Resistance of CePUB34 gene to taro epidemic disease and application of CePUB34 gene

Abstract

The invention provides application of a CePUB34 gene in treatment and / or prevention of taro blight in plants and cultivation of taro blight resistant transgenic plants. It is found for the first time that the CePUB34 gene from the Huai Kui taro variety has obvious resistance to taro epidemic diseases. The resistance of taro leaves of the transient overexpression CePUB34 gene to taro epidemic diseases is remarkably enhanced, and the scab area after inoculation is remarkably reduced compared with that of a control group. Therefore, the CePUB34 gene can be used for treating and / or preventing taro epidemic diseases in plants and culturing taro epidemic disease resistant transgenic plants, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular biology and plant genetic engineering, and particularly relates to the resistance of CePUB34 gene to taro blight and its application. Background Art

[0002] Taro blight is an oomycete disease caused by the pathogen Phytophthora colocasiae Racib., which primarily infects taro leaves, petioles, and corms. Initially, small yellow-brown circular spots appear on leaves, gradually expanding into concentric brown lesions surrounded by a dark green, water-soaked halo. In high humidity, white mold (sporangia and sporangiophores) and a honey-yellow dripping fluid develop on the surface of the lesions. Later, the lesions become perforated in the center, leaving only the leaf veins in severe cases. Dark brown, oblong lesions with blurred edges and white mold may form on the petioles. In severe cases, the petioles rot and fall over. Later, the corm can infect, resulting in brown, rotten patches and even the death of the entire plant. Taro can be affected throughout its growth period. Once infected, chemical treatments are difficult to control, resulting in significant economic losses. Breeding resistant varieties is an economical, environmentally friendly, and effective approach to controlling taro blight, and the identification of genes (or proteins) responsible for its resistance is fundamental to breeding for resistance. Summary of the Invention

[0003] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the purpose of the present invention is to provide the application of the CePUB34 gene in resistance to taro blight. The CePUB34 (protein U-box domain-containing protein 34) gene is a U-BOX family gene, which was obtained by transcriptome analysis of Huaijikui taro and Paodan taro. The CDS sequence of the CePUB34 gene (accession number: C_AA107791, publicly available at the China National Center for Bioinformation https: / / ngdc.cncb.ac.cn / genbase) is shown in SEQ ID NO: 1, and the amino acid sequence of the CePUB34 protein is shown in SEQ ID NO: 2.

[0004] The first aspect of the present invention provides the use of the CePUB34 gene in treating and / or preventing taro blight in plants and cultivating taro blight-resistant transgenic plants.

[0005] In some embodiments of the present invention, the CePUB34 gene comprises the nucleotide sequence shown in SEQ ID NO: 1.

[0006] In some embodiments of the present invention, the CePUB34 gene comprises a complementary sequence or a reverse sequence of the nucleotide sequence shown in SEQ ID NO: 1.

[0007] In some embodiments of the present invention, the CePUB34 gene has a nucleotide sequence as shown in SEQ ID NO: 1.

[0008] In some embodiments of the present invention, the CePUB34 gene is a complementary sequence or a reverse sequence of the nucleotide sequence shown in SEQ ID NO: 1.

[0009] In some embodiments of the present invention, the amino acid sequence expressed by the CePUB34 gene includes the amino acid sequence shown in SEQ ID NO: 2.

[0010] In some embodiments of the present invention, the amino acid sequence expressed by the CePUB34 gene is the amino acid sequence shown in SEQ ID NO: 2.

[0011] In some embodiments of the present invention, the taro blight is a disease caused by Phytophthora colocasiae Racib.

[0012] In some embodiments of the invention, the plant comprises taro.

[0013] In some embodiments of the present invention, the taro includes Huaijikui taro and Paodan taro.

[0014] The second aspect of the present invention provides a biomaterial containing the CePUB34 gene, characterized in that the biomaterial comprises at least one of the following (1) to (4):

[0015] (1) an expression cassette containing the CePUB34 gene comprising the nucleotide sequence shown in SEQ ID NO: 1;

[0016] (2) an expression vector comprising the expression cassette described in (1);

[0017] (3) A host bacterium containing the expression cassette described in (1) or the expression vector described in (2);

[0018] (4) A transgenic cell line comprising the expression cassette described in (1) or the expression vector described in (2).

