CN110862975A

CN110862975A - Citrus pectin acetyl esterase CsPAE and coding gene and application thereof

Info

Publication number: CN110862975A
Application number: CN201911308648.4A
Authority: CN
Inventors: 李强; 胡安华; 祁静静; 何永睿; 陈善春
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2019-12-18
Filing date: 2019-12-18
Publication date: 2020-03-06
Anticipated expiration: 2039-12-18
Also published as: CN110862975B

Abstract

The invention discloses citrus pectin acetyl esterase CsPAE and a coding gene and application thereof. The invention discloses a citrus susceptibility protein CsPAE shown as SEQ ID NO.1 and a coding frame nucleic acid sequence thereof shown as SEQ ID NO. 2. The gene belongs to a member of citrus pectin acetyl esterase family. The invention also provides a breeding method for resisting bacterial canker of citrus, which comprises the following steps: and reducing the content and/or activity of CsPAE protein in the target plant by using an RNAi mode, so that the disease resistance of the target citrus to citrus canker is improved. The method has great application value for breeding the citrus canker resistant gene, lays a foundation for breeding the citrus canker resistant gene, and greatly promotes the development and application of the citrus canker resistant gene engineering.

Description

Citrus pectin acetyl esterase CsPAE and coding gene and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to citrus pectin acetyl esterase CsPAE and a coding gene and application thereof.

Background

Citrus canker (CBC), caused by the Xanthomonas citri subsp. citri, severely hampers the healthy development of the Citrus industry (fennel et al, 2015). At present, more than 30 countries and regions around the world list citrus canker as an important plant quarantine disease. The first generation citrus has been long-term crossbred in disease-resistant breeding, but the breeding efficiency is very low due to the long crossbred period. Breeding experts have made various attempts in anti-ulcer studies by means of genetic engineering (Yongjie et al, 2016; Girard et al, 2017) to obtain transgenic materials resistant to ulcer diseases. For example, the strains of the malorange, the Xinhui orange and the navel orange which are transferred with the tussah silkworm antibacterial peptide D gene are obtained in Chenoponchun et al (Chenoponchun et al, 1996); li obtains a CsBZIP transcription factor-transformed citrus variety (Li et al, 2019); peng et al performs CRISPR/Cas9 targeted knockout on a citrus canker susceptibility gene CsLOB1 promoter so as to obtain a plant with improved resistance to citrus canker, and the like. However, at present, candidate genes which can be used for breeding ulcer disease molecules are limited, multi-gene synergy for resisting ulcer disease is difficult to achieve, and more effective candidate genes are urgently needed.

The plant cell wall is the first physical barrier against infection by pathogenic bacteria. Wherein pectin is the main component. Acetylation of pectin changes the physicochemical properties of the pectin components, affecting cell adhesion and thus the cell wall structure. The degree of Pectin acetylation is regulated and controlled by Pectin Acetylesterase (PAE), and PAE has the function of cutting acetyl ester bond to deacetylate Pectin. PAEs have been reported in the literature to be involved in plant growth, development and propagation processes, fruit firmness changes, and plant defense against biotic and abiotic stresses (Gou et al, 2012; Bonnin et al, 2008; orifia et al, 2012). Pogorelko et al found that transgenic Arabidopsis thaliana and brachypodium distachyon overexpressing the nidulans Aspergillus nidulans PAE had higher resistance to pathogenic bacteria (Pogorelko et al, 2013); the Arabidopsis PAE 7T-DNA insertion mutant was found to be more sensitive to Phytophthora sojae (Yangyuan et al, 2016). Therefore, PAEs are possible pathogenic bacteria related genes and participate in the process of resisting pathogenic attack of plants. The PAEs gene is utilized to carry out disease-resistant molecular breeding, and has great potential. At present, no research and application for improving the resistance of citrus to citrus bacterial canker by using PAEs is available.

Disclosure of Invention

In order to solve the technical problems, the invention discloses a citrus pectin acetyl esterase CsPAE and a coding gene and application thereof, and provides a new choice for improving the resistance of citrus canker.

The invention is realized by the following technical scheme:

the citrus pectin acetylesterase CsPAE, the CsPAE protein is (a1) or (a2) or (a3) or (a 4):

(a1) protein with amino acid sequence shown as SEQ ID No. 1;

(a2) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1);

(a3) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a1) and related to citrus canker;

(a4) a protein derived from citrus, having 98% or more identity to (a1) and associated with citrus canker.

The coding gene of the citrus pectin acetylesterase CsPAE, the coding gene of the CsPAE protein is (b1) or (b 2):

(b1) has a nucleotide sequence shown as SEQ ID No. 2;

(b2) a nucleotide sequence derived from citrus and having 95% or more identity to (b1) and encoding said PAE-like protein.

