Application of PbPI16-1 protein and / or coding gene PbPI16-1 thereof in prevention of rape diseases

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of PbPI16-1 protein and / or an encoding gene PbPI16-1 thereof in prevention of rape diseases. The invention provides an application of a PbPI16-1 protein and / or an encoding gene PbPI16-1 thereof in prevention of rape diseases. The amino acid sequence of the PbPI16-1 protein is as shown in SEQ ID NO. 1; the diseases comprise clubroot and / or sclerotiniose. The PbPI16-1 is overexpressed in the rape, and the resistance of the rape to plasmodiophora brassicae and sclerotinia sclerotiorum can be enhanced. Therefore, the coding gene PbPI16-1 of the PbPI16-1 protein can be used as a gene resource for green prevention and control of the rape diseases, and a new method is provided for green prevention and control of the rape diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to the application of PbPI16-1 protein and / or its encoding gene PbPI16-1 in preventing rapeseed diseases. Background Art

[0002] Clubroot fungi are the pathogens that cause clubroot disease in cruciferous plants. They are one of the biggest threats to the rapeseed and Brassica vegetable industries worldwide. Clubroot disease is widely distributed worldwide. Its dormant spores survive in the soil for a long time, making it difficult to control. Therefore, exploring gene resources for resistance to clubroot disease is an urgent issue to be addressed.

[0003] Peptidase inhibitors (PIs) are substances that regulate the catalytic activity of proteolytic enzymes. Some PIs can protect plants not only from pests but also from pathogenic bacteria. However, the role of peptidase inhibitor 16 (PI16) in plants or pathogens is still unclear. Summary of the invention

[0004] The object of the present invention is to provide the use of PbPI16-1 protein and / or its encoding gene PbPI16-1 in preventing rapeseed diseases, wherein the PbPI16-1 protein and / or its encoding gene PbPI16-1 can enhance rapeseed's resistance to clubroot and / or sclerotinia.

[0005] The present invention provides the use of PbPI16-1 protein and / or its encoding gene PbPI16-1 in preventing rapeseed diseases; the amino acid sequence of the PbPI16-1 protein is shown in SEQ ID NO.1; the diseases include clubroot and / or sclerotinia.

[0006] As a preferred embodiment, the nucleotide sequence of the coding gene PbPI16-1 is shown as SEQ ID NO.2.

[0007] As a preferred solution, the rapeseed includes Zhongshuang No. 6.

[0008] The present invention provides the use of PbPI16-1 protein and / or its encoding gene PbPI16-1 in improving rapeseed disease resistance, the amino acid sequence of the PbPI16-1 protein is shown in SEQ ID NO.1; the diseases include clubroot and / or sclerotinia.

[0009] As a preferred embodiment, the nucleotide sequence of the coding gene PbPI16-1 is shown as SEQ ID NO.2.

[0010] The present invention provides the use of PbPI16-1 protein and / or its encoding gene PbPI16-1 in cultivating disease-resistant rapeseed, the amino acid sequence of the PbPI16-1 protein is shown in SEQ ID NO.1; the disease resistance includes resistance to clubroot and / or resistance to sclerotinia.

[0011] The present invention provides a method for improving disease resistance of rapeseed, which comprises overexpressing a gene PbPI16-1 encoding a PbPI16-1 protein in rapeseed; the amino acid sequence of the PbPI16-1 protein is shown in SEQ ID NO.1; and the disease comprises clubroot and / or sclerotinia.

[0012] As a preferred scheme, the overexpression of the gene PbPI16-1 encoding the PbPI16-1 protein in rapeseed includes the following steps: constructing a recombinant expression vector containing the gene PbPI16-1 encoding the PbPI16-1 protein; transforming the recombinant expression vector into Agrobacterium to obtain a transformant; and transforming the transformant into rapeseed to obtain transgenic rapeseed.

[0013] As a preferred embodiment, the nucleotide sequence of the coding gene PbPI16-1 is shown as SEQ ID NO.2.

[0014] As a preferred solution, the rapeseed includes Zhongshuang No. 6.

[0015] Beneficial effects:

[0016] The present invention provides the use of PbPI16-1 protein and / or its encoding gene PbPI16-1 in preventing rapeseed diseases, wherein the amino acid sequence of the PbPI16-1 protein is shown in SEQ ID NO.1; the diseases include clubroot and / or sclerotinia. The present invention transforms the encoding gene PbPI16-1 of the PbPI16-1 protein into rapeseed ZS6, and screens three transgenic families. The results show that overexpression of PbPI16-1 in rapeseed can enhance rapeseed's resistance to clubroot and sclerotinia. It can be seen that the encoding gene PbPI16-1 of the PbPI16-1 protein can be used as a gene resource for green prevention and control of rapeseed diseases, providing a new method for green disease prevention and control of rapeseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 Graph showing the expression level of the PbPI16-1 gene in wild-type rapeseed and T1 transgenic rapeseed in Example 3; Pb16-1 in the graph is the PbPI16-1 gene;

[0019] Figure 2 This is a diagram of different rapeseed growth phenotypes in Example 3, scale bar = 10 cm;

[0020] Figure 3 The results of rapeseed inoculated with root knot fungi 30 days after Example 4; A is a representative diseased root image of ZS6 and T1 transgenic rapeseed OE-8, OE-9 and OE-12 after inoculation with root knot fungi, scale = 1 cm; B is a full diseased root image of ZS6 and T1 transgenic rapeseed after inoculation with root knot fungi;

