CePUB34 gene for resistance to bacterial wilt of taro and application

Abstract

The application provides application of CePUB34 gene in treating and / or preventing taro blight in plants and cultivating taro blight-resistant transgenic plants. The CePUB34 gene derived from Huaiji Kui taro variety is found to have obvious resistance to taro blight for the first time. The taro leaf with transient overexpression of the CePUB34 gene has significantly enhanced resistance to taro blight, and the lesion area after inoculation is significantly reduced compared with the control. Therefore, the CePUB34 gene can be used for treating and / or preventing taro blight in plants and cultivating taro blight-resistant transgenic plants, and has wide application prospects.

Description

TECHNICAL FIELD

[0001] The present application 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 application thereof. BACKGROUND

[0002] Taro blight is an oomycete disease, and the pathogen is Phytophthora colocasiae Racib. It mainly invades taro leaves, petioles and corms. When the leaves are diseased, yellow-brown round spots are formed at the initial stage, and then gradually expand into concentric ring-shaped brown lesions with a deep green water immersion halo around the edge. Under high humidity environment, white mold-like substances (sporangia and sporangium stalks) and honey yellow exudate are produced on the surface of the lesions, and the central part of the lesions is perforated in the later stage, and the leaf veins are left in severe cases. When the petiole is diseased, dark brown oblong lesions appear with a blurred edge, and white mold-like substances can be produced on the surface, and the petiole can be severely rotted and collapsed. In the later stage, the corms are infected, leading to brown rot patches, and even the whole plant dies. Taro can be harmed throughout its growth period, and once it is diseased, it is difficult to control with chemical agents, which can cause serious economic losses. Breeding disease-resistant varieties is one of the economic, environmentally friendly and effective ways to control taro blight, and tapping disease-resistant genes (or proteins) is the basis for disease-resistant breeding. SUMMARY

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

[0004] In a first aspect of the present application, the application of CePUB34 gene in treating and / or preventing taro blight in plants and cultivating taro blight-resistant transgenic plants is provided.

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

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

[0007] In some embodiments of the present application, the CePUB34 gene is a nucleotide sequence as set forth in SEQ ID NO: 1.

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

[0009] In some embodiments of the present application, the amino acid sequence expressed by the CePUB34 gene comprises an amino acid sequence as set forth in SEQ ID NO: 2.

[0010] In some embodiments of the present application, the amino acid sequence expressed by the CePUB34 gene is an amino acid sequence as set forth in SEQ ID NO: 2.

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

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

[0013] In some embodiments of the present application, the taro comprises Huiji Kui taro and Paolong taro.

[0014] In a second aspect of the present application, a biological material containing a CePUB34 gene is provided, characterized in that the biological material comprises at least one of the following (1) to (4):

[0015] (1) an expression cassette containing a CePUB34 gene comprising a nucleotide sequence as set forth in SEQ ID NO: 1;

[0016] (2) an expression vector containing the expression cassette as set forth in (1);

[0017] (3) a host bacterium containing the expression cassette as set forth in (1) or the expression vector as set forth in (2);

[0018] (4) a transgenic cell line containing the expression cassette as set forth in (1) or the expression vector as set forth in (2).

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

[0020] In some embodiments of the present application, the viral vector comprises an adenovirus vector and a lentivirus vector.

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

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

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

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

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

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

[0027] In a third aspect of the present application, there is provided a use of a biological material containing a CePUB34 gene as described in the second aspect of the present application for treating and / or preventing a Phytophthora colocasiae Racib. disease in a plant and cultivating a transgenic plant resistant to the Phytophthora colocasiae Racib. disease.

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

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

[0030] In some embodiments of the present application, the taro comprises Huaji Kui taro and Cannonball taro.

[0031] In a fourth aspect of the present application, there is provided a use of an agent for modulating the expression of a CePUB34 gene in a host for treating and / or preventing a Phytophthora colocasiae Racib. disease in a plant and cultivating a transgenic plant resistant to the Phytophthora colocasiae Racib. disease.

[0032] In a fifth aspect of the present application, there is provided a use of an agent for identifying the expression of a CePUB34 gene in a host for treating and / or preventing a Phytophthora colocasiae Racib. disease in a plant and cultivating a transgenic plant resistant to the Phytophthora colocasiae Racib. disease.