The application of the citrus pectin acetyl esterase CsPAE and the coding gene thereof in improving the resistance of citrus canker diseases is realized, and the resistance of citrus plants to the citrus canker diseases is improved by reducing the content and/or activity of CsPAE protein in the citrus plants.

Further, a way to reduce the content and/or activity of CsPAE protein in citrus is RNAi interfering with expression.

The step of reducing the content and/or activity of CsPAE protein in the citrus plant comprises:

(1) cloning a citrus CsPAE gene fragment sequence, then constructing an RNAi expression vector, and transforming agrobacterium;

(2) and (3) transforming the citrus by the RNAi expression vector to obtain a transgenic plant with improved canker resistance.

The cloned citrus CsPAE gene fragment is used as an RNAi fragment, and the RNAi fragment is shown as SEQ ID No. 3.

Wherein, in the step (1), the primers adopted for cloning the CsPAE gene segment are R-CsPAE-F and R-CsPAE-R, and respectively have the sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5.

Further, in the step (1), the cloning method of the citrus CsPAE gene fragment comprises the following steps: extracting total citrus RNA, then carrying out reverse transcription to obtain cDNA, and finally adopting high-fidelity enzyme PCR to clone a CsPAE gene segment.

Further, in the step (1), the method for constructing the interference expression vector comprises the following steps: designing specific primers (R-CsPAE-F) respectively containing XbaI and SwaI enzyme cutting sites and protective bases at the upstream according to the CsPAE coding sequence, respectively containing BamHI and AscI enzyme cutting sites and protective bases at the downstream (R-CsPAE-R), performing PCR amplification by taking citrus cDNA as a template to obtain interference fragments, recovering the product, dividing the product into two groups, performing double enzyme digestion on the first group by SwaI and AscI, performing double enzyme digestion on the second group by BamHI and XbaI, simultaneously connecting the two groups of fragments recovered by enzyme digestion to corresponding parts of a pUCr interference vector to obtain an intermediate vector pUCr-CsPAE-RNAi for sequencing verification, then, the correctly sequenced product and the pLGNe carrier are subjected to double enzyme digestion by Kpn I and SalI respectively, and the enzyme digestion product containing the interference fragment is connected to the pLGNe expression vector to construct a final interference expression vector pLGNe-CsPAE-RNAi.

Further, in the step (1), the pLGNe vector has a CaMV 35S promoter and a GUS coding gene, the 35S promoter is a cauliflower mosaic virus promoter, and GUS is used for transgenic plant detection and has SEQ ID NO: 6 and SEQ ID NO: 7.

Further, in the step (2), the method for transforming citrus by interfering with the expression vector comprises the following steps: the interference expression vector is transformed into agrobacterium by an electric shock method, then the agrobacterium is used for mediating and transforming the citrus explant, and the explant cell after genetic transformation is subjected to in vitro culture, dyeing identification and grafting to obtain a transgenic plant.

Further, after the transgenic plant is obtained in the step (2), resistance evaluation is carried out on the transgenic plant, and the relevance of CsPAE gene silencing and citrus canker is judged.

Further, before resistance evaluation is carried out on the transgenic plant, the transgenic plant is verified through PCR, and the adopted primers are ID-CsPAE-F and ID-CsPAE-R which respectively have the sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9.

Further, after PCR verification, verifying the transgenic plant again by using qRT-PCR, wherein the adopted primers are RT-CsPAE-F and RT-CsPAE-R which respectively have the nucleotide sequences shown as SEQ ID NO: 10 and SEQ ID NO: 11. The qRT-PCR internal reference is a citrus Actin gene, and primers are RT-CsActin-F and RT-CsActin-R which respectively have the sequences shown in SEQ ID NO: 12 and SEQ ID NO: 13, or a nucleotide sequence as set forth in seq id no.

The invention obtains a PAE gene which is induced and reduced by citrus canker in a disease-resistant variety according to genes which are differentially expressed before and after citrus is infected with bacterial canker, the expression of the PAE gene in a disease-sensitive variety is up-regulated, an RNAi expression vector is constructed, and the obvious resistance to the citrus canker is expressed after the citrus is transformed through agrobacterium-mediated transformation.

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

the citrus pectin acetyl esterase CsPAE, the coding gene and the application thereof are the application of CsPAE protein and the coding gene thereof in improving the resistance of citrus canker, the coding sequence of the citrus CsPAE gene is cloned, RNAi expression vector construction is carried out, agrobacterium is transformed, and then citrus is transformed, the scab area of the obtained transgenic plant canker can be reduced to 63% of that of the existing citrus to the maximum extent, the morbidity degree of the canker can be obviously reduced, the phenotype of the citrus is not influenced, so the silent expression CsPAE is obtained, the resistance of the citrus to the canker can be greatly improved, and the molecular breeding can be carried out in cooperation with other disease-resistant genes or disease-susceptible genes in the future.