[0021] Figure 4 The results of the inoculation of root knot fungi in Example 4 are shown in Figure 4; wherein A is a disease index diagram after the inoculation of root knot fungi; and B is a diagram showing the results of the biomass of root knot fungi;

[0022] Figure 5 This is a picture of rapeseed leaves after inoculation with Sclerotinia sclerotiorum in Example 5;

[0023] Figure 6 This is a graph showing the lesion area and biomass after inoculation with Sclerotinia sclerotiorum in Example 5. DETAILED DESCRIPTION

[0024] The present invention provides the use of PbPI16-1 protein and / or its encoding gene PbPI16-1 in preventing rapeseed diseases; the amino acid sequence of the PbPI16-1 protein is shown in SEQ ID NO.1; the diseases include clubroot and / or sclerotinia. The amino acid sequence of the PbPI16-1 protein is as follows: MLGAKSIVL AVALVSAARAQLTPDDINAILSAHNQARGVVSPTAANMMALQWDNTIAASAQAYANTCPTKHSGNPSYGENLSWGWPTMTPAQAVKEWTNEQPAYSFASNACVNSKVCNHYTQLVWSNSTKVGCGKMACQNLFGNPKVDAWVCQYDPPGNYVGELPYVSGPSCSQCPSGTTCSNNICVPGGATTPAPTSAPAAGSSPAAGSSTPAASPAAGSSPAAGSTPAASPAAGSTTPAGAGAAPAAGAAAPAPAGAAPSATGAAPSAAGAAPATATAPAPVGSVPAKAVPAGDAPAASSTSTSTPGVEFATTGHLQKPHPVNETRPRTGIAINPSMSNAANATNSTMPATAMPAATMMPTATMMPTATMMPSTAAPATSADQAPFVTVIGFPKSSTTN*.

[0025]

[0026] As an embodiment, the rapeseed includes Zhongshuang No. 6.

[0027] The present invention also provides the use of PbPI16-1 protein and / or its encoding gene PbPI16-1 in improving rapeseed disease resistance, wherein the amino acid sequence of the PbPI16-1 protein is shown in SEQ ID NO.1; the disease includes clubroot and / or sclerotinia. As an embodiment, the nucleotide sequence of the encoding gene PbPI16-1 is shown in SEQ ID NO.2.

[0028] The present invention also provides the use of PbPI16-1 protein and / or its encoding gene PbPI16-1 in cultivating disease-resistant rapeseed, the amino acid sequence of the PbPI16-1 protein is shown in SEQ ID NO.1; the disease resistance includes resistance to clubroot and / or resistance to sclerotinia.

[0029] The present invention also provides a method for improving rapeseed disease resistance, overexpressing the coding gene PbPI16-1 of the PbPI16-1 protein in rapeseed; the amino acid sequence of the PbPI16-1 protein is shown in SEQ ID NO.1; the disease includes clubroot and / or sclerotinia.

[0030] As an embodiment, the method for overexpressing the gene PbPI16-1 encoding the PbPI16-1 protein in rapeseed includes the following steps: constructing a recombinant expression vector containing the gene PbPI16-1 encoding the PbPI16-1 protein; transforming the recombinant expression vector into Agrobacterium to obtain a transformant; transforming the transformant into rapeseed to obtain transgenic rapeseed. As an embodiment, the nucleotide sequence of the coding gene PbPI16-1 is shown in SEQ ID NO.2. As an embodiment, the basic vector of the recombinant expression vector includes the pCAMBIA2300 vector. As an embodiment, the Agrobacterium includes GV3101. As an embodiment, the rapeseed includes Zhongshuang No. 6.

[0031] To further illustrate the present invention, the application of the PbPI16-1 protein and / or its encoding gene PbPI16-1 provided by the present invention in preventing rapeseed diseases is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] (1) Extraction of total RNA from root knotweed bacteria

[0034] ① Wash the freshly diseased rapeseed root material (public document: Research on the biological function of PbPI16s, a secretory peptidase inhibitor of root-knot bacteria, Chen Yimin, master's thesis of Huazhong Agricultural University, 2022), grind the tissue sample into powder in a mortar with liquid nitrogen, quickly transfer 100 mg of frozen tissue powder into a 2 mL centrifuge tube, add 1 mL of RNA extraction solution TRIpure, immediately shake vigorously to mix, and place the homogenized sample on ice for 10 minutes.

[0035] ② Add 0.2 mL of chloroform to every 1 mL of TRIpure, cap the tube tightly, shake vigorously to mix immediately, and place on ice for 10 min.

[0036] ③ High-speed refrigerated centrifugation at 12000r / min for 20 minutes at 4°C. After centrifugation, the mixture is divided into three layers: a lower red organic phenol chloroform layer, a middle layer, and an upper colorless water layer. RNA exists in the water layer.

[0037] ④ Transfer the supernatant of the aqueous layer to a clean 1.5mL centrifuge tube, add an equal volume of isopropanol, invert and mix, place in a -20℃ refrigerator for 30min, centrifuge at 4℃12000r / min for 20min, and discard the supernatant.

