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Alfalfa trypsin inhibitor MT-mth2-89i19 as well as encoding gene and application thereof

A coding and protein technology, applied in protease inhibitors, applications, genetic engineering, etc., can solve the problems of slow dissociation speed and cracking speed, and achieve a wide range of insect resistance

Active Publication Date: 2018-07-24
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

Different from the substrates of general enzymes, the peptide chain of the active center of the inhibitor is not cleaved or the cleavage speed is very slow after the inhibitor is combined with the enzyme. Therefore, although the complex can be decomposed into free enzymes and denatured or undenatured inhibitors, but very slow dissociation

Method used

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  • Alfalfa trypsin inhibitor MT-mth2-89i19 as well as encoding gene and application thereof
  • Alfalfa trypsin inhibitor MT-mth2-89i19 as well as encoding gene and application thereof
  • Alfalfa trypsin inhibitor MT-mth2-89i19 as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1, the acquisition of MT-mth2-89i19 protein and its coding gene

[0067] 1. Alfalfa planting

[0068] 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.

[0069] 2. RNA extraction

[0070] 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:

[0071] 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;

[0072] 2) Stand at room temperature for 5-10 minu...

Embodiment 2

[0092] Embodiment 2, preparation of trypsin inhibitor MT-mth2-89i19

[0093] 1. Construction of recombinant expression vector

[0094] 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-mth2-89i19 gene fragment. The primer sequences are as follows (the underlined bases are restriction sites):

[0095] Restriction Primer F:5'-CCG GGATCC ATGAAAACCTCACTC-3';

[0096] Restriction Primer R:5'-GC CTCGAG AGCCCTTTTAAACACAA-3'.

[0097] 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;

[0098] 3) Connecting the MT-mth2-89i19 gene fragment and the vector backbone to obtain the recombinant expression vector pSyno-1-MT-mth2-89i19 and verify it by sequencing;

[0099] The results showed that the recombinant expression vector pSyno-1-M...

Embodiment 3

[0110] Example 3, Functional verification of MT-mth2-89i19 protein

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Abstract

The invention discloses an alfalfa-trypsin inhibitor MT-mth2-89i19 as well as an encoding gene and application thereof. The alfalfa-trypsin inhibitor MT-mth2-89i19 provided by the invention is as thefollowing protein of a), b), c) and d): a) a protein with an amino acid sequence shown as a sequence 2; b) a fusion protein obtained by connecting an N terminal and / or C terminal of the protein shownas the sequence 2 with a tag; c) protein with the same function, obtained by substitution, deletion and / or addition of one or more amino acid residues of the amino acid sequence shown as the sequence2; and d) a protein with the homology being 75 percent or above with the amino acid sequence shown as the sequence 2 and with the same function. Shown through experiments, the protein has very good inhibition effect to therioaphis trifolii, has the inhibition effect to both of the survival rate and the reproductive rate of aphids and can be used for preventing and controlling the aphids.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an alfalfa trypsin inhibitor MT-mth2-89i19 and its coding gene and application. Background technique [0002] Protease inhibitors can be divided into serine, cysteine, aspartate and metalloproteinase inhibitors according to the type of protease they inhibit. Protease inhibitors are a kind of substrate of proteases, and their active centers work by combining with proteases to form a stable complex. The interaction between protease inhibitors and target enzymes is usually complementary, just like the interaction between enzymes and substrates. mechanism of action. When the two react, the exposed active center of the inhibitor and the active center of the target enzyme are connected by hydrogen bonds to form a stable covalent complex, which leads to the blockage of the active center of the enzyme and the loss of the activity of the target enzyme. Compared with the usual en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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