Drawings

The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:

FIG. 1 is a schematic diagram of the domain of the CsPAE protein of the present invention: tm & signal peptide, signal peptide and transmembrane domain; PAE: a pectin acetyl esterase domain; the website used for the alignment was PFAM.

FIG. 2 is a schematic diagram of the sequence and secondary structure of the CsPAE protein of the present invention, including helix, α helix structure and arrow, β fold structure.

FIG. 3 is an analysis of the evolutionary tree of CsPAE protein and Arabidopsis PAEs of the present invention: the evolutionary tree is constructed for maximum likelihood, using software MEGA7, and the numbers on the branches represent the cluster support rate.

FIG. 4 is an expression diagram of CsPAE protein induced by canker pathogen infection.

FIG. 5 is a flow chart of the present invention for using CsPAE silencing to increase citrus resistance to canker diseases.

FIG. 6 is a flow chart of construction of RNAi expression vector of CsPAE plant of the present invention, GUS, &lTtTtransformation = beta "&gTtbeta&lTt/T &gTt-glucoronidase gene, 35S, plant constitutive promoter derived from cauliflower mosaic virus, NOS, opine synthase gene terminator, and vector pLGNe has GUS gene under the control of CaMV 35S promoter, which is convenient for GUS staining screening of transformants in the process of plant genetic transformation.

FIG. 7 is a final structural diagram of the RNAi expression vector of CsPAE plant of the present invention, including GUS, &. lTtT translation = β "&. gTtbeta&. lTtTtTtTtgTt-glucoronidase gene, CaMV 35S, a plant constitutive promoter derived from cauliflower mosaic virus, NOS, opine synthase gene terminator, and a vector pLGNe having a GUS gene under the control of CaMV 35S promoter.

FIG. 8 is a GUS staining map of transgenic plants of the present invention.

FIG. 9 is a PCR assay of transgenic plants of the invention.

FIG. 10 is a phenotype of transgenic plants of the present invention.

FIG. 11 is a CsPAE expression analysis chart in transgenic plants of the present invention: indicates that the difference between them was significant compared to the wild type (P ═ 0.01), (the same below).

FIG. 12 is a statistical plot of lesion sizes of transgenic citrus leaves according to the present invention.

FIG. 13 is a statistical chart of the degree of disease (disease index) of transgenic citrus leaves according to the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

In the embodiment of the invention, the late orange is taken as a test object, and in practical application, the method can be used for improving the resistance of other citrus varieties to canker.

Example 1

Clonal informatics analysis of citrus CsPAE

Total RNA from citrus (late orange) leaves was extracted using an RNA extraction kit (Elder Lei, CAT: RN09), the RNA quality was verified by agarose gel electrophoresis, and the concentration was measured using a densitometer. cDNA (TAKARA) was synthesized using Recombinant DNase I and stored at-20 ℃ until needed. The CsPAE coding cassette (SEQ ID No.2) was obtained by amplifying CsPAE from the above cDNA using the primers CL-CsPAE-F (SEQ ID No.14) and CL-CsPAE-R (SEQ ID No.15) and sequencing.

The domain of the CsPAE protein was analyzed using PFAM website and found to have a typical pectin acetyl esterase domain and to contain N pieces of signal peptide and transmembrane domain (fig. 1). secondary structure has 11 α helices and 9 β folds (fig. 2). the software MEGA7 was used to construct the maximum likelihood tree of CsPAE protein and arabidopsis PAEs, which was found to be closely related to AtPAE7 and AtPAE8 (fig. 3).

Example 2

Canker pathogen infection induction expression of citrus CsPAE

1. Material treatment

Citrus leaves were Xcc treated according to the method of Huanhua et al (Huanhua et al, 2019). Briefly described as follows: collecting mature leaves of the late orange and the Chinese holly, wiping the mature leaves with 75% ethanol, washing the leaves with sterile water to remove residual ethanol, and placing the leaves in a culture dish; inoculating ulcer bacteria liquid (OD600 ═ 0.5) to the lower epidermis by injection method, and taking the leaf inoculated with culture medium only as control; and culturing at 28 deg.C for 0, 12, 24, 36 and 48 hr, and cutting to obtain lesion.

RNA extraction, cDNA Synthesis and qRT-PCR

RNA of the above-described material was extracted with an RNA extraction kit (Idelay, CAT: RN 09). Synthesis of cDNA (TAKARA) using Recombinant DNase I.

