Cloning method and application of tomato late blight resistant gene CDPK12

Abstract

The invention provides a cloning method and application of a tomato late blight resistant gene CDPK12. The cloning method comprises the following steps: carrying out PCR (Polymerase Chain Reaction) amplification by taking cDNA (Complementary Deoxyribonucleic Acid) of tomato early powder No. 2 as a template, connecting an obtained PCR product with a double-enzyme-digested pBI-121 cloning vector, transforming escherichia coli CDPK12, selecting a single colony to extract plasmids, carrying out plasmid PCR, sequencing the plasmids, and carrying out screening to obtain the tomato late blight resistant gene CDPK12. The plasmid which is consistent with the CDPK12 is the plasmid which is correctly connected; the invention also provides application of the tomato late blight resistant gene CDPK12 in tomato late blight resistance. After the anti-late blight gene CDPK12 is over-expressed, the resistance of tomatoes to late blight can be enhanced. In addition, overexpression of the CDPK12 can also up-regulate the expression quantity of the PR gene, and the invention lays a foundation for revealing a molecular mechanism that CDPK participates in tomato late blight resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to a cloning method and application of a tomato late blight resistance gene CDPK12. Background Art

[0002] Tomato is an annual or perennial herbaceous plant of the genus Tomato in the Solanaceae family. The fruit of tomato is rich in nutrients and has a special flavor. Late blight of Phytophthora infestans (P. infestans) and soil salt stress are important factors that limit the normal growth and development of tomatoes, because both will affect the quality of tomatoes by affecting related pathways, thereby acting on hormone synthesis and defense-related gene expression. Due to the fixation of plants, plants have evolved adaptive regulatory mechanisms and immune mechanisms to cope with salt stress and late blight infection in their long evolutionary history. At present, spraying pesticides is one of the most effective ways to prevent and control late blight, but a series of environmental pollution, pathogen resistance and other problems caused by this still make agricultural production face great difficulties. Therefore, studying the molecular mechanism of disease resistance of tomatoes and using biological methods to improve the resistance of tomatoes to late blight has become a key issue.

[0003] Calcium-dependent protein kinase (CDPK / CPK) consists of four domains, namely the variable N-terminal domain (VNTD), serine / threonine protein kinase domain (PKD), autoinhibitory linker domain (JD) and C-terminal regulatory domain (CaM-LD). The N-terminus of most CDPKs has myristoylation or palmitoylation sites involved in protein membrane binding, suggesting that the VNTD region is involved in the CDPKs membrane binding process; the PKD region of CDPKs protein is highly conserved and contains Ser / Thr phosphorylation sites, and key amino acid mutations often inactivate the kinase; while the JD region on CDPKs is highly conserved and mainly binds to the kinase region of CDPKs as a pseudosubstrate. The PKD region is divided into two globular structures, namely N-lobe and C-lobe. The former is sensitive to Ca 2+ The affinity of the latter is lower than that of the latter. Under normal circumstances, the JD region binds to the catalytic site of C-lobe and acts as a pseudosubstrate to maintain CDPKs in a low basal kinase activity state. When plants are stimulated by light, low or high temperature, high salt, drought, hormones or even pathogens, specific Ca 2+ Signal, Ca 2+ Direct binding to the EF-hand structure changes the conformation of CDPKs, exposing the kinase active site and activating the kinase activity. The uniqueness of CDPKs lies in the presence of CaM-LD. Due to its degradation, the EF-hand structure is not directly regulated by calcium ions, allowing CDPK to bind to Ca ions independently of CaM.2+ High affinity. 2+ When the concentration is low, CDPKs are in a self-inhibited state. 2+ After the concentration increases, the conformation of CDPKs changes, self-inhibition is released, and its kinase activity is exercised. The important role played by CDPKs in plant disease resistance has been widely reported. In Arabidopsis, four homologous calcium-dependent protein kinases, CPK4, CPK5, CPK6 and CPK11, are involved in promoting the production of ROS and transcriptional reprogramming induced by flg22; overexpression of AtCPK1 will produce broad-spectrum resistance to bacteria and fungi; Arabidopsis CPK28 enhances its E3 ubiquitin ligase activity by phosphorylating PUB25 / 26, thereby degrading the key immune protein BIK1, playing a negative regulatory role. In recent years, it has been found that CPK28 is controlled by the proteasome and interacts with the ubiquitin ligases ATL31 and ATL6 on the plasma membrane. ATL31 / 6 directly ubiquitinates CPK28, resulting in the degradation of CPK28 through the 26S proteasome, which can fine-tune immune signal transduction; the cpk5 mutant is resistant to the fungal gray mold (B. botrytis cinerea (B. cinerea) and plays a role upstream of salicylic acid (SA) synthesis, perception and signal transduction in local basal resistance; in systemic tissues, CPK5 signaling leads to the accumulation of systemic acquired immunity (SAR)-inducing metabolite N-hydroxy-L-piperidinic acid (NHP) and SAR marker genes, including systemic acquired resistance defect 1 (SARD1) expression, thus contributing to local immunity and SAR; recent studies have reported that CPK5 can phosphorylate CAMTA3 at serine 964, leading to reduced protein accumulation, thereby weakening its new mechanism of transcriptional inhibition of downstream resistance-related genes, forming a TN2-CPK5-CAMTA3 immune module involved in regulating exo70B1-mediated plant resistance.

