CN117363629B - Citrus CsGATA gene and method for enhancing citrus canker resistance by using same - Google Patents
Citrus CsGATA gene and method for enhancing citrus canker resistance by using same Download PDFInfo
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Abstract
The invention discloses a citrus CsGATA gene and a method for enhancing citrus canker resistance thereof, which relate to the technical field of plant disease resistance molecular breeding, and the nucleotide sequence of the citrus CsGATA gene is SEQ ID NO:1, a nucleotide sequence shown in 1; the method specifically comprises the following steps: (1) cloning a VIGS fragment of the citrus CsGATA gene; (2) constructing an expression vector of VIGS; (3) The VIGS vector converts citrus to yield a VIGS plant with the citrus CsGATA gene silenced. The invention clones the citrus CsGATA gene for the first time, reduces the transcription level of citrus CsGATA by using VIGS silencing, greatly enhances the resistance to canker of citrus, remarkably reduces the incidence of canker, verifies the function of CsGATA in the canker resistance of citrus, and has great application value in the breeding of canker resistance of citrus.
Description
Technical Field
The invention relates to the technical field of plant disease-resistant molecular breeding, in particular to a citrus CsGATA gene and a method for enhancing citrus canker resistance by using the same.
Background
Citrus canker (Citrus bacterial canker, CBC) is a bacterial disease caused by xanthomonas carpet grass subspecies citrus (Xanthomonas citri subsp. Citri, xcc) that jeopardizes most current main-cultivated citrus varieties. Therefore, strengthening the research on prevention and control of citrus canker is an urgent need for the development of the citrus industry. Traditional prevention and control means of citrus canker, such as disease tree incineration, pesticide use and the like, require a great deal of manpower and material resources, and cause huge environmental hazards. Therefore, the prevention and treatment of citrus canker is more expected to be used for breeding new disease-resistant germplasm.
Molecular breeding is rapidly developed and widely used at present because of the capability of directionally and efficiently breeding new disease-resistant germplasm. In recent years, some citrus resources for resisting canker, such as orange, xinhui orange and navel orange strains transformed with tussah antibacterial peptide D genes, have been obtained through biotechnology means; a late brocade orange strain overexpressing CsBZIP 40; and editing the citrus canker gene CsLOB1 promoter at a fixed point to obtain plants with improved resistance to citrus canker. However, the candidate genes with high quality are still deficient, and the research on functions and action mechanisms is not deep, so that more genes closely related to citrus canker are purposefully excavated, and the functions and mechanisms of the candidate genes are deeply analyzed and used for molecular breeding of the anti-canker.
Transcription factor GATA is a group of transcription regulatory factors involved in plant growth, development and metabolism, named because of its ability to bind to the W-GATA-R (w=t/a, r=g/a) sequence on the target gene promoter (Shi et al 2022). In recent years, the understanding of the regulation and biological effects of plant GATA has been greatly improved. Members of the GATA family in plants are involved in photomorphogenetic growth, chloroplast development, chlorophyll biosynthesis, root leaf and flower development, regulation of stomata formation and photosynthesis, and the like (SCHWECHHEIMER ET al., 2022). For example, pdGATA19 can directly activate the promoter of the pdnrt2.4b gene (a high affinity nitrate transporter) in poplar, significantly increasing nitrate uptake at low nitrate supply levels in poplar, playing An important role in regulating and increasing nitrogen utilization efficiency and growth (An et al 2020). Wheat TaGATA1 can enhance dormancy of wheat seeds by directly modulating TaABI5 expression, thereby increasing spike resistance (Wei et al 2023). GATA family members also play an important role in plant abiotic stress. For example, osGATA16 improves the cold tolerance of young rice plants by inhibiting some genes associated with cold, such as OsWRKY45-1, osSRFP, osCYL and OsMYB30, as active modulators of cold tolerance (Zhang et al, 2021). GATA family members also play a critical role in plant biotic stress. For example, taGATA.sup.1 transcriptional activators positively regulate the defense response of wheat against banded sclerotial blight by binding to and activating some of the jasmonate signal-mediated defense genes (Liu et al 2020). Although GATA transcription factor regulation has been partially applied in plant disease resistance, no related studies have been reported in the field of citrus canker.
