Citrus canker resistance regulation gene AbCOMT1 and application thereof

Abstract

The invention discloses a citrus canker resistance regulation gene AbCOMT1 and application thereof.According to the citrus canker resistance regulation gene AbCOMT1, due to the fact that different citrus varieties have significant difference on citrus canker resistance, canker resistance evaluation is conducted on multiple citrus varieties, and wild citrus resource Acanthopanax trifoliatus with significant resistance to citrus canker is identified; the AbCOMT1 gene is screened and identified by applying transcriptomics, and the CDS sequence of the AbCOMT1 gene is as shown in SEQ ID No. 1. The function of the AbCOMT1 gene in citrus canker resistance is verified, and valuable gene resources are provided for canker-resistant variety cultivation in a molecular breeding mode. According to the invention, the AbCOMT1 gene is introduced into the sweet orange, and the resistance of the transgenic plant to the citrus canker is obviously increased.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and particularly relates to a citrus canker resistance regulation gene AbCOMT1 and its application. Background Art

[0002] Citrus is a fruit widely cultivated globally, and China ranks first in both the cultivation area and output of citrus in the world. Citrus canker is the second major quarantine disease in the citrus industry, with extremely strong transmissibility and great destructiveness, severely restricting the development of the citrus industry. Citrus canker can damage the leaves, shoots, and fruits of most cultivated citrus varieties, with seedlings and young trees being the most severely affected, seriously affecting the yield and quality. Therefore, cultivating new citrus varieties resistant to canker is an important way to solve the problem of citrus canker.

[0003] Citrus canker occurs in various citrus-producing areas in China, such as Jiangxi, Guangxi, Yunnan, and Sichuan. After citrus is infected with canker, symptoms such as necrotic lesions, premature leaf and fruit drop will appear, and in severe cases, it will cause the destruction of the orchard. All commercial citrus varieties, such as sweet oranges and grapefruits, are susceptible to canker. However, some wild resources show strong resistance to canker, such as kumquat, calamondin, citron, and Atalantia buxifolia.

[0004] In recent years, genes related to the resistance of citrus canker have been continuously discovered. For example, WRKY22 and WRKY50 in the WRKY transcription factor family, salicylic acid-related genes CsSAMT and CsMES1, antibacterial peptide genes SHivaA, cecropinB, and AttacinA, etc. After these genes are expressed in citrus, they enhance the resistance to canker to a certain extent, but cannot completely inhibit the proliferation of citrus canker bacteria, and there is still a gap from practical application. Therefore, using citrus canker resistance resources, identifying potent resistance genes, and analyzing the disease resistance mechanism can provide gene resources and theoretical basis for citrus canker resistance breeding. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a citrus canker resistance regulation gene AbCOMT1 and its application. In view of the significant differences in the resistance of different citrus varieties to citrus canker, the present invention evaluated the disease resistance of multiple citrus varieties to canker, identified the wild citrus resource Atalantia buxifolia with significant resistance to citrus canker, and used transcriptomics to screen and identify the AbCOMT1 gene, providing gene resources and key targets for citrus canker resistance molecular breeding.

[0006] To achieve the above purpose, the technical solutions designed by the present invention are as follows:

[0007] The present invention provides a citrus canker resistance regulatory gene AbCOMT1, and the CDS sequence (AbCOMT1-CDS) of the regulatory gene AbCOMT1 is as shown in SEQ ID No.1.

[0008] The present invention also provides a protein AbCOMT1 encoded by the above-mentioned citrus canker resistance gene AbCOMT1, and the amino acid sequence of the protein AbCOMT1 is as shown in SEQ ID No.2.

[0009] The present invention also provides a primer pair for obtaining the above-mentioned citrus canker resistance gene AbCOMT1, and the primer pair is AbCOMT1-F / R, which are respectively:

[0010] AbCOMT1 primer F: 5’-ATGGCAAATTCCAAGCCCAAG-3’, as shown in SEQ ID No.3;

[0011] AbCOMT1 primer R: 5’-CATTTGTGAAACTCCATTAC-3’, as shown in SEQ ID No.4.

[0012] The present invention also provides an overexpression vector pK7WG2D-AbCOMT1 of citrus AbCOMT1, and the overexpression vector pK7WG2D-AbCOMT1 contains the above-mentioned AbCOMT1 gene.

[0013] Use of any one of the following in citrus canker resistance breeding, wherein,

[0014] (1) The above-mentioned citrus canker resistance regulatory gene AbCOMT1;

[0015] (2) The above-mentioned overexpression vector pK7WG2D-AbCOMT1.