[0019] In some embodiments of the present invention, the expression vector includes a plasmid, a viral vector, an artificial chromosome, and a transposon.

[0020] In some embodiments of the present invention, the viral vector includes an adenoviral vector and a lentiviral vector.

[0021] In some embodiments of the present invention, the artificial chromosome includes a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a P1-derived artificial chromosome (PAC), a mammalian artificial chromosome (MAC) and a human episomal artificial chromosome (HAEC).

[0022] In some embodiments of the present invention, the expression vector is a plasmid.

[0023] In some embodiments of the present invention, the vector backbone of the plasmid includes pGWB5 and pK7FWG2.0.

[0024] In some embodiments of the present invention, the plasmid uses pGWB5 as a vector backbone.

[0025] In some embodiments of the present invention, the host bacteria include Agrobacterium GV3101, Agrobacterium EHA105 and Agrobacterium LBA4404.

[0026] In some embodiments of the present invention, the transgenic cell line comprises taro leaf epidermal cells and tobacco leaf epidermal cells.

[0027] The third aspect of the present invention provides the use of the biological material containing the CePUB34 gene as described in the second aspect of the present invention in treating and / or preventing taro blight in plants and cultivating taro blight-resistant transgenic plants.

[0028] In some embodiments of the present invention, the taro blight is a disease caused by Phytophthora colocasiae Racib.

[0029] In some embodiments of the invention, the plant comprises taro.

[0030] In some embodiments of the present invention, the taro includes Huaijikui taro and Paodan taro.

[0031] A fourth aspect of the present invention provides the use of an agent for regulating the expression of the CePUB34 gene in a host in treating and / or preventing taro blight in plants and cultivating taro blight-resistant transgenic plants.

[0032] A fifth aspect of the present invention provides the use of an agent for identifying CePUB34 gene expression in a host in treating and / or preventing taro blight in plants and cultivating taro blight-resistant transgenic plants.

[0033] A sixth aspect of the present invention provides a method for treating and / or preventing taro blight in plants, the method comprising upregulating the expression of the CePUB34 gene and / or promoting the production of its encoded product in the plant.

[0034] In some embodiments of the present invention, the upregulation of CePUB34 expression and / or promotion of the production of its encoded product includes upregulating the DNA molecule expression of the CePUB34 gene, upregulating the mRNA molecule expression of the CePUB34 gene, and upregulating the protein molecule expression of the translation product of the CePUB34 gene.

[0035] In some embodiments of the present invention, the method comprises the following steps: introducing the CePUB34 gene and / or the biological material containing the CePUB34 gene as described in the second aspect of the present invention into the plant, wherein the CePUB34 gene comprises the nucleotide sequence shown in SEQ ID NO: 1.

[0036] In some embodiments of the present invention, the taro blight is a disease caused by Phytophthora colocasiae Racib.

[0037] In some embodiments of the invention, the plant comprises taro.

[0038] In some embodiments of the present invention, the taro includes Huaijikui taro and Paodan taro.

[0039] A seventh aspect of the present invention provides a method for cultivating taro blight-resistant transgenic plants, the method comprising upregulating the expression of the CePUB34 gene and / or promoting the production of its encoded product in the plant.

[0040] In some embodiments of the present invention, the upregulation of CePUB34 expression and / or promotion of the production of its encoded product includes upregulating the DNA molecule expression of the CePUB34 gene, upregulating the mRNA molecule expression of the CePUB34 gene, and upregulating the protein molecule expression of the translation product of the CePUB34 gene.

[0041] In some embodiments of the present invention, the method includes the following steps: introducing the CePUB34 gene and / or the biological material containing the CePUB34 gene as described in the second aspect of the present invention into the plant, wherein the CePUB34 gene includes the nucleotide sequence shown in SEQ ID NO: 1, to obtain the taro blight-resistant transgenic plant.

[0042] In some embodiments of the present invention, the taro blight is a disease caused by Phytophthora colocasiae Racib.

[0043] In some embodiments of the invention, the plant comprises taro.

[0044] In some embodiments of the present invention, the taro includes Huaijikui taro and Paodan taro.