[0004] CDPK12 (So1yc04g009800) is a member of the calcium-dependent kinase gene family in tomato. In recent years, more and more studies have shown that CDPKs are involved in plant disease resistance, antibacterial and other biological stress response processes. After the fungus gray mold infects Micro-Tom (MT), the expression level of CDPK12 is upregulated (Tian, ​​S., Liu, B, et al.. Unraveling the Molecular Mechanisms of Tomatoes' Defense against Botrytis cinerea: Insights from Transcriptome Analysis of Micro-Tom and Regular Tomato Varieties. Plants (Basel, Switzerland), 2023, 12 (16), 2965.); the commercial variety M82 was treated with salt stress, and transcriptome sequencing found that CDPK12 responded to salt stress (Wang, B., Wang, J., et al. The transcriptional regulatory network of hormones and genes under salt stress in tomato plants (Solanum lycopersicum L.). 2023, Frontiers in plant science, 14, 1115593.).

[0005] At present, most of the functional studies of CDPKs are limited to abiotic stress, and the research on biotic stress is not in-depth enough. In addition, CDPKs have been studied in other Solanaceae plants, but there are fewer studies in tomatoes, and most of them are focused on bacteria and fungi. There are no reports on oomycetes. Summary of the invention

[0006] The purpose of the present invention is to provide a cloning method and application of a tomato late blight resistance gene CDPK12, study the disease resistance molecular mechanism of tomatoes, use biological methods to improve the resistance of tomatoes to late blight, apply the function of CDPKs to the resistance of tomatoes to late blight, screen and obtain the late blight resistance gene CDPK12 through the method of the present invention, and increase the resistance of tomatoes to late blight by overexpressing the gene CDPK12, which lays a foundation for revealing the molecular mechanism of CDPK involved in the resistance of tomatoes to late blight.

[0007] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0008] The first aspect of the present invention provides a method for cloning a tomato late blight resistance gene CDPK12, comprising the following steps:

[0009] Sa1, using the cDNA of Tomato Zaofen 2 as a template and specific primers, PCR amplification was performed;

[0010] The specific primers are as follows:

[0011] CDPK12-FP: gcccttgctcaccatggatccATGGGGAACACTTGTGTTGGAC

[0012] CDPK12-RP: agctttcgcgagctcggtaccCTAAAGTTTTAGAACGTCTCTAAATCCG.

[0013] Sa2, connect the PCR product obtained in Sa1 with the successfully double-digested pBI121 cloning vector; the connection is carried out by homologous recombination;

[0014] Sa3. The ligation product pBI121-CDPK12 obtained in Sa2 was transformed into Escherichia coli DH5α and spread on LB solid culture medium containing kanamycin. Single colonies were picked, and the plasmid was extracted after shaking. After plasmid PCR, the plasmid was sent for sequencing. The plasmid that was consistent with the CDPK12 sequence was the correctly connected plasmid, which was recorded as pBI121-CDPK12 plasmid.