The detection primers are RT-CsPAE-F (SEQ ID No.10) and RT-CsPAE-R (SEQ ID No. 11); the detection primers of the reference gene Actin are RT-CsActin-F (SEQ ID No.12) and RT-CsActin-F (SEQ ID No. 13). Reaction volume 20 μ L, reaction conditions: 95 ℃ for 3min and 94 ℃ for 10 s; at 56 ℃ for 10s, at 72 ℃ for 10s, and for 40 cycles; 10min at 72 ℃. The experiment was repeated three times. By using 2^-△△CtCalculating the relative expression quantity of the CsPAE gene in the citrus plant by the method: the result shows that CsPAE is induced to be up-regulated and expressed by canker bacteria in late orange which is sensitive to canker, and is induced to be down-regulated by canker bacteria in citrus fruit which is resistant to canker (figure 4), so that the CsPAE is inferred to be possible canker disease genes. Silencing expression thereof may enhance resistance of citrus to canker.

Example 3

CsPAE silencing is used to enhance resistance of citrus to canker. The important application of the gene is to enhance the resistance of citrus to canker by silencing, and the specific implementation steps are shown in figure 5.

PCR amplification and recovery of CsPAE gene fragment

The CsPAE fragment (SEQ ID No.3) was obtained from the amplification of citrus cDNA using primers R-CsPAE-F (SEQ ID No.4) and R-CsPAE-F (SEQ ID No.5), and the fragment length was 300 bp. The PCR kit used was PrimeSTAR mastermix (Takara Shuzo). An amplification system: 10X PCR mix: 2.5 mu L; primer RNAi-CsPAE-F (5. mu. mol/L): 1 mu L of the solution; primer RNAi-CsPAE-R (5. mu. mol)L): 1 mu L of the solution; about 60ng of cDNA; add ddH₂O to 25. mu.L. And (3) amplification procedure: 94 ℃ for 5 min; 94 ℃, 30s, 56 ℃, 30s, 72 ℃, 1.5min, 35 cycles; extension at 72 ℃ for 10 min. The agarose gel block containing the desired fragment was cut with a clean blade under an ultraviolet lamp. The recovery method was carried out with reference to the instructions of the kit (Edley) and the fragments recovered were quantified on a concentration tester.

2. Construction of CsPAE interference expression vector and transformation of Agrobacterium

Vector construction flow diagram as in FIG. 6, all restriction enzymes were purchased from (THERMO) and operated according to the instructions. The specific operation is as follows: after the recovery of the PCR product of the CsPAE gene fragment, the PCR product is divided into two groups, the first group is subjected to double enzyme digestion by SwaI and AscI, the second group is subjected to double enzyme digestion by BamHI and XbaI, the two groups of fragments recovered by enzyme digestion are simultaneously connected to the corresponding parts of a pUCr interference vector to obtain an intermediate vector pUCr-CsPAE-RNAi for sequencing verification, then the product with correct sequencing and a pLGNe vector are subjected to double enzyme digestion by KpnI and SalI respectively, and the enzyme digestion product containing the interference fragment is connected to a pLGNe expression vector to construct a final interference expression vector pLGNe-CsPAE-RNAi (figure 7). Plasmid extraction was performed using a kit (Edley).

The constructed RNAi vector was introduced into Agrobacterium tumefaciens EHA105 by electric stimulation. Frozen EHA105 Agrobacterium competent cells (50. mu.L) were taken in advance and thawed on ice; adding 2 μ L of the constructed RNAi vector into competent cells, blowing, mixing, and standing on ice for 5 min. The procedure was performed according to the instrument instructions. Adding 1mL of LB liquid culture medium into an electric shock cup, blowing and uniformly mixing by a liquid transfer gun, transferring into a sterile centrifuge tube, and carrying out shaking culture on a shaking table at 28 ℃ for 40min at 260 r/min; 10000r/min for 1min, discarding the supernatant (about 100. mu.L of the resuspended thallus), after resuspension, using a pipette to spray on LK solid medium (the expression vector contains kanamycin resistance), evenly spreading, and performing inversion dark culture at 28 ℃ for 2 days. After the bacterial plaque grows out, single bacterial colony is picked up to LK liquid culture medium and shaken on a constant temperature shaking bed (28 ℃) overnight.

3. Genetically transformed citrus (late golden orange)

Obtaining an epicotyl of the citrus seedling: cleaning fresh Mandarin orange (Citrus reticulata Blanco), sterilizing with 70% ethanol, taking out seed under aseptic condition, peeling off seed coat, inoculating on seed germination culture medium for germination, culturing at 28 deg.C in dark for 2 weeks, and culturing in 16h light/8 h dark photoperiod for 1 week. Taking the epicotyl of the germinated seedling under aseptic condition, cutting the epicotyl into stem segments of about 1cm, and using the stem segments for genetic transformation of the agrobacterium tumefaciens.

