Plant immune activator protein PlAvh23 secreted by peronophythora litchii and application of plant immune activator protein PlAvh23

A litchi downy mildew and immune activation technology, applied in the biological field, can solve problems such as economic loss, impact on litchi yield and quality, disease incidence, etc., and achieve the effect of reducing infection

Active Publication Date: 2022-05-20
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Litchi frost blight often causes a large number of flower and fruit drop, seriously affecting the yield and quality of litchi
In addition, e...

Method used

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  • Plant immune activator protein PlAvh23 secreted by peronophythora litchii and application of plant immune activator protein PlAvh23
  • Plant immune activator protein PlAvh23 secreted by peronophythora litchii and application of plant immune activator protein PlAvh23
  • Plant immune activator protein PlAvh23 secreted by peronophythora litchii and application of plant immune activator protein PlAvh23

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Cloning of coding genes and construction of expression vectors

[0044] 1. Cloning of the gene encoding PlAvh23

[0045] According to the instructions of the All-In-One DNA / RNA / Protein Mini-Preps Kit (Shenggong BBI), the total mRNA of Phytophthora peronosa was extracted and analyzed with PrimeScript TM RT reagent kit with gDNA Eraser (Takara) was reverse-transcribed into cDNA, using cDNA as a template, and PCR amplified the PlAvh23 coding gene after removing the signal peptide (the protein sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO .2). Wherein, the PCR amplification primer sequence is as follows:

[0046] Upstream primer PVX-PlAvh23-F:

[0047] 5'-CAGCTAGCATCGATTCCCGGGATCGACTCGAAGATCGCTGT-3';

[0048] Downstream primer PVX-PlAvh23-R:

[0049] 5'-AATCTCTAGAGGATCCCCGGGCTACGAGCGTCTAGGGGCTA-3'.

[0050]PCR amplification reaction system (50 μL): 2×Phanta Max Buffer 25 μL, dNTP Mix (10Mm each) 1 μL, upstream primer (...

Embodiment 2

[0058] Example 2: Transient expression of PlAvh23 on tobacco leaves induces plant defense responses

[0059] (1) Cultivation of Agrobacterium

[0060] Transform the PVX::PlAvh23-HA plasmid into Agrobacterium GV3101, smear it on LB (containing Kanamycin 50 μg / mL) plate, and culture it at 28°C for 2-3 days, then use Green Tag Mix (Vazyme) for colony PCR verification, pick Correctly cloned for subsequent experiments.

[0061] In addition, negative clones were obtained—transfected with PVX::GFP-HA plasmid (PVX::GFP-HA plasmid construction method refers to Example 1, the RFP gene sequence shown in SEQ ID NO.3 is constructed on the PVX vector, A single colony of Agrobacterium GV3101 with restriction sites as above), and a positive clone—a single colony of Agrobacterium GV3101 transfected with an INF1 plasmid (GenBank: AY830094.1).

[0062] RFP base sequence (SEQ ID NO.3):

[0063] ATGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCG...

Embodiment 3

[0088] Example 3PlAvh23 63-331 Induces the production of plant defense responses

[0089] (1) Cloning of deletion mutant fragments

[0090] Using the full length of PlAvh23 (SEQ ID NO.2) as a template, respectively amplify PlAvh23 24-331 , PlAvh23 24-62 , PlAvh23 63-331 , PlAvh23 24-65，134-331 , PlAvh23 24-132，225-331 , PlAvh23 24-224、292-331 sequence.

[0091] The PCR amplification primer sequences are as follows:

[0092] PlAvh23 24-331 Upstream primer F:

[0093] 5'-AGAGGATCCGTCGACCCCGGGATCGACTCGAAGATCGCTGT-3';

[0094] PlAvh23 24-331 Downstream primer R:

[0095] 5'-CTGTACAAGGGTACCCCCGGGCTACGAGCGTCTAGGGGCTA-3';

[0096] PlAvh23 24-62 Upstream primer F:

[0097] 5'-AGAGGATCCGTCGACCCCGGGATCGACTCGAAGATCGCTGT-3';

[0098] PlAvh23 24-62 Downstream primer R:

[0099] 5'-CTGTACAAGGGTACCCCCGGGCTACCTCTCATCGTCATTTT-3';

[0100] PlAvh23 63-331 Upstream primer F:

[0101] 5'-AGAGGATCCGTCGACCCCGGGATGGCGTTTCCTGGTTTGGA-3';

[0102] PlAvh23 63-331 Downstream primer ...

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Abstract

The invention discloses a plant immune activator protein PlAvh23 secreted by peronophythora litchii and an application of the plant immune activator protein PlAvh23. A novel peronophythora litchii RXLR type effect protein PlAvh23 is identified, and the amino acid sequence of the novel peronophythora litchii RXLR type effect protein PlAvh23 is selected from any one of the following sequences: (a) an amino acid sequence as shown in SEQ ID NO: 1; (b) an amino acid sequence as shown in SEQ ID NO: 4; and (c) an amino acid sequence as shown in SEQ ID NO: 6. The plant immune activator protein PlAvh23 can induce plants to generate immune response, reduces infection of pathogenic bacteria, provides a new way for improving plant resistance, and can be applied to prevention and treatment of litchi downy blight.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a plant immune activation protein P1Avh23 secreted by Pythophthora litchie and its application. Background technique [0002] Litchi frost blight caused by Peronophythora litchii is the most serious disease in litchi production, storage and transportation. The disease is currently mainly distributed in Thailand, Australia, Vietnam, Papua New Guinea and other countries. Litchi frost blight often causes a large number of flower and fruit drop, which seriously affects the yield and quality of litchi. In addition, even fruits that appear to be healthy and intact when picked may become diseased during transportation, causing serious economic losses. [0003] Effector proteins are a kind of virulence molecules secreted by pathogenic bacteria in the process of infecting host plants, which can destroy the immune system of plant cells, thereby promoting the infection of pathogeni...

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12N15/82C12N15/84A01H5/00A01H6/82
CPCC07K14/37C12N15/8282C12N15/8205Y02A50/30
Inventor 孔广辉黄琳晶司徒俊键张梓敬张心宁连帅利习平根姜子德
Owner SOUTH CHINA AGRI UNIV
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