In view of this, the present application has been made.
Disclosure of Invention
The invention aims to solve the technical problem of insufficient resistance of the existing citrus canker, and aims to provide a citrus CsGATA gene and a method for enhancing the resistance of the citrus canker, so as to solve the problem of insufficient resistance of the existing citrus canker.
The invention is realized by the following technical scheme:
In one aspect, the application provides a citrus CsGATA gene, the nucleotide sequence of the citrus CsGATA gene is SEQ ID NO: 1.
In another aspect, the application provides a method for enhancing citrus canker resistance by using a citrus CsGATA gene, reducing the transcription level of CsGATA gene in citrus, wherein the nucleotide sequence of the citrus CsGATA gene is SEQ ID NO: 1.
Further, the transcript level of CsGATA gene in citrus is reduced by means of VIGS silencing.
Further, the method for enhancing citrus canker resistance by citrus CsGATA gene comprises the following steps:
step 1: cloning a VIGS fragment of the citrus CsGATA gene;
step 2: constructing a CsGATA VIGS vector;
step 3: csGATA 12A vector of VIGS converts citrus to obtain a VIGS plant with the gene of citrus CsGATA silenced.
Further, in step 1, the cloning method of the VIGS fragment of the citrus CsGATA gene is as follows: extracting total RNA of citrus, reversely transcribing the total RNA into cDNA, and carrying out PCR amplification by using high-fidelity enzyme by taking the cDNA as a template to obtain the VIGS fragment of the citrus CsGATA gene.
Further, the nucleotide sequence of the VIGS fragment is SEQ ID NO: 2.
Further, in step 1, the primers used for PCR amplification are CsGATA-VIGS-F and CsGATA-VIGS-R, and the nucleotide sequences of the primers are SEQ ID NOs: 3 and SEQ ID NO:4.
Further, in step 2, the construction method of the VIGS gene fragment expression vector comprises the following steps: and (3) carrying out digestion on the VIGS fragment of the citrus CsGATA gene obtained in the step (1) by EcoR I and Sal I, recovering, connecting with the same digested TRV2 vector, transforming competent cells of escherichia coli, and extracting plasmids to obtain the VIGS vector of the CsGATA gene.
Further, in step 3, the method for transforming citrus with VIGS vector comprises the following steps: and (3) transforming the VIGS vector obtained in the step (2) into agrobacterium, preparing agrobacterium liquid containing the VIGS vector, infecting aseptic citrus seedlings, and obtaining a VIGS plant with the citrus CsGATA gene silenced after fluorescent observation, PCR and qRT-PCR verification.
Further, after the VIGS plant is obtained in the step 3, citrus canker resistance evaluation is performed on the VIGS plant, and it is determined that citrus CsGATA gene silencing can enhance citrus canker resistance.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) According to the citrus CsGATA gene provided by the invention, the early-stage downregulation degree of the citrus canker infection in the disease-resistant variety obtained from the genes differentially expressed before and after the citrus canker infection is continuously increased, and the early-stage downregulation degree of the citrus canker infection in the disease-resistant variety is firstly increased and then reduced to the degree when induction starts, so that the CsGATA gene can be presumed to be a disease-sensitive gene of the citrus canker;
(2) According to the method for enhancing the citrus canker resistance by silencing the citrus CsGATA gene, provided by the invention, the citrus plant obtained by constructing the VIGS carrier of the citrus CsGATA gene and carrying out agrobacterium-mediated transformation on citrus can show obvious resistance to canker, the disease spot area is reduced by 52.0% at most, the disease index is reduced by 56.5%, and the disease degree of canker is obviously reduced;
(3) According to the method for enhancing the citrus canker resistance by silencing the citrus CsGATA gene, the disease degree of the citrus canker can be remarkably reduced by silencing the citrus CsGATA gene VIGS, and the phenotype of a citrus plant is not affected by silencing the citrus CsGATA gene VIGS;
(4) The method for enhancing the canker resistance of the citrus by silencing the citrus CsGATA gene provided by the invention can be used for carrying out canker resistance breeding by using the candidate gene and a plurality of canker resistance and disease-sensing genes in cooperation with the technologies of VIGS silencing, RNA interference, gene editing and the like, and has great application value in the canker resistance breeding of the citrus.