[0016] Use of any one of the following in cultivating new citrus varieties resistant to citrus canker, wherein,

[0017] (1) The above-mentioned citrus canker resistance regulatory gene AbCOMT1;

[0018] (2) The above-mentioned overexpression vector pK7WG2D-AbCOMT1.

[0019] Advantages of the present invention:

[0020] The present invention verifies the function of the citrus canker resistance gene AbCOMT1 (hereinafter referred to as the AbCOMT1 gene) in citrus canker resistance, and provides valuable gene resources for citrus canker resistance breeding through molecular breeding methods.

[0021] The present invention introduces the citrus canker resistance gene AbCOMT1 into citrus, and the transgenic plants show significantly increased resistance to citrus canker. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a diagram showing the phenotypes and gene expression differences of Atalantia buxifolia and sweet orange inoculated with citrus canker;

[0023] In the figure, in A, the upper part shows a schematic diagram of the identified citrus canker-resistant resource Atalantia buxifolia, and the lower part shows a schematic diagram of the susceptible cultivar sweet orange.

[0024] B is a heat map of differentially expressed genes at different time points after Atalantia buxifolia and sweet orange were inoculated with citrus canker.

[0025] C is a schematic diagram for qRT-PCR verification of the expression of the AbCOMT1 gene in Atalantia buxifolia and sweet orange.

[0026] Figure 2 It is a phenotypic diagram of sweet orange overexpressing the AbCOMT1 gene inoculated with citrus canker bacteria;

[0027] In the figure, A is a verification diagram of the overexpression effect in the AbCOMT1 overexpression line.

[0028] B is a phenotypic diagram of the AbCOMT1 overexpression line inoculated with citrus canker.

[0029] C is a statistical chart of the lesion area of the AbCOMT1 overexpression line inoculated with citrus canker.

[0030] D is a statistical chart of the pathogen copy number at different time points after the AbCOMT1 overexpression line was inoculated with citrus canker.

[0031] Figure 3 It is a schematic diagram for detecting the expression of immune-related genes in sweet orange overexpressing the AbCOMT1 gene;

[0032] A is a schematic diagram of the relative expression level of the RLP12 gene in sweet orange overexpressing the AbCOMT1 gene.

[0033] B is a schematic diagram of the relative expression level of the GST1 gene in sweet orange overexpressing the AbCOMT1 gene.

[0034] C is a schematic diagram of the relative expression level of the LRR8 gene in sweet orange overexpressing the AbCOMT1 gene.

[0035] D is a schematic diagram of the relative expression level of the PR1 gene in sweet orange overexpressing the AbCOMT1 gene.

[0036] E is a schematic diagram of the relative expression level of the PR2 gene in sweet orange overexpressing the AbCOMT1 gene.

[0037] Figure showing the relative expression level of the FRK1 gene in sweet oranges overexpressing the AbCOMT1 gene. Detailed implementation manners

[0038] The present invention will be further described in detail below in conjunction with specific embodiments for the understanding of those skilled in the art.

[0039] Example 1 Obtaining of the citrus canker resistance regulation gene AbCOMT1

[0040] Through the evaluation of citrus canker resistance, a material Atalantia buxifolia ( Figure 1 A)) with extremely strong resistance to citrus canker was screened. Therefore, this material and susceptible sweet oranges were selected. By measuring the transcriptomes of the resistant resource Atalantia buxifolia and susceptible sweet oranges at different time points after inoculation with citrus canker bacteria, the AbCOMT1 gene ( Figure 1 B)) was identified. The primer pair designed was AbCOMT1-F / R, which were respectively:

[0041] The primer pair was AbCOMT1-F / R, which were respectively:

[0042] AbCOMT1 primer F: 5’-ATGGCAAATTCCAAGCCCAAG-3’, as shown in SEQ ID No.3;

[0043] AbCOMT1 primer R: 5’-CATTTGTGAAACTCCATTAC-3’, as shown in SEQ ID No.4.

[0044] The CDS sequence (AbCOMT1-CDS) of the AbCOMT1 gene amplified was as shown in SEQ ID No.1; the amino acid sequence of the protein AbCOMT1 encoded by the AbCOMT1 gene was as shown in SEQ ID No.2.

