Female root-knot nematode esophageal gland gene in-situ hybridization technology and application thereof

Abstract

The invention discloses a female root-knot nematode esophageal gland gene in-situ hybridization technology and application thereof and belongs to the technical field of female root-knot nematode gene expression detection. According to the characteristic that a female root-knot nematode body is prone to fracture, an acupuncture needle is adopted in the in-situ hybridization process to gently make 1 or 2 small holes in the front body part of a female root-knot nematode adult, and then body fracture is avoided. The invention further provides a female root-knot nematode fixing technology. A 96-hole plate is used in an experiment, so body movement can be reduced, and deformation caused by gathering and squeezing of a large number of female root-knot nematodes in a centrifuge tube is avoided; a female root-knot nematode is large in size and weight and can sink to the bottom without centrifugation, so centrifugation is omitted, and the body can be better prevented from getting deformed. Through technological innovation, in-situ hybridization of genes is achieved in an esophageal gland of the female adult of the root-knot nematode.

Description

technical field [0001] The invention relates to a technology for detecting gene expression of root-knot nematode, in particular to an in-situ hybridization technology of root-knot nematode esophageal gland gene and its application. Background technique [0002] In situ hybridization (Insituhybridization, ISH for short) is the use of nucleic acid (DNA or RNA) probes with markers to detect the expression and distribution of nucleic acids to be tested in tissues or cells, and probes with known sequences It binds to the nucleic acid to be tested in the tissue or cell through complementary base pairing, and the specific marker on the probe can form a colored marker in situ on the nucleic acid to be tested by histochemical or immunohistochemical methods with the corresponding detection system. Hybridization signals; especially in gene analysis and diagnosis, it can be used for qualitative, localization and quantitative analysis. It is an important technology for analyzing low-abun...

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Problems solved by technology

[0003] Root-knot nematode (Meloidogynespp.) can parasitize more than 2,000 kinds of plants, causing serious losses to crop production; root-knot nematode life cycle is about 28 days in 25; including eggs, first-instar larvae, second-instar larvae, third-instar larvae, Larval and adult stages; the second instar larvae are linear, and the female adults are enlarged to be spherical or lemon-shaped ( figure 1 ); the female root-knot nematode has the largest body size and lives longer in the host body

[0004] Root-knot nematodes can reproduce 3-4 generations in one growing season, and the reproductive capacity of each generation is very large. On average, each female can produce 300-500 eggs. Female root-knot nematodes can successfully lay eggs, which is the foundation of continuous cropping. The key factor for the increasing population density of knot nematodes; but because the female root-knot nematode is in the plant, it is difficult to control it with conventional control techniques; the gene silencing technology and transgenic technology developed in recent years are expected to create root-knot nematode-resistant diseased plant material

[0005] There have been many reports on the study of gene expression sites in root-knot nematodes by using in situ hybridization technology, and a variety of esophageal gland secretion proteins have been successfully located on the second instar larvae of root-knot nematode; In situ hybridization technique of second instar larvae of root-knot nematode

In situ hybridization of root-knot nematode without spherical shape

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