[0033] In a sixth aspect of the present application, there is provided a method for treating and / or preventing a Phytophthora colocasiae Racib. disease in a plant, the method comprising upregulating the expression of a CePUB34 gene and / or promoting the production of a product encoded thereby in the plant.

[0034] In some embodiments of the present application, the up-regulating expression of CePUB34 and / or promoting production of its encoded product comprises up-regulating expression of a DNA molecule of CePUB34 gene, up-regulating expression of a mRNA molecule of CePUB34 gene, and up-regulating expression of a protein molecule of translation product of CePUB34 gene.

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

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

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

[0038] In some embodiments of the present application, the taro comprises Huaji Kui taro and Paolong taro.

[0039] In a seventh aspect of the present application, there is provided a method for breeding a taro blight-resistant transgenic plant, the method comprising up-regulating expression of a CePUB34 gene and / or promoting production of its encoded product in a plant.

[0040] In some embodiments of the present application, the up-regulating expression of CePUB34 and / or promoting production of its encoded product comprises up-regulating expression of a DNA molecule of CePUB34 gene, up-regulating expression of a mRNA molecule of CePUB34 gene, and up-regulating expression of a protein molecule of translation product of CePUB34 gene.

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

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

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

[0044] In some embodiments of the present application, the taro includes Huaiji Kui taro and Cannonball taro.

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

[0046] The present application first discovers that the CePUB34 gene derived from the Huaiji Kui taro variety has obvious resistance to taro blight. The taro leaves with transient overexpression of the CePUB34 gene have significantly enhanced resistance to taro blight, and the lesion area after inoculation is significantly reduced compared with the control. The CePUB34 gene can be used for taro products resistant to taro blight and cultivating taro transgenic plants resistant to blight, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 The transcriptional expression of the CePUB34 gene induced by Phytophthora colocasiae;

[0048] Figure 2 The plasmid map of pGWB5;

[0049] Figure 3 The transient overexpression of the CePUB34 gene in the leaves of Cannonball taro;

[0050] Figure 4 The resistance detection of taro leaves expressing the CePUB34 gene. DETAILED DESCRIPTION

[0051] The content of the present application will be further described in detail through specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments of the present application will be described clearly and completely. The experimental methods not specified in the following examples are usually carried out according to the conventional conditions, for example, 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. Various common chemical reagents used in the examples are commercially available products. The taros used in the examples are Huaiji Kui taro (blight-resistant material) and Cannonball taro (blight-susceptible material) (Guo Jushen et al., 2022), which are provided by the Facility Agriculture Institute of Guangdong Academy of Agricultural Sciences. The Phytophthora colocasiae is derived from taro diseased plants in Zhongluotan experimental field in Guangzhou.

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

[0053] This embodiment uses Huaji Kuiyutou as anti-bacterial wilt material and Cannon Potato as bacterial wilt material to detect the transcription expression of CePUB34 gene in the two materials, and the specific steps are as follows:

[0054] 1. The RNA of Huaji Kuiyutou and Cannon Potato leaves was extracted by CTAB method. That is, the leaves of Huaji Kuiyutou and Cannon Potato were ground into powder with liquid nitrogen, and 0.3 g of the powder was transferred to a 2 mL centrifuge tube, and 1 mL of 65℃

[0055] CTAB (Cetyltrimethylammonium Bromide) buffer was added, shaken and mixed, and then incubated in a 65℃ metal water bath for 15 min. Then 1 mL of chloroform:isopropyl alcohol (v / v=24:1) extraction solution was added, shaken well, and then centrifuged at 4℃, 12000 rpm for 10 min. The supernatant was transferred to a new 2 mL centrifuge tube, and the above shaking and centrifugation steps were repeated. The supernatant was transferred to a new 2 mL centrifuge tube and 1 / 3 volume of 10M LiCl was added, and the RNA was precipitated at 4℃ overnight. The next day, centrifugation was performed at 4℃, 10000 rpm for 20 min, and the supernatant was removed to obtain the RNA precipitate.

[0056] 2. The RNA was purified by ethanol method. That is, the precipitate was washed with -20℃ pre-cooled 70% ethanol (treated with DEPC), centrifuged at 4℃, 13000 rpm for 2 min, and then the supernatant was removed. After being placed on ice for 10 min, the ethanol was completely volatilized; 30 μL of double distilled water (treated with DEPC) was used to fully dissolve the RNA precipitate.

