CN115820685B

CN115820685B - Citrus CsGSTF1 gene and application thereof

Info

Publication number: CN115820685B
Application number: CN202211004475.9A
Authority: CN
Inventors: ***; 傅佳; 樊捷; 喻奇缘; 杨雯; 黄馨; 张晨希; 线宝航; 何永睿; 陈善春; 姚利晓
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2022-08-22
Filing date: 2022-08-22
Publication date: 2023-09-12
Anticipated expiration: 2042-08-22
Also published as: CN115820685A

Abstract

The application discloses a citrus CsGSTF1 gene and application thereof, and the nucleotide sequence of the gene is SEQ ID NO:1 is shown in the specification; the application method specifically comprises the following steps: (1) cloning a VIGS fragment of the citrus CsGSTF1 gene; (2) constructing an expression vector of VIGS; (3) The VIGS expression vector converts citrus to obtain a VIGS plant with the citrus CsGSTF1 gene silenced. The application reduces the transcription level of the orange CsGSTF1 by the VIGS silencing for the first time, and verifies the function of the CsGSTF1 in the citrus canker resistance; h of the obtained VIGS-silenced plants ₂ O ₂ The content is obviously increased, and the incidence degree of the ulcer disease is obviously reduced.

Description

Citrus CsGSTF1 gene and application thereof

Technical Field

The application relates to the technical field of molecular biology, in particular to a citrus CsGSTF1 gene and application thereof.

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.

The traditional prevention and control means of citrus canker, such as disease tree burning, 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 applied at present because of the capability of directionally and efficiently cultivating 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 strain of late orange 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.

glutathione-S-transferase, which is commonly found in animals, plants and microorganisms, is a superfamily of enzymes encoded by multiple genes and having multiple functions. The GST family is divided into 8 subfamilies, F (Phi), U (Tau), T (Theta), Z (Zeta), L (Lambda), DHAR, EF1bγ and TCHQD, based on the homology and genetic structural characteristics of plant proteins, with the F and U subfamilies being plant-specific, most members and most abundant compared to other subfamilies (Jain et al, 2010). At present, plant GST proteins are successively discovered in plants such as corn, arabidopsis thaliana, soybean, rice, tobacco and the like, and have the functions of detoxification, metabolite transportation, stress regulation, growth regulation, signal transduction and the like in the plants. Although GST regulation has been partially applied in plant disease resistance, there has been no study report in the field of citrus canker.

In view of this, the present application has been made.

Disclosure of Invention

The application provides a citrus CsGSTF1 gene and application thereof in enhancing the resistance of citrus to canker, wherein the citrus CsGSTF1 gene is obtained by cloning for the first time, and the transcription level of the citrus CsGSTF1 is reduced by transferring a VIGS vector of the citrus CsGSTF1 gene into the citrus, so that the resistance of the citrus to canker can be obviously improved, the incidence of canker is reduced, and the phenotype of transgenic plants is not influenced.

The application is realized by the following technical scheme:

a citrus CsGSTF1 gene, wherein the nucleotide sequence of the citrus CsGSTF1 gene is SEQ ID NO:1 or in SEQ ID NO:1 with one or several nucleotides and with the nucleotide sequence encoding the same active protein.

The application also provides an application of the citrus CsGSTF1 gene in enhancing the resistance of citrus to canker, and the application method is to reduce the transcription level of the CsGSTF1 gene in citrus plants.

Further, the means for reducing the transcript level of CsGSTF1 gene in citrus plants employs VIGS silencing.

Further, the application method specifically comprises the following steps:

(1) Cloning a VIGS fragment of a citrus CsGSTF1 gene;

(2) Constructing an expression vector of VIGS;

(3) The VIGS expression vector converts citrus to obtain a VIGS plant with the citrus CsGSTF1 gene silenced.

Further, in the step (1), the cloning method of the VIGS fragment of the citrus CsGSTF1 gene comprises the following steps: 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 citrus CsGSTF1 gene, wherein the nucleotide sequence is SEQ ID NO: 2.