Preparation of agrobacterium tumefaciens: the agrobacterium liquid (containing CsPAE interference expression vector) for transfection is added with 30% of sterile glycerol and stored in an ultra-low temperature incubator at-70 ℃. Before transfection, the cells were streaked on LK solid medium containing 50mg/L kanamycin. A single colony of Agrobacterium was picked, inoculated into 25ml of LK broth containing the same antibiotic, and shake-cultured overnight at 28 ℃. The next day, the bacterial solution diluted to OD 0.1 after concentration measurement was shaken twice, and after 3 hours, when the bacterial solution was in logarithmic phase (OD 0.5 or so), it was centrifuged at 5000r/min for 10min, the supernatant was discarded, and it was resuspended in MS liquid medium of pH 5.4 for transfection.

Transformation of citrus epicotyl stem segments: soaking the epicotyl segment of Citrus (Citrus aurantium) cut to about 1cm in Agrobacterium for 13min while slightly shaking. Taking out the stem section and then sucking the bacteria liquid on the surface; the stem sections were transferred to co-cultivation medium and cultured at 26 ℃ for 2 days. After co-cultivation was completed, the epicotyls were transferred to selection medium and cultured in dark at 28 ℃ for 7d, and explants were cultured at 28 ℃ for 16h light/8 h dark, subcultured every two weeks. When the seedlings grow to be more than 1cm, cutting off the seedlings, grafting the cut seedlings to sterile test tube late golden orange seedlings, and culturing in a seedling culture medium; grafting the seedlings onto the immature bitter orange seedlings when the seedlings grow to about 5cm, and culturing in a greenhouse.

The media used were as follows:

seed germination culture medium: MS +30g/L sucrose +2.5g/L Gelrite, pH 5.8.

Co-culture medium: MS +2mg/L BA +0.5mg/L IAA +1 mg/L2, 4-D + 100. mu. mol AS +30g/L sucrose +2.5g/L Gelrite, pH 5.8.

Screening a culture medium: MS +2mg/L BA +0.5mg/L IAA +500mg/L Cef +50mg/L Kan +30g/L sucrose +2.5g/L Gelrite, pH 5.8.

Seedling culture medium: MS +30g/L sucrose, pH 5.8.

4. Transgenic plant validation

The transgenic plant is verified by GUS staining, PCR detection and qRT-PCR modes, and the specific method comprises the following steps:

GUS staining identification: the leaves of the transgenic plants were cut into leaf disks with a diameter of 7mm, GUS histochemical staining was carried out, the edges of the leaf disks of the positive plants appeared blue, and the leaf disks of the wild type plants did not appear color (FIG. 8).

And (3) PCR detection: when the plant leaves grow large enough, 100mg of the plant leaves obtained by primary screening are taken, genomic DNA is extracted by using an Edley reagent kit (CAT: DN15), and the integration of the CsPAE interference fragment is detected by PCR. And (3) PCR reaction conditions: 3min at 94 ℃; 30 cycles of 94 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 30 s; 10min at 72 ℃. The detection primers are ID-CsPAE-F (SEQ ID No.8) and ID-CsPAE-R (SEQ ID No. 9). The positive plants can obtain 1454bp amplified fragment without amplification of the control plants (FIG. 9).

qRT-PCR analysis: transgenic citrus leaves were extracted, total RNA from leaves was extracted using an EASYspin plant RNA rapid extraction kit (Eldella, CAT No: RN09), and cDNA (Takara) was synthesized using Recombinant DNase I. The detection primers of the target gene are RT-CsPAE-F (SEQ ID No.10) and RT-CsPAE-R (SEQ ID No. 11); the detection primers of the reference gene Actin are RT-CsActin-F (SEQ ID No.12) and RT-CsActin-R (SEQ ID No. 13). Reaction volume 20 μ L, reaction conditions: 95 ℃ for 3min and 94 ℃ for 10 s; at 56 ℃ for 10s, at 72 ℃ for 10s, and for 40 cycles; 10min at 72 ℃. The experiment was repeated three times. By using 2^-△△CtCalculating the relative expression quantity of the CsPAE gene in the transgenic plant by the method: defining the water-treated sample as a reference factor, i.e., the CsPAE expression level is 1, and calculating the multiple 2 of the gene expression in transgenic citrus relative to the reference factor^-△△CtThe relative expression amount is shown. The results are shown in FIG. 10. The result shows that the CsPAE gene is greatly silenced in transgenic plants compared with wild plants, and the expression level is up to 13% of that of the existing citrus.