[0045] The AbCOMT1 gene was significantly up-regulated within 48 hours after inoculation with citrus canker bacteria in Atalantia buxifolia, while there was no obvious difference in sweet oranges. To further verify this result, we sampled Atalantia buxifolia and sweet oranges at different time points after inoculation with canker disease, extracted RNA to detect the expression level of the AbCOMT1 gene, and found that it was significantly up-regulated in Atalantia buxifolia ( Figure 1 C)), verifying the results of the transcriptome.

[0046] Example 2 Construction of the AbCOMT1 gene overexpression vector

[0047] Using the expression vector pDONR221 as an intermediate vector and pK7WG2D (kindly provided by Professor Andrew C. Allan of Plant & Food Research, New Zealand) as the final vector, the CDS fragment of the AbCOMT1 gene was inserted into the pK7WG2D vector through gateway recombination technology and driven by the 35S promoter for expression; electrophoresis detection and sequencing analysis showed that the overexpression vector of citrus AbCOMT1, namely pK7WG2D-AbCOMT1, was successfully obtained. The specific experimental steps are as follows:

[0048] 1. Amplification of citrus AbCOMT1 gene

[0049] Primers for gateway cloning technology were designed according to the CDS sequence of the AbCOMT1 gene for PCR amplification. The primer sequences are as follows:

[0050] Primers for the first round of PCR amplification:

[0051] AttB1-AbCOMT1-F:

[0052] 5’-AAAAAGCAGGCTTCATGGCAAATTCCAAGCCCAAGAGG-3’,

[0053] AttB1-AbCOMT1-R:

[0054] 5’-AGAAAGCTGGGTGTCATTTGTGAAACTCCATTACCC-3’;

[0055] Primers for the second round of PCR amplification:

[0056] Adapter attB primer F:

[0057] 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3’,

[0058] Adapter attB primer R:

[0059] 5’-GGGGACCACTTTGTACAAGAAAGCTGGGT-3’.

[0060] Vazyme Phanta Max Super-Fidelity DNA polymerase was used for 2 rounds of PCR amplification to obtain the PCR product.

[0061] The reaction system for the first round of PCR is as follows (total volume 20 μl):

[0062] 2×Phanta Max Buffer 10 μl dNTP Mix 0.4 μl AttB1-AbCOMT1-F 0.8 μl AttB1-AbCOMT1-R 0.8 μl Phanta Max Super-Fidelity DNA Polymerase 0.4 μl cDNA (Aegle marmelos) 0.5 μl <![CDATA[ddH 2 O]]> 7.1 μl

[0063] The reaction procedure is as follows: 95°C, 3 min; 95°C, 15 s; 55°C, 15 s; 72°C, 30 s; 72°C, 5 min; 35 cycles;

[0064] The reaction system for the second round of PCR is as follows (total volume 50 μl)

[0065] 2×Phanta Max Buffer 25 μl dNTP Mix 1 μl Adapter attB-F 2 μl Adapter attB-R 2 μl Phanta Max Super-Fidelity DNA Polymerase 1 μl The first-round PCR product 1 μl <![CDATA[ddH 2 O]]> 18 μl

[0066] The reaction procedure is as follows: 95°C, 3 min; 95°C, 15 s; 55°C, 15 s; 72°C, 30 s; 72°C, 5 min; 35 cycles;

[0067] 2. Detection and recovery of PCR products by gel:

[0068] Detect the PCR products using agarose gel electrophoresis. Recover the target fragment using the BIOMIGA gel extraction kit;

[0069] 3. BP reaction:

[0070] Perform the BP reaction using the BP clonase enzyme from Thermofisher; the reaction system is as follows:

[0071]

[0072] Gently pipette and mix well, and collect the reaction solution to the bottom of the centrifuge tube after brief centrifugation; incubate in a water bath at 25°C for 12 h;

[0073] 4. Product transformation:

[0074] Transform the product into DH5α competent cells, referring to the molecular cloning experimental guide.

[0075] 5. Sequencing and identification:

[0076] After identifying positive clones by PCR, perform sequencing analysis. For the successfully sequenced single clones

[0077] extract plasmids, which are the intermediate vectors of pDONR221-AbCOMT1. The primers for PCR positive detection and sequencing are as follows:

[0078] pDONR221 primer F: 5’-GTAAAACGACGGCCAG-3’,

[0079] pDONR221 primer R: 5’-CAGGAAACAGCTATGAC-3’

[0080] 6. LR reaction:

[0081] Perform the BP reaction using the LR clonase enzyme from Thermofisher; the reaction system is as follows:

[0082]

[0083] Use a pipette to gently pipette and mix well, and collect the reaction solution to the bottom of the centrifuge tube after brief centrifugation; incubate in a water bath at 25 °C for 12 h;

[0084] 7. Product transformation:

[0085] Transform the product into DH5α competent cells, referring to the Molecular Cloning Experiment Guide.

