Medicago sativa trypsin inhibitor MT-mth2-36p5 as well as encoding gene and application thereof

A coding and protein technology, applied in the direction of protease inhibitors, applications, genetic engineering, etc., can solve the problems of environmental pollution, unreasonable aphid drug resistance, etc., and achieve the effect of a wide range of insect resistance

Active Publication Date: 2018-05-22
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

[0006] Aphid alfalfa belongs to Aphididae and Hemiptera. It is an important piercing-sucking pest in agriculture and animal husbandry. It mainly harms leguminous plants alfalfa. Due to the strong reproductive ability of aphids, most of the gen

Method used

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  • Medicago sativa trypsin inhibitor MT-mth2-36p5 as well as encoding gene and application thereof
  • Medicago sativa trypsin inhibitor MT-mth2-36p5 as well as encoding gene and application thereof
  • Medicago sativa trypsin inhibitor MT-mth2-36p5 as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, the acquisition of MT-mth2-36p5 protein and its coding gene

[0069] 1. Alfalfa planting

[0070] Soak alfalfa seeds (purchased from Beijing Zhengdao Ecological Technology Co., Ltd.) in warm water (50°C) for 10 minutes to soften the seed coat and increase the germination rate of the seeds; then sow the seeds in nutrient soil: vermiculite = 3:1 mixture Plant and cultivate in a 30cm-caliber flowerpot at room temperature (about 25°C) under light for 16 hours / darkness for 8 hours, and keep a small amount of water in the soil during the cultivation process.

[0071] 2. RNA extraction

[0072] The alfalfa grows to more than the three-leaf stage, and the alfalfa leaves are taken to extract the alfalfa RNA. Specific steps are as follows:

[0073] 1) Take out the alfalfa leaves from -80°C and grind them to powder under liquid nitrogen, add the powder to a 1.5ml centrifuge tube, add 1ml Ttrizol, shake and mix;

[0074] 2) Stand at room temperature for 5-10 minut...

Embodiment 2

[0094] Embodiment 2, the preparation of MT-mth2-36p5 protein

[0095] 1. Construction of recombinant expression vector

[0096] 1) Using the recovered and purified PCR product in Step 4 of Example 1 as a template, PCR amplification was performed using restriction primers F and R to obtain the MT-mth2-36p5 gene fragment. The primer sequences are as follows (the underlined bases are restriction sites):

[0097] Restriction Primer F: 5'-CCG GGATCC AAGCATC TTTTATC-3';

[0098] Restriction Primer R: 5'-GC CTCGAG AACAACAGAC-3'.

[0099] 2) Digest pSyno-1 vector (purchased from Suzhou Synbio Biotechnology Co., Ltd., catalog number: SGSGC19) with restriction endonucleases BamHI and XhoII to obtain the vector backbone;

[0100]3) connecting the MT-mth2-36p5 gene fragment and the vector backbone to obtain the recombinant expression vector pSyno-1-MT-mth2-36p5 and verify it by sequencing;

[0101] The results showed that the recombinant expression vector pSyno-1-MT-mth2-36p5 was ...

Embodiment 3

[0112] Example 3, Functional verification of MT-mth2-36p5 protein

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Abstract

The invention discloses a medicago sativa trypsin inhibitor MT-mth2-36p5 as well as an encoding gene and application thereof. The medicago sativa trypsin inhibitor MT-mth2-36p5 is a protein of a), b),c) or d), namely, a) a protein with the amino acid sequence shown in the sequence 2, b) a fusion protein obtained by connecting labels to the N end and/or the C end of the protein shown in the sequence 2, c) a protein with the same functions obtained by performing replacement and/or deletion and/or addition of one or more amino acid residues on the amino acid sequence shown in the sequence 2 andd) a protein with 75% or more homology as the amino acid sequence shown in the sequence 2 and the same functions. Experiments show that the protein has a good inhibition effect on therioaphis trifoliiand an inhibition effect on the survival rate and the reproduction rate of aphids and can be used for controlling and preventing the aphids.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an alfalfa trypsin inhibitor MT-mth2-36p5 and its coding gene and application. Background technique [0002] Tens of millions of years of evolutionary plants have produced mechanisms to resist herbivores, such as the formation of protease inhibitors. Plant-derived protease inhibitors are a class of substances that inhibit the activity of proteolytic enzymes. They regulate the physiological and biochemical activities that proteases participate in plays an important role. Proteases are involved in the food digestion of insects, the infection and expansion of pathogenic bacteria, etc. Therefore, the application of plant-derived protease inhibitors in disease and insect resistance has been widely developed. [0003] The anti-insect mechanism of protease inhibitors is still not very clear. Hahnenberger et al. believed that the anti-insect effect may be mainly due to the exis...

Claims

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Application Information

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IPC IPC(8): C07K14/81C12N15/29A01N47/44A01P7/04
CPCA01N47/44C07K14/8117
Inventor 涂雄兵张泽华赵海龙
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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