[0015] According to the preferred embodiment, the method for screening the tomato late blight resistance gene CDPK12 comprises the following steps:

[0016] Sb1. Preliminary screening of target CDPK

[0017] 1) Bioinformatics prediction analysis identified CDPKs gene family members, compared them with the transcriptome library of tomatoes infected with late blight, and preliminarily screened out CDPKs that could respond to late blight infection;

[0018] 2) Bioinformatics prediction was used to analyze species conservation and select CDPKs specifically expressed in tomato leaves from the CDPKs obtained in 1).

[0019] Specifically, first, promoter cis-acting element analysis was performed on the 28 identified CDPK gene families to screen out CDPKs gene family members that respond to hormone, low temperature or trauma stress; then, bioinformatics prediction analysis was performed to determine the selected CDPKs gene family members, and then they were compared with the transcriptome library of tomatoes infected with late blight fungus constructed in the laboratory in the early stage (for the transcriptome library, see reference: Cui, J., Xu, P., et al. Transcriptome signatures of tomato leaf induced by Phytophthora infestans and functional identification of transcription factor SpWRKY3.2018. Theoretical and applied genetics. 131(4), 787–800.), and CDPKs that can respond to late blight fungus infection were preliminarily screened out; finally, bioinformatics prediction analysis was performed on species conservation, and CDPKs with high homology to disease-resistant CDPKs reported in Arabidopsis, potato, pepper, cotton, etc. were further screened out from the preliminarily screened CDPKs.

[0020] Tomato plants treated with Sb2 and late blight

[0021] The late blight fungus solution was sprayed on tomato plants at the five-leaf stage, and after culturing in the dark for 12 to 14 hours, light-dark alternating culture was performed, and tomato leaves after the light-dark alternating culture for 0 hours, 6 hours, 12 hours, 24 hours, and 48 hours were taken and placed in liquid nitrogen for rapid freeze-drying; the light-dark alternating culture was 16 hours of light / 12 hours of darkness alternating culture.

[0022] Extraction and reverse transcription of Sb3 and RNA

[0023] The freeze-dried samples obtained at different time points by Sb2 were placed in RNAiso Plus, and the RNA was extracted and then reverse transcribed into cDNA.

[0024] Initial identification of Sb4 and CDPK12

[0025] Using the cDNA obtained from Sb3 as a template, we designed the preliminarily screened CDPKs-specific primers, and detected the gene expression levels of the target CDPKs at different time points by real-time fluorescence quantitative PCR. Finally, we determined So1yc04g009800 (the nucleotide sequence of So1yc04g009800 can be found in the reference: Hu, Z., Lv, X., et al. Genome-Wide Identification and Expression Analysis of Calcium-dependent Protein Kinase in Tomato. 2016. Frontiers in plant science, 7, 469.) that responded to the infection of late blight, and recorded it as CDPK12.

[0026] In a preferred embodiment, in step Sb2, the late blight bacteria liquid is obtained by culturing the late blight bacteria on an oat culture medium, and the tomato plants are tomato plants susceptible to late blight.

[0027] Specifically, the method for obtaining tomato plants and the process for cultivating late blight bacteria include the following steps:

[0028] 1) Obtaining tomato plants: Soak Zaofen No. 2 tomato seeds in water overnight and then place them on a culture plate with moist filter paper at 28°C for germination. After sowing in soil and germination, culture them at 28°C, 16 h light / 12 h dark. When they grow to the five-leaf stage, they can be used for subsequent experiments.

[0029] 2) Inoculation of late blight: Inoculate late blight into oat culture medium (80 g / L oats, 20 g / L sucrose, 1 g / L CaCO 3 , 15g / L agar powder, the balance is water) and cultured at 20℃ for about one month. The mycelium was scraped and placed in a 4℃ refrigerator to promote the release of zoospores. The filtrate was collected by 3 layers of filter cloth and the number of spores was counted with a hemocytometer under a microscope. The concentration was diluted to 1×10 6 Spores / mL.

[0030] The second aspect of the present invention provides an application of a tomato late blight resistance gene CDPK12 in tomato late blight resistance.

