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Ha-62292 Protein, encoding gene and application of Heterodera avenae

A cereal cyst nematode, ha-62292 technology, applied in the application, nematicide, genetic engineering and other directions, can solve the problems of backward pathogenic mechanism research, narrow host range of cereal cyst nematode, unclear research background, etc.

Inactive Publication Date: 2017-09-22
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the narrow host range of cereal cyst nematodes, long infection cycle, and unclear research background, the research on its pathogenic mechanism is relatively backward

Method used

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  • Ha-62292 Protein, encoding gene and application of Heterodera avenae
  • Ha-62292 Protein, encoding gene and application of Heterodera avenae
  • Ha-62292 Protein, encoding gene and application of Heterodera avenae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Isolation of Ha-62292 protein and its coding gene

[0025] Cereal cyst nematode total RNA was extracted using TRIzol (Ambion) kit, and used after DNase Treatment and Removal Reagents (Ambion) treatment III First-Strand Synthesis System for RT-PCR Reverse Transcription Kit (invitrogen) was used to synthesize cDNA. And using this cDNA as a template, PCR amplifies the gene sequence, the reagent used is Max DNA Polymerase high-fidelity enzyme (Takara), the primer sequence is: 5'-GATTTGTTGTCCGCAGTGCCTTTG-3', 5'-GCCAAAGGCACTGCGGACAACAAA-3'. The amplified fragments were purified and ligated for sequencing. The sequencing results showed that it contained the open reading frame shown in SEQ ID NO: 2 in the sequence list, encoding the protein sequence of SEQ ID NO: 1, and named the protein sequence and gene sequence as Ha-62292 protein and Ha-62292 gene respectively .

Embodiment 2

[0026] Example 2: Developmental expression changes of the Ha-62292 gene

[0027] Cereal cyst nematodes at different developmental stages were collected by enzymatic hydrolysis, total RNA was extracted using TRIzol (Ambion) kit, and used after treatment with DNase Treatment and Removal Reagents (Ambion) cDNA was synthesized with IIIFirst-Strand Synthesis System for RT-PCR Reverse Transcription Kit (invitrogen). And using this cDNA as a template, real-time PCR was used to detect the expression level changes at different stages, and the PCR reagent used was SelectMaster Mix (Applied Biosystems), the primers are 5'-CTACAAAGCGGCGGGAAAT-3', 5'-ATGCACCAGAATAATCTGAACAGC-3', the internal reference gene is GAPDH, and the internal reference gene primers are 5'-AGCGGCACAGAACATCATCC-3', 5'-GGTCCTCCGTGTAGCCCAAA-3'. The results showed that the expression level of this gene was mainly concentrated in the second instar before infection and the first day of the second instar after infection. ...

Embodiment 3

[0028] Example 3: Tissue expression localization of Ha-62292 gene

[0029] Using the cereal cyst nematode cDNA as a template, Max DNA Polymerase high-fidelity enzyme (Takara) and primers 5'-CTTCTTGCTCATTGCCCTAA-3', 5'-AAATTCTCGCCCATCCCT-3'PCR amplification, recovery and purification of the amplified fragment, and using this as a template, PCR DIG Probe Synthesis Kit( Roche) and antisense primers were used as a single primer for amplification, and the amplified product was used as a hybridization probe. Hybridization method reference de J.M.Boer YY, Geert Smant, Eric L.Davis, Thomas J.Baum (1998) In-situ Hybridization to Messenger RNA inHeterodera glycines. Journal of Nematology 30:309-312. Hybridization results show that the gene expression Localized in the esophageal gland cells of cereal cyst nematodes.

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Abstract

The invention discloses Ha-62292 protein, encoding gene and application of Heterodera avenae. The invention provides a protein acquired from Heterodera avenae, and named Ha-62292 protein, having an amino acid sequence shown as in SEQ ID NO: 1, and a gene sequence for encoding the protein is shown as in SEQ ID NO: 2. Experiments show that the Ha-62292 gene plays an important role in infective parasitism of nematodes, and may act as a target gene for plant engineering for nematode resistance. The Ha-62292 protein, encoding gene and application of Heterodera avenae are of great significance to the study on pathogenesis of Heterodera avenae and the development of nematode control plants.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a Ha-62292 protein derived from cereal cyst nematode, coding gene and application thereof. Background technique [0002] Cereal cyst nematode (Heterodera avenae) is a sessile endoparasitic nematode that mainly damages the roots of grasses such as wheat, barley and oats, causing serious yield loss and is widely distributed. At present, it has been found and reported in 13 provinces and cities including Henan, Anhui, Shandong, Qinghai, Ningxia, and Shaanxi (Su Zhiheng, Huang Wenkun, Zheng Guodong, Zhang Hongjia, Liu Shuyan, Peng Deliang. Occurrence dynamics of wheat cereal cyst nematode in Beijing area Investigation. Plant Protection, 2013, (01): 116-120.) (Zhao Honghai, Yang Yuanyong, Peng Deliang, Liu Feng. New reports on the distribution and occurrence characteristics of wheat cereal cyst nematodes in Shandong Province. Journal of Qingdao Agricultural University ( Natural Science Edi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/113A01N57/16A01P5/00
CPCA01N57/16C07K14/4354C12N15/113
Inventor 彭德良罗书介彭焕黄文坤孔令安崔江宽王高峰刘敬乔芬
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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