Further, in the step (1), primers used for PCR amplification are CsGSTF1-VIGS-F and CsGSTF1-VIGS-R, and the nucleotide sequences of the primers are SEQ ID NO:3 and SEQ ID NO:4.

further, in the step (2), the construction method of the VIGS gene fragment expression vector comprises the following steps: and (3) carrying out enzyme digestion on the VIGS fragment of the PCR product CsGSTF1 gene obtained in the step (1) by using Xba I and Sma I, recovering the fragment, connecting the fragment with the same digested TRV2 vector, converting competent cells of escherichia coli, and extracting plasmids to obtain the VIGS expression vector of the CsGSTF1 gene.

Further, in the step (3), the method for transforming citrus with the VIGS expression vector comprises the following steps: and (3) transforming agrobacterium with the VIGS expression vector obtained in the step (2), preparing agrobacterium liquid containing the VIGS expression vector, infecting citrus aseptic seedlings, and obtaining the VIGS plant with the citrus CsGSTF1 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 CsGSTF1 gene silencing can enhance citrus canker resistance.

Compared with the prior art, the application has the following advantages and beneficial effects:

1. the glutathione-S-transferase CsGSTF1 gene obtained from the genes differentially expressed before and after the canker of the citrus infection is subjected to down-regulation of the expression of the canker of the citrus in the early infection stage of the disease-resistant variety and up-regulation of the expression of the canker of the citrus in the early infection stage of the disease-resistant variety can reduce H in citrus cells during transient over-expression ₂ O ₂ Content, it shows that CsGSTF1 gene is a disease-sensing gene of citrus canker;

2. according to the citrus CsGSTF1 gene and the application thereof in enhancing the resistance of citrus to canker, provided by the embodiment of the application, through constructing a VIGS carrier of the citrus CsGSTF1 gene, agrobacterium-mediated transformation of citrus is carried out, the obtained citrus plant can show obvious resistance to canker, the disease spot area is reduced by 25%, the disease index is reduced by 21%, and the incidence degree of canker is obviously reduced;

3. according to the citrus CsGSTF1 gene and the application thereof in enhancing the resistance of citrus to canker, the disease degree of citrus canker can be remarkably reduced through the VIGS silencing of the citrus CsGSTF1 gene, and the phenotype of a citrus plant is not affected by the VIGS silencing of the citrus CsGSTF1 gene;

4. the embodiment of the application provides a citrus CsGSTF1 gene and application thereof in enhancing the resistance of citrus to canker, and the gene is prepared by inhibiting glutathione-S-transferase activity and H of a plant with VIGS silence ₂ O ₂ Content detection shows that the gene silencing of the citrus CsGSTF1 can be realized by increasing H ₂ O ₂ The content of the citrus CsGSTF1 silencing plant is further enhanced;

5. the citrus CsGSTF1 gene and the application thereof in enhancing the canker resistance of citrus provided by the embodiment of the application can be used as candidate genes to cooperatively carry out canker resistance breeding with a plurality of canker resistance and disease susceptibility genes by utilizing technologies such as VIGS silencing, RNA interference, gene editing and the like, and have great application value in the canker resistance breeding of citrus.

Drawings

In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present application, 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 application 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.

Fig. 1 is a diagram showing the bioinformatics characteristics of citrus CsGSTF1 according to an embodiment of the present application: chromosome localization of the citrus CsGSTF1 gene; b, gene structure of citrus CsGSTF 1; c, a conserved domain of citrus CsGSTF 1;

FIG. 2 is a diagram showing the induction expression of citrus canker pathogen of citrus CsGSTF1 according to the embodiment of the application: letters on the data columns represent significance of difference (P < 0.05);

FIG. 3 is a diagram showing hormone-induced expression of citrus CsGSTF1 according to the embodiment of the present application: letters on the data columns represent significance of difference (P < 0.05);

FIG. 4 is a diagram of a transient overexpression vector of citrus CsGSTF1 constructed according to the embodiment of the application: GUS: a beta-glucuronidase gene; NPTII, kanamycin resistance gene; 35S, a plant constitutive promoter derived from cauliflower mosaic virus; NOS, opine synthase gene terminator; LB, left homology arm; RB, right homology arm;

FIG. 5 shows the relative expression levels of the citrus CsGSTF1 in the transient over-expression material according to the embodiment of the application: * Difference significance (p=0.05);

