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Heterodera avenae derived Ha-18764 protein and coding gene and application thereof

A cyst nematode and coding technology, applied in application, nematicide, genetic engineering and other directions, can solve problems such as large side effects, insufficient research on the pathogenic mechanism of cereal cyst nematodes, and limited control effect.

Active Publication Date: 2017-03-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to insufficient research on the pathogenic mechanism of cereal cyst nematodes, traditional control methods have outstanding problems such as poor specificity, large side effects, and limited control effects.

Method used

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  • Heterodera avenae derived Ha-18764 protein and coding gene and application thereof
  • Heterodera avenae derived Ha-18764 protein and coding gene and application thereof
  • Heterodera avenae derived Ha-18764 protein and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1, Discovery of Ha-18764 protein and Ha-18764 gene

[0044] 1. Pick fresh single-grain cyst nematode cysts, freeze in liquid nitrogen, crush the sample with a tissue grinder, extract total RNA by magnetic bead method (QIAGEN), and reverse transcribe to obtain cDNA.

[0045] 2. Using cDNA as a template, PCR amplification was performed using a primer pair composed of Ha-18764F and Ha-18764R.

[0046] Ha-18764F: 5'-ATGGCTTCTTCAGCATCCTTTTC-3';

[0047] Ha-18764R: 5'-GAGTTTGTGTGATCCGCTTTTG-3'.

[0048] PCR amplification reaction system (25.00μL): 5×Q5Buffer 5.00μL, Q5High-Fidelity DNAPolymerase 0.25μL, Ha-18764F 1.25μL, Ha-18764R 1.25μL, template 1.00μL, use ddH 2 O make up.

[0049] PCR reaction program: 98°C for 30s; 35 cycles of 98°C for 10s, 55°C for 30s, 72°C for 40s; 72°C for 10min; 4°C for storage.

[0050] 3. Linearize the pGR107 vector (37°C water bath for 3-4 hours), recover the linearized plasmid, connect it with the PCR amplification product in step 2...

Embodiment 2

[0052] Embodiment 2, tissue localization of Ha-18764 gene

[0053] DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) was used to conduct in situ hybridization experiments on the second instar larvae of the cereal cyst nematode.

[0054] 1. Using the cDNA of cereal cyst nematode as a template, a primer pair composed of Ha-18764-situ-S and Ha-18764-situ-A is used to amplify the target fragment (282bp) of the probe, and recover the target fragment.

[0055] Ha-18764-situ-S: 5'-AACACATTGTCTCGCTGAT-3';

[0056] Ha-18764-situ-A: 5'-AGTTGGTCCAGTCGTTGT-3'.

[0057] Reaction system (25μL): Phusion High-Fidelity DNA Polymerase 0.5μL, dNTPMixture (10mM each) 1.0μL, Ha-18764-situ-S 2.5μL, Ha-18764-situ-A 2.5μL, template 2μL, 5×Phusion HF Buffer 10μL,ddH 2 O make up.

[0058] Reaction conditions: 94°C for 4min; 35 cycles of 94°C for 30s, 52°C for 30s, 72°C for 1min; 72°C for 10min; 15°C for 5min.

[0059] 2. Asymmetric PCR is used to prepare sense probes or antisense pr...

Embodiment 3

[0065] Embodiment 3, the expression profile of Ha-18764 gene

[0066] Real-time fluorescence quantitative PCR was used to analyze the expression of Ha-18764 gene in each developmental stage of cereal cyst nematode (egg, pre-parasitic second-instar larvae, parasitic second-instar larvae, third-instar larvae, fourth-instar larvae, mature females and mature males) ) relative expression level. The GAPDH-1 gene was used as an internal reference. use Premix Ex Taq TM Kit (Takara), for Real time RT-PCR detection on ABI PRISM7000 fluorescent quantitative PCR instrument. The primer pair used to detect the Ha-18764 gene consisted of qHa-18764S and qHa-18764AS. The primer pair used to detect GAPDH-1 gene consisted of GAPDH-1S and GAPDH-1AS. The template is cDNA obtained by reverse transcription of the total RNA of cereal cyst nematodes at various developmental stages.

[0067] qHa-18764S (upstream primer): 5'-CCGCAGTGTCGGCTTTG-3';

[0068] qHa-18764AS (downstream primer): 5'-TTTG...

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Abstract

The invention discloses a heterodera avenae derived Ha-18764 protein and a coding gene and application thereof. The protein is obtained from heterodera avenae and named as the Ha-18764 protein and is (a1) or (a2), (a1) refers to a protein formed by an amino sequence shown as a sequence 1 in a sequence table, and (a2) refers to a (a1) derived protein associated with parasitism and / or pathopoiesia and / or development of heterodera avenae and is obtained by subjecting the (a1) to replacement and / or deficiency and / or addition of one or two amino acid residues. The coding gene of the Ha-18764 protein is named as a Ha-18764 gene which plays an important role in a nematode parasitism process and can serve as a target gene for plant nematode resistance engineering. The heterodera avenae derived Ha-18764 protein and the coding gene and application thereof have a significant value in heterodera avenae pathogenesis research and nematode-resistant plant preparation.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a Ha-18764 protein derived from cereal cyst nematode, its coding gene and application. Background technique [0002] Cereal cyst nematode (Heterodera avenae) is a kind of sessile endoparasitic plant nematode, which mainly invades the root system of wheat plants, inhibits the normal growth of roots, and causes great losses to the yield of wheat, barley and other crops. According to research, in my country, the area of ​​cereal cyst nematode (H. avenae) harming wheat has reached 4 million hm 2 Above, and in an increasing trend year by year, causing 15.5%-55.0% yield loss of wheat, has become a major problem in my country's wheat production. [0003] Due to insufficient research on the pathogenic mechanism of cereal cyst nematodes, traditional control methods have outstanding problems such as poor specificity, large side effects, and limited control effects. As a new control str...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N5/10C12N7/01C12N15/63A01N47/44A01P5/00
CPCA01N47/44C07K14/4354
Inventor 简恒戴依然刘倩杨姗姗
Owner CHINA AGRI UNIV
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