Pick a single colony and incubate in 2 mL of liquid medium containing Kana antibiotic at 30°C and 150 rpm for 12 hours.

[0082] Centrifuge the cultured bacterial culture at 4000×g for 10 min and discard the supernatant. Resuspend the bacterial cells in 1 mL of transformation buffer, centrifuge at 4000×g for 10 min, and discard the supernatant. Finally, resuspend the pellet in 1 mL of buffer, and then take a small amount of bacterial cells in a separate 1.5 mL centrifuge tube to adjust the OD. 600 The value is approximately 0.6–0.8. Tobacco leaves that have grown for about 4 weeks are selected for injection. Using a syringe (without the needle), an appropriate amount of Agrobacterium suspension is drawn and injected into the tobacco leaves to moisten them; subcellular localization of the protein can be observed after approximately 36–48 hours; the situation is as follows. Figure 4 As shown.

[0083] Transcriptional levels of AcoCPK4, AcoCPK8, and AcoCPK15 in pineapple fruits with different degrees of watercore disease

[0084] Normal pineapple pulp (fruit without lesions), mildly watercore pineapple pulp (water core in the eye or core, scattered, not contiguous, lesion area ≤30% of the whole fruit area), moderately watercore pineapple pulp (water core in and around the eye, contiguous, water core not extending to the peel and core, lesion area ≤60% of the whole fruit area), and severely watercore pineapple pulp (water core widely distributed, water core symptoms extending to the peel and core, lesion area >60% of the whole fruit area) were selected and flash-frozen in liquid nitrogen. The pineapple fruit materials were ground in liquid nitrogen, and total RNA was extracted using the RNA extraction kit (catalog number: 0416-100GK) from Beijing Huayueyang Biotechnology Co., Ltd. The obtained total RNA was used as a template to synthesize cDNA double strands using the reverse transcription kit (catalog number: M20105) from Beijing Quanshijin Biotechnology Co., Ltd. under the action of reverse transcriptase and polymerase. Quantitative PCR was performed using cDNA as a template with the PerfectStart GreenQpcrSuperMix (catalog number: TG-AQ601-02-V2) from Beijing TransGen Biotech Co., Ltd. to detect the transcriptional levels of AcoCPK4, AcoCPK8, and AcoCPK15. Figure 5As shown, the transcription level of AcoCPK4 (orange line) gradually increases with the severity of watercore disease, while the transcription levels of AcoCPK8 and AcoCPK15 genes gradually decrease with the severity of watercore disease.

[0085] Transient conversion of pineapple fruits with AcoCPK4, AcoCPK8, and AcoCPK15 can significantly induce watercore disease in pineapples.

[0086] Single colonies of Agrobacterium transformed with pCOMBIA2300 and P2300-AcoCPK4, P2300-AcoCPK8, and P2300-AcoCPK15 were picked and incubated in 10 mL of liquid medium containing Kana antibiotic at 30°C and 150 rpm for 12–24 h. The cultured bacterial suspension was centrifuged at 4000×g for 10 min, and the supernatant was discarded. The bacterial cells were resuspended in 2 mL of transformation buffer (see Table 3), centrifuged at 4000×g for 10 min, and the supernatant was discarded. The final precipitate was resuspended in 2 mL of transformation buffer. Then, in a separate 15 mL centrifuge tube, 10 mL of transformation buffer was added, and a small amount of bacterial cells was added to the 15 mL tube. The OD was adjusted. 600 The value was 0.6-0.8. Take 200 μL of the adjusted bacterial solution and inject it into the gap between the eyes of a mature pineapple using a 1 mL syringe. Cut the pineapple 50 h after injection, take a picture, and use ImageJ software to count the area of ​​the lesions.

[0087] Table 3 Conversion Buffer

[0088] <![CDATA[1MMgCl2]]> 0.5mL 0.1 MME S pH 5.6 5mL 0.1MMAS 7.5.6 Ultrapure water Volume adjustment refers to 50 mL

[0089] See Figure 6 Compared with the transient expression empty vector control (EV), transient transformation of pineapple fruits with AcoCPK4, AcoCPK8, and AcoCPK15 significantly inhibited pineapple watercore disease.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The application of the pineapple calcium-dependent protein kinase gene in the prevention and control of pineapple watercore disease or in the preparation of reagents for the prevention and control of pineapple watercore disease, characterized in that, The pineapple calcium-dependent protein kinase gene includes AcCPK4 , AcCPK8 and / or AcCPK15 Genes; the stated AcCPK4 The CDS sequence of the gene is shown in SEQ ID NO. 1; AcCPK8 The CDS sequence of the gene is shown in SEQ ID NO. 2; AcCPK15 The CDS sequence of the gene is shown in SEQ ID NO.

3.

2. Application of the pineapple calcium-dependent protein kinase gene in the breeding of watercore-resistant pineapple varieties, characterized in that... The pineapple calcium-dependent protein kinase gene includes AcCPK4, AcCPK8 and / or AcCPK15 Genes; the stated AcCPK4 The CDS sequence of the gene is shown in SEQ ID NO. 1; AcCPK8 The CDS sequence of the gene is shown in SEQ ID NO. 2; AcCPK15 The CDS sequence of the gene is shown in SEQ ID NO.

3.

3. A method for inhibiting watercore disease in pineapples, characterized in that, include: An expression vector for inhibiting pineapple watercore disease was transformed into pineapple fruit by infecting it with Agrobacterium tumefaciens; the expression vector for inhibiting pineapple watercore disease included the pineapple calcium-dependent protein kinase gene as described in claim 1.

4. The method for inhibiting pineapple watercore disease according to claim 3, characterized in that, The AcCPK4 The amplification primers for the gene are shown in SEQ ID NO. 7-SEQ ID NO. 8; AcCPK8 The amplification primers for the gene are shown in SEQ ID NO. 9-SEQ ID NO. 10; AcCPK15 The primers for gene amplification are shown in SEQ ID NO. 11-SEQ ID NO.

12.

5. The application of an expression vector for inhibiting pineapple watercore disease or an engineered bacterium for inhibiting pineapple watercore disease in the prevention and control of pineapple watercore disease or in the preparation of reagents for the prevention and control of pineapple watercore disease; wherein the expression vector for inhibiting pineapple watercore disease or the engineered bacterium for inhibiting pineapple watercore disease includes the pineapple calcium-dependent protein kinase gene as described in claim 1.

6. The application of an expression vector for inhibiting pineapple watercore disease or an engineered bacterium for inhibiting pineapple watercore disease in the breeding of pineapple varieties resistant to watercore disease; wherein the expression vector for inhibiting pineapple watercore disease or the engineered bacterium for inhibiting pineapple watercore disease includes the pineapple calcium-dependent protein kinase gene as described in claim 1.

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