[0038] ⑤ Add 1 mL of 75% ethanol prepared with DEPC water to the precipitate to wash it, centrifuge at 4°C 12000r / min for 5 min, discard the supernatant, and pay attention to not losing the RNA precipitate.

[0039] ⑥Repeat step ⑤ twice.

[0040] ⑦ Place the centrifuge tube in a centrifuge at 12000r / min for 1 minute, then carefully aspirate the remaining liquid with a pipette and let it stand at room temperature for 10 minutes to dry the RNA precipitate;

[0041] ⑧ Add 50 μL of DEPC water to each tube to fully dissolve the RNA. After measuring the concentration of the obtained RNA using Nanodrop2000, store it at -80°C to prevent degradation for future use.

[0042] (2) Cloning of target gene

[0043] ①Use the reverse transcription kit (TransGene, Code#AE311-03) to synthesize the first-strand cDNA from the RNA extracted in step (1). Total reaction system: Total RNA 1-4μg, Anchored Oligo(dT)18 Primer (0.5μg / μL) 1μL, gDNA Remover 1μL, EasyScript RT / RI Enzyme Mix 1μL, 2×ES Reaction Mix 10μL, add RNase-free water to make up to 20μL. Mix gently and incubate at 42℃ for 30min. Heat at 85℃ for 5 seconds to inactivate EasyscriptRT / RI and gDNA Remover.

[0044] ② Based on the coding sequence (CDS, as shown in SEQ ID NO.2) of the PbPI16-1 gene obtained from the genome sequencing results of the root knotweed monospore isolation strain ZJ-1 (published in Proto-oncogenes in a eukaryotic unicellular organism play essential roles in plasmodial growth in host cells, BMC Genomics, 2018 Dec 6; 19(1):881.doi:10.1186 / s12864-018-5307-4.) and the multiple cloning site of the vector, the cloning primers PbPI16-1-F and PbPI16-1-R of the gene were designed. The design information of the primer sequence includes the vector terminal homologous sequence (lowercase font), the restriction site (lowercase font underlined part) and the gene-specific sequence (uppercase font) from 5' to 3'.

[0045] PbPI16-1-F (SEQ ID NO.3): 5'-ctccccttgctccgt ggatcc ATGCTGGGCGCCA AGTCT-3′;

[0046] PbPI16-1-R (SEQ ID NO.4): 5'-gatgagcttctgctc aggcct GTTCGTCGTCGAAGACTTGGG-3'.

[0047] ③Use the synthesized root knotweed cDNA as a template and use the high-fidelity DNA polymerase KOD One for PCR amplification. The total PCR amplification system is 40μL: 10μM Primer F 1μL, 10μM Primer R1μL, 2×KOD One PCR MasterMix 20μL, cDNA 2μL, ddH2O 16μL.

[0048] The PCR cycle conditions were as follows: pre-denaturation, 98°C, 2 min; denaturation, 98°C, 10 sec; annealing, 58°C, 5 sec; extension, 68°C, 5 sec / kb, cycle number 35; final extension, 68°C, 30 sec; storage at 8°C, 5 min.

[0049] After the reaction, add 4.4 μL 10× DNA loading buffer to the PCR product and mix well. Then run the product through 1% (w / v) agarose gel electrophoresis at 130V for 20 min with nucleic acid dye (Biosharp, BS354B; added at a volume ratio of 1:10000). Use Tanon 1600 fully automatic digital gel imager to detect whether the PCR product is correct. Cut the gel with the correct size band, and then purify and recover it using a gel recovery kit.

[0050] (3) Enzyme digestion vector:

[0051] Linearize the pCAMBIA2300-Myc tag vector (public literature: Peipei Qi, et, al. Ubiquitinat ion and degradation of plant helper NLR by the Ralstonia solanacearum effector rRipV2 overcome tomato bacterial wilt resistance. Cell Reports, 2024.) with two restriction endonucleases, Bam HI and Stu Ⅰ. Enzyme digestion system 70 μL: plasmid 50 μL, Bam HI-HF 1 μL, Stu Ⅰ-HF 1 μL, 10× rCutsmart buffer (purchased from NEB) 7 μL, dd H2O 11 μL. Mix well and incubate in a 37℃ water bath for 2 hours. Then add 7.7 μL 10× DNA loading buffer and mix well. Run the gel at 130 V for 20 min on 1% (w / v) agarose gel with nucleic acid dye (Biosharp, BS354B; added at a volume ratio of 1:10000). Use Tanon 1600 fully automatic digital gel imager to detect whether the enzyme cleavage bands are correct. Cut the target band from the gel and purify and recover it using a gel recovery kit.

[0052] (4) Vector construction:

[0053] The target gene fragment obtained by gel recovery and purification in step (2) is cloned into the linearized vector obtained in step (3) by seamless cloning technology. Use VAZYME's kit (Cat. No. C112-01) to prepare 10 μL of the following reaction system on ice: 2 μL of 5×CEⅡbuffer, 1 μL of ExnaseⅡ, 2 μL of linearized vector, 1 μL of inserted fragment, and 4 μL of ddH2O. Use a pipette to gently mix, and centrifuge briefly to collect the reaction solution to the bottom of the PCR tube. Place in a PCR instrument at 37°C for 30 minutes.