FIG. 6 shows GST enzyme activity and H in the transient overexpression material according to the embodiment of the application ₂ O ₂ The content is as follows: a, GST enzyme activity; b, H ₂ O ₂ The content is as follows; pLGNe, material transiently transformed with empty vector; material of pLGNe-GSTF1 for transient transformation of CsGSTF1 over expression vector;

FIG. 7 is a flowchart showing an embodiment of the present application for performing VIGS silencing using the citrus CsGSTF1 gene to increase citrus canker resistance;

FIG. 8 is a diagram of a vector of the citrus CsGSTF1 gene VIGS according to an embodiment of the present application: GFP, green fluorescent protein; rdRp, NA-dependent RNA polymerase; CP, coat protein; 35S, a plant constitutive promoter derived from cauliflower mosaic virus; NOS, opine synthase gene terminator; LB, left homology arm; RB, right homology arm;

FIG. 9 is a PCR detection chart of VIGS plants according to the embodiment of the application: positive control +; negative control; m, molecular weight standard; VIGS plants of 1, csgstf1; TRV1 and TRV2, and detection result diagrams of the two vectors;

FIG. 10 is a qRT-PCR assay of CsGSTF1 expression in VIGS plants according to the examples of the present application: * The difference was significant (p=0.05) (the same applies below); TRV2, plants with empty vector (below); plants transformed with the VIGS vector of CsGSTF1 with TRV2-GSTF1 (hereinafter);

FIG. 11 is a phenotype diagram of a VIGS plant according to an embodiment of the present application: WT, untranslated plants;

FIG. 12 shows the onset of disease after the leaf blades of the VIGS plants of the example of the application are inoculated with Umbelliferae;

FIG. 13 is a chart showing the statistics of the size of lesions after the inoculation of leaf blades of VIGS plants with Umbelliferae according to the embodiment of the present application;

FIG. 14 is a statistical chart of disease index after the leaf blades of the VIGS plants are inoculated with the Umbelliferae bacteria according to the embodiment of the present application;

FIG. 15 shows GST enzyme activity and H activity of VIGS plants according to the embodiment of the application ₂ O ₂ The content is as follows: a, GST enzyme activity; b, H ₂ O ₂ The content is as follows.

Detailed Description

For the purpose of making apparent the objects, technical solutions and advantages of the present application, the present application will be further described in detail with reference to the following examples and the accompanying drawings, wherein the exemplary embodiments of the present application and the descriptions thereof are for illustrating the present application only and are not to be construed as limiting the present application.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one of ordinary skill in the art that: no such specific details are necessary to practice the application. In other instances, well-known materials or methods have not been described in detail in order to avoid obscuring the present application.

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 application. 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 CsGSTF1

As shown in FIG. 1, the citrus CsGSTF1 gene is located between 27945446bp and 27946977bp of the citrus chromosome 3 (FIG. 1A), contains 2 introns and 3 exons, encodes 221 amino acids (FIG. 1B), and contains GST functional domains at the N-and C-termini (FIG. 1C), respectively.

CsGSTF1 gene nucleotide sequence SEQ ID NO:1:

ATGGCAACTCCAGTAAAAGTGTACGGTCCGCCACTCTCTACTGCCGTGTGCAGGGTCGTAGCCTGTCTCCTGGAGAAAGATGTGGAGTTTCAGCTCATTTCCCTCAACATGGCTAAAGGCGATCACAAGAAACCTGATTTTCTGAAGATCCAGCCCTTTGGCCAAGTACCAGCATTTCAGGATGAGAAAATCTCCCTCTTTGAGTCTCGAGCTATATGCCGCTATGTTTGTGAGAATTATCCAGAAAAAGGAAACAAGGGATTATTTGGAACAAATCCGTTGGCAAAAGCTTCAATAGATCAGTGGCTGGAAGCCGAGGGGCAAAGCTTTAACCCGCCAAGCTCTGCTCTAGTGTTTCAACTAGCACTCGCTCCTCGAATGAACATCAAGCAAGACGAAGGAGTAATCAAACAGAATGAAGAAAAGCTGGCAAAAGTGCTCGATGTTTATGAGAAGAGGCTGGGGGAGAGTCGGTTCTTGGCTGGGGATGAATTTTCTTTGGCTGATCTTTCACACTTGCCTAATGCGCATTATTTGGTGAATGCAACTGATAGAGGAGAGATTTTAACTTCCAGGGATAATGTAGGGAGATGGTGGGGTGAGATTTCGAACAGAGATTCATGGAAGAAGGTGGTTGATATGCAGAAACAGCAGCACAGTCCTTGA。