Drawings
In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the drawings that are needed in the examples will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and that other related drawings may be obtained from these drawings without inventive effort for a person skilled in the art. In the drawings:
FIG. 1 is a diagram showing the bioinformatics and the induction expression of citrus canker pathogens of example citrus CsGATA according to the present invention: chromosome localization of the a-citrus CsGATA gene; the genetic structure of B-citrus CsGATA; a conserved domain of C-citrus CsGATA; d-citrus canker pathogen induced expression of citrus CsGATA, data source transcriptome sequencing;
FIG. 2 is a diagram of a transient over-expression vector for constructing citrus CsGATA according to an example of the present invention: GUS- -beta-glucuronidase gene; NPTII, kanamycin resistance gene; 35S-a plant constitutive promoter derived from cauliflower mosaic virus; NOS-nopaline synthase gene terminator; LB-left homology arm; RB-right homology arm;
FIG. 3 is a graph showing the relative expression levels of citrus CsGATA12 in the transient over-expression material according to the example of the present invention; pLGNe-material of the transiently empty carrier (vide infra); pLGNe-CsGATA 12-material of the overexpression vector of transient CsGATA (vide infra);
FIG. 4 is a graph showing the evaluation of resistance of the transient over-expression material inoculated with Umbelliferae bacteria according to example CsGATA of the present invention: a-ulcerous disease onset symptoms; b-area of ulcer lesions; c-ulcerative disease index;
FIG. 5 is a flowchart showing an embodiment of the present invention for performing VIGS silencing using the citrus CsGATA gene to increase citrus canker resistance;
FIG. 6 is a diagram of a vector of the citrus CsGATA gene VIGS according to an embodiment of the present invention: GFP-green fluorescent protein; rdRp-NA dependent RNA polymerase; CP-coat protein; 35S-a plant constitutive promoter derived from cauliflower mosaic virus; NOS-nopaline synthase gene terminator; LB-left homology arm; RB-right homology arm;
FIG. 7 is a PCR detection chart of VIGS plants according to the embodiment of the invention: positive control +; negative control; m-molecular weight standard; plants of TRV 2-empty vector (below); plants of the VIGS vector TRV 2-CsGATA-CsGATA (supra);
FIG. 8 is a graph showing qRT-PCR detection of CsGATA expression in VIGS plants according to the example of the present invention;
FIG. 9 is a phenotype diagram of a VIGS plant according to an embodiment of the present invention;
FIG. 10 is a graph showing the evaluation of resistance of the leaf blades of VIGS plants of example CsGATA of the present invention after inoculation with Umbelliferae bacteria: a-ulcerous disease onset symptoms; b-area of ulcer lesions; c-ulcer disease index.
Detailed Description
For the purpose of making apparent the objects, technical solutions and advantages of the present invention, the present invention will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present invention and the descriptions thereof are for illustrating the present invention only and are not to be construed as limiting the present invention.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that: no such specific details are necessary to practice the invention. In other instances, well-known materials or methods have not been described in detail in order to avoid obscuring the present invention.