[0031] In a preferred embodiment, the above application comprises the following steps:

[0032] Sc1. The recombinant expression vector pBI121-CDPK12 containing the tomato late blight resistance gene CDPK12 was introduced into Agrobacterium GV3101 competent cells and spread on YEB solid culture medium containing kanamycin and rifampicin, and a single colony was picked for bacterial liquid PCR verification.

[0033] Sc2. Take five-leaf-stage tomatoes as the treatment object, and inject the positive engineered bacterial solution obtained in Sc1 into the leaves of the five-leaf-stage tomatoes to achieve overexpression of the gene CDPK12.

[0034] In a preferred embodiment, the recombinant expression vector containing the tomato late blight resistance gene CDPK12 is obtained by inserting the tomato late blight resistance gene into the pBI121 expression vector.

[0035] More preferably, the method for constructing a recombinant expression vector containing the tomato late blight resistance gene CDPK12 comprises the following steps: recovering the PCR product (target fragment) obtained by the above-mentioned cloning method Sa1 by gel reversal and then connecting the purified target fragment with the pBI121 expression vector that has been successfully double-enzyme-cut, transforming the obtained connection product into Escherichia coli DH5α and spreading it on an LB solid culture medium containing kanamycin, picking a single colony and shaking the bacteria to extract the plasmid, performing plasmid PCR identification, and sending the plasmid for sequencing. The one with correct sequencing is the recombinant expression vector pBI121-CDPK12.

[0036] The beneficial effects of the present invention are as follows:

[0037] 1. There is no relevant report on CDPKs and tomato late blight. The tomato late blight resistance gene CDPK12 provided by the present invention can enhance the resistance of tomato to late blight after overexpression.

[0038] 2. CDPKs can not only act as Ca 2+ Sensor proteins that also act on Ca 2+ The effector protein plays a role, but its molecular mechanism is still unclear. In the present invention, overexpression of CDPK12 will also upregulate the expression of PR genes, and the present invention lays a foundation for revealing the molecular mechanism of CDPKs involved in tomato resistance to late blight.

[0039] 3. There are many members in the CDPKs gene family. Bioinformatics prediction is used for a preliminary screening in order to narrow the scope of the research object, which is low-cost and greatly reduces the workload. The final determination of the target gene cannot rely solely on bioinformatics analysis, but must be combined with experiments to be more convincing. The present invention sprays five-leaf tomato seedlings with Phytophthora infestans, takes samples at different time points, extracts RNA and reverse transcribes it into cDNA, and then performs real-time fluorescence quantitative PCR analysis. After Phytophthora infestans infects tomato leaves, whether the expression level of the target gene changes significantly over time, thereby judging whether the target gene responds to the infection of late blight. Based on the above results, the research object is finally determined. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the expression level of CDPK12 gene at different time points after infection by Phytophthora infestans.

[0041] Figure 2 Overexpression of CDPK12 expression and disease resistance.

[0042] Figure 3 is the PR gene expression level. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0044] Example 1

[0045] A method for screening a tomato late blight resistance gene CDPK12 comprises the following steps:

[0046] 1. Preliminary screening of target CDPKs

[0047] 1) Promoter cis-acting element analysis: Hidden Markov model (HMM) files were used to download the CDPK-specific EF-hand (PF00036) and CDPK Ser / Thr (PF00069) hidden Markov models, and candidate genes were searched in the tomato database (E-value < 1 × 10 -7 , similarity>50%). The retrieved SlCDPKs candidate members were analyzed for conserved domains using InterProScan (http: / / www.ebi.ac.uk / Tools / InterProScan) and SMART (http: / / smart.embl-heidelberg.de). SlCDPKs with 4 EF-hands and SlCIPKs with a kinase domain (PF00069) were screened. Subsequently, the CDPKs members in Arabidopsis were used to blast out the corresponding CDPKs in tomato using the bidirectional blast of Tbtools software (www.tbtools.com). The genes obtained by the above two methods were then intersected according to the sequence numbers to obtain 28 CDPKs. The promoter cis-acting elements of the 28 identified CDPK gene families were analyzed, and CDPKs that responded to hormones, low temperature or wound stress were preliminarily screened out;

[0048] 2) Bioinformatics prediction analysis was used to identify the CDPKs gene family members obtained in 1) and compared them with the transcriptome library of tomatoes infected with late blight constructed in the laboratory in the early stage to screen for CDPKs that can respond to late blight infection;

[0049] 3) Bioinformatics prediction and analysis of species conservation, and further screening out CDPKs with high homology to disease-resistant CDPKs reported in other species from the CDPKs obtained in 2).