5. Phenotypic observation of transgenic plants

The phenotype of 3 transgenic plants is observed and analyzed, and no obvious difference in appearance is found. Because the transgenic plant obtained by the invention is grafted, is influenced by factors such as affinity difference with the rootstock or wound healing and the like to a greater or lesser extent, and the development time is shorter at present, the invention considers that the not very obvious difference in the development aspect is completely negligible, the phenotype is not changed with the wild type control, and the interference expression CsPAE gene does not generate direct and obvious change on the phenotype and development of the plant (figure 11).

6. Evaluation of resistance of transgenic plants

Collecting mature leaves, cleaning, disinfecting with 75% alcohol, washing in ultrapure water, and placing in a super clean bench; needling with vein as center; using a pipette to sample the liquid of the ulcer disease, wherein each pinhole samples 1 mu L (1X 10)⁵CFU/mL). Culturing in a constant-temperature light incubator at 28 ℃ (16h light/8 h dark). After the leaf is inoculated, the leaf is cultured for 10 days for photographing, and the size (mm) of the lesion is counted by using Image J V1.47 software²). The degree of onset (disease index) is calculated according to the disease index formula. Dividing the disease condition into 0-7 grades according to the lesion area, and using letter R to represent the lesion area, 0 grade (R is less than or equal to 0.25 mm)²) Class 1 (0.25 mm)²＜R≤0.5mm²) Class 2 (0.5 mm)²＜R≤0.75mm²) Class 3 (0.75 mm)²＜R≤1mm²) Class 4 (1.0 mm)²＜R≤1.25mm²) Grade 5 (1.25 mm)²＜R≤1.5mm²) Class 6 (1.5 mm)²＜R≤1.75mm²) Class 7 (R > 1.75 mm)²) (ii) a Calculating the degree of disease according to the formula: DI is 100X Σ (the number of disease spots at each stage X corresponds to the number of stages) (the total number of disease spots X is the maximum stage). After inoculating canker pathogen for 10 days, after counting the size of lesion spots on the citrus leaves and disease index, the area (figure 12) and the disease degree (figure 13) of the lesion spots on the transgenic plant leaves are obviously smaller than those of wild citrus, especially R2, the lesion spots are only 62.6% of the wild citrus leaves, and the disease index is 63.1% of the wild citrus leaves.

Therefore, the silencing of CsPAE can reduce the lesion area of the ulcer to a certain extent and reduce the incidence degree of the ulcer. The gene can be independently used for breeding disease-resistant molecules, and can also be used for breeding disease resistance together with other disease-resistant or disease-susceptible genes.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> university of southwest

<120> citrus pectin acetyl esterase CsPAE and coding gene and application thereof

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Ala Gln Ala Cys Phe Asn Val Ser Ile Thr Tyr Val Glu Asp Ala Val

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Ala Lys Gly Ala Val Cys Leu Asp Gly Ser Pro Pro Ala Tyr His Phe

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Asp Lys Gly Phe Gly Ala Gly Ile Asn Asn Trp Leu Val His Ile Asp

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Gly Gly Ala Trp Cys Asn Asn Val Ala Asp Cys Ser Asn Arg Arg Asp

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Ser Ser His Gly Ser Ser Lys Tyr Met Val Lys Glu Ala Asn Phe Thr

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Gly Ile Leu Ser Asn Glu Gln Lys Phe Asn Pro Asp Phe Tyr Asp Trp

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Asn Arg Val Lys Val Arg Tyr Cys Asp Gly Ala Ser Phe Thr Gly Asp

115 120 125

Val Glu Ala Val Asn Pro Glu Thr Asn Leu His Phe Arg Gly Ala Arg

130 135 140

Val Phe Glu Ala Val Met Glu Asp Leu Leu Ala Lys Gly Met Lys Asn

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Ala Gln Asn Ala Ile Leu Thr Gly Cys Ser Ala Gly Gly Leu Thr Ser

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Ile Leu His Cys Asp Asn Phe Arg Ala Leu Phe Pro Val Asp Thr Arg

180 185 190

Val Lys Cys Phe Ala Asp Ala Gly Tyr Phe Val Asn Val Lys Gly Val

195 200 205

Ser Gly Ala Ser His Ile Glu Glu Phe Tyr Lys Gln Val Val Ala Leu

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His Gly Ser Ala Lys His Leu Pro Ala Ser Cys Thr Ser Arg Leu Ser

225 230 235 240

Pro Gly Leu Cys Phe Phe Pro Gln Asn Val Ala Gly Gln Val Lys Thr

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Pro Leu Phe Ile Ile Asn Ser Ala Tyr Asp Ser Trp Gln Ile Arg Asn