Throughout the specification, references to "one embodiment," "an embodiment," "one example," or "an example" mean: a particular feature, structure, or characteristic described in connection with the embodiment or example is included within at least one embodiment of the invention. Thus, the appearances of the phrases "in one embodiment," "in an example," or "in an example" in various places throughout this specification are not necessarily all referring to the same embodiment or example. Furthermore, the particular features, structures, or characteristics may be combined in any suitable combination and/or sub-combination in one or more embodiments or examples. Moreover, those of ordinary skill in the art will appreciate that the illustrations provided herein are for illustrative purposes and that the illustrations are not necessarily drawn to scale. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Example 1
Bioinformatics analysis of citrus CsGATA12
As shown in FIG. 1, the gene CsGATA of citrus is located between 6189073bp and 6191751bp of chromosome 4 of citrus (FIG. 1A), contains 2 introns and 3 exons (FIG. 1B), encodes 319 amino acids, and contains a GATA functional domain at the C-terminus (FIG. 1C). Wherein, csGATA gene nucleotide sequence SEQ ID NO:1 is as follows:
ATGGCTCCAATGTATTTGAACCCAGCACAGGACCACTCTGATCCTTTTCGACTTGCG GAGGCGCAAAAACGTGATCATCAACGTTTGCAGGTATTACACTCTTCATCACATAATCGACCTGCTGCTTCTGTTTCTCGCCCTATTTTTCTTAACTCATCAACTCAAGATCAAGGAATGATCAAATTGGAAGGATCACAGCAACATGATCAAAAGATTGATCAGCGCATAGCTGGCGGTGGATCAAGCGATCTTCAATCTTCAATGTCTCAACCTAAGACGATGACCAATAAACTGGCCATTCGTAGAAGGGAAGTGGGAGAAGGAAGCACAAGCGACAACAGTTCATATACATCATCATCATCAGGCGAATCTATGTCCTCCAAAATGAGGCTAGCGAATAAAATAATCAACTCATCATCGGTTAGCACAGGAACCCATGATGAATCAGTAAAAGTTGCAGAAAAGCTTCTGCATGAGCATGACAATATCGAAGTCCATTATTTTACTACGAACAGTAGTAACAGCAACAACACCGTTAGAATCTGCTCAGACTGCAACACGACCACAACACCCCTTTGGAGAAGCGGTCCTCGTGGTCCCAAGTCGCTGTGCAACGCTTGTGGCATTCGGCAGAGGAAGGCGAGAAAGGCTATGCAAGCAGCAGCTGAAAGTGGCACCACAACGGCCAAGGACAATTCTTCATTTTCGAAGATTAAGTTGCAAAACAATATGGAAAAGAAACCGCGGACGAGCCATGTGGCTCAGTACAAGAAAGTACAGTGCAACACTCCTGATCCTGATCCTCCTCACCATGAGTATCGGAGTCAAAGAAAGCTTTGTTTCAAAGATTTTGCTTTAAGTTTGAGCAGTAATTCTGCTCTTAAACAAGTGTTTCCACGGGATGTTGAAGAAGCTGCCATCCTGCTAATGGAACTTTCTTGTGGTTTTAGTCATACTTAG.
Example 2
Analysis of citrus canker pathogen induced expression of citrus CsGATA12
To verify the relationship between citrus CsGATA and canker pathogen infection, RNA-seq was performed by injecting and inoculating canker pathogen into kumquat, a disease resistant variety, and orange, a disease susceptible variety, evening primrose, and extracting mRNA at different time points. Analysis CsGATA of the induced expression profile of infection by citrus canker pathogen as shown in figure 1D: the early stage downregulation degree of the disease-resistant varieties infected by the citrus canker is continuously increased, and the early stage downregulation degree of the disease-resistant varieties is firstly increased and then reduced to the degree at the beginning of induction. From the above results, csGATA was estimated that the possible disease gene of citrus canker was a disease gene whose expression level was inversely related to canker resistance.
Example 3
Transient overexpression analysis of citrus CsGATA12
To analyze the effect of CsGATA on ulcerative disease, a transient over-expression analysis was performed. As shown in FIG. 2, csGATA gene was inserted into pLGNe vector to construct CsGATA over-expression vector pLGNe-CsGATA12, and the late orange leaf was injected. qRT-PCR was performed using primers CsGATA-RT-F and CsGATA-RT-R to verify whether CsGATA was successfully overexpressed. It was verified that CsGATA a12 was significantly overexpressed in the transient over-expression material, as shown in figure 3. The development of the ulcer disease of the over-expression material is obviously increased (shown in figure 4A), the area of the disease spots is increased (shown in figure 4B), and the disease index is increased (shown in figure 4C). The above results indicate that CsGATA gene is a disease-causing gene for citrus canker.
