Cereal cyst nematode ha-63744 protein, coding gene and its application

A technology of ha-63744, cereal cyst nematode, which is applied in the fields of application, nematicides, genetic engineering, etc., can solve the problems of lack of control methods, soil pollution, and not being economical and effective

Active Publication Date: 2019-12-06
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

At present, the pathogenic mechanism of wheat cereal cyst nematode is still unclear, and there is a lack of economical and effective control methods. Although crop rotation and the application of biocontrol bacteria have certain control effects, they are not economical and effective, and chemical insecticides are easy to Pollution of the soil, causing environmental problems

Method used

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  • Cereal cyst nematode ha-63744 protein, coding gene and its application
  • Cereal cyst nematode ha-63744 protein, coding gene and its application
  • Cereal cyst nematode ha-63744 protein, coding gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. Discovery of Ha-63744 protein and Ha-63744 gene

[0019] 1. Collect nematode larvae (about 5000 heads), wash 2-3 times with DEPC water, transfer to 1.5ml centrifuge tube, add 1ml Trizol (Invitrogen), quick freeze in liquid nitrogen for 30S, 37℃ water bath for 30S, repeat freezing and thawing 4-5 times, then let stand at room temperature for 5 minutes, extract total RNA, use DNA-free TM The DNA Removal Kit removes the remaining DNA in the total RNA, and reverse transcribes the cDNA.

[0020] 2. Using cDNA as a template, the upstream primer HaFull-F: 5'-ATGCGCGCCATCCTCTTCCT-3'; the downstream primer HaFull-R1: 5'-CTAATTTGTCGGTTCATTAATCTG-3' for PCR amplification.

[0021] Upstream primer HaFull-F: 5’-ATGCGCGCCATCCTCTTCCT-3’;

[0022] Downstream primer HaFull-R1: 5’-CTAATTTGTCGGTTCATTAATCTG-3’

[0023] The amplification system is 10×PCR Ex Buffer (Mg2+), 5μl; dNTP (10mM), 4μl; forward primer (10mM), reverse primer (10mM) each 1μl; Ex Taq, 0.5μl; cDNA first strand templa...

Embodiment 2

[0025] Example 2. In situ hybridization location analysis of Ha-63744 gene

[0026] Using the Ha-63744 gene cloning vector as a template, the target sequence was amplified by conventional PCR, and the upstream primer HaH-F: 5'-GATGCGCACAAAGGAGCAAG-3'; the downstream primer HaH-R: 5'-TCTACTGGTGCACTGCTTGG-3' was used to perform the PCR reaction. The system is as follows: 10×PCR Ex Buffer(Mg 2+ ), 2μl; dNTP (10mM), 1μl; forward primer (10mM), reverse primer (10mM) each 1μl; Ex Taq, 0.3μl; plasmid template, 1μl; deionized water, 14.2μl, total volume 20μl. The amplification program was denatured at 94°C for 5 minutes; the next 34 cycles of 94°C, 30sec, 60°C, 30sec, 72°C extension for 1 min; the last 72°C extension for 10 minutes, stored at 4°C. The PCR products were separated and purified by 1% agarose gel electrophoresis. Digoxin-labeled positive-strand probe and reverse-strand probe were synthesized by asymmetric PCR. Use HaH-F or HaH-R as primers and use the target fragment recov...

Embodiment 3

[0027] Example 3 Developmental expression analysis of Ha-63744 gene

[0028] Refer to Long et al., the method [Long H, Peng H, Huang W, Wang G, Gao B, Moens M, and Peng D. Identification and molecular characterization of a new β-1,4-endoglucanase gene (Ha-eng-1a) In the cereal cyst nematode Heteroderaavenae.European journal of plant pathology.2013,134:391-400.], the roots of Wenmai 19 susceptible wheat were inoculated with CCN second instar larvae for 5d, 10d, 20d and 30d, and then the enzyme lysis method was adopted. J2, J3, J4 and females after infection were separated, and J2 and eggs before infection were collected at the same time. Using magnetic bead method to extract mRNA of 6 ages, reverse transcribed into first-strand cDNA as template, and use primer 5 software to design Ha-63744 gene specific primer upstream primer HaQ-F: 5'-TCGCCTCAAAAGCAGTTGTC-3'; Downstream primer HaQ-R: 5'-TCTGCCAAATCGCCATTGTC-3', using Realtime PCR relative quantitative technology to detect the ex...

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Abstract

The invention relates to a heterodera avenae sensu lato Ha-63744 protein, an encoding gene and the application of the heterodera avenae sensu lato Ha-63744 protein. A sequence of the heterodera avenae sensu lato Ha-63744 protein is shown as SEQ ID NO: (sequence identifier number) 1, and a gene sequence encoding the protein is shown as SEQ ID NO: 2. The Ha-63744 gene is expressed in an esophageal gland of heterodera avenae sensu lato, and an expression level is higher in third and fourth instar larva periods after parasitism of the heterodera avenae sensu lato. After the silent Ha-63744 gene is treated by dsRNA (double-stranded ribonucleic acid), the quantity of the heterodera avenae sensu lato at a wheat root is obviously less than that of a control, which shows that the gene plays an important role in a parasitism pathogenesis process of the heterodera avenae sensu lato and can serve as a target gene of a heterodera avenae sensu lato resistance project of a plant. The protein and the gene have a great value for pathogenic mechanism research of the heterodera avenae sensu lato and preparation of the heterodera avenae sensu lato resistance plant.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and relates to a Ha-63744 protein derived from Cereal cyst nematode, coding gene and application thereof. Background technique [0002] Wheat is one of the three important food crops in China and plays a pivotal role in ensuring my country's food security. In 2015, the sown area and output of wheat in my country accounted for 14.51% and 20.95% of the national grain crops respectively. Cereal cyst nematodes (CCNs) are parasitic nematodes in plants. CCNs are harmful in 38 countries around the world, mainly to wheat (T. aestivum), barley (Hordeum vulgare), and oats (Avena sativa) And important cereal crops such as pasture. Since my country was first discovered in Hubei in 1991, in less than 30 years, it has been discovered in 16 provinces, cities and regions including Henan, Hebei, Tibet, and Xinjiang [Peng DL, Nicol JM, Li H, HouS, Li H, Chen S, Ma P, Li H, and Riley I T. Current knowledge of cereal cystn...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/113A01N57/16A01P5/00
CPCA01N57/16C07K14/4354C12N15/113C12N2310/14
Inventor 彭德良乔芬彭焕黄文坤孔令安崔江宽王高峰刘敬罗书介
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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