[0054] (5) Transformation of Escherichia coli:

[0055] After the reaction in step (4) is completed, the recombinant product is heat-shocked to transform Escherichia coli DH5α, and the specific steps are as follows: take out the competent DH5α from the -80°C refrigerator and thaw it on ice; take 10 μL of the recombinant product and add it to 30 μL of competent cells, gently tap the tube wall to mix, and let it stand on ice for 30 minutes; after heat shock in a 42°C water bath for 45 seconds, immediately place it on ice to cool for 2 minutes; add 1 mL of liquid LB culture medium (without adding antibiotics), shake in a 37°C shaker at 220 r / min for 1 hour; centrifuge at 5000 r / min for 5 minutes, and discard 800 μL of supernatant; resuspend the bacteria with the remaining culture medium, and gently spread it on an LB plate containing Kana resistance with a sterile spreading stick, wherein the final concentration of Kana in the LB plate is 50 μg / mL; place the LB plate in an incubator at 37°C and incubate it upside down.

[0056] (6) Identification of recombinant plasmid

[0057] ① After a single colony grows on the LB plate in step (5), pick several single colonies for PCR identification. Pick a small amount of bacteria and place them in 10μL of the prepared PCR Mix, which contains 0.5μL of 10μM Vector Primer F, 0.5μL of 10μM Vector Primer R, 5μL of 2×Rapid Taq Master Mix, and 4μL of ddH2O.

[0058] PbPI16-1-F1 (SEQ ID NO.5): 5'-ATGCTGGGCGCCAAGTCT-3';

[0059] PbPI16-1-R2 (SEQ ID NO. 6): 5'-GTTCGTCGTCGAAGACTTGGG-3'.

[0060] The PCR cycle conditions are as follows: pre-denaturation, 95°C, 3 min; denaturation, 95°C, 15 sec; annealing, 58°C, 15 sec; extension, 72°C, 15 sec / kb, cycle number 35; final extension, 75°C, 5 min; storage at 8°C, 5 min.

[0061] ② The PCR product obtained in step ① was run on a 1% (w / v) agarose gel electrophoresis at 130 V for 20 min with nucleic acid dye (Biosharp, BS354B; added at a volume ratio of 1:10000), and the PCR product band size was detected using a Tanon 1600 fully automatic digital gel imager.

[0062] ③ Select a single colony with the correct PCR band and continue to inoculate it into 2 mL of liquid culture medium containing Kana antibiotics (final concentration of 50 μg / mL). After culturing at 37°C and 220 r / min for 12 hours, a small amount of bacterial liquid was taken and sent to Beijing Aoke Dingsheng Biotechnology Co., Ltd. for sequencing to further verify whether the recombinant plasmid is correct.

[0063] ④ For the bacterial solution with correct sequencing, collect the remaining bacteria and refer to the small amount extraction method of plasmid (published in the literature: Research on the molecular mechanism of CPK18 gene regulating ethylene and disease resistance, Li Hong, Ph.D. dissertation of Huazhong Agricultural University, 2024) to extract the plasmid, and store the extracted plasmid at -20℃.

[0064] Example 2

[0065] Agrobacterium-mediated genetic transformation of rapeseed

[0066] (1) Seed disinfection and germination:

[0067] ① In a 10mL EP tube, immerse the medium double 6 (ZS6) rapeseed seeds in 75% (v / v) alcohol for 1 minute, and discard the alcohol; ② Wash once with sterilized ddH2O, and discard the water; ③ Soak in 50% (v / v) 84 disinfectant for 10 minutes, and discard the disinfectant. For seeds with heavy pollution, the sterilization time can be extended to 20 minutes; ④ Wash the seeds 5 times with sterilized ddH2O; ⑤ Sow the rapeseed seeds treated in step ④ into M0 culture medium with sterile tweezers, sow 25 seeds per dish, and then put the culture dish into a sterile culture box and culture it at 24°C for 6 days under dark conditions. 1L of the M0 culture medium contains: MS2.22g, agar 8g, and the pH is adjusted to 5.8.

[0068] (2) Plasmid transformation of Agrobacterium:

[0069] ① Take out the competent Agrobacterium GV3101 from the -80℃ refrigerator and thaw it on ice; ② Take 2μL of the correctly sequenced plasmid in Example 1 and add it to 100μL of competent Agrobacterium GV3101 cells, and flick the side of the tube wall with your finger to mix the plasmid and competent cells thoroughly; ③ Add the mixed liquid to a clean and dried electroporation cup, and put the electroporation cup into the Bio-rad electroporator; select the program for electroporation transformation, then take out the electroporation cup, add 500μL of liquid LB culture medium (without adding antibiotics) to the cup, and transfer the mixed solution to a new 1.5mL centrifuge tube; ④ Place the centrifuge tube in step ③ in a 28℃ The culture medium was shaken at 220 r / min for 1 h, centrifuged at 5000 r / min for 5 min, and 400 μL of supernatant was discarded; the bacteria were resuspended with the remaining culture medium, and gently spread on the LB plate containing Kana (final concentration of 50 μg / mL) and Rif (final concentration of 50 μg / mL) with a sterile coating stick; the culture was inverted and placed in a 28°C incubator for 48 h; ⑤ After the single colonies grew out, several single colonies were picked for PCR identification. The PCR system and cycle program were the same as those for recombinant plasmid identification in step (6) of Example 1; ⑥ A small amount of colonies identified correctly by PCR was dipped into a white pipette tip and placed in a 10 mL EP tube that had been added with 2 mL LB liquid culture medium, and shaken overnight. The LB liquid culture medium contained Kana and Rif with final concentrations of 50 μg / mL respectively; after the shaking culture was completed, 1 mL of the bacterial solution was added with 1 mL of 50% glycerol and added to a 2 mL seed tube, and stored in a -20°C refrigerator.