[0086] 8. Sequencing identification:

[0087] After PCR identification of positive clones, perform sequencing analysis. For the successfully sequenced single clones,

[0088] extract plasmids, which are the overexpression vectors of pK7WG2D-AbCOMT1. The primers for PCR positive detection and sequencing are as follows:

[0089] pK7WG2D primer F: 5’-GACGCACAATCCCACTATCC-3’,

[0090] pK7WG2D primer R: 5’-TAGAGGGCCCGACGTCGCAT-3’.

[0091] Example 3 Obtaining of AbCOMT1 gene overexpressing citrus positive lines

[0092] 1. Transformation of citrus epicotyl stem segments

[0093] Transform the constructed overexpression vector into the Agrobacterium strain EHA105, and obtain transgenic positive lines through the citrus epicotyl stem segment infection method. The specific method is as follows: Disinfect the seeds of Anliu sweet orange fruits, sow them in MT medium, and place them in a 28 °C incubator for dark culture for 30 days. Then transfer them to 16 h / 8 h (Light / Dark) light culture. When the epicotyl turns light green, the infection experiment can be carried out.

[0094] Streak the Agrobacterium strain EHA105 carrying the target vector on a plate and incubate it upside down at 28 °C for 2 days. Then resuspend the Agrobacterium in citrus suspension medium to OD 600nm = 0.6, add acetosyringone to make its final concentration 20 mg / L, and then let it stand and activate at 28 °C for 1 h.

[0095] Cut the greening epicotyls into 1-cm-long stem segments, transfer them to the activated Agrobacterium suspension, and incubate them in a shaker at 28 °C and 200 rpm / min for 20 min, then apply a vacuum for 5 min. Then transfer the infected stem segments to the citrus co-culture medium and incubate them in the dark at 22 °C for 3 days. Then transfer them to the selection medium and incubate them in the dark at 25 °C for 7 days. Then transfer the stem segments to a light incubator at 28 °C with a 16 h / 8 h (Light / Dark) photoperiod for bud induction. Screen for positive seedlings using the green fluorescent protein (GFP) screening marker, and then graft and culture the positive seedlings.

[0096] 2. Screening of positive lines

[0097] For the screening of AbCOMT1 gene overexpressing citrus positive lines, first use a handheld fluorometer to screen for citrus plants with green fluorescence, and then extract DNA and RNA from the leaves of the preliminarily screened citrus plants, and use PCR amplification and qRT-PCR quantification to identify positive lines ( Figure 2 A).

[0098] The PCR amplification primers and qRT-PCR quantification primers are as follows:

[0099] PCR amplification primers:

[0100] pK7WG2D primer F: 5’-GACGCACAATCCCACTATCC-3’,

[0101] AttB1-AbCOMT1-R:

[0102] 5’-AGAAAGCTGGGTGTCATTTGTGAAACTCCATTACCC-3’;

[0103] qRT-PCR quantification primers:

[0104] qRT-PCR-AbCOMT1-F: 5’-CCGTTCTTCCGATGGTGCTAA-3’,

[0105] qRT-PCR-AbCOMT1-R: 5’-TGCGTCTGGATTCTTGGTAGG-3’.

[0106] Example 4 Evaluation of citrus canker resistance of AbCOMT1 gene overexpressing citrus positive lines

[0107] Inoculate the AbCOMT1 gene overexpressing citrus positive line sweet orange and wild-type sweet orange with Xanthomonas citri subsp. citri (10 8 CFU / ml), take pictures of the leaves 10 days after inoculation, and calculate the lesion area using ImageJ 2.0 software.

[0108] To determine the bacterial content of the inoculated leaves, leaf samples (0.50 cm) at 1, 4, 7, and 10 days after inoculation were collected. 0.10 g of each sample was taken for the extraction of plant DNA, and 50 ng of the extracted DNA was used for the determination of bacterial growth. The copy number of the pthA gene of the citrus canker pathogen was determined by quantitative polymerase chain reaction (qPCR), and the calculation formula was based on: copy number (pthA) = 10 (38.3-ct) / 3.56 .

