Protein BxCDP1 of bursaphelenchus-xylophilus pathogen-associated molecular patterns and application thereof

A related model, the technology of pine wood nematode, applied in the biological field to achieve the effect of improving resistance and good development and application prospects

Active Publication Date: 2019-08-23
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a mobile plant parasitic nematode, pine wood nematode has not b

Method used

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  • Protein BxCDP1 of bursaphelenchus-xylophilus pathogen-associated molecular patterns and application thereof
  • Protein BxCDP1 of bursaphelenchus-xylophilus pathogen-associated molecular patterns and application thereof
  • Protein BxCDP1 of bursaphelenchus-xylophilus pathogen-associated molecular patterns and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1.1 Transcriptome sequencing at the early stage of pine wood nematode infection

[0029] The pine wood nematode virulent strain AMA3 (isolated from the naturally infected Pinus massoniana in Maanshan City, Anhui Province) was selected to infect the 3-year-old Japanese black pine, and the tree body was cut into small sections when the pine wood nematode was infected for 6h, 12h and 24h, respectively. Nematodes were isolated using the Baermann funnel method. After that, the pine wood nematode samples were sent to Novogene for transcriptome sequencing. Experimental results: B. xylophilus collected at 3 early stages of infection (6h, 12h and 24h) and on Botrytis cinerea (0h) were successfully sequenced for transcriptome and subsequently analyzed.

[0030] 1.2 Screen the genes up-regulated in the early stage of B. xylophilus infection to screen for candidate effectors

[0031] Genes whose up-regulation times were greater than 2 in the early stage of B. xylophilus infection...

Embodiment 2B

[0042] Example 2 Expression of BxCDP1 in Nicotiana benthamiana that silences BAK1, SOBIR1 and GFP

[0043] In the experiment of transiently expressing Nicotiana benthamiana in Example 1, it was found that the BxCDP1 protein could cause the necrosis of Nicotiana benthamiana leaves. Using virus-induced gene silencing (VIGS) technology, co-receptor brassinosteroidinsensitive 1-associated kinase 1 (BAK1) and suppressor of BIR1-1 (SOBIR1) in Nicotiana benthamiana were sequentially silenced, and GFP-silenced Nicotiana benthamiana was used as a control. The viral expression bodies pTRV1, pTRV2: BAK1, pTRV2: SOBIR1 and pTRV2: GFP plasmids were electroporated into Agrobacterium competent GV3101 according to the method 1.3 in Example 1. The Agrobacterium carrying the above-mentioned carrier was adjusted to OD with a spectrophotometer according to the 1.3 method in Example 1. 600 The value is 2. The Agrobacterium carrying pTRV2: BAK1, pTRV2: SOBIR1, pTRV2: GFP and the Agrobacterium ca...

Embodiment 3B

[0045] Expression and purification of embodiment 3BxCDP1 pure protein

[0046] After the open reading frame (Open Reading Frame, ORF) of BxCDP1 was amplified by PCR, the purified product was connected to the yeast expression vector pPICZαA, and the pPICZaA vector carrying BxCDP1 was heat-shock transformed into the large intestine according to the method 1.3 in Example 1 In the Bacillus Competent Top10, the plasmid was extracted according to the method 1.3 in Example 1, and sent to the company for sequencing. After the sequencing is correct, linearize the plasmid and then electrotransfer it into the competent yeast KM71H according to the method 1.3 in Example 1. Immediately after the electrotransfer is completed, add 500 μL of sorbitol to the competent yeast. The successfully transformed transformants were placed on a solid plate of Yeast extract peptone dextrose medium (YPD) containing 100 μg / mL bleomycin at 30°C for 3 days. Buffered glycerol-complex medium (BMGY) containing ...

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Abstract

The invention discloses protein BxCDP1 of bursaphelenchus-xylophilus pathogen-associated molecular patterns and application thereof. The amino acid sequence of the protein BxCDP1 of the pathogen-associated molecular patterns (PAMPS) is shown in SEQID NO.2. From effectors secreted by bursaphelenchus xylophilus and the PAMP, the defensive responses of a host plant, namely, a pine tree, to bursaphelenchus xylophilus invasion are studied, the protein BxCDP1 of the pathogen-associated molecular patterns is obtained from the bursaphelenchus xylophilus, it is proven through experiments that the protein BxCDP1 can trigger cell necrosis of various plants including the host plant, has a certain broad spectrum in cell necrosis triggering, and stimulates the defensive response of the host plant. The BxCDP1 triggered cell necrosis depends on a co-receptor BAK1 of pattern recognition receptors, the BxCDP1 can stimulate the accumulation of nicotiana benthamiana ROS and up regulation of PTI Marker genes, and the immunoreaction of the nicotiana benthamiana is stimulated. It can be seen that the BxCDP1 is a PAMP secreted by the bursaphelenchus xylophilus, and has important theoretical and practicalsignificance for revealing the pathogenic mechanism of the bursaphelenchus xylophilus and improving the resistance of the pine tree to the bursaphelenchus xylophilus in a targeted mode.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a pathogen-associated pattern (PAMP) molecular protein BxCDP1 of pine xylophilus and its application. Background technique [0002] In nature, plants are attacked by many pathogenic microorganisms (including fungi, bacteria, nematodes, etc.) and insects. However, due to the innate immunity of plants, they are resistant to most microorganisms. The plant's immune system includes two levels, namely a "Z" pattern: the first defense response is that the pattern recognition receptors (PRRs) on the plant cell membrane can recognize pathogens or microorganism-associated pattern molecules (Pathogen- or microbial-associated molecular patterns, PAMPs or MAMPs), thereby stimulating the basic defense response of plants, inducing the activation of some signaling pathways, and hindering the further expansion of pathogens, that is, PAMP-triggered immunity (PTI). At this time, in order to further in...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/82A01H5/00A01H6/82
CPCC07K14/4354C12N15/8285
Inventor 吴小芹胡龙娇叶建仁黄鑫李煜
Owner NANJING FORESTRY UNIV
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