example 2

Expression analysis of citrus CsGSTF1

1. Citrus canker pathogen induced expression of citrus CsGSTF1

In order to verify the relation between the citrus CsGSTF1 and the infection of the citrus canker, the citrus canker is inoculated by injecting in the disease-resistant variety kumquat and the disease-resistant variety night orange, and qRT-PCR primers CsGSTF1-RT-F and CsGSTF1-RT-R are designed in the specific area of the citrus CsGSTF 1.

qRT-PCR reaction conditions: 3min at 95℃and 10s at 95 ℃; 10s at 56 ℃, 10s at 72 ℃ and 40 times of circulation; 72 ℃ for 10min; utilization 2 ^-ΔΔCt The relative expression level of CsGSTF1 gene in the plant was calculated by the method.

The nucleotide sequence of the primer CsGSTF1-RT-F is shown in SEQ ID NO:5 are as follows:

TCTCGAGCTATATGCCGCTATG

primer CsGSTF1-RT-R nucleotide sequence SEQ ID NO:6 are as follows:

TTGGCGGGTTAAAGCTTTGC。

analysis of the inducible expression characteristics of CsGSTF1 infected by canker citri shows that as shown in fig. 2: in the late citrus reticulata, the expression level of CsGSTF1 is obviously increased in the early stage of the infection of the citrus canker, while in the kumquat, the expression level of CsGSTF1 is reduced in the early stage of the infection of the citrus canker. From the above results, it was estimated that CsGSTF1 might be a disease-causing gene of citrus canker, and its expression level was inversely related to canker resistance.

2. Hormone-induced expression of citrus CsGSTF1

To further determine the relevance of CsGSTF1 to citrus canker, the plant biotic stress related hormones Abscisic acid (ABA), jasmonic Acid (JA) and Salicylic Acid (SA) were analyzed for the induced expression of CsGSTF1, as shown in fig. 3, and as a result, it was found that after 3 hormones induced by the late orange and kumquat, csGSTF1 had different expression patterns, indicating that CsGSTF1 responds to the signal transduction pathways of these 3 hormones to different extents, and it was further verified that CsGSTF1 is related to citrus resistance and canker.

3. Transient overexpression of citrus CsGSTF1

To analyze CsGSTF1 in citrus cell H ₂ O ₂ Function in content control as shown in fig. 4, csGSTF1 gene was inserted into pLGNe vector to construct CsGSTF1 over-expression vector pLGNe-GSTF1, and late orange leaf was transiently injected. As shown in FIG. 5, csGSTF1 is proved to be obviously over-expressed in the material which is transiently over-expressed for 5 days, and as can be seen from FIG. 6, the GST enzyme activity of the over-expressed material is obviously increased (FIG. 6A), H ₂ O ₂ The content was significantly reduced (fig. 6B), further indicating that CsGSTF1 gene is a disease-susceptibility gene for citrus canker.

Example 3

Enhancement of citrus canker resistance using citrus CsGSTF1 silencing

As shown in FIG. 7, specific embodiments of the application utilizing CsGSTF1 silencing to increase citrus canker resistance include 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 an 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 the CsGSTF1 gene, the nucleotide sequence of which was SEQ ID NO:2 is as follows:

CTCGCTCCTCGAATGAACATCAAGCAAGACGAAGGAGTAATCAAACAGAATGAAGAAAAGCTGGCAAAAGTGCTCGATGTTTATGAGAAGAGGCTGGGGGAGAGTCGGTTCTTGGCTGGGGATGAATTTTCTTTGGCTGATCTTTCACACTTGCCTAATGCGCATTATTTGGTGAATGCAACTGATAGAGGAGAGATTTTAACTTCCAGGGATAATGTAGGGAGATGGTGGGGTGAGATTTCGAACAGAGATTCATGGAAGAAGGTGGTTGATATGCAGAAACAGCAGCACAGTCCTTGA。