[0057] 3. The RQ1 RNase-Free DNase (Promega, Madison, Wisconsin, USA) reagent was used to remove the genomic DNA in the total RNA (for specific operation, refer to the reagent instruction); the quality of the RNA was detected by 1.5% agarose gel electrophoresis, and then stored in a -80℃ refrigerator for standby after concentration and quality detection.

[0058] 4. The MLV Reverse Transcriptase (Promega, Madison, Wisconsin, USA) kit was used to reverse transcribe the first strand of cDNA (for specific operation, refer to the kit instruction); the synthesized first strand of cDNA can be directly used for real-time quantitative qRT-PCR reaction.

[0059] 5. The expression of CePUB34 gene was detected by real-time quantitative qRT-PCR experiment. That is, the reaction was carried out using the CFX96 fluorescent quantitative PCR instrument of Bio-Rad Company of the United States, the fluorescent dye used was ABsolute Blue QPCR SYBR Green Low ROX Mix of Thermo Fisher Company of the United States, and the reaction template used was cDNA diluted according to a volume ratio of 1:32. The qRT-PCR reaction system and reaction conditions are as follows: qRT-PCR reaction system (10 μL): 5 μL AB gene QPCR SYBR green ROX mix, 1 μL 2 μM forward primer, 1 μL 2 μM reverse primer, 1 μL cDNA, 2 μL ddH2O, wherein the sequence of the forward primer is as follows: 5'-AAGTCATCCCCGAGCTTGAA-3' (SEQ ID NO: 3), and the sequence of the reverse primer is as follows: 5'-GTGTGCTGGAGTCTGAGTCT-3' (SEQ ID NO:

[0060] 4). The qRT-PCR reaction conditions are as follows: 95 °C for 15 min, 95 °C for 15 s, 60 °C for 20 s, 72 °C for 20 s, and then PCR reaction for 40 cycles from 95 °C for 15 s to 72 °C for 20 s, and finally 72 °C for 5 min.

[0061] 15s to 72 °C for 20 s, and finally 72 °C for 5 min.

[0062] The experimental results are shown in Figure 1 At 72 hours after inoculation of Phytophthora colocasiae, the transcription expression of CePUB34 gene in taro resistant material rapidly increased, while the transcription in susceptible material was severely inhibited.

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

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

[0065] The CDS sequence of 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] MLSAECSEESRKVKDALQREEVFKRIAAEEKAKHLKALMEVKEARQLLAKETMDRHRAEIDATKESSKKLKMVDALFSSDKRYRKYSRNEIEVATDNFSVDKKIGEGGFGNVYKGILDHTPVAVKVLREDAIERQKEFLKEVEVLSQLHHPHMLLLLGACPEIGCLVYEYMENGSLEDRLFCRGGTPPLPWFVRFRIIFEVACGLAFLHGSKPEPIVHRDLKPGNILLDRNYVSKIGDVGLAKLMSNVVPEGITEYKETVLAGTLYYMDPEYQRTGTLRPKSDLFAFGVIVLQLVTGRPPHGLLVTVEEAISSGTFVSILDKSISDWPIAESQKLAQIALGCTQLKCRDRPDLESEVIPELEELSKMADTFSKSRRSHVNAPAHFFCPILQALMDDPYIAADGFSYEYVAIEAWFEKHSISPVTRLRLQHTAVIPNHSLRAAIQEWKSELIGRHS (SEQ ID NO: 2).

[0071] Example 3: Construction of a model of taro leaf with transient overexpression of CePUB34

[0072] (1) The overexpression vector pGWB5-CePUB34 obtained in Example 2 was used to transform Agrobacterium GV3101, which was inverted and cultured on lysogeny broth (LB) with kanamycin and rifampicin resistance for 48 h.

[0073] (2) Single colonies were picked and added to 4 mL of LB medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin, and cultured at 28°C and 180 rpm for 24 h.

[0074] (3) Fresh LB medium containing kanamycin and rifampicin was added at a ratio of 1:100, and the culture was shaken at 28°C and 180 rpm until the OD600 value reached about 3.0.

[0075] (4) Collect the bacteria by centrifugation at 3000 rpm for 5 min, resuspend the bacteria with suspension [10 mM 2-morpholinoethanesulfonic acid (MES), 10 mM MgCl2], and adjust the OD600 value to about 0.4, and then add 200 mM acetyl-syringone.