[0050] 2. Obtaining tomato plants and cultivating late blight pathogens

[0051] 1) Soak Zaofen No. 2 tomato seeds in water overnight and germinate them on a culture plate with moist filter paper at 28°C. Sow them in soil and culture them at 28°C, 16h light / 12h dark. When they grow to the five-leaf stage, they can be used for subsequent experiments.

[0052] 2) Inoculation of late blight: Inoculate late blight onto oatmeal medium and culture at 20°C for about a month. Scrape the mycelium and place it in a 4°C refrigerator to promote the release of zoospores. Collect the filtrate with three layers of filter cloth and count the number of spores with a hemocytometer under a microscope. Dilute with sterile water to a concentration of 1×10 6 Spores / mL.

[0053] 3. Treat tomato plants with late blight

[0054] The bacterial solution inoculated with late blight bacteria was evenly sprayed onto five-leaf tomato plants, first cultured in the dark overnight, and then cultured under 16h light / 12h dark conditions. Tomato leaves were taken at different time points of 0h, 6h, 12h, 24h, and 48h and quickly frozen in liquid nitrogen.

[0055] 4. RNA extraction and reverse transcription

[0056] The sample obtained in step 3 was ground with liquid nitrogen and placed in RNAiso Plus, and RNA was extracted (see the relevant steps of Example 2 below for details) and then reverse transcribed into cDNA;

[0057] 5. Initial identification of CDPK12

[0058] Using the cDNA obtained in step 4 as a template, the preliminarily screened CDPK12-specific primers were designed, and the gene expression of CDPK12 at different time points was detected by real-time fluorescence quantitative PCR ( Figure 1 ), it can be seen that the gene expression levels at different time points are different, indicating that CDPK12 responds to pathogen infection.

[0059] Example 2

[0060] A method for cloning a tomato late blight resistance gene CDPK12 comprises the following steps:

[0061] 1. Extraction and reverse transcription of tomato RNA

[0062] (1) Grind about 0.1 g of tomato leaves into powder in a mortar containing liquid nitrogen. Transfer the sample to a 1.5 mL RNase-free centrifuge tube, place in 1 mL pre-cooled RNAiso Plus at room temperature for 5 min, and centrifuge at 4°C and 12,000 rpm for 5 min.

[0063] (2) Keep the supernatant, add 200 μL of chloroform, mix by inversion, and centrifuge at 4°C and 12,000 rpm for 10 min.

[0064] (3) Transfer 300 μL of the supernatant to a new RNase-free centrifuge tube, add an equal amount of isopropanol, mix well, let stand at room temperature for 5 min, and centrifuge at 4°C and 12,000 rpm for 10 min.

[0065] (4) Retain the precipitate, add 1 mL of 75% ethanol (freshly prepared and used) to wash the precipitate, and centrifuge at 4°C and 12,000 rpm for 5 min.

[0066] (5) Discard the supernatant, dry for 5 min, and add 25 μL enzyme-free ddHO 2 O to dissolve RNA.

[0067] (6) The RNA obtained above was reverse transcribed into cDNA, and the reaction system was as follows:

[0068]

[0069] The reaction conditions were 37°C, 15 min, and 85°C, 5 s.

[0070] 2. PCR amplification of CDPK12 gene

[0071] Using the cDNA of Tomato Zaofen 2 as template, specific primers were designed and PCR amplification was performed.

[0072] The specific primers used for cloning are as follows:

[0073] CDPK12-FP:gcccttgctcaccatggatccATGGGGAACACTTGTGTTGGAC

[0074] CDPK12-RP: agctttcgcgagctcggtaccCTAAAGTTTTAGAACGTCTCTAAATCCG

[0075] The reaction system is as follows:

[0076]

[0077] The reaction conditions are as follows:

[0078]

[0079] 3. Obtaining double-enzyme-digested plasmid

[0080]

[0081] The enzyme digestion was performed at 30°C for 10 min and then at 37°C for 20 min. The digestion products were detected by 1% agarose gel electrophoresis.

