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Meloidogyne jauanica effect gene Mj-ttl, encoded protein and application of meloidogyne jauanica effect gene Mj-ttl

A Java root-knot nematode and effector gene technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of lack of similarity in function, TTL protein cannot achieve TTR, etc., and achieves significant theoretical value, good theory and guiding significance Effect

Active Publication Date: 2015-09-09
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Saverwyns confirmed through experiments that the TTL protein of A. austrii can not realize the function of TTR (Saverwyns et al., 2008), and Sauer predicted through the method of bioinformatics that although the TTL protein has homology with TTR and TRP, But there seems to be no similarity in their functions (Sauer-Eriksson et al., 2009)
[0005]Therefore, the current understanding of nematode TTL protein is almost a blank

Method used

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  • Meloidogyne jauanica effect gene Mj-ttl, encoded protein and application of meloidogyne jauanica effect gene Mj-ttl

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Mj-ttl Acquisition of full-length gene sequence and sequence analysis

[0054] 1, Mj-ttl Acquisition of mid-gene fragments

[0055] According to the homologous genes in the NCBI gene bank, degenerate primers ttlestF (sequence shown in SEQ ID NO: 5) and ttlestR (sequence shown in SEQ ID NO: 6) were designed, and the cDNA of M. javanica was used as a template for PCR amplification. increase.

[0056] The PCR reaction system is 25 μL: cDNA 1 μL, ddH 2 O 17.875μL, dNTP Mixture(2.5mmoL / L each) 2.5μL, 10×PCR Buffer 2.5μL, littlestF(10μmoL / L) 0.5μL, littlestR(10μmoL / L) 0.5μL, Ex Taq(5U / μL) 0.125μL .

[0057] The PCR reaction program was: 94°C for 1min; 30 cycles of 94°C for 30s, 60°C for 50s, 72°C for 1min; 72°C for 10min, 4°C, ∞.

[0058] 2, Mj-ttl Amplification of gene 3'RACE

[0059] (1) Primers: according to the amplified Mj-ttlTwo specific primers ttl3r1 (sequence shown in SEQ ID NO: 7) and ttl3r2 (sequence shown in SEQ ID NO: 8) were designed for the...

Embodiment 2

[0074] Example 2 Mj-ttl Immunological localization of genes in C. elegans

[0075] The primary antibody used for immunolocalization in this example is the MJ-TTL protein obtained by prokaryotic expression and purification using pET28a, and the antiserum obtained by immunizing rabbits. The secondary antibody was HRP-labeled goat anti-rabbit IgG antibody (provided by Beijing Boaosen Biotechnology Co., Ltd.).

[0076] 1. Preparation of primary antibody:

[0077] (1) Vector construction: amplify using primers ttlFYEcorI (sequence shown in SEQ ID NO: 13) and ttlRYHind (sequence shown in SEQ ID NO: 14) Mj-ttl fragment, using EcroR I and Hind III was digested, connected to the pET28a vector that had been digested with the same enzyme, and the pET28-ttl vector was obtained after sequencing verification, and then transformed into Escherichia coli BL21 (DE3).

[0078] (2) Protein purification: using Ni 2+ -NTA gel to purify the obtained total protein, the specific method refe...

Embodiment 3

[0098] Example 3 Mj-ttl Developmental expression analysis of genes in C. elegans

[0099] 1. Collection of root-knot nematode javanese at different ages

[0100] 2nd instar larvae: Take fresh root knots, use a dissecting microscope to pick out a single egg mass with light yellow or yellow color, culture it in a 28°C incubator for 7 days, transfer it to a test tube and centrifuge to collect nematodes for later use.

[0101] Root-knot nematodes at different developmental stages: Inoculate freshly hatched 2nd instar larvae on 4-week-old tomato roots, pull out the roots of the tomatoes after 24 hours, wash the roots with clean water, and replant the tomato seedlings into new sterilized soil. Pull out the tomatoes at different time periods, collect nematodes and store them in centrifuge tubes for later use.

[0102] 2. Template cDNA: Extract total RNA by referring to the method for extracting total RNA from animal tissue in TIANGEN's RNAprep Micro Kit kit. cDNA was synthesized...

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Abstract

The invention discloses a meloidogyne jauanica effect gene Mj-ttl, encoded protein and application of the meloidogyne jauanica effect gene Mj-ttl, and belongs to the technical field of biology gene engineering. The sequence of the effect gene Mj-ttl is shown in SEQ ID NO:1-2. The amino acid sequence of the encoded protein MJ-TTL of the meloidogyne jauanica effect gene Mj-ttl is shown in SEQ ID NO:3. The expression of Mj-ttl gene specificity in meloidogyne sub-abdomen esophageal glands is determined. The effect protein of meloidogyne jauanica is achieved and interacts with FTRc in the plants, so that the degradation capacity of plants on ROS is enhanced. Accumulation of the ROS in plant cells in the initial stage of meloidogyne infection is restrained. Then the occurrence of plant PTI reactions is restrained. Therefore, the plants can have the higher susceptibility to the meloidogyne jauanica. New targets are provided for culturing plants of varieties resistant to the meloidogyne jauanica. The good theoretical and guide significance in culturing crops resistant to the meloidogyne jauanica in practice is achieved.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering. More specifically, a root-knot nematode effector gene Mj-ttl , encoding protein and its application. Background technique [0002] Root-knot nematode is an important obligate parasitic plant nematode, and it is also one of the most serious plant parasitic nematodes. It can infect more than 3000 species of plants of different families worldwide (Sasser, 1980), and cause huge Economic losses. Among them, root-knot nematode javanica is one of the most widely distributed root-knot nematodes in the world, and it is an important pest in agricultural production in many countries (Sasser, 1980). Therefore, the research on the characteristics of M. javanica and its interaction with the host is a very important and urgent research topic. [0003] In the study of root-knot nematode parasitism, the study of its effector proteins is a very critical and significant subject. The TTL ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C12N15/84C12N1/21A01H5/00
Inventor 廖金铃林柏荣卓侃
Owner SOUTH CHINA AGRI UNIV
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