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

[0077] (6) Use a syringe needle to make a hole on each side of the main vein of the leaf blade of the Xanthosoma violaceum.

[0078] (7) Use a 1 mL syringe to suck an equal amount of the bacterial solution after standing in step (5), and inject the bacterial solution through the hole on the back, with the front blocked by hand.

[0079] (8) After injection, cultivate the Xanthosoma violaceum seedlings in darkness for 12 h, and then cultivate under light at 22°C for 3-4 days.

[0080] (9) After detaching the Xanthosoma violaceum cotyledon, inoculate the Xanthosoma violaceum cotyledon with the Pythium aphanidermatum mycelium, and then cultivate the inoculated Xanthosoma violaceum cotyledon in a 28°C culture dish in darkness for 24 h.

[0081] (10) Collect the leaves of the treatment group (transient expression group) and the control group (wild type, WT) to detect the gene expression level (for details, see Example 1).

[0082] The experimental results show that the CePUB34 gene has a high expression level in the Xanthosoma violaceum leaves with transient overexpression of the CePUB34 gene, and the wild type control (WT) group has no expression Figure 3 , indicating that the Xanthosoma violaceum leaf model with transient overexpression of the CePUB34 gene is successfully constructed.

[0083] Example 4: Disease resistance experiment of Xanthosoma violaceum leaves

[0084] Inoculate the Xanthosoma violaceum leaves with transient overexpression of the CePUB34 gene successfully constructed in Example 3 and the Xanthosoma violaceum leaves of the wild type (WT) with the Pythium aphanidermatum, and 72 h after inoculation, judge the resistance function of the target gene to the Pythium aphanidermatum according to the lesion size of the Xanthosoma violaceum leaves of different treatments.

[0085] Statistically analyze the lesion areas of 30 leaves of the transient overexpression group and the wild type group (control group) after inoculation with the Pythium aphanidermatum, calculate the mean value, and perform inter-group significance difference analysis.

[0086] The experimental results show that the Xanthosoma violaceum leaves with transient overexpression of the CePUB34 gene (pGWB5-CePUB34) can significantly enhance the resistance of the Xanthosoma violaceum leaves to the disease caused by the Pythium aphanidermatum compared with the wild type (WT) Figure 4 .

[0087] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. CePUB34 Use of a gene in the treatment and / or prevention of fusarium wilt in plants, including taro, and in the breeding of fusarium wilt resistant transgenic plants, said plants comprising taro, said CePUB34 The nucleotide sequence of the gene is set forth in SEQ ID NO:

1.

2. Upregulate the host CePUB34 The application of gene expression reagents in the treatment and / or prevention of taro blight in plants and in the breeding of transgenic plants resistant to taro blight, wherein the plants include taro. CePUB34 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

3. A method for treating and / or preventing taro blight in plants and for breeding transgenic taro blight-resistant plants, said method comprising upregulating in plants... CePUB34 The expression of genes and / or the promotion of the production of their encoded products, the plant including taro, the CePUB34 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

4. The method of claim 3, wherein, The method includes the steps of introducing into the plant CePUB34 a gene, and / or a biological material containing CePUB34 a gene, the nucleotide sequence of the CePUB34 gene being as shown in SEQ ID NO: 1; the biological material including at least one of the following (1)~(4): (1) an expression cassette containing a gene comprising a nucleotide sequence as set forth in SEQ ID NO: 1 CePUB34 a nucleotide sequence as set forth in SEQ ID NO: 1 (2) an expression vector containing the expression cassette as described in (1); (3) a host bacterium containing the expression cassette as described in (1) or the expression vector as described in (2); (4) a transgenic cell line containing the expression cassette as described in (1) or the expression vector as described in (2).

5. The method of claim 4, wherein, The expression vector is selected from the group consisting of a plasmid, a viral vector, an artificial chromosome, and a transposon.

6. The method of claim 4, wherein, The host bacterium includes Agrobacterium GV3101, Agrobacterium EHA105, and Agrobacterium LBA4404.

7. Use according to claim 1, use according to claim 2, or a method according to any one of claims 3 to 6, characterized in that, The taro blight is a disease caused by Phytophthora colocasiae (Racib.) Phytophthora colocasiae Racib. ).

Images