[0082] 4. Recovery and purification of PCR amplification products and restriction enzyme-cut plasmids

[0083] After detecting the above PCR products and the successfully digested plasmid by 1% agarose gel electrophoresis, the PCR products and digested plasmid that matched the size of the target fragment CDPK12 were recovered using a gel recovery kit (purchased from Takara).

[0084] 5. Connect the target fragment to the restriction plasmid

[0085] The target fragment recovered above was connected with the cloning vector pBI121 (purchased from Takara) that was successfully digested with double enzymes. The reaction system was as follows:

[0086]

[0087] The ligation product pBI121-CDPK12 was obtained by ligation at 50°C for 15 min.

[0088] 6. Transformation of ligation products into E. coli

[0089] (1) Add 5 μL of the ligation product reaction solution to 50 μL of E. coli DH5α competent cells, mix gently and place on ice for 30 min. Then transfer to a 42°C constant temperature water bath and heat shock for 45 s. After taking out, immediately place on ice for 2 to 3 min.

[0090] (2) Add 700 μL of LB liquid medium without antibiotics, mix thoroughly, and culture at 37°C, 180 rpm, with shaking for 1 h.

[0091] (3) Centrifuge at 4000 rpm for 5 min at room temperature, discard the supernatant, suspend the remaining approximately 100 μL of liquid and spread it on an LB solid culture medium plate (containing 50 mg / L kanamycin), and place it in a 37°C incubator for 14 to 16 h after the liquid is dried.

[0092] (4) Pick a single white colony and inoculate it into LB liquid culture medium containing 50 mg / L kanamycin. Incubate the culture overnight at 37°C in a constant temperature shaker at 180 rpm.

[0093] 6. Extraction of pBI121-CDPK12 plasmid

[0094] According to the instructions of the plasmid mini-extraction kit (purchased from TIANGEN), the pBI121-CDPK12 plasmid contained in the above bacterial solution was extracted. 3 μL of the plasmid sample was taken for 1% agarose gel electrophoresis detection.

[0095] 7. Plasmid PCR detection

[0096] The extracted pBI121-CDPK12 plasmid was used as a template and CDPK12-FP and CDPK12-RP were used as primers for PCR. The reaction conditions and reaction system were the same as those in “Example 2, Step 2”.

[0097] 8. Sequencing

[0098] The obtained plasmid was sent to Sangon Biogene (Changchun) Co., Ltd. for sequencing. The sequencing results were analyzed and the plasmid that was consistent with the CDPK12 sequence was the correct plasmid for connecting to pBI121.

[0099] Example 3

[0100] Application of Tomato Late Blight Resistance Gene CDPK12

[0101] 1. Preparation of pBI121-CDPK12 Agrobacterium engineering bacteria

[0102] (1) Add 2 μL of pBI121-CDPK12 plasmid to 50 μL of Agrobacterium GV3101 competent cells, mix thoroughly by pipetting, freeze in liquid nitrogen for 2 min, and heat in a 37°C water bath for 5 min;

[0103] (2) Add 1 mL of sterile YEB liquid medium and culture at 28°C with shaking at 180 rpm for 3 h;

[0104] (3) Centrifuge at 12,000 rpm at room temperature, discard the supernatant, suspend the remaining cells and spread them on YEB solid culture medium (containing 100 mg / L rifampicin and 50 mg / L kanamycin), and culture inverted in a 28°C incubator for 48 h.

[0105] 2. Identification of Agrobacterium engineering bacteria

[0106] (1) Pick a single colony obtained from the above bacterial plate after antibiotic screening and add it to 1 mL of YEB liquid medium (100 mg / L rifampicin and 50 mg / L kanamycin) and culture it at 28°C and 180 rpm for 14 to 16 hours;

[0107] (2) Bacterial liquid PCR

[0108] The cultured bacterial solution was subjected to PCR verification, and the reaction system was as follows:

[0109]

[0110] After detecting the above PCR products by 1% agarose gel electrophoresis, the correct bacterial solution with a band size of about 1600 bp was added to a suitable sterile tube, mixed with an equal volume of 50% glycerol, and stored at -80°C.