[0070] (3) Activation and preparation of Agrobacterium:

[0071] ① Activate the glycerol bacteria stored in step ② by drawing lines on a dual-resistance LB plate containing Kana (final concentration of 50 μg / mL) and Rif (final concentration of 50 μg / mL), and first incubate the bacteria overnight with 2 mL of dual-resistance liquid LB medium. ② Then inoculate 100 mL of dual-resistance liquid LB medium at a ratio of 1:100 (v / v), and culture at 28°C with shaking at 220 r / min until OD 600 = about 0.8; ③ Pour the cultured Agrobacterium culture solution equally into two 50mL sterile centrifuge tubes (operated on a clean bench), balance and centrifuge at 3000r / min for 20min; ④ Take the sterile centrifuge tube after centrifugation in step ③ to the clean bench, pour out the supernatant, gently wash the bacteria with 1mL DM culture solution (with acetosyringone, AS), and then pour out the waste liquid; ⑤ Add 1mL DM culture solution and use a pipette to suspend and mix to obtain Agrobacterium culture solution. The OD of the Agrobacterium culture solution is 600=0.8; after the bacterial solution is prepared, place it on ice for activation. 1L of the DM culture solution contains: MS 4.43g, sucrose 30g, pH adjusted to 5.8, and after sterilization, add AS 1mL, 2,4-dichlorophenoxyacetic acid (2,4-D) 2mL, and kinetin (KT) 1.5mL.

[0072] (4) Infection and co-cultivation of explants:

[0073] ① Use sterile tweezers and a scalpel to vertically cut the hypocotyl of the seedling obtained by dark culture in step (1) as an explant, and the cutting operation is performed in DM culture medium, and the length of the hypocotyl is 0.8 cm. ② Place the explant cut in step ① into the Agrobacterium bacterial solution prepared in step (3), and immerse for 15 minutes. The number of explants in each dish is 150; ③ Shake once every 3 minutes to make the Agrobacterium bacterial solution and the explant fully contact, and shake 5 times in total; ④ After the immersion is completed, use sterile tweezers to gently clip the explant, place it on sterile filter paper to absorb the excess bacterial solution on the surface, and then use sterile tweezers to place the explant on M1 culture medium, and co-culture at 24°C under dark conditions for 48 hours. 1L of the M1 culture medium contains: MS 4.43g, sucrose 30g, mannitol 18g, 2,4-D 2mL, KT 1.5mL, pH adjusted to 5.8, and AS1mL added after sterilization.

[0074] (5) Selective training:

[0075] After the co-cultivation in step (4) is completed, the explants are transferred to M2 medium for selection culture for 20 days. The conditions of the selection culture are: 24°C light culture, 16 hours during the day and 8 hours at night. 1L of the M2 medium contains: MS 4.43g, sucrose 30g, mannitol 18g, 2,4-D 2mL, KT 1.5mL, pH adjusted to 5.8, and after sterilization, 13μL of AgNO32, 1mL of Timentin, and 1mL of glufosinate (Basta, purchased from Sigma, CAS: 77182-82-2).

[0076] (6) Differentiation culture:

[0077] After the selection culture in step (5) is completed, the explants are transferred to M3 medium for differentiation culture, and subcultured every 20 days or so until budding. The conditions of the differentiation culture are: 24°C light culture, 16 hours during the day and 8 hours at night. 1L of the M3 medium contains: MS 4.43g, glucose 10g, xylose 0.25g, MES 0.6g, pH adjusted to 5.9, and after sterilization, 4mL of zeatin (ZT), 200μL of indoleacetic acid (IAA), 1mL of Timentin, and 1mL of Basta are added.

[0078] (7) Rooting culture and transplanting:

[0079] After the buds are differentiated and cultured in step (6) and obvious growth points can be seen, the buds are carefully cut off from the callus with sterile tweezers and a scalpel to avoid involving excess callus tissue and damaging the growth points, and then transferred to the M4 medium for rooting; the vitrified buds need to be cultured for a period of time before they can return to normal and then take root; finally, the rooted resistant seedlings are transferred to the culture room for transplanting; the rooting culture conditions are: 24°C light culture, 16 hours during the day and 8 hours at night. 1L of the M4 medium contains: MS 2.22g, sucrose 10g, indolebutyric acid (IBA) 5mL, agar 8g, and Timentin 500μL after sterilization.

