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HaGLAND5 protein of cereal cyst nematode, encoding gene of protein and application

A technology for cereal cyst nematodes and cyst nematodes, which is applied in the fields of application, genetic engineering, and plant genetic improvement, and can solve problems such as large side effects, limited control effects, and poor specificity

Inactive Publication Date: 2018-11-02
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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  • HaGLAND5 protein of cereal cyst nematode, encoding gene of protein and application
  • HaGLAND5 protein of cereal cyst nematode, encoding gene of protein and application
  • HaGLAND5 protein of cereal cyst nematode, encoding gene of protein and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, discovery of HaGLAND5 protein and HaGLAND5 gene

[0056] 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, and reverse transcribe to obtain cDNA.

[0057] 2. Using cDNA as a template, PCR amplification was performed using a primer pair consisting of HaGLAND5-F and HaGLAND5-R.

[0058] HaGLAND5-F: 5'-ATGTCGTCTTTCTCCTTCCTCC-3';

[0059] HaGLAND5-R: 5'-TTGTTTGTGCGGGCCC-3'.

[0060] PCR amplification reaction system (50 μL): 5×Q5Buffer 10.00 μL, Q5High-Fidelity DNA Polymerase 0.5 μL, HaGLAND5-F 2.5 μL, HaGLAND5-R 2.5 μL, template 2.00 μL, use ddH 2 O make up. The reaction program of PCR amplification: 98°C for 30s; 98°C for 10s, 56°C for 30s, 72°C for 40s, 35 cycles; 72°C for 10min; 4°C for storage.

[0061] 3. Linearize the pGR107 vector (37°C water bath for 3-4 hours), recover the linearized plasmid, connect it with the PCR amplification product...

Embodiment 2

[0063] Embodiment 2, tissue localization of HaGLAND5 gene

[0064] 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.

[0065] 1. Using the cDNA of cereal cyst nematode as a template, a primer pair composed of HaGLAND5-situ-F and HaGLAND5-situ-R is used to amplify the target fragment (291bp) of the probe, and recover the target fragment.

[0066] HaGLAND5-situ-F: 5'-GCTCCAATGCCCAAAGTGT-3';

[0067] HaGLAND5-situ-R: 5'-GGTCGATGAGCACTGTTCCAA-3'.

[0068] Reaction system (25μL): Phusion High-Fidelity DNA Polymerase 0.5μL, dNTPMixture (10mM each) 1.0μL, HaGLAND5-situ-F 2.5μL, HaGLAND5-situ-R 2.5μL, template 2μL, 5×Phusion HF Buffer 10μL, ddH 2 O make up.

[0069] Reaction conditions: 94°C for 4min; 35 cycles of 94°C for 30s, 59°C for 30s, 72°C for 1min; 72°C for 10min; 15°C for 5min.

[0070] 2. Asymmetric PCR is used to prepare sense probes or antisense...

Embodiment 3

[0076] Embodiment 3, the developmental expression pattern of HaGLAND5 gene

[0077] Real-time fluorescent quantitative PCR was used to analyze the expression of HaGLAND5 gene in each developmental stage of cereal cyst nematodes (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 TM Kit (Takara), for Real time RT-PCR detection on ABI PRISM7500 fluorescent quantitative PCR instrument. The primer pair used to detect the HaGLAND5 gene consisted of qRT-HaGLAND5-F and qRT-HaGLAND5-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.

[0078] qRT-HaGLAND5-F (upstream primer): 5'-CTGTCCACTACTTCTTCTGCTCCT-3';

[0079] qRT-...

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Abstract

The invention discloses HaGLAND5 protein of cereal cyst nematode as well as an encoding gene and an application of protein. The protein is obtained from the cereal cyst nematode, is named as HaGLAND5protein and is protein formed by an amino acid sequence shown in a sequence 1 in a sequence list. The gene for encoding the HaGLAND5 protein is named as HaGLAND5 gene and also belongs to the protection range of the invention. The invention also protects an application of the HaGLAND5 protein. The application comprises regulation of parasitic ability of the cyst nematode; regulation of pathogenic ability of the cyst nematode; regulation of development of the cyst nematode. The invention also protects a method for cultivating a plant with improved resistance to the cyst nematode. The method comprises steps as follows: a substance for inhibiting HaGLAND5 gene expression is guided into a target plant, and the plant with improved resistance to the cyst nematode is obtained. The protein, the gene and the application have great value for research of the pathogenesis mechanism of the cereal cyst nematode and preparation of plants resisting nematode.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to the HaGLAND5 protein of cereal cyst nematode and its coding gene and application. Background technique [0002] Cereal cyst nematode is a type 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. [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 and technology, RNA interference has brought new breakthroughs in nematode resistance genetic engineering. The main scheme of RNA interference against nematodes is as follows: construct an RNA interference vector targeting nematode parasitic pathogenicity-related genes, introduce t...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/82A01H5/00A01H6/82
CPCC07K14/4354C12N15/8218C12N15/8285
Inventor 简恒杨姗姗刘倩
Owner CHINA AGRI UNIV
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