The nucleotide sequence of primer CsGATA-RT-F is SEQ ID NO:5 are as follows:
AACTCATCAACTCAAGATCAAGGA。
The nucleotide sequence of primer CsGATA-RT-R is SEQ ID NO:6 are as follows:
CCAGCTATGCGCTGATCAAT。
qRT-PCR reaction conditions: 3min at 95℃and 10s at 95 ℃; 10s at 56 ℃, 10s at 72 ℃ and 40 times of circulation; and at 72℃for 10min.
Example 4
Use of citrus CsGATA to silence to improve citrus canker resistance
Given that CsGATA gene is a disease-susceptibility gene for citrus canker, we increased canker resistance by CsGATA silencing. As shown in FIG. 5, the specific implementation of the present invention using CsGATA silencing to increase citrus canker resistance includes cloning of the VIGS fragment, vector construction, citrus transformation, positive identification and resistance evaluation.
Cloning of the VIGS fragment
1) RNA extraction and cDNA Synthesis
Total RNA of the evening primrose orange was extracted with RNA extraction kit (Edley, CAT: RN 09) and cDNA was synthesized by reverse transcription using Recombinant DNase I (TAKARA) to obtain a VIGS fragment of CsGATA gene, the nucleotide sequence of which was SEQ ID NO:2 is as follows:
CAACGGCCAAGGACAATTCTTCATTTTCGAAGATTAAGTTGCAAAACAATATGGAAAAGAAACCGCGGACGAGCCATGTGGCTCAGTACAAGAAAGTACAGTGCAACACTCCTGATCCTGATCCTCCTCACCATGAGTATCGGAGTCAAAGAAAGCTTTGTTTCAAAGATTTTGCTTTAAGTTTGAGCAGTAATTCTGCTCTTAAACAAGTGTTTCCACGGGATGTTGAAGAAGCTGCCATCCTGCTAATGGAACTTTCTTGTGGTTTTAGTCATACTTAG.
2) VIGS fragment amplification of CsGATA Gene
1.2.1 Cloning of the VIGS fragment of the CsGATA gene with PRIMESTAR MASTER mix (TAKARA) kit, length 281bp;
1.2.2 PCR amplification with primers CsGATA-VIGS-F and CsGATA-VIGS-R;
1.2.3 cutting agarose gel containing the desired fragment, and recovering RNAi fragment using DNA gel recovery kit (Edley).
PCR reaction conditions: 98 ℃ for 5min;98 ℃,30s,56 ℃,30s,72 ℃,1.5min,35 cycles; extending at 72℃for 10min.
The nucleotide sequence of primer CsGATA-VIGS-F is SEQ ID NO:3 are as follows:
GAATTCCAACGGCCAAGGACAATT。
The nucleotide sequence of primer CsGATA-VIGS-R is SEQ ID NO:4 are as follows:
GAGCTCGTCGACTCATAAAGTGA。
Construction of the VIGS vector
As shown in FIG. 6, the VIGS fragments of the genes TRV2 and CsGATA of the VIGS vector were digested with EcoR I and Sal I, recovered in gel, and then the two fragments were ligated and transformed into competent cells of E.coli to extract plasmids, thereby obtaining the VIGS vector TRV 2-CsGATA; wherein, GFP: green fluorescent protein; rdRp: NA-dependent RNA polymerase; CP: a coat protein; 35S: a plant constitutive promoter derived from cauliflower mosaic virus; and (2) NOS: nopaline synthase gene terminators; LB: a left homology arm; RB: right homology arm. The vector TRV2-CsGATA12 is transformed into agrobacterium by a freeze thawing method to prepare agrobacterium liquid containing the VIGS vector.
Vigs vector transformation
1) Agrobacterium activation
Respectively taking 500 mu L of TRV1, TRV2 and TRV2-CsGATA of agrobacterium tumefaciens liquid, adding the TRV1 and the TRV2-CsGATA of agrobacterium tumefaciens liquid into 50mL of liquid LB culture medium (containing kanamycin), and culturing at 28 ℃ for 200 r.min -1 until OD 600 =1; the cells were collected, resuspended in MMA (10 mM MgCl 2, 10nM MES, 100. Mu.M acetosyringone) liquid and OD 600 = 0.8 was adjusted; mixing TRV1 with TRV2 and TRV2-CsGATA12 respectively according to volume ratio of 1:1, and culturing at room temperature for 3 hr.