[0080] Example 3

[0081] Molecular Detection of Genetically Modified Rapeseed

[0082] After the resistant seedlings transplanted in Example 2 grew leaves, fresh leaves were taken to extract RNA. The specific extraction steps were the same as the extraction of total RNA of root knot fungi in Example 1, and then reverse transcribed into cDNA. The reverse transcription method was the same as the reverse transcription step in the cloning of the target gene in Example 1. The cDNA was diluted 2 times as a template, and rape ACTIN7 was used as an internal reference gene. qRT-PCR was used to detect the expression level of the target gene PbPI16-1 in transgenic rapeseed. The primer sequence information for qRT-PCR detection is as follows:

[0083] qBnACTIN7-F (SEQ ID NO.7): 5'-CCCTGGAATTGCTGACCGTA-3';

[0084] qBnACTIN7-R (SEQ ID NO.8): 5'-TGGAAAGTGCTGAGGGATGC-3';

[0085] qPbPI16-1-F (SEQ ID NO.9): 5'-GCAATCACCCGAGCATGTC-3';

[0086] qPbPI16-1-R (SEQ ID NO. 10): 5'-ACAGTGACGAATGGAGCCTG-3'.

[0087] The qRT-PCR system is 10 μL: cDNA 2 μL, 10 μM Primer F 0.5 μL, 10 μM Primer R 0.5 μL, 2×PerfectStart Green qPCR SuperMix 5 μL, ddH2O 2 μL.

[0088] The instrument used for real-time fluorescence quantitative PCR was BioRad CFX96, and the reaction was amplified using a three-step method: pre-denaturation at 95°C for 30 seconds, cycling at 95°C for 5 seconds, 60°C for 30 seconds, and 72°C for 10 seconds, for 35 cycles. -ΔΔCT The relative expression of the target gene was calculated by the method of . 19 positive seedlings were screened by qRT-PCR, among which the target gene expression levels of plants numbered 8, 9 and 12 were higher, and they were recorded as OE-8, OE-9 and OE-12 respectively. The above three families were selected to collect the T1 generation for the experiment.

[0089] The three families of T1 generation OE-8, OE-9 and OE-12 were screened for positive seedlings on 1 / 2MS plates supplemented with Kana (final concentration of 50 μg / mL), and 20 strains were planted in each family. The three strains with higher expression levels, OE-8-9, OE-9-9 and OE-12-8, were selected by qRT-PCR and continued to be planted for T2 generation ( Figure 1 The growth phenotypes of the three families of OE-8-9, OE-9-9 and OE-12-8 grown in the culture room for 3 months are as follows Figure 2 As shown, ZS6 is wild-type rapeseed.

[0090] Example 4

[0091] Transgenic rapeseed inoculated with clubroot

[0092] (1) Preparation of crude extract of dormant spores of Arabidopsis thaliana: Take out the swollen rape root sample stored in a -30°C refrigerator (public literature: Ying Zhao, Kai Bi et al. Transcriptome Analysis of Arabidopsis thaliana in Response to Plasmodiophorabrassicae during Early Infection. Frontiers in Microbiology, 2017), wash it with water, cut the root sample into pieces with a kitchen knife, put it into a juicer, add an appropriate amount of water to grind it, and after it is fully ground, filter it with 8 layers of gauze to obtain the crude extract of dormant spores of Arabidopsis thaliana. Then, take 100 μL of the crude extract and dilute it 1000 times with ddH2O. Count the spores under a microscope with a hemocytometer to calculate the concentration of the spore suspension. Dilute the crude spore extract to a concentration of 1×10 7 spores / mL.

[0093] (2) Inoculation of rapeseed with root knot fungi: 16 to 26 T1 rapeseed plants of the three families OE-8, OE-9 and OE-12 obtained in Example 3 and the wild-type rapeseed family ZS6 were randomly selected, and each rapeseed plant was inoculated with 1 mL of the crude spore extract obtained in step (1) by root irrigation. After 30 days of inoculation, pictures of each family after inoculation with root knot fungi were taken, as shown in FIG. Figure 3 As shown, according to Figure 3 It can be seen that the rapeseed plants in each family of OE-8, OE-9, OE-12 and ZS 6 were successfully inoculated with root knot fungi.

[0094] Statistical disease index: Statistical standard of rapeseed clubroot disease disease index: According to the degree of swelling of rapeseed roots, it is divided into five levels: Level 0, no disease symptoms; Level 1, there are very small tumors on the lateral roots, and no disease symptoms on the main root and hypocotyl; Level 3, the number of lateral roots is reduced, and there are small tumors on the main root; Level 5, medium to large irregularly shaped tumors are distributed on the hypocotyl and main root; Level 7, the tumors on the hypocotyl and main root are very large. Disease index (DI) is calculated according to the following formula: DI = (1×n1+3×n3+5×n5+7×n7)×100 / 7×n t , where n1, n3, n5, n7 represent the number of rapeseed seedlings corresponding to each level, n t represents the total number of experimental rapeseed. The disease index statistics are as follows Figure 4 As shown in A and Table 1, Figure 4 The numbers 0, 1, 3, 5, and 7 in A represent the degree of swelling of the rapeseed roots.

[0095] Table 1 Statistical results of rapeseed clubroot disease

[0096] Rapeseed family ZS6 OE-8 OE-9 OE-12 0 0 0 0 0 1 4 13 7 11 3 6 10 6 7 5 6 2 1 2 7 9 1 2 0 Disease index 65.71 32.96 39.28 30.00

[0097] according to Figure 4 As shown in Figure A and Table 1, after inoculation with root knot fungus, the three transgenic lines OE-8, OE-9 and OE-12 were more resistant to the disease than the wild type ZS6. The disease index of the wild type ZS6 was 65.71, and the disease indexes of the transgenic lines OE-8, OE-9 and OE-12 were 32.96, 39.28 and 30.00, respectively, which were all lower than the wild type ZS6.