2) VIGS fragment amplification of CsGSTF1 Gene

1.2.1 cloning of the VIGS fragment of the CsGSTF1 gene with a PrimeSTAR master mix (TAKARA) kit, 300bp in length;

1.2.2 PCR amplification with primers CsGSTF1-VIGS-F and CsGSTF 1-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 the primer CsGSTF1-VIGS-F is shown in SEQ ID NO:3 are as follows:

GCTCTAGACTCGCTCCTCGAATGAACATC

the nucleotide sequence of the primer CsGSTF1-VIGS-R is shown in SEQ ID NO:4 are as follows:

GCCCCGGGTCAAGGACTGTGCTGCTG。

construction of the VIGS vector

As shown in fig. 8, the VIGS fragments of the VIGS vectors TRV2 and CsGSTF1 gene are digested with Xba I and Sma I, recovered in gel, and then the two fragments are ligated and transformed into competent cells of escherichia coli, and plasmids are extracted to obtain the VIGS expression vector TRV2-GSTF1; 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: opine synthase gene terminator; LB: a left homology arm; RB: right homology arm. The vector TRV2-GSTF1 is transformed into agrobacterium by an electric shock method to prepare agrobacterium tumefaciens liquid containing the VIGS expression vector.

Vigs vector transformation

1) Agrobacterium activation

500. Mu.L of TRV1 and TRV2, TRV2-GSTF1 agrobacteria liquid are respectively taken and added into 50mL of liquid LB medium (containing a card)Natamycin) at 28℃200 r.min ^-1 Culturing to OD ₆₀₀ =1; the cells were collected and treated with MMA (10 mM MgCl) ₂ 10nM MES, 100. Mu.M acetosyringone) liquid was resuspended and OD adjusted ₆₀₀ =1; mixing TRV1 with TRV2 and TRV2-GSTF1 carrier at volume ratio of 1:1, and incubating at room temperature for 3h.

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 darkness, observing that if green fluorescence is emitted, transferring to soil for culturing, culturing at 25deg.C for 16h/8h, and watering periodically.

Vigs positive identification and phenotypic observation

The seedlings showing green fluorescence were positive seedlings, and then transferred to nutrient soil medium, after one month, tissues were collected to extract DNA and total RNA, and PCR was performed using two pairs of primers CsGSTF1-ID1-F, csGSTF1-ID1-R and CsGSTF1-ID2-F, csGSTF-ID 2-R, to obtain TRV1 and TRV2 vector fragments, respectively, as shown in FIG. 9.

The nucleotide sequence of primer CsGSTF1-ID1-F is SEQ ID NO:7 are as follows:

TTGGGTTGCTACTGATTCGACT

the nucleotide sequence of the primer CsGSTF1-ID1-R is shown in SEQ ID NO:8 are as follows:

CTGTAAGGACCATCATACTTCGC

nucleotide sequence SEQ ID NO of primer CsGSTF1-ID 2-F: 9 are as follows:

CTGCCCGACAACCACTACCT

the nucleotide sequence of primer CsGSTF1-ID2-R is SEQ ID NO:10 is shown below:

CTTGTACAGCTCGTCCATGCC。

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 CsGSTF1-RT-F and CsGSTF1-RT-R to verify that CsGSTF1 was successfully silenced. Setting the gene expression level of the TRV1 and TRV2 empty vector plants as 1, and if the gene expression level of the CsGSTF1 of the plant containing the target fragment vector is less than 1, generating gene silencing. It was identified that CsGSTF1 transcript levels were reduced by 48% as shown in figure 10.

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. 11, 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 CsGSTF1 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 pipette to obtain solution of ulcer bacteria, and spotting 1 μl (1×10) per pinhole ⁵ CFU·mL ^-1 ) Culturing in a constant temperature illumination incubator at 28 ℃ (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.