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Ile Leu Val Pro Asp Asn Ala Asp Pro Lys Gly Thr Trp Arg Arg Cys

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Lys Lys Asp Ile Lys Thr Cys Ser Ser Thr Gln Leu Gln Thr Met Gln

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Gly Phe Arg Val Gln Phe Leu Asn Ala Leu Ala Gly Leu Gly Asn Ser

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Ser Ser Arg Gly Met Leu Ile Asp Ser Cys Tyr Thr His Cys Arg Thr

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Val Phe Gln Glu Ala Trp Leu Ser Ala Asp Ser Pro Val Leu Asp Lys

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Thr Pro Ile Ala Lys Ala Val Gly Asp Trp Tyr Tyr Asp Arg Asn Pro

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gtgcacattg atggaggagc atggtgtaac aatgtcgcag attgttctaa ccgaagggat 240

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tattttgtga atgtgaaggg tgtttctgga gcaagtcaca ttgaggagtt ttataaacaa 660

gtggtcgcat tacatggatc ggctaagcat ttgccagcat cctgcacttc gagattgagt 720

ccaggattgt gtttcttccc acaaaacgta gcagggcaag tcaagacacc gctatttatc 780

atcaactcag catacgattc atggcagata aggaacattt tggttccgga caatgctgat 840

ccgaaaggaa cttggaggcg ctgcaagaag gatataaaaa cgtgctcatc tactcagctg 900

caaactatgc aaggttttag ggtgcaattc ctgaatgcat tggccggact tggcaactct 960

tcatccagag gaatgcttat agactcttgt tatactcact gccgaaccgt gtttcaggag 1020

gcttggttga gtgctgactc tccggtgctg gataaaacgc ctattgcaaa ggcagtggga 1080

gactggtatt acgacaggaa tccattccaa aagattgatt gcccttaccc ctgcaaccca 1140

ttgccagaga gttgcttttg a 1161

<210>3

<211>300

<212>DNA

<213> Artificial sequence (3)

<400>3

aatgagcaga aatttaaccc agacttctac gattggaata gagtcaaggt tagatactgc 60

gatggggcat cgtttactgg agacgtagag gctgtcaatc cagaaactaa ccttcacttc 120

agaggagcaa gggtttttga agctgtcatg gaggatctat tggctaaagg aatgaaaaat 180

gctcaaaatg ctatacttac tggttgttcc gctgggggtt tgacttcgat tttgcattgc 240

gataacttcc gagctctctt tcctgttgat actagagtaa aatgctttgc agatgctggc 300

<210>4

<211>37

<212>DNA

<213> Artificial sequence (4)

<400>4

gctctagagg cgcgccaatg agcagaaatt taaccca 37

<210>5

<211>37

<212>DNA

<213> Artificial sequence (5)

<400>5

cgggatccat ttaaatgcca gcatctgcaa agcattt 37

<210>6

<211>345

<212>DNA

<213> Artificial sequence (6)

<400>6

gagacttttc aacaaagggt aatatccgga aacctcctcg gattccattg cccagctatc 60

tgtcacttta ttgtgaagat agtggaaaag gaaggtggct cctacaaatg ccatcattgc 120

gataaaggaa aggccatcgt tgaagatgcc tctgccgaca gtggtcccaa agatggaccc 180

ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg 240

gattgatgtg atatctccac tgacgtaagg gatgacgcac aatcccacta tccttcgcaa 300

gacccttcct ctatataagg aagttcattt catttggaga gaaca 345

<210>7

<211>1812

<212>DNA

<213> Artificial sequence (7)

<400>7

atgttacgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg cctgtgggca 60

ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag cgcgttacaa 120

gaaagccggg caattgctgt gccaggcagt tttaacgatc agttcgccga tgcagatatt 180

cgtaattatg cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa aggttgggca 240

ggccagcgta tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt gtgggtcaat 300

aatcaggaag tgatggagca tcagggcggc tatacgccat ttgaagccga tgtcacgccg 360

tatgttattg ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga actgaactgg 420

cagactatcc cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa gcagtcttac 480