[0109] The primers used were as follows:

[0110] qPCR-pthA-F: 5'-GCATTTGATGACGCCATGAC-3',

[0111] qPCR-pthA-R: 5'-TCCCTGATGCCTGGAGGATA-3'.

[0112] The inoculation evaluation of the canker resistance of the obtained positive lines of sweet orange showed that:

[0113] The canker resistance of the sweet orange overexpressing the AbCOMT1 gene was significantly higher than that of the wild-type sweet orange ( Figure 2 B), and the lesion area used was significantly reduced compared to the wild-type sweet orange ( Figure 2 C), and the pathogen copy number was significantly reduced compared to the wild-type sweet orange ( Figure 2 D).

[0114] Example 5 The immune response of the positive line of citrus overexpressing the AbCOMT1 gene was activated

[0115] To further verify the reason for the improvement of the canker resistance of the positive line of citrus overexpressing the AbCOMT1 gene, RNA of the positive line of citrus overexpressing the AbCOMT1 gene and wild-type citrus was extracted and reverse-transcribed, and the relative expression levels of the resistance-related genes in citrus were detected by qRT-PCR.

[0116] The primers used were as follows:

[0117] qRT-PCR-RLP12-F: 5'-ACGGATTCCTCAACAACTGG-3',

[0118] qRT-PCR-RLP12-R: 5'-TTGTTGCAGCTTCACCACTC-3';

[0119] qRT-PCR-GST1-F: 5'-AGACATGAAGGCTGGTGAAC-3',

[0120] qRT-PCR-GST1-R: 5’-AACTCCTGGGCGATGTATTG-3’;

[0121] qRT-PCR-LRR8-F: 5’-TGCACCAGAGCTAGCCTACA-3’,

[0122] qRT-PCR-LRR8-R: 5’-GATGGATATGGAAGCCGAGA-3’;

[0123] qRT-PCR-PR1-F: 5’-AAATGTGGGTGAATGAGAAAGC-3’,

[0124] qRT-PCR-PR1-R: 5’-ATTATTGTTGCACGTCACCTTG-3’;

[0125] qRT-PCR-PR2-F: 5’-TTCCACTGCCATCGAAACTG-3’,

[0126] qRT-PCR-PR2-R: 5’-GTAATCTTGTTTAAATGAGCCTCTTG-3’;

[0127] qRT-PCR-FRK1-F: 5’-AGTGACGTGTACAGCTTTGG-3’,

[0128] qRT-PCR-FRK1-R: 5’-TCTGATATTTCCCTCGGCAAG-3’.

[0129] According to the qRT-PCR detection results, it was found that compared with wild-type citrus, the genes related to immune response, RLP12, GST1, LRR8, PR1, PR2, and FRK1, were significantly up-regulated in the AbCOMT1 gene overexpressing citrus positive line. The results indicate that after overexpression of the AbCOMT1 gene in citrus, it may enhance the resistance to citrus canker by activating the citrus immune response.

[0130] Other parts not described in detail are prior art. Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A citrus canker resistance regulatory gene AbCOMT1, characterized in that: The CDS sequence of the regulatory gene AbCOMT1 is shown in SEQ ID No.

1.

2. A protein AbCOMT1 encoded by the citrus canker resistance gene AbCOMT1 according to claim 1, characterized in that: The amino acid sequence of the protein AbCOMT1 is shown in SEQ ID No.

2.

3. A primer pair for obtaining the citrus canker resistance gene AbCOMT1 according to claim 1, characterized in that: The primer pair is AbCOMT1-F / R, which are: AbCOMT1 Primer F: 5'-ATGGCAAATTCCAAGCCCAAG-3', AbCOMT1 Primer R: 5′-CATTTGTGAAACTCCATTAC-3′.

4. A citrus AbCOMT1 overexpression vector pK7WG2D-AbCOMT1, characterized in that: The overexpression vector pK7WG2D-AbCOMT1 contains the AbCOMT1 gene according to claim 1.

5. Use of any one of the following in breeding for resistance to citrus canker, characterized in that: (1) The citrus canker resistance regulatory gene AbCOMT1 according to claim 1; (2) The overexpression vector pK7WG2D-AbCOMT1 according to claim 4.

6. Use of any one of the following in breeding new citrus varieties resistant to citrus canker, characterized in that: (1) The citrus canker resistance regulatory gene AbCOMT1 according to claim 1; (2) The overexpression vector pK7WG2D-AbCOMT1 according to claim 4.

Images