[0045] The beneficial effects of the present invention are:

[0046] This study, published in Nature Communications, shows for the first time that the CePUB34 gene, derived from the Huaijikui taro variety, confers significant resistance to taro blight. Taro leaves transiently overexpressing the CePUB34 gene exhibited significantly enhanced resistance to taro blight, with the inoculated lesion area significantly reduced compared to controls. The CePUB34 gene has broad potential applications in developing taro products resistant to taro blight and cultivating transgenic taro plants resistant to the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is the transcriptional expression of CePUB34 gene induced by Phytophthora taro;

[0048] Figure 2 is the plasmid map of pGWB5;

[0049] Figure 3 This is the transient overexpression of CePUB34 gene in the leaves of Taro taro;

[0050] Figure 4 Resistance detection in taro leaves expressing CePUB34 gene. DETAILED DESCRIPTION

[0051] The contents of the present invention are further described in detail below through specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The experimental methods for which specific conditions are not specified in the following examples are generally carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products. The taro used in the examples is Huaijikui taro (anti-disease material) and Paodan taro (susceptible to disease material) (Guo Juxian et al., 2022), and the materials are provided by the Institute of Facility Agriculture, Guangdong Academy of Agricultural Sciences. Taro Phytophthora taro is derived from diseased taro plants in the Zhongluotan experimental field in Baiyun District, Guangzhou.

[0052] Example 1: Transcriptional expression of CePUB34 gene in disease-resistant and disease-susceptible materials

[0053] This example uses Huaijikui taro as a disease-resistant material and Paodan taro as a disease-susceptible material, and detects the transcriptional expression of the CePUB34 gene in the two materials. The specific steps are as follows:

[0054] 1. RNA was extracted from the leaves of Huaijikui taro and Paodan taro using the CTAB method. The leaves of Huaijikui taro and Paodan taro were ground into powder using liquid nitrogen, and 0.3 g of the powder was transferred to a 2 mL centrifuge tube and 1 mL of 65°C

[0055] Preheat CTAB (Cetyltrimethylammonium Bromide) buffer, vortex to mix, and incubate in a 65°C metal water bath for 15 minutes. Add 1 mL of chloroform:isoamyl alcohol (v / v = 24:1) extraction solution and shake thoroughly. Centrifuge at 12,000 rpm at 4°C for 10 minutes. Transfer the supernatant to a new 2 mL centrifuge tube. Repeat the vortexing and centrifugation steps. Transfer the supernatant to a new 2 mL centrifuge tube and add 1 / 3 the volume of 10 M LiCl. Precipitate RNA at 4°C overnight. The next day, centrifuge at 10,000 rpm at 4°C for 20 minutes. Remove the supernatant to obtain the RNA precipitate.

[0056] 2. Purify RNA using the ethanol method. Wash the precipitate with -20°C pre-cooled 70% ethanol (DEPC-treated), centrifuge at 4°C, 13,000 rpm for 2 minutes, remove the supernatant, and place on ice for 10 minutes to allow the ethanol to evaporate completely. Dissolve the RNA precipitate in 30 μL of double-distilled water (DEPC-treated).

[0057] 3. Use RQ1 RNase-Free DNase (Promega, Madison, Wisconsin, USA) reagent to remove genomic DNA from total RNA (refer to the reagent instructions for specific procedures); check the quality of RNA by 1.5% agarose gel electrophoresis and store it in a -80°C refrigerator after concentration and quality testing.

[0058] 4. Use the MLV Reverse Transcriptase kit (Promega, Madison, Wisconsin, USA) to reverse transcribe and synthesize the first-strand cDNA (refer to the kit instructions for specific procedures); the synthesized first-strand cDNA can be directly used for real-time quantitative qRT-PCR reaction.

