Plant expression vector for antimicrobial peptide induced expression and application thereof

Abstract

The invention belongs to the technical field of citrus quality improvement, and particularly relates to a plant expression vector for antimicrobial peptide induced expression and application thereof. The invention aims to provide a new option for improving citrus canker resistance. The technical scheme is an expression cassette for antimicrobial peptide induced expression. The invention is characterized in that the citrus canker pathogen is adopted to efficiently induce the expression of the expression promoter control cecropin antimicrobial peptide. The invention also relates to a plant expression vector containing the expression cassette and a host cell containing the plant expression vector. The invention also relates to a method for enhancing citrus canker resistance. The antimicrobial peptide gene mAATCB under the control of the canker high-efficiency promoter is integrated into the citrus genome, thereby implementing quick high-level expression of the antimicrobial peptide gene when the citrus is infected by the canker pathogen, and further enhancing the citrus canker resistance.

Description

technical field

[0001] The invention belongs to the technical field of citrus quality improvement, and in particular relates to a plant expression vector for inducing the expression of antimicrobial peptides and an application thereof. Background technique

[0002] Citrus canker is a worldwide disease that occurs in almost every citrus producing country in Asia and other regions except the Mediterranean countries. Citrus canker bacteria mainly harm new leaves, twigs and immature fruits. In severe cases, leaves, dead branches and fruits will fall, the tree will weaken, the yield will decrease, and the quality of fruits will deteriorate. Once the epidemic spreads in the main producing areas of citrus, It has a devastating and catastrophic blow to the citrus industry. At present, countries in the world cannot completely eradicate citrus canker disease through medicaments or manual leaf picking, but can only take a series of measures such as eradicating the diseased plants and ...

Examples

Embodiment 1

[0046] Embodiment 1 vector construction

[0047] 1. Acquisition of a specific promoter

[0048] According to the tobacco pathogen-induced promoter PPP1 (GenBank accession number: AF469482.1), primers (SEQ ID No.1, SEQ ID No.2) were designed, and a fragment of about 1300 bp was obtained by PCR amplification from the tobacco genome. Sequencing analysis after cloning the amplified DNA fragment shows that it is a tobacco PPP1 specific promoter, see SEQ ID No.3.

[0049] According to the potato pathogen-induced promoter gst1 (GenBank accession number: J03679.1), primers (SEQ ID No.4, SEQ ID No.5) were designed, and a fragment of about 1600bp was amplified from the potato genome by PCR. Sequencing analysis after cloning the amplified DNA fragment shows that it is the gst1 specific promoter of potato, see SEQ ID No.6.

[0050] According to the tobacco pathogen-induced promoter hsr203J (GenBank accession number: X77136.1), primers (SEQ ID No.7, SEQ ID No.8) were designed, and a frag...

Embodiment 2

[0058] Example 2 Genetic transformation of citrus by the method mediated by Agrobacterium tumefaciens

[0059] The constructed plant expression vector plasmid was introduced into Agrobacterium EHA105 by electric shock method. Referring to the user manual of Bio-RAD MicroPulser, the vector described in Example 1 was introduced into Agrobacterium EHA105 by electroporation.

[0060] The above-mentioned expression vector was introduced into Jincheng through the method of Agrobacterium-mediated epicotyl, and the medium used is shown in Table 1. The specific method is as follows:

[0061] 1. Acquisition of epicotyls from Jincheng seedlings

[0062] Take fresh Jincheng and wash it, disinfect the surface with 70% alcohol, take out the seeds under aseptic conditions, peel off the seed coat, inoculate on MS solid medium, culture in dark at 28°C for 2 weeks, and then in 16h / d Cultured under photoperiod for 1 week. The epicotyls of germinated seedlings were taken and cut into stem seg...