[0098] (3) Extraction of total DNA of rapeseed and root knot fungus: Four diseased roots from each family of OE-8, OE-9, OE-12 and ZS6 in step (2) were mixed as a sample, and the total DNA of the diseased roots of the transgenic plants was extracted using the CTAB method. The specific steps are as follows: ① Heat the CTAB extraction buffer in a 65°C water bath; ② Take fresh diseased roots of rapeseed, clean them, quickly put them into liquid nitrogen, and quickly grind them into powder in a mortar with liquid nitrogen; ③ Take 100g of tissue powder prepared in step ②, add it to a 2mL centrifuge tube, add 700μL of the preheated CTAB extraction buffer in step ①, vortex mix, and place it in a 65°C water bath for 20 minutes, shaking it vigorously every 5 minutes; ④ Take the centrifuge tube out of the water bath and cool it to room temperature, add 350μL of chloroform and 350μL of chloroform, respectively; Vortex the Tris-saturated phenol solution to mix well; ⑤ Centrifuge at 12000r / min for 10min at room temperature, transfer 700μL of supernatant to a new 1.5mL centrifuge tube, add an equal volume of isopropanol, mix well by inversion, and place in a -20℃ refrigerator for 30min; ⑥ Centrifuge at 12000r / min for 10min at room temperature; discard the supernatant, add 1mL 75% ethanol to wash the DNA precipitate, centrifuge at 12000r / min for 5min, discard the supernatant, and repeat this step twice; ⑦ Centrifuge empty for 1min, use a pipette to aspirate the remaining ethanol, wait for the precipitate to dry to a transparent state at room temperature, add 50μL TE buffer with RNase added to fully dissolve the DNA, determine the concentration by Nanodrop, and store in a -20℃ refrigerator for later use.

[0099] (4) Detection of root knot fungus biomass in diseased roots: The total DNA concentration of rapeseed and root knot fungus extracted in step (3) was diluted to 500 ng / μL as a template, and rapeseed ACTIN7 was used as an internal reference gene. qRT-PCR was used to detect the expression of root knot fungus PbACTIN1 relative to rapeseed ACTIN7, with 3 technical replicates for each sample. The system, procedure steps and calculation method of the qRT-PCR were the same as the method for molecular detection of transgenic rapeseed in Example 3; the primers for qRT-PCR detection of the internal reference gene ACTIN7 are shown in SEQ ID NO.7 and SEQ ID NO.8, and the primers for the sclerotinia PbACTIN1 are qPbACTIN1-F and qPbACTIN1-R, as follows:

[0100] qPbACTIN1-F (SEQ ID NO.11): 5'-CACCGACTACCTGATGAA-3';

[0101] qPbACTIN1-R (SEQ ID NO. 12): 5'-CAGCTTCTCCTTGATGTC-3'.

[0102] The biomass detection results of PbACTIN1 in the root diseased roots are as follows Figure 4 As shown in Figure B and Table 2, different lowercase letters indicate significant differences in the data, p < 0.05.

[0103] Table 2 Detection results of rapeseed root knot fungus biomass

[0104] strain ZS6 OE-8 OE-9 OE-12 Biomass of root knotweed fungi 1.00 0.19 0.07 0.04

[0105] according to Figure 4 As shown in Figure B and Table 2, the biomass of root knot fungi in the three transgenic lines OE-8, OE-9 and OE-12 was significantly lower than that in the wild-type line ZS6. It can be seen that overexpressing PbPI16-1 in rapeseed can enhance the resistance of rapeseed to root knot fungi.

[0106] Example 5

[0107] Inoculation of Sclerotinia sclerotiorum and detection of Sclerotinia sclerotiorum biomass in transgenic rapeseed

[0108] (1) Transgenic rapeseed inoculated with Sclerotinia sclerotiorum

[0109] ① One week before inoculation, take a small mycelium block from the 1980 strain of Sclerotinia sclerotioides stored at 4°C (public literature: Yongkun Hu ang, Zhima Zha Xi, et, al. The Transcription Factor SsZNC1 Mediates Virulence, Sclerotial Development, and Osmotic Stress Response in Sclerotinia sclerotio. J Fungi, 2024), transfer it to a PDA plate, put it in a 22°C incubator for activation for 2 days, wait for new mycelium to grow, use a sterilized yellow gun tip to punch a 6 mm diameter bacterial cake at the edge of the colony, and then transfer it to a new PDA plate, and subculture and activate it three times; finally, the 6 mm diameter bacterial cake was activated on a 2×SY plate, put it in a 22°C incubator for activation for 2 days, and a 3 mm puncher was used to obtain the bacterial cake at the edge of the colony. The PDA formula 1L: 200g potato, 20g glucose, 15g agar; the 2×SY formula 1L: 10g sucrose, 1g yeast extract, 15g agar.

