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Cereal cyst nematode Ha34609 protein, encoding gene and application of encoding gene

A cereal cyst nematode and protein technology, applied in the fields of application, nematicides, genetic engineering, etc., can solve the problems of lack of control measures, threats to food security, loss of wheat yield, etc.

Active Publication Date: 2017-07-18
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

Due to the lack of effective, rapid and economical control measures, if the outbreak of wheat cyst nematode becomes a disaster, it will cause serious losses to my country's wheat production, thus posing a major threat to my country's food security

Method used

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  • Cereal cyst nematode Ha34609 protein, encoding gene and application of encoding gene
  • Cereal cyst nematode Ha34609 protein, encoding gene and application of encoding gene
  • Cereal cyst nematode Ha34609 protein, encoding gene and application of encoding gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1, Discovery of Ha34609 protein and Ha34609 gene

[0025] 1. Collect the 2nd instar larvae of cereal cyst nematodes (about 5,000), wash with DEPC for 2-3 times, transfer to a 1.5ml centrifuge tube, add 1ml Trizol (Invitrogen), quick freeze in liquid nitrogen for 30S, 37℃ water bath for 30S , repeated freezing and thawing 4-5 times, then standing at room temperature for 5 minutes, extracting total RNA, using DNA-free TM The DNA Removal Kit kit removes the residual DNA in the total RNA, and obtains cDNA by reverse transcription.

[0026] 2. Using cDNA as a template, use primers Ha34609-F and Ha34609-R for PCR amplification. The specific sequence is as follows:

[0027] Upstream primer Ha34609-F: 5'-ATGAGTTTTATCAATATTTTTTGTGGTTGT-3',

[0028] Downstream primer Ha34609-R: 5'-TCAAGTGCAAAATGGCCAGAA-3';

[0029] The amplification system is 10×PCR Ex Buffer (Mg 2+ ), 5 μl; dNTP (10 mM), 4 μl; upstream primer (10 mM), downstream primer (10 mM), 1 μl each; Ex Taq, 0....

Embodiment 2

[0030] Embodiment 2, tissue localization analysis of Ha34609 gene

[0031] 1. Use the Ha34609 gene cloning vector as a template to amplify the target sequence by conventional PCR, using the upstream primer Ha34609-yw-F: 5'-AAGGCGTTGAAAATGGGTGC-3' and the downstream primer Ha34609-yw-R: 5'-CCCAGGGCCTTGTTCGATAA-3', Carry out PCR reaction, the system is as follows 10×PCR Ex Buffer (Mg 2+ ), 2.5 μl; dNTP (10 mM), 4 μl; upstream primer (10 mM), downstream primer (10 mM), 1 μl each; Ex Taq, 0.3 μl; plasmid template, 1 μl; deionized water, 16.2 μl, total volume 25 μl. The amplification program was denatured at 94°C for 5 minutes; the next 34 cycles were extended at 94°C, 30sec, 57°C, 30sec, and 72°C for 30sec; the final extension was at 72°C for 10min, and stored at 4°C. PCR products were separated and purified by 1% agarose gel electrophoresis. 2. Synthesize DIG-labeled forward-strand probe and anti-strand probe by single-primer PCR. Use Ha34609-yw-F or Ha34609-yw-R as primers, a...

Embodiment 3

[0032] Embodiment 3, developmental expression analysis of Ha34609 gene

[0033] Refer to Long et al (Long H B, Peng D L, Huang W K, Peng H, Wang G F. Molecular characterization and functional analysis of two new β-1,4-endoglucanase genes (Ha-eng-2, Ha-eng-3) from the cereal Cyst nematode Heterodera avenae[J].PlantPathology,2013,62(4):953-960.) The method described in, a large amount of inoculation of graminearum cyst nematode 2nd instar larvae at the roots of warm wheat 19 susceptible wheat, combined by controlling the inoculation time Six species of cereal cyst nematodes at different developmental stages (2nd instar larvae before infection, 2nd instar larvae after infection, 3rd instar larvae, 4th instar larvae, females and eggs) were obtained by enzymatic lysis treatment. The mRNAs of 6 ages were extracted by magnetic bead method, and reverse-transcribed into the first-strand cDNA as a template. The Ha34609 gene-specific upstream primer Ha34609-q-F: 5'-TTTACTACATCCGCCGCTTC-3...

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Abstract

The invention relates to a cereal cyst nematode Ha34609 protein, an encoding gene and application of the encoding gene. The amino acid sequence of the cereal cyst nematode Ha34609 protein is shown as SEQ ID NO: 1. The nucleotide sequence of the encoding gene is shown as SEQ ID NO: 2. After a silent Ha34609 gene is treated by adopting dsRNA (double-stranded ribonucleic acid), compared with eGFP (enhanced green fluorescent protein) dsRNA, the length and the width of a white female worm are remarkably reduced (t-test, confidence interval: 95%), which indicates that the gene plays an important role in parasitic and pathogenic processes of cereal cyst nematodes and can serve as a target gene of plant nematode resistance engineering. The cereal cyst nematode Ha34609 protein, the encoding gene and the application of the encoding gene have significant values in a study on a nosogenesis of the cyst nematodes and preparation of nematode resistance plants.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a Ha34609 protein derived from cereal cyst nematodes, a coding gene and applications thereof. Background technique [0002] Wheat is one of the three important food crops in my country, and ensuring the safe production of wheat is of great importance to my country's food security. Wheat cereal cyst nematodes (Cereal cyst nematodes, referred to as CCNs) is an important pathogenic nematode that seriously damages wheat (Triticum aestivum), barley (Hordeum vulgare) and oats (Avena sativa) and other cereal crops (Meagher J W. World dissemination of the cereal-cyst nematode (Heterodera avenae) and its potential as a pathogen of wheat. Journal of Nematology, 1977, 9(1):9-15.). Since it was first discovered in Germany in 1874, it has occurred and harmed in more than 40 countries and regions in Asia, Africa, Europe, the United States, and Australia (Rivoal R, Nicol J M. Past research on the ce...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/113A01N57/16A01P5/00
CPCA01N57/16C07K14/4354C12N15/113C12N2310/14
Inventor 彭德良李新彭焕黄文坤孔令安王高峰崔江宽刘敬乔芬罗书介
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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