HaHSP4 protein of cereal cyst nematode and coding gene and application thereof

A cereal cyst nematode, cyst nematode technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of limited control effect, large side effects, insufficient research on the pathogenic mechanism of cereal cyst nematode, etc.

Inactive Publication Date: 2019-04-16
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • HaHSP4 protein of cereal cyst nematode and coding gene and application thereof
  • HaHSP4 protein of cereal cyst nematode and coding gene and application thereof
  • HaHSP4 protein of cereal cyst nematode and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1, discovery of HaHSP4 protein and HaHSP4 gene

[0070] 1. Pick fresh cysts of single-grain cyst nematode, 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.

[0071] 2. Using cDNA as a template, PCR amplification was performed using a primer pair composed of HaHSP4-F and HaHSP4-R.

[0072] HaHSP4-F: 5'-ATGCATGTGCAAACTGTTTTCC-3';

[0073] HaHSP4-R: 5'-TCAGTTGTTGGCCAGCCC-3'.

[0074] PCR amplification reaction system (50 μL): 5×Q5Buffer 10.00 μL, Q5 High-Fidelity DNA Polymerase 0.5 μL, HaHSP4-F 2.5 μL, HaHSP4-R 2.5 μL, template 2.00 μL, supplemented with ddH2O.

[0075] PCR reaction program: 98°C for 30s; 35 cycles of 98°C for 10s, 60°C for 30s, 72°C for 1min; 72°C for 10min; store at 4°C.

[0076] 3. Linearize the pGR107 vector (a gift from the laboratory of Professor Wang Yuanchao of Nanjing Agricultural University) (37°C water bath for 3-4 hours), r...

Embodiment 2

[0078] Embodiment 2, tissue localization of HaHSP4 gene

[0079] 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.

[0080] 1. Using the cDNA of cereal cyst nematode as a template, a primer pair composed of HaHSP4-situ-F and HaHSP4-situ-R is used to amplify the target fragment (263bp) of the probe, and recover the target fragment.

[0081] HaHSP4-situ-F: 5'-GAAGGAAGCCATTGAGGAG-3';

[0082] HaHSP4-situ-R: 5'-CCGTATCGGTGTAGTAGCG-3'.

[0083] Reaction system (25μL): Phusion High-Fidelity DNA Polymerase 0.5μL, dNTPMixture (10mM each) 1.0μL, HaHSP4-situ-F 2.5μL, HaHSP4-situ-R 2.5μL, template 2μL, 5×Phusion HF Buffer 10μL, ddH2O make up.

[0084] Reaction conditions: 94°C for 4min; 35 cycles of 94°C for 30s, 54°C for 30s, 72°C for 1min; 72°C for 10min; 15°C for 5min.

[0085] 2. Asymmetric PCR is used to prepare sense probes or antisense probes.

[0086]...

Embodiment 3

[0091] Embodiment 3, the developmental expression pattern of HaHSP4 gene

[0092] Real-time fluorescence quantitative PCR was used to analyze the expression of HaHSP4 gene in each developmental stage of cereal cyst nematode (egg, pre-parasitic second instar larvae, parasitic second instar larvae, parasitic third instar larvae, parasitic fourth instar larvae and mature female) relative expression levels. The GAPDH-1 gene was used as an internal reference. use Premix Ex Taq TMKit (Takara), for Real time RT-PCR detection on ABI PRISM7500 fluorescent quantitative PCR instrument. The primer pair used to detect HaHSP4 gene consisted of qRT-HaHSP4-F and qRT-HaHSP4-R. The primer pair for detecting GAPDH-1 gene consisted of GAPDH-1-F and GAPDH-1-R. The template is cDNA obtained by reverse transcription of the total RNA of cereal cyst nematodes at various developmental stages.

[0093] qRT-HaHSP4-F (upstream primer): 5'-ACCTGTTCGTTCCGTTGTT-3';

[0094] qRT-HaHSP4-R (downstream p...

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Abstract

The invention discloses a HaHSP4 protein of cereal cyst nematode and a coding gene and application thereof. The protein is obtained from the cereal cyst nematode and named the HaHSP4 protein which isthe protein consisting of the amino acid sequence shown in SEQ ID NO:1 in the Sequence Listing. A gene encoding the HaHSP4 protein is named the HaHSP4 gene, and also belongs to the scope of the invention. The invention also provides the application of the HaHSP4 protein. The application includes regulating the host tropism of the cyst nematode; regulating the parasitic capability of the cyst nematode; regulating the pathogenic capability of the cyst nematode; regulating the development of the cyst nematode. The invention also provides a method for cultivating a plant with improved resistance to the cyst nematode. The method comprises the steps of introducing a substance inhibiting the expression of the HaHSP4 protein into a target plant to obtain the plant with the improved resistance to the cyst nematode. The protein has great value for research on the pathogenic mechanism of the cyst nematode and the preparation of anti-nematode plants.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to cereal cyst nematode HaHSP4 protein and 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. Studies have shown that in my country, the cereal cyst nematode (H. avenae) damages wheat in an area of ​​4 million hectares 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 strategy a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/82A01H5/00A01H6/46C12N15/113
CPCC07K14/4354C12N15/113C12N15/8285C12N2310/10
Inventor 简恒杨姗姗刘倩
Owner CHINA AGRI UNIV
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