Calculating disease index according to disease index formula, classifying disease condition into 0-7 according to disease area, and expressing disease area by letter R, wherein 0 grade (R is less than or equal to 0.25 mm) ² ) Grade 1 (0.25 mm) ² ＜R≤0.5mm ² ) Grade 2 (0.5 mm) ² ＜R≤0.75mm ² ) Grade 3 (0.75 mm) ² ＜R≤1mm ² ) Grade 4 (1.0 mm) ² ＜R≤1.25mm ² ) Grade 5 (1.25 mm) ² ＜R≤1.5mm ² ) Grade 6 (1.5 mm) ² ＜R≤1.75mm ² ) Grade 7 (R > 1.75 mm) 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. 12-14, after 10d inoculation with canker bacteria, the VIGS plant symptoms of CsGSTF1 were significantly reduced; the area of the lesion is reduced by 25%; the disease index is reduced by 21 percent. Thus, csGSTF1 gene silencing may enhance citrus canker resistance.

Example 4

Analysis of mechanisms of citrus CsGSTF1 silencing to increase citrus canker resistance

As shown in FIG. 15, GST enzyme activity and H on CsGSTF 1-silenced plants ₂ O ₂ The content is detected, and the GST enzyme activity is obviously reduced, while H is detected ₂ O ₂ The content is obviously increased. H ₂ O ₂ Is an important signal molecule of plants against biological stress, thus it can be seen that CsGSTF1 regulates H ₂ O ₂ The content affects citrus canker resistance.

In conclusion, the application can greatly reduce the disease spot area of citrus canker and lighten the incidence of the canker through silencing CsGSTF 1. The CsGSTF1 gene provided by the application 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 application, and is not meant to limit the scope of the application, but to limit the application to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the application are intended to be included within the scope of the application.

Claims

1. The application of the citrus CsGSTF1 gene in enhancing the resistance of the citrus CsGSTF1 to canker is characterized in that the application method is to reduce the transcription level of the CsGSTF1 gene in the citrus CsGSTF1 gene by adopting a VIGS silencing technology, and the nucleotide sequence of the citrus CsGSTF1 gene is SEQ ID NO: 1.

2. Use of the citrus CsGSTF1 gene according to claim 1 for enhancing the resistance of citrus sinensis to canker, characterized in that the method of application comprises in particular the following steps:

(1) Cloning a VIGS fragment of a citrus CsGSTF1 gene, wherein the nucleotide sequence of the VIGS fragment is SEQ ID NO:2 is shown in the figure;

(2) Constructing an expression vector of the VIGS, wherein the expression vector also comprises green fluorescent protein GFP;

(3) And the VIGS expression vector is used for transforming the late orange to obtain a VIGS plant with the orange CsGSTF1 gene silenced.

3. The use of the citrus CsGSTF1 gene according to claim 2 for enhancing the resistance of citrus CsGSTF1 to canker, wherein in step (1), the cloning method of the VIGS fragment of the citrus CsGSTF1 gene is: extracting total RNA of the citrus aurantium, 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 citrus CsGSTF1 gene.

4. The use of CsGSTF1 gene according to claim 3, wherein in step (1), the primers used for PCR amplification are CsGSTF1-VIGS-F and CsGSTF1-VIGS-R, the nucleotide sequences of which are SEQ ID NOs: 3 and SEQ ID NO:4.

5. the use of the citrus CsGSTF1 gene according to claim 2 for enhancing the resistance of citrus to canker, wherein in step (2), the construction method of the VIGS gene fragment expression vector is as follows: and (3) carrying out enzyme digestion on the VIGS fragment of the PCR product CsGSTF1 gene obtained in the step (1) by using Xba I and Sma I, recovering the fragment, connecting the fragment with the same digested TRV2 vector, converting competent cells of escherichia coli, and extracting plasmids to obtain the VIGS expression vector of the CsGSTF1 gene.

6. The use of the citrus CsGSTF1 gene according to claim 2 for enhancing the resistance of oranges to canker, wherein in step (3), the method for transforming oranges with VIGS expression vector comprises: and (3) transforming agrobacterium with the VIGS expression vector obtained in the step (2), preparing agrobacterium liquid containing the VIGS expression vector, infecting aseptic seedlings of the citrus fruit, and obtaining the VIGS plant with the orange CsGSTF1 gene silenced after fluorescent observation, PCR and qRT-PCR verification.

7. The use of the citrus CsGSTF1 gene according to claim 2, wherein after the VIGS plant is obtained in step (3), the VIGS plant is evaluated for citrus canker resistance, and it is determined that citrus CsGSTF1 gene silencing can enhance the resistance to citrus canker.