[0110] ② The three transgenic families OE-8, OE-9 and OE-12 obtained in Example 3 and the wild-type family ZS6 were selected for the experiment. When the rapeseed seedlings in each family grew to 44 days, three rapeseed leaves with uniform growth and health were selected from the roots of each family, and the bacterial cake obtained in step ① was inoculated on one side of the rapeseed leaves; after inoculation, sterile water was evenly sprayed on the surface of the rapeseed leaves until the surface of the leaves was moistened; the rapeseed leaves were placed in a sealed culture box and cultured at 22°C in a dark and moisturizing environment for 48 hours; then the two groups of diameters of the lesions were measured using a vernier caliper by the cross method, and the elliptical area, i.e., the lesion area, was calculated. The results are as follows: Figure 5 , Figure 6 As shown in Table 3, different capital letters indicate significant differences in the data, p < 0.05.

[0111] (2) Detection of Sclerotinia biomass:

[0112] ① The diseased area of ​​the rapeseed leaves in step (1) was cut off with scissors, and three leaves from each family were mixed as a sample. The samples were weighed on a balance to ensure that the weight of each sample remained consistent. The total DNA of rapeseed and Sclerotinia sclerotiorum was extracted using the CTAB method. The extraction method was the same as the total DNA extraction step of rapeseed and root knot fungus in Example 4.

[0113] ② The extracted total DNA concentration of rapeseed and Sclerotinia sclerotiorum was diluted to 500 ng / μL as a template, and rapeseed ACTIN7 was used as an internal reference gene. qRT-PCR was used to detect the expression of SsTub1 of Sclerotinia sclerotiorum relative to ACTIN7 of rapeseed; the system, procedure steps and calculation method of the qRT-PCR were the same as the method for molecular detection of transgenic rapeseed in Example 3. The primers of the internal reference gene ACTIN7 are shown in SEQ ID NO.7 and SEQ ID NO.8, and the primers of SsTub1 of Sclerotinia sclerotiorum are qSsTub-F and qSsTub-R, which are as follows:

[0114] qSsTub-F (SEQ ID NO. 13): 5'-ACCTCCATCCAAGAACTC-3';

[0115] qSsTub-R (SEQ ID NO. 14): 5'-GAACTCCATCTCGTCCAT-3'.

[0116] The biomass detection results of PbACTIN1 of Sclerotinia sclerotiorum in diseased roots are as follows Figure 6 As shown in Table 3, different lowercase letters indicate significant differences in the data, p < 0.05.

[0117] Table 3 Detection results of rapeseed inoculated with Sclerotinia sclerotiorum

[0118] Family lineage ZS6 OE-8 OE-9 OE-12 <![CDATA[Lesion area (cm 2 )]]> 800 425 500 463 Sclerotinia biomass 1.00 0.09 0.04 0.08

[0119] according to Figure 6 As shown in Table 3, the biomass of Sclerotinia sclerotiorum in the three transgenic lines OE-8, OE-9 and OE-12 was significantly lower than that in the wild-type line ZS6. It can be seen that overexpression of PbPI16-1 in rapeseed can enhance the resistance of rapeseed to Sclerotinia sclerotiorum.

[0120] In summary, the present invention transforms the root knot fungus gene PbPI16-1 into rapeseed ZS6, screens three transgenic families, and overexpresses the root knot fungus gene PbPI16-1 in rapeseed, which can enhance the resistance of rapeseed to root knot fungus and sclerotinia. The root knot fungus gene PbPI16-1 can be used as a gene resource for green prevention and control of rapeseed diseases, providing a new method for green disease prevention and control of rapeseed.

[0121] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of PbPI16-1 protein and / or its encoding gene PbPI16-1 in preventing rapeseed diseases; the amino acid sequence of the PbPI16-1 protein is shown in SEQ ID NO.1; the diseases include clubroot and / or sclerotinia.

2. The use according to claim 1, characterized in that: The nucleotide sequence encoding the gene PbPI16-1 is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: The rapeseed includes Zhongshuang No.

6.

4. Use of PbPI16-1 protein and / or its encoding gene PbPI16-1 in improving rapeseed disease resistance, characterized in that: The amino acid sequence of the PbPI16-1 protein is shown in SEQ ID NO.1; the diseases include clubroot and / or sclerotinia.

5. The use according to claim 4, characterized in that: The nucleotide sequence encoding the gene PbPI16-1 is shown in SEQ ID NO.

2.

6. Use of PbPI16-1 protein and / or its encoding gene PbPI16-1 in cultivating disease-resistant rapeseed, characterized in that: The amino acid sequence of the PbPI16-1 protein is shown in SEQ ID NO.1; the disease resistance includes resistance to clubroot and / or resistance to sclerotinia.

7. A method for improving rapeseed disease resistance, characterized in that: The gene PbPI16-1 encoding the PbPI16-1 protein is overexpressed in rapeseed; the amino acid sequence of the PbPI16-1 protein is shown in SEQ ID NO.1; and the disease includes clubroot and / or sclerotinia.

8. The method according to claim 7, characterized in that The method of overexpressing the gene PbPI16-1 encoding the PbPI16-1 protein in rapeseed includes the following steps: constructing a recombinant expression vector containing the gene PbPI16-1 encoding the PbPI16-1 protein; transforming the recombinant expression vector into Agrobacterium to obtain a transformant; and transforming the transformant into rapeseed to obtain transgenic rapeseed.

9. The method according to claim 8, characterized in that The nucleotide sequence encoding the gene PbPI16-1 is shown in SEQ ID NO.

2.

10. The method according to any one of claims 7 to 9, characterized in that: The rapeseed includes Zhongshuang No. 6.

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