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A kind of root-knot nematode effector gene mj-1-1, related protein and application thereof

A Java root-knot nematode and effector gene technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems such as chemical control of environmental pollution and lack of nematode-resistant germplasm resources

Active Publication Date: 2018-11-27
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, root-knot nematode control methods mainly include crop rotation, chemical control and selection of disease-resistant varieties, but in actual production, due to the limitations of crop rotation and the huge pollution caused by chemical control to the environment, as well as the lack of nematode-resistant germplasm resources, looking for Efficient and sustainable root-knot nematode control methods are currently the focus of plant parasitic nematode research, and nematode-resistant transgenic engineering that relies on molecular biology is also a hot spot in plant nematode research

Method used

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  • A kind of root-knot nematode effector gene mj-1-1, related protein and application thereof
  • A kind of root-knot nematode effector gene mj-1-1, related protein and application thereof
  • A kind of root-knot nematode effector gene mj-1-1, related protein and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Cloning of embodiment 1 root-knot nematode javanica MJ-1-1 protein and its coding gene

[0083] 1. Use the TRIZOL method to extract the RNA of the second instar larvae of Meloidogyne javanica, and reverse transcribe it into cDNA.

[0084] 2. Design the downstream primer DC6R2 (sequence shown in SEQ ID NO.5), paired with the trans-splicing sequence SL1 (sequence shown in SEQ ID NO.6), amplify with the cDNA obtained in step 1 as a template, The cDNA coding sequence of Mj-1-1 was obtained.

[0085] PCR amplification system: cDNA 2 μL, two primers 3 μL each, 10×KOD plus Buffer 5 μL, MgSO 4 2 μL, dNTP 5 μL, Kod plus Neo DNA polymerase 1 μL, ddH 2 O 34 μL. PCR amplification program: 94°C for 3min, 30×(94°C for 30s, 55°C for 30s, 68°C for 2min), 68°C for 5min, and store at 20°C. Agarose electrophoresis detection amplification results are attached figure 1 shown.

[0086] 3. Using DNA as a template, use primers to amplify Mj1DNAF (sequence shown in SEQ ID NO.7) and Mj1D...

Embodiment 2

[0088] Example 2 Expression Analysis of Mj-1-1 Gene in Different Developmental Stages of Meloidogyne javanica

[0089] 1. Extract the RNA of M. javanica at different developmental stages and reverse transcribe it into cDNA.

[0090] 2. Using primers qMj1F (sequence shown in SEQ ID NO.9) and qMj1R (sequence shown in SEQ ID NO.10) to perform fluorescence quantitative PCR analysis on gene Mj-1-1. The root-knot nematode javanica β-actin gene was used as an internal reference, and the primers were qActinF (sequence shown in SEQ ID NO.11) and qActinR (sequence shown in SEQ ID NO.12). Reaction conditions: 94°C for 30s, 40×(94°C for 30s, 55°C for 30s, 72°C for 30s).

[0091] 3. The results are attached image 3 shown. The results showed that the Mj-1-1 gene was expressed in all developmental stages of M. javanica, the expression level was the lowest in the second instar larvae before infection, and the expression level increased significantly after infection, and the expression lev...

Embodiment 3

[0092] Example 3 Exogenous dsRNA soaking method to silence the Mj-1-1 gene and analyze its function

[0093] 1. The exogenous dsRNA was obtained according to the method of Li et al. (2011).

[0094] (1) Synthesis of the sense and antisense strands of the Mj-1-1 gene: use the primer pair Mj1ST7 (sequence such as SEQ ID NO.15) and Mj1A (sequence such as SEQ ID NO.16), and Mj1S (sequence such as SEQ ID NO.17 ) and Mj1AT7 (sequence such as SEQ ID NO.18); PCR amplification annealing temperature 55 ° C, 1 min extension time.

[0095] (2) Synthesis of positive control GFP gene sense and antisense strands: use primer pair GFPST7 (sequence such as SEQ ID NO.19) and GFPA (sequence such as SEQ ID NO.20), and GFPS (sequence such as SEQ ID NO.21) and GFPAT7 (sequence as SEQ ID NO.22); PCR annealing temperature 55° C., extension time 1 min.

[0096] (3) The synthesis of dsMj-1-1 and dsGFP was performed according to the instructions of ScriptMAX Thermo T7 Transcription Kit.

[0097] 2. Th...

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Abstract

The invention discloses a meloidogyne javanica effect gene Mj-1-1, related protein and application of the effect gene Mj-1-1. The nucleotide sequence of the effect gene Mj-1-1 is shown in SEQ ID NO.1-3, and the amino acid sequence of the related protein is shown in SEQ ID NO.4. It is proved that the gene Mj-1-1 plays an important role in parasitism of meloidogyne javanica, an RNAi plant expression vector is successfully built, after converting agrobacterium EHA105, the vector is transferred into a plant through a tobacco tissue culture method, dsRNA of Mj-1-1 is generated in the plant, the plant can remarkably restrain parasitism of meloidogyne javanica, and the plant with good meloidogyne javanica resistance is obtained. A new target is provided for meloidogyne javanica prevention and control, and the meloidogyne javanica effect gene Mj-1-1 is of great theoretical and guiding significance in cultivating plants with meloidogyne javanica resistance in practice.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering. More specifically, it relates to a root-knot nematode effector gene Mj-1-1, related proteins and applications thereof. Background technique [0002] Root-knot nematode is an important plant pathogen with worldwide distribution. It has a wide parasitic range and strong environmental adaptability, and can harm almost all crops. With the development of modern agriculture, the agricultural damage caused by root-knot nematodes continues to increase. my country is located in the temperate zone and subtropical zone, with good climatic conditions, suitable for the activities and reproduction of root-knot nematodes. World-wide important root-knot nematode diseases occur in our country, and the fixative endoparasitic root-knot nematode (Meloidogyne javanica) is a hazard. One of the most serious root-knot nematode species. At present, root-knot nematode control methods mainly includ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/84C12N15/83C12N15/89C12N15/90C12N15/66C12N1/21A01H5/00A01H6/82
Inventor 廖金铃扈丽丽卓侃林柏荣
Owner SOUTH CHINA AGRI UNIV
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