[0111] 3. Establishment of transient infection system and related tests

[0112] (1) Add 2-3 mL of sterile water to the oatmeal medium plate cultured with late blight fungus that has been cultured in an incubator at 20°C for 21 days, gently scrape off the hyphae and spores on the surface of the medium, filter with sterile gauze, transfer the obtained bacterial solution to a 50 mL conical flask, and adjust the final concentration of the bacterial solution to 1×10 6 spores / mL, and placed it in a 4°C refrigerator for about 3 h to fully release the spores to swim;

[0113] (2) The Agrobacterium culture liquid containing the pBI121 empty vector (EV) and pBI121-CDPK12 was inoculated into 5 mL of YEB (100 mg / L rifampicin and 50 mg / L kanamycin) liquid culture medium, and cultured at 28°C with shaking at 180 rpm for 12 to 16 h;

[0114] (3) Take 1 mL of each of the two bacterial suspensions obtained above and transfer them into 50 mL of YEB liquid medium (containing 100 mg / L rifampicin and 50 mg / L kanamycin, 200 μmol / L acetosyringone, 2 mmol / L magnesium sulfate, and 10 mmol / L morpholineethanesulfonic acid monohydrate), shake culture for 20 h, centrifuge at 4000 rpm for 15 min at 4°C, discard the supernatant, and collect the bacteria;

[0115] (4) Resuspend the cells and adjust the OD with infection solution MMA (containing 10 mmol / L morpholineethanesulfonic acid monohydrate, 200 μmol / L acetosyringone and 10 mmol / L magnesium chloride, pH = 5.6). 600 to 1.0, and culture at 28°C and 180 rpm for 3 to 4 hours to activate Agrobacterium.

[0116] (5) Select several five-leaf tomato plants with the same growth trend and place them under weak light for 1 to 2 hours. Use a disposable syringe tip that has been polished with fine sandpaper to remove the needle, and inject 200 μL of the above-mentioned activated Agrobacterium solution containing pBI121-CDPK12 (positive engineering bacteria solution) and the empty Agrobacterium solution. The treated tomato plants are cultured in the dark for 12 hours and then cultured in 16 hours of light / 12 hours of darkness for 3 days.

[0117] (6) The treated EV and CDPK12 tomato leaves were picked and tested for transient expression of the gene CDPK12 (TOE-CDPK12). The specific steps included: grinding the tomato leaves in a mortar with liquid nitrogen, adding them to an RNase-free centrifuge tube containing 1 mL RNAisoPlus, extracting RNA and reversely transcribing it into cDNA, and detecting the expression of CDPK12 by real-time fluorescence quantitative PCR to ensure the successful transfer of the target gene. Figure 2 As shown, the expression level of CDPK12 (TOE-CDPK12) was almost 3.4 times that of the empty vector (EV) transiently expressing PBI121 in tomato leaves.

[0118] (7) Take 20 μL of the spore suspension of Phytophthora infestans obtained in (3) and drip it onto the wound of the tomato leaf treated in (6), place it on a plate with moist filter paper, seal it with plastic wrap, and culture it at 20°C in the dark for 5 days. Observe the disease situation, detect the lesion area and the expression level of the PR gene of the protein related to the course of disease. Figure 2 and 3 As shown, compared with the control group, the lesions of tomato leaves overexpressing CDPK12 after inoculation with late blight were smaller than those of the control group, and the expression of PR genes was upregulated.

[0119] The late blight resistance gene CDPK12 of the present invention can enhance the resistance of tomatoes to late blight after overexpression. In addition, overexpression of CDPK12 will also increase the expression of PR genes. The present invention lays a foundation for revealing the molecular mechanism of CDPK's involvement in tomato's resistance to late blight.