[0059] 5. Real-time quantitative qRT-PCR was used to detect the expression of the CePUB34 gene. The reaction was carried out using a CFX96 fluorescent quantitative PCR instrument from Bio-Rad, USA. The fluorescent dye used was ABsolute BlueQPCRSYBR Green Low ROX Mix from Thermo Fisher Scientific, USA. The reaction template used was cDNA diluted at a volume ratio of 1:32. The qRT-PCR reaction system and reaction conditions were as follows: qRT-PCR reaction system (10 μL): 5 μL AB geneQPCR SYBR green ROX mix, 1 μL 2 μM forward primer, 1 μL 2 μM reverse primer, 1 μL cDNA, 2 μL ddH2O, wherein the forward primer sequence is as follows: 5'-AAGTCATCCCCGAGCTTGAA-3' (SEQ ID NO: 3), and the reverse primer sequence is as follows: 5'-GTGTGCTGGAGTCTGAGTCT-3' (SEQ ID NO:

[0060] 4). qRT-PCR reaction conditions were: 95℃ for 15 min, 95℃ for 15 s, 60℃ for 20 s, 72℃ for 20 s, and then 95℃ for 15 min.

[0061] The PCR reaction was performed for 40 cycles of heating at 72°C for 15 s and 20 s, and finally at 72°C for 5 min.

[0062] The experimental results are as follows Figure 1 As shown, 72 hours after inoculation with Phytophthora taro, the transcriptional expression of CePUB34 gene increased rapidly in the taro disease-resistant materials, while the transcription was severely inhibited in the susceptible materials.

[0063] Example 2: Construction of CePUB34 gene transient overexpression vector

[0064] The full-length CDS sequence of the CePUB34 gene (1368 bp, accession number: C_AA107791, publicly available at the China National Center for Bioinformation https: / / ngdc.cncb.ac.cn / genbase) was constructed into pGWB5. The plasmid map of pGWB5 is shown in Figure 2 As shown, the specific position is between attR1 and attR2 after the 35S promoter (ccdB gene is replaced by gene recombination exchange), and an overexpression vector (pGWB5-CePUB34) is obtained. The expression vector is constructed using conventional construction methods in the field of molecular biology.

[0065] The CDS sequence of the CePUB34 gene is as follows:

[0066] >C_AA107791.1

[0067]

[0068] The amino acid sequence of the CePUB34 protein is as follows:

[0069] >C_AAK92803.1

[0070] MLSAECSEESRKVKDALQREEVFKRIAAEEKAKHLKALMEVKEARQLLAKETMDRHRAEIDATKESSKKLKMVDALFSSDKRYRKYSRNEIEVATDNFSVDKKIGEGGFGNVYK GILDHTPVAVKVLREDAIERQKEFLKEVEVLSQLHHPHMLLLLGACPEIGCLVYEYMENGSLEDRLFCRGGTPPLPWFVRFRIIFEVACGLAFLHGSKPEPIVHRDLKPGNILLD RNYVSKIGDVGLAKLMSNVVPEGITEYKETVLAGTLYYMDPEYQRTGTLRPKSDLFAFGVIVLQLVTGRPPHGLLVTVEEAISSGTFVSILDKSISDWPIAESQKLAQIALGCTQ LKCRDRPDLESEVIPELEELSKMADTFSKSRRSHVNAPAHFFCPILQALMDDPYIAADGFSYEYVAIEAWFEKHSISPVTRLRLQHTAVIPNHSLRAAIQEWKSELIGRHS(SEQ ID NO: 2).

[0071] Example 3: Construction of a taro leaf model transiently overexpressing CePUB34

[0072] (1) Agrobacterium GV3101 was transformed using the overexpression vector pGWB5-CePUB34 obtained in Example 2 and cultured in lysate broth (LB) with kanamycin and rifampicin resistance for 48 h.

[0073] (2) Pick a single clone and add it to 4 mL of LB medium containing 50 μg / ml kanamycin and 25 μg / ml rifampicin, and shake the culture at 28°C and 180 rpm for 24 h.

[0074] (3) Add the solution to fresh LB medium containing kanamycin and rifampicin at a ratio of 1:100 and shake at 28°C and 180 rpm until the OD600 value reaches approximately 3.0.

[0075] (4) The cells were collected by centrifugation at 3000 rpm for 5 min, and the cells were resuspended in a suspension solution [10 mM 2-morpholineethanesulfonic acid (MES), 10 mM MgCl2], and the OD600 value was adjusted to about 0.4. 200 mM acetosyringone was added.

[0076] (5) Let the bacterial solution stand at room temperature for 3 hours.