ttccatgatt tctttaacta tgccggaatc catcgcagcg taatgctcta caccacgccg 540

aacacctggg tggacgatat caccgtggtg acgcatgtcg cgcaagactg taaccacgcg 600

tctgttgact ggcaggtggt ggccaatggt gatgtcagcg ttgaactgcg tgatgcggat 660

caacaggtgg ttgcaactgg acaaggcact agcgggactt tgcaagtggt gaatccgcac 720

ctctggcaac cgggtgaagg ttatctctat gaactgtgcg tcacagccaa aagccagaca 780

gagtgtgata tctacccgct tcgcgtcggc atccggtcag tggcagtgaa gggcgaacag 840

ttcctgatta accacaaacc gttctacttt actggctttg gtcgtcatga agatgcggac 900

ttgcgtggca aaggattcga taacgtgctg atggtgcacg accacgcatt aatggactgg 960

attggggcca actcctaccg tacctcgcat tacccttacg ctgaagagat gctcgactgg 1020

gcagatgaac atggcatcgt ggtgattgat gaaactgctg ctgtcggctt taacctctct 1080

ttaggcattg gtttcgaagc gggcaacaag ccgaaagaac tgtacagcga agaggcagtc 1140

aacggggaaa ctcagcaagc gcacttacag gcgattaaag agctgatagc gcgtgacaaa 1200

aaccacccaa gcgtggtgat gtggagtatt gccaacgaac cggatacccg tccgcaaggt 1260

gcacgggaat atttcgcgcc actggcggaa gcaacgcgta aactcgaccc gacgcgtccg 1320

atcacctgcg tcaatgtaat gttctgcgac gctcacaccg ataccatcag cgatctcttt 1380

gatgtgctgt gcctgaaccg ttattacgga tggtatgtcc aaagcggcga tttggaaacg 1440

gcagagaagg tactggaaaa agaacttctg gcctggcagg agaaactgca tcagccgatt 1500

atcatcaccg aatacggcgt ggatacgtta gccgggctgc actcaatgta caccgacatg 1560

tggagtgaag agtatcagtg tgcatggctg gatatgtatc accgcgtctt tgatcgcgtc 1620

agcgccgtcg tcggtgaaca ggtatggaat ttcgccgatt ttgcgacctc gcaaggcata1680

ttgcgcgttg gcggtaacaa gaaagggatc ttcactcgcg accgcaaacc gaagtcggcg 1740

gcttttctgc tgcaaaaacg ctggactggc atgaacttcg gtgaaaaacc gcagcaggga 1800

ggcaaacaat ga 1812

<210>8

<211>27

<212>DNA

<213> Artificial sequence (8)

<400>8

tgcagatgct ggcatttaaa tgtgtaa 27

<210>9

<211>28

<212>DNA

<213> Artificial sequence (9)

<400>9

ctacgaccgg gatccaaata cctgcaaa 28

<210>10

<211>25

<212>DNA

<213> Artificial sequence (10)

<400>10

actcttcatc cagaggaatg tttgt 25

<210>11

<211>20

<212>DNA

<213> Artificial sequence (11)

<400>11

caccggagag tccgcactaa 20

<210>12

<211>18

<212>DNA

<213> Artificial sequence (12)

<400>12

catccctcag caccttcc 18

<210>13

<211>19

<212>DNA

<213> Artificial sequence (13)

<400>13

ccaaccttag cacttctcc 19

<210>14

<211>23

<212>DNA

<213> Artificial sequence (14)

<400>14

atgggccaat ggttcaatct ttt 23

<210>15

<211>23

<212>DNA

<213> Artificial sequence (15)

<400>15

tcaaaagcaa ctctctggca atg 23

Claims

1. Citrus pectin acetylesterase CsPAE, characterized in that the CsPAE protein is (a1) or (a2) or (a3) or (a 4):

(a1) protein with amino acid sequence shown as SEQ ID No. 1;

2. The gene encoding a citrus pectin acetylesterase CsPAE according to claim 1, wherein the CsPAE protein encoding gene is (b1) or (b 2):

(b1) has a nucleotide sequence shown as SEQ ID No. 2;

3. The application of the citrus pectin acetyl esterase CsPAE and the coding gene thereof in improving the resistance of citrus canker is characterized in that the content and/or activity of CsPAE protein in citrus plants are reduced, so that the resistance of the citrus plants to the citrus canker is improved.

4. The use of the citrus pectin acetylesterase CsPAE and the gene coding therefor according to claim 3 for increasing resistance to citrus canker, characterized in that the means for reducing the CsPAE protein content and/or activity in citrus is RNAi-mediated expression.

5. The use of the citrus pectin acetylesterase CsPAE and the gene coding therefor according to claim 4 for increasing the resistance to citrus canker, characterized in that the CsPAE protein content and/or activity in citrus is reduced by: cloning a citrus CsPAE gene segment as an RNAi segment, wherein the RNAi segment is shown as SEQ ID No.3, then constructing an RNAi expression vector, and transforming the RNAi expression vector into the citrus after transforming agrobacterium.

6. The application of the citrus pectin acetylesterase CsPAE and the coding gene thereof in improving the resistance of citrus canker, according to claim 5, characterized in that primers adopted for cloning CsPAE gene segments are R-CsPAE-F and R-CsPAE-R, and have the nucleotide sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5.