Embodiment 3

[0074] Example 3 PCR detection of exogenous gene integration

[0075] Take 100 mg of transgenic citrus leaves, use Aidlab company kit (cat.No.DN15) to extract genomic DNA, and detect the integration of antimicrobial peptide gene mAATCB by PCR. The reaction volume was 25 μL, and the reaction conditions were: 94°C for 3 minutes; 30 cycles of 94°C for 30s, 58°C for 30s, and 72°C for 30s; 72°C for 10 minutes. For detection primers, see SEQ ID No.11 and SEQ ID No.12. The detection target fragment is about 100bp mAATCB antimicrobial peptide gene. Such as Figure 4 As shown, a specific fragment of the mAATCB antimicrobial peptide gene was detected in the genome of the transgenic plant, indicating that the antimicrobial peptide gene had been successfully integrated into the citrus genome.

technical field

[0001] The invention belongs to the technical field of citrus quality improvement, and in particular relates to a plant expression vector for inducing the expression of antimicrobial peptides and an application thereof. Background technique

[0002] Citrus canker is a worldwide disease that occurs in almost every citrus producing country in Asia and other regions except the Mediterranean countries. Citrus canker bacteria mainly harm new leaves, twigs and immature fruits. In severe cases, leaves, dead branches and fruits will fall, the tree will weaken, the yield will decrease, and the quality of fruits will deteriorate. Once the epidemic spreads in the main producing areas of citrus, It has a devastating and catastrophic blow to the citrus industry. At present, countries in the world cannot completely eradicate citrus canker disease through medicaments or manual leaf picking, but can only take a series of measures such as eradicating the diseased plants and ...

Examples

Embodiment 1

[0046] Embodiment 1 vector construction

[0047] 1. Acquisition of a specific promoter

[0048] According to the tobacco pathogen-induced promoter PPP1 (GenBank accession number: AF469482.1), primers (SEQ ID No.1, SEQ ID No.2) were designed, and a fragment of about 1300 bp was obtained by PCR amplification from the tobacco genome. Sequencing analysis after cloning the amplified DNA fragment shows that it is a tobacco PPP1 specific promoter, see SEQ ID No.3.

[0049] According to the potato pathogen-induced promoter gst1 (GenBank accession number: J03679.1), primers (SEQ ID No.4, SEQ ID No.5) were designed, and a fragment of about 1600bp was amplified from the potato genome by PCR. Sequencing analysis after cloning the amplified DNA fragment shows that it is the gst1 specific promoter of potato, see SEQ ID No.6.

[0050] According to the tobacco pathogen-induced promoter hsr203J (GenBank accession number: X77136.1), primers (SEQ ID No.7, SEQ ID No.8) were designed, and a frag...

Embodiment 2

[0058] Example 2 Genetic transformation of citrus by the method mediated by Agrobacterium tumefaciens

[0059] The constructed plant expression vector plasmid was introduced into Agrobacterium EHA105 by electric shock method. Referring to the user manual of Bio-RAD MicroPulser, the vector described in Example 1 was introduced into Agrobacterium EHA105 by electroporation.

[0060] The above-mentioned expression vector was introduced into Jincheng through the method of Agrobacterium-mediated epicotyl, and the medium used is shown in Table 1. The specific method is as follows:

[0061] 1. Acquisition of epicotyls from Jincheng seedlings

[0062] Take fresh Jincheng and wash it, disinfect the surface with 70% alcohol, take out the seeds under aseptic conditions, peel off the seed coat, inoculate on MS solid medium, culture in dark at 28°C for 2 weeks, and then in 16h / d Cultured under photoperiod for 1 week. The epicotyls of germinated seedlings were taken and cut into stem seg...

Embodiment 3

[0074] Example 3 PCR detection of exogenous gene integration

[0075] Take 100 mg of transgenic citrus leaves, use Aidlab company kit (cat.No.DN15) to extract genomic DNA, and detect the integration of antimicrobial peptide gene mAATCB by PCR. The reaction volume was 25 μL, and the reaction conditions were: 94°C for 3 minutes; 30 cycles of 94°C for 30s, 58°C for 30s, and 72°C for 30s; 72°C for 10 minutes. For detection primers, see SEQ ID No.11 and SEQ ID No.12. The detection target fragment is about 100bp mAATCB antimicrobial peptide gene. Such as Figure 4 As shown, a specific fragment of the mAATCB antimicrobial peptide gene was detected in the genome of the transgenic plant, indicating that the antimicrobial peptide gene had been successfully integrated into the citrus genome.

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