2) Agrobacterium infection
Immersing sterile seedlings with radicle length of 3cm into agrobacterium tumefaciens bacteria liquid, and vacuumizing for 1min by a vacuum pump; washing 3-5 times with sterile water, inserting into seed culture medium, culturing for 2-3 d at room temperature in dark, observing that if green fluorescence is emitted, transferring into soil for culturing, culturing at 25deg.C for 16h in light/8 h in dark, and watering periodically.
Vigs positive identification and phenotypic observation
The seedlings showing green fluorescence under ultraviolet light are positive seedlings, then the seedlings are transferred to nutrient soil culture medium, after one month, tissues are collected to extract DNA and total RNA, and PCR verification is carried out by using primer pairs TRV1-F/R, TRV-F/R and TRV2-F/TRV 2-CsGATA-R respectively, and the results are shown in FIG. 7: the primer pair TRV1-F/R, TRV2-F/R can be respectively amplified in TRV2 and TRV2-CsGATA plants to obtain a 150bp band; the primer pair TRV2-F/TRV 2-CsGATA-R had no amplified band in the TRV2 plant, while the 447bp band was amplified in the TRV2-CsGATA plant.
Nucleotide sequence SEQ ID NO of primer TRV 1-F: 7 are as follows:
TTGGGTTGCTACTGATTCGACT。
Nucleotide sequence SEQ ID NO of primer TRV 1-R: 8 are as follows:
CTGTAAGGACCATCATACTTCGC。
Nucleotide sequence SEQ ID NO of primer TRV 2-F: 9 are as follows:
ATTCACTGGGAGATGATACGCT。
Nucleotide sequence SEQ ID NO of primer TRV 2-R: 10 is shown below:
GAATCTAAGTCCACTCGTCCGT。
the nucleotide sequence of the primer TRV 2-CsGATA-R is SEQ ID NO:11 are as follows:
CTAAGTATGACTAAAACCACAAGAAAG。
PCR reaction conditions: 94 ℃ for 3min;94℃for 30s,58℃for 30s,72℃for 30s,30 cycles; and at 72℃for 10min.
QRT-PCR was performed using primers CsGATA-RT-F and CsGATA-RT-R to verify that CsGATA was successfully silenced. Setting the gene expression level of the TRV1 and TRV2 empty vector plants to be 1, and if the gene expression level of the plant CsGATA containing the target fragment vector is less than 1, then gene silencing occurs. It was identified that transcript levels of CsGATA in 3 groups CsGATA of 12-silenced plants were reduced by 60.0%, 63.5% and 70.0%, respectively, as shown in fig. 8.
QRT-PCR reaction conditions: 3min at 95℃and 10s at 95 ℃; 10s at 56 ℃, 10s at 72 ℃ and 40 times of circulation; and at 72℃for 10min.
As shown in FIG. 9, the phenotype of the positive plants after fluorescent observation, PCR identification and qRT-PCR identification is not obviously different from that of the wild type, which indicates that the silencing of CsGATA does not adversely affect the growth and development of citrus.
5. Resistance evaluation
The evaluation of the canker resistance of the VIGS plants is carried out by an in vitro needling method, and the specific operation is as follows:
Collecting mature leaves, cleaning, sterilizing with 75% alcohol, and washing with ultrapure water; needling with vein as center, spotting with a pipette to obtain 1 μl (1× 5CFU·mL-1) of ulcer bacteria liquid, and culturing in a constant temperature illumination incubator at 28deg.C (16 h illumination/8 h darkness); leaf spot was cultivated for 10d to photograph after the leaf spot was inoculated, and the size of the lesion was counted with Image J V1.47.
