Meloidogyne incognita Chitwood prevention and control related gene Misp12 and use thereof

A technology of root-knot nematode incognita and sequence, which is applied in application, nematicides, genetic engineering, etc., can solve the problems of unknown function of Misp12 and no homologous genes retrieved, so as to achieve environmental protection and safety off-target effects, reduce the number of invasions, and avoid The effect of off-target effects

Inactive Publication Date: 2017-03-15
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The applicant searched the existing patent and non-patent literature, and the results showed that Misp12 is still a gene with unknown function, and no related homologous genes were found in other species

Method used

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  • Meloidogyne incognita Chitwood prevention and control related gene Misp12 and use thereof
  • Meloidogyne incognita Chitwood prevention and control related gene Misp12 and use thereof
  • Meloidogyne incognita Chitwood prevention and control related gene Misp12 and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cloning of Misp12 gene related to developmental pathogenicity of Meloidogyne incognita

[0034] The applicant obtained the candidate secreted protein sequence by analyzing and comparing the genome data and proteome data of Meloidogyne incognita through bioinformatics technology, and isolated and cloned the gene Misp12 through molecular biology technology. Through genome database and proteome database sequence comparison, it is found that Misp12 is highly specific and conserved in M. The growth and development of root-knot nematodes may be related to the pathogenic process and play an important role ( figure 1 ).

[0035] 1.1 Bioinformatics analysis of genome and proteome data of M. incognita

[0036] Download the genome and proteome data of M. incognita through NCBI ( http: / / meloidogyne.toulouse.inra.fr ). Use SignalP 3.0 and THMM2.0 to analyze the signal peptide structure and transmembrane structure of its proteome to obtain secretome proteins, and then use variou...

Embodiment 2

[0044] Bioinformatic analysis of Misp12 protein and its homologous proteins in other fungi

[0045] The amino acid sequence putatively encoded by the Misp12 gene was analyzed through the NCBI BLAST database and the Swissprot database, and it was found that this sequence only exists in Meloidogyne incognita, and there are no corresponding homologous sequences and homologous proteins in other animals, plants, fungi and bacteria. Motif analysis of the protein using MotifScan shows that it contains 2 predicted N-glycosylation sites, 2 predicted protein kinase C phosphorylation sites, 1 cAMP and cGMP dependent phosphorylation site and 1 N- Myristoylation site ( Figure 4 ).

Embodiment 3

[0047] Localization of Misp12 gene in Meloidogyne incognita and its protein localization in plant cells

[0048] The expression position of Misp12 gene in Meloidogyne incognita was detected by in situ hybridization, and Misp12 gene fused with gfp gene was transferred into onion epidermal cells to observe its localization in plant cells.

[0049] 3.1 In situ hybridization test

[0050] 3.1.1 Solution preparation

[0051] (1) M9Buffer: 22mM KH 2 PO 4 , 42.3mM NaHPO 4 , 85.6mM NaCl, 1mM MgSO 4 . 7H2O

[0052] (2) Fixative: 3% paraformaldehyde, prepared with 1×M9Buffer

[0053] (3) Proteinase K: 2mg / ml, prepared with 10mM Tris-HCl (PH 7.5)

[0054] (4) For the preparation of other hybridization reagents, refer to the instructions of the Digoxigenin Labeling Kit (purchased from Roche)

[0055] 3.1.2 Digoxigenin-labeled DNA single-strand probe synthesis

[0056] The single-stranded sense probe and antisense probe of the target gene were synthesized by using the digoxin lab...

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Abstract

The invention discloses a Meloidogyne incognita Chitwood prevention and control related gene Misp12 and a use thereof. The gene Misp12 related to the growth and development of Meloidogyne incognita Chitwood has a gene sequence shown in the formula of SEQ ID NO. 1. The encoded protein of the gene Misp12 is shown in the formula of SEQ ID NO. 2. The gene Misp12 is increased sharply in maturation of the Meloidogyne incognita Chitwood. Through use of VIGS silencing of the gene, Meloidogyne incognita Chitwood-caused damage to a host plant is reduced. The gene Misp12 can be used as a candidate gene for preventing and controlling Meloidogyne incognita Chitwood invasion, is used for controlling Meloidogyne incognita Chitwood damage and provides a reference for development of a novel Meloidogyne incognita Chitwood prevention and control agent.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and specifically relates to the isolation, cloning, functional verification and application of a gene Misp12 related to the growth and development of Meloidogyne incognita. Background technique [0002] Southern root-knot nematode (Meloidogyne incognita) is a type of plant parasitic nematode that seriously damages agricultural production. It has strong adaptability and diverse transmission routes. It can parasitize more than 5,000 kinds of plants, harm plant roots and form root knots, and cause major economic losses to crops worldwide. (Blok et al., 2008). According to statistics, the annual loss to the fruit and vegetable industry alone is as high as 70 billion US dollars (Caboni P et al., 2012). At the same time, root-knot nematode damage has aggravated the occurrence of soil-borne fungal diseases and some bacterial diseases such as Fusarium wilt and root rot, and increas...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C12N15/63A01N47/44A01N57/16A01P5/00
Inventor 肖炎农谢家廉肖雪琼莫陈汨李少军王高峰谢冲
Owner HUAZHONG AGRI UNIV
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