[0077] (6) Use a syringe needle to poke a hole on each side of the main vein of the leaf of the taro.

[0078] (7) Use a 1 mL syringe to draw an equal amount of the bacterial solution after standing in step (5), align the syringe with the needle hole on the back, block the front with your hand, and inject the bacterial solution.

[0079] (8) The injected taro seedlings were cultured in the dark for 12 h and then cultured in the light at 22°C for 3-4 days.

[0080] (9) After the taro cotyledons were separated from the body, they were inoculated with the fungus block of Phytophthora taro, and then the inoculated taro cotyledons were placed in a culture dish at 28°C and cultured in the dark for 24 hours.

[0081] (10) Leaves from the treatment group (transient expression group) and the control group (wild type, WT) were collected and gene expression levels were detected (for detailed operation steps, please refer to Example 1).

[0082] The experimental results showed that CePUB34 gene was highly expressed in the leaves of the taro plant with transient overexpression of CePUB34 gene, while the wild type control (WT) group had no expression ( Figure 3 ), indicating that the taro leaf model with transient overexpression of CePUB34 gene was successfully constructed.

[0083] Example 4: Disease resistance experiment of taro leaves

[0084] The taro leaf model with transient overexpression of the CePUB34 gene successfully constructed in Example 3 and wild-type (WT) taro leaves were inoculated with Phytophthora taro, and the resistance function of the target gene to Phytophthora taro was determined based on the size of the lesions on the taro leaves with different treatments 72 hours after inoculation.

[0085] The lesion areas of 30 leaves in the transient overexpression group and the wild type group (control group) after inoculation with Phytophthora were counted, the means were calculated, and significant differences between the groups were analyzed.

[0086] The experimental results showed that transient overexpression of CePUB34 gene (pGWB5-CePUB34) in the leaves of susceptible taro material Paodan taro could significantly enhance the resistance of taro leaves to blight caused by Phytophthora taro compared with the wild type (WT) ( Figure 4 ).

[0087] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Application of the CePUB34 gene in treating and / or preventing taro blight in plants and cultivating taro blight-resistant transgenic plants.

2. The use according to claim 1, characterized in that The CePUB34 gene includes the nucleotide sequence shown in SEQ ID NO:

1.

3. A biomaterial containing the CePUB34 gene, characterized in that: The biomaterial includes at least one of the following (1) to (4): (1) an expression cassette containing the CePUB34 gene comprising the nucleotide sequence shown in SEQ ID NO: 1; (2) an expression vector comprising the expression cassette described in (1); (3) A host bacterium containing the expression cassette described in (1) or the expression vector described in (2); (4) A transgenic cell line comprising the expression cassette described in (1) or the expression vector described in (2).

4. The biomaterial according to claim 3, characterized in that The expression vector is selected from the group consisting of a plasmid, a viral vector, an artificial chromosome and a transposon.

5. The biomaterial according to claim 3, characterized in that The host bacteria include Agrobacterium GV3101, Agrobacterium EHA105 and Agrobacterium LBA4404.

6. Use of an agent for regulating the expression of the CePUB34 gene in a host in treating and / or preventing taro blight in plants and cultivating taro blight-resistant transgenic plants.

7. Use of an agent for identifying CePUB34 gene expression in a host in treating and / or preventing taro blight in plants and cultivating taro blight-resistant transgenic plants.

8. A method for treating and / or preventing taro blight in plants and cultivating taro blight-resistant transgenic plants, the method comprising upregulating the expression of the CePUB34 gene and / or promoting the production of its encoded product in the plant.

9. The method according to claim 8, characterized in that The method comprises the following steps: introducing the CePUB34 gene and / or the biological material containing the CePUB34 gene according to any one of claims 3 to 5 into the plant, wherein the CePUB34 gene comprises the nucleotide sequence shown in SEQ ID NO:

1.

10. The use according to any one of claims 1 to 2, the biomaterial containing the CePUB34 gene according to any one of claims 3 to 5, the use according to claim 6, the use according to claim 7, or the method according to claims 8 to 9, characterized in that: The taro blight is a disease caused by Phytophthora colocasiae Racib., and the plant includes taro.

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