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Peronophythora litchii secreting type protein elicitor PlPeL8 and application thereof

A technology of litchi downy mildew and secreted protein, which is applied in the field of molecular phytopathology, can solve the problems of few resistance resources, difficulty in litchi breeding, and high risk of drug resistance, so as to improve disease resistance and plant disease resistance , Improve the effect of resistance

Active Publication Date: 2022-05-06
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the control of litchi downy mildew is still mainly based on pesticide control and agricultural control, and there are several difficulties in the following aspects: difficulty in litchi breeding, few resistance resources, and high risk of drug resistance

Method used

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  • Peronophythora litchii secreting type protein elicitor PlPeL8 and application thereof
  • Peronophythora litchii secreting type protein elicitor PlPeL8 and application thereof
  • Peronophythora litchii secreting type protein elicitor PlPeL8 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: Cloning and acquisition of PlPeL8 gene

[0043]Most plant pathogenic organisms secrete a large number of cell wall degrading enzymes (CWDEs), which degrade plant cell walls to obtain nutrients needed for growth and complete infection. At the same time, some CWDEs or molecules released by CWDEs degrading cell wall polysaccharides can act as inducers of plant immune responses. Among them, pectate lyase (Pectate Lyase, PL) is the main member of cell wall degrading enzymes. Based on the completion of genome and transcriptome sequencing of P. litchie, we predicted that P. litchie may encode a total of 19 pectin lyase genes. We designed full-length specific primers for pectin lyase 8 (PlPeL8), the primers contain homologous sequences at both ends of the vector cloning site, forward primer PlPeL8-F: 5'- GATAGCCGGTACCCCCC ATGGCTCGTATCATCTCAC-3', reverse primer PlPeL8-R: 5'- CTCTAGAGGATCCCC GTAGAACTCGATCTTGTCC-3' (the underlined sequence is introduced to be co...

Embodiment 2

[0052] Example 2: Prediction of homologous proteins of protein elicitor PlPeL8 in oomycetes

[0053] Use SeqHunter2.0 software, JGI genome database (https: / / genome.jgi.doe.gov / ), NCBI (https: / / www.ncbi.nlm.nih.gov / ) genome database as the basis, and perform PlPeL8 by Blast Homologous protein analysis. Three PlPeL8 homologous proteins: Ps_141345 (NCBI accession number: XP_009520586.1), Pc_15766 (NCBI accession number: KAG1685646.1), PITG_GN244_ATG14395 (NCBI accession number: KAF4033686.1), respectively from Phytophthora sojae, P.capsici and P.infestans have a homology rate of more than 90% with PlPeL8 and are highly conserved; Pythium ultimum homolog protein PYU1_T002542 (NCBI accession number: KAF1335911. 1) The homology rate is 53.1%.

[0054] Multiple sequence alignment of PlPeL8 and Ps_141345, Pc_15766, PITG_GN244_ATG14395, PYU1_T002542 was performed using BioEdit software, and it was found that the sequences were highly conserved in P. litchi and Phytophthora ( figure ...

Embodiment 3

[0055] Example 3: Transient expression of PlPeL8 and its homologous proteins in Nicotiana benthamiana induces allergic cell necrosis

[0056] The recombinant plasmid pBin-PlPeL8-HA obtained in Example 1 was transferred into 100 μL of Agrobacterium GV3101 competent, and the LB solid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampicin was used for screening. After the clones were verified by colony PCR, they were shaken in liquid LB and cultured at 28° C. with shaking at 180 rpm for 1 to 2 days (d).

[0057] The bacteria were centrifuged at 4000rpm for 4min, the supernatant was discarded, and 10mmol / L MgCl 2 Suspended bacteria, washed 3-4 times in total, and OD 600 Adjust to 0.4~0.6. Use a 1mL sterile syringe without a needle to inject the Agrobacterium suspension on the back of vigorously growing Nicotiana benthamiana leaves at the 5-6 leaf stage, so that the target gene can be expressed transiently in Nicotiana benthamiana cells; at the same time, each leaf is injec...

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Abstract

The invention discloses a peronophythora litchii secreting type protein elicitor PlPeL8 and an application thereof. The amino acid sequence of the protein elicitor PlPeL8 is as shown in SEQ ID NO.1, the coding gene of the protein elicitor PlPeL8 is constructed into a plant expression vector, then agrobacterium tumefaciens is transformed, transient expression is carried out in nicotiana benthamiana cells, and it is found that the protein elicitor PlPeL8 can induce plant defense reactions including causing plant cell allergic necrosis, inducing plant cell apoptosis and inducing plant cell apoptosis. The gene can induce active oxygen bursting and up-regulated expression of marker genes of plant immune related pathways such as salicylic acid and jasmonic acid pathways, can significantly improve the resistance of plants to phytophthora, provides a new way for improving plant disease resistance and reducing diseases, and has a wide application prospect in green development of modern agriculture.

Description

technical field [0001] The invention belongs to the technical field of molecular phytopathology, and in particular relates to a secreted protein elicitor PlPeL8 of Phytophthora litchie and its application. Background technique [0002] Litchi downy mildew (Peronophythora litchii Chen ex Ko et al.) is a semi-biotrophic oomycete, and the litchi frost blight caused by it is one of the most serious diseases threatening litchi production, and no effective disease-resistant litchi cultivars have been found yet. . Litchi may suffer from litchi frost blight from flower spikes to fruit ripening, as well as postharvest storage and transportation, which will cause a large number of flower rot, fruit drop and rotten fruit, and the economic loss rate can reach 30% to 50%. [0003] At present, the control of downy mildew on litchi is still dominated by pesticide control and agricultural control, and there are several difficulties in the following aspects: difficulty in litchi breeding, f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/31C12N15/82A01H5/00A01H6/82A01H6/00
CPCC07K14/37C12N15/8282
Inventor 孔广辉李雯司徒俊键周文喆宋雨李鹏连帅利习平根姜子德
Owner SOUTH CHINA AGRI UNIV
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