The disease index is calculated according to the disease index formula, the disease is divided into 0-7 grades according to the disease area, the disease area is expressed by letter R, grade 0 (R is less than or equal to 0.5mm 2), grade 1 (0.5 mm 2＜R≤1.0mm2), grade 2 (1.0 mm 2＜R≤1.5mm2), grade 3 (1.5 mm 2＜R≤2.0mm2), grade 4 (2.0 mm 2＜R≤2.5mm2), grade 5 (2.5 mm 2＜R≤3.0mm2), grade 6 (3.0 mm 2＜R≤3.5mm2), grade 7 (R is more than 3.5mm 2). Calculating the morbidity degree according to the formula: di=100×Σ [ number of lesions at each stage×number of corresponding stage ]/(total number of lesions×maximum number of stages).
As shown in FIG. 10, the symptoms of the VIGS plants of CsGATA.sup.12 were significantly reduced after 10d inoculation with the Umbelliferae bacteria; the area of the lesion is reduced by 52.0% at maximum; the disease index is reduced by 56.5 percent at maximum. Thus, csGATA gene silencing can enhance citrus canker resistance.
In conclusion, the invention can greatly reduce the area of the disease spots of the citrus canker and lighten the incidence of the canker through the silencing of CsGATA. The CsGATA gene provided by the invention can be silenced by various technologies, can be used for molecular breeding of canker resistance together with other disease resistance or disease susceptibility genes, and has great application value in the molecular breeding of canker resistance of oranges.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (8)
1. A method for enhancing citrus canker resistance by using a citrus CsGATA gene, wherein the transcription level of the CsGATA gene in citrus is reduced, and wherein the nucleotide sequence of the citrus CsGATA gene is SEQ ID NO: 1.
2. A method of enhancing citrus canker resistance with the CsGATA gene according to claim 1, wherein the transcript level of the CsGATA gene in citrus is reduced by VIGS silencing.
3. A method for enhancing citrus canker resistance with the CsGATA gene as claimed in claim 1, wherein the method comprises the steps of:
step 1: cloning a VIGS fragment of the citrus CsGATA gene;
step 2: constructing a CsGATA VIGS vector;
step 3: csGATA 12A vector of VIGS converts citrus to obtain a VIGS plant with the gene of citrus CsGATA silenced.
4. A method for enhancing citrus canker resistance by citrus CsGATA gene according to claim 3, wherein in step 1, the VIGS fragment of the citrus CsGATA gene is cloned by: extracting total RNA of citrus, reversely transcribing the total RNA into cDNA, and carrying out PCR amplification by using high-fidelity enzyme by taking the cDNA as a template to obtain a VIGS fragment of the CsGATA gene of citrus;
the nucleotide sequence of the VIGS fragment is SEQ ID NO: 2.
5. A method for enhancing citrus canker resistance by citrus CsGATA gene according to claim 3, wherein in step 1, primers used for PCR amplification are CsGATA-VIGS-F and CsGATA-VIGS-R, the nucleotide sequences of which are SEQ ID NOs: 3 and SEQ ID NO:4.
6. A method for enhancing citrus canker resistance by citrus CsGATA gene according to claim 3, wherein in step 2, the VIGS gene fragment expression vector is constructed by: and (3) carrying out digestion on the VIGS fragment of the citrus CsGATA gene obtained in the step (1) by EcoR I and Sal I, recovering, connecting with the same digested TRV2 vector, transforming competent cells of escherichia coli, and extracting plasmids to obtain the VIGS vector of the CsGATA gene.
7. A method for enhancing citrus canker resistance by citrus CsGATA gene according to claim 3, wherein in step 3, the VIGS vector is transformed into citrus fruit by: and (3) transforming the VIGS vector obtained in the step (2) into agrobacterium, preparing agrobacterium liquid containing the VIGS vector, infecting aseptic citrus seedlings, and obtaining a VIGS plant with the citrus CsGATA gene silenced after fluorescent observation, PCR and qRT-PCR verification.
8. A method of enhancing citrus canker resistance as claimed in claim 3, wherein after obtaining VIGS plants in step 3, evaluation of citrus canker resistance is performed on VIGS plants, and it is determined that citrus CsGATA gene silencing can enhance citrus canker resistance.
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