[0120] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for cloning a tomato late blight resistance gene CDPK12, characterized in that: The steps include: Sa1, using the cDNA of Tomato Zaofen 2 as a template and specific primers, PCR amplification was performed; The specific primers are as follows: CDPK12-FP: gcccttgctcaccatggatccATGGGGAACACTTGTGTTGGAC CDPK12-RP: agctttcgcgagctcggtaccCTAAAGTTTTAGAACGTCTCTAAATCCG Sa2, connect the PCR product obtained in Sa1 with the successfully double-digested pBI121 cloning vector by homologous recombination; Sa3. The ligation product pBI121-CDPK12 obtained in Sa2 was transformed into Escherichia coli DH5α and spread on LB solid culture medium containing kanamycin. Single colonies were picked, and the plasmid was extracted after shaking. After plasmid PCR, the plasmid was sent for sequencing. The plasmid that was consistent with the CDPK12 sequence was the correctly connected plasmid, which was recorded as pBI121-CDPK12 plasmid.

2. The method for cloning the tomato late blight resistance gene CDPK12 according to claim 1, characterized in that: The method for screening the tomato late blight resistance gene CDPK12 comprises the following steps: Sb1. Preliminary screening of target CDPK 1) Promoter cis-acting element analysis: Promoter cis-acting element analysis was performed on the 28 CDPK gene families identified, and CDPKs that responded to hormone, low temperature or trauma stress were preliminarily screened out; 2) Bioinformatics prediction analysis was used to identify the CDPKs gene family members obtained in 1) and compared them with the transcriptome library of tomatoes infected with late blight constructed in the laboratory in the early stage to screen for CDPKs that can respond to late blight infection; 3) Bioinformatics prediction and analysis of species conservation, and further screening CDPKs with high homology to disease resistance CDPKs in plant varieties other than tomatoes from the CDPKs obtained in 2); the plant varieties other than tomatoes include Arabidopsis, potato, pepper, and cotton; Tomato plants treated with Sb2 and late blight Spraying the late blight fungus solution on tomato plants at the five-leaf stage, culturing in the dark for 12 to 14 hours, and then performing light-dark alternating culturing, taking tomato leaves after 0 hours, 6 hours, 12 hours, 24 hours, and 48 hours of light-dark alternating culturing, and placing them in liquid nitrogen for rapid freeze-drying; the light-dark alternating culturing is 16 hours of light / 12 hours of darkness alternating culturing; Extraction and reverse transcription of Sb3 and RNA The freeze-dried samples obtained at different time points from Sb2 were placed in RNAiso Plus, and RNA was extracted and reverse transcribed into cDNA; Initial identification of Sb4 and CDPK12 Using the cDNA obtained from Sb3 as a template, we designed specific primers for the preliminarily screened CDPKs, and detected the gene expression levels of the target CDPKs at different time points by real-time fluorescence quantitative PCR. Finally, So1yc04g009800, which responded to the infection of late blight fungus, was determined and recorded as CDPK12.

3. Use of the tomato late blight resistance gene CDPK12 according to claim 1 or 2 in tomato late blight resistance.

4. The use according to claim 3, characterized in that: The steps include: Sc1, introducing the recombinant expression vector containing the tomato late blight resistance gene CDPK12 into Agrobacterium GV3101 competent cells and spreading them on YEB solid culture medium containing kanamycin and rifampicin, picking a single colony for bacterial liquid PCR verification; Sc2. Take five-leaf-stage tomatoes as the treatment object, and inject the positive engineered bacteria solution obtained in Sc1 into the leaves of the five-leaf-stage tomatoes to overexpress the tomato late blight resistance gene CDPK12.

5. The use according to claim 4, characterized in that: The recombinant expression vector containing the tomato late blight resistance gene CDPK12 is obtained by inserting the tomato late blight resistance gene CDPK12 into the pBI121 expression vector.

6. The use according to claim 5, characterized in that: The method for constructing a recombinant expression vector containing a tomato late blight resistance gene CDPK12 comprises the following steps: The PCR product obtained by Sa1 in claim 1 or 2 is gel-recovered and purified, and the purified target fragment is then connected to the pBI121 expression vector that has been successfully double-enzyme-cut. The connection product pBI121-CDPK12 is transformed into Escherichia coli DH5α and spread on LB solid culture medium containing kanamycin. A single colony is picked and shaken to extract the plasmid. After plasmid PCR identification, the plasmid is sent for sequencing. The one with correct sequencing is the recombinant expression vector pBI121-CDPK12.

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