阅读说明：本技术 一种解决杀虫毒素被昆虫肠道消化酶过度酶解的毒素改造方法 (Toxin modification method for solving excessive enzymolysis of insecticidal toxin by insect intestinal digestive enzyme ) 是由 吴松青 张飞萍 郭雅洁 于 2020-05-18 设计创作，主要内容包括：本发明提供了一种解决杀虫毒素被昆虫肠道消化酶过度酶解的毒素改造方法。其是利用PeptideCutter或荧光多肽酶活测定或多重底物质谱分析法(MSP-MS)对昆虫肠道消化酶的特异性切割位点进行测定；然后利用Swiss Model和PyMOL软件相结合筛选出位于毒素非功能区域的改造位点；最后将筛选到的多个位点同时突变为不被肠道蛋白酶识别的氨基酸。本发明改造方法能够对毒素上的多个蛋白酶切割位点同时进行准确的突变,显著的提高对靶标昆虫的杀虫活性,具有特异性强,操作简单等优点。这为农、林业害虫的生物防治以及抗性的治理提供了一条有利的途径。(The invention provides a toxin modification method for solving the problem that insecticidal toxin is excessively enzymolyzed by digestive enzyme of insect intestinal tracts. The specific cleavage site of the insect intestinal digestive enzyme is determined by PeptideCutter or fluorescence polypeptide enzyme activity determination or multiple substrate mass spectrometry (MSP-MS); then, screening a modification site in a toxin non-functional area by combining Swiss Model and PyMOL software; finally, the screened multiple sites are mutated into amino acids which are not recognized by the intestinal protease at the same time. The modification method can simultaneously and accurately mutate a plurality of protease cleavage sites on the toxin, obviously improve the insecticidal activity to target insects, and has the advantages of strong specificity, simple operation and the like. It provides a favorable way for the biological control and resistance treatment of agricultural and forestry pests.)

1. A toxin modification method for solving the problem that insecticidal toxin is excessively enzymolyzed by insect intestinal digestive enzyme is characterized by comprising the following steps: the method comprises the following steps: 1) recognition of insect gut protease cleavage sites; 2) screening protease recognition sites on toxins; 3) molecular modification of the toxin.

2. The toxin modification method of claim 1, wherein the insecticidal toxin is a parasporal crystal protein Cry and Cyt toxoid produced by Bacillus thuringiensis during the spore phase.

3. The toxin engineering method of claim 1, wherein the insect comprises coleoptera, lepidoptera, diptera, hymenoptera, homoptera, hemiptera, orthoptera, and nematodes, mites.

4. The method of claim 1, wherein the insect gut protease cleavage site is identified using PeptideCutter or fluorescent Peptidezyme assay or multiplex bottom mass spectrometry.

5. The method of claim 1, wherein the protease recognition sites on the toxin are selected by a combination of Swiss Model and PyMOL software prediction.

6. The method of claim 1, wherein the toxin is molecularly engineered by biosynthesizing or point mutation to modify the protease cleavage site on the surface of the toxin protein.

7. The toxin engineering method of claim 4, wherein PeptideCutter software is used to obtain all potential protease cleavage sites on the toxin protein; the enzyme activity determination of the fluorescent polypeptide substrate is used for determining the species and the activity of the insect intestinal protease; multiple-substrate mass spectrometry is used to identify specific cleavage sites for insect gut proteases.

8. The method of toxin engineering according to claim 5, wherein the protease cleavage sites distributed on the surface of the toxin protein and in the non-functional region are selected by using Swiss Model software and PyMOL software.

9. The method of toxin engineering according to claim 8, wherein the toxin protein surface non-functional region is a region excluding loop1, loop2, loop3 and loop α -8 of domain II and other non-functional region excluding β 16 of domain III.

Technical Field

The invention belongs to the field of biological control of pests, and particularly relates to a toxin modification method for solving the problem that insecticidal toxin is excessively enzymolyzed by digestive enzymes of insect intestinal tracts.

Background

Bacillus thuringiensis (B.thuringiensis) (B.thuringiensis)Bacillus thuringiensisBt) is the most widely used microbial insecticide (Adang et al, 2014) currently, bacillus thuringiensis is mainly characterized by being capable of synthesizing crystal proteins during spore production, virulence factors in the crystal proteins are endotoxins, and the toxins are mainly divided into two families including Cry and Cyt (H ö fte et al, 1989), while protoxins need to be activated by specific proteases to generate active fragments before exerting insecticidal toxicity (Pardo-L pez et al, 2013).

At present, the main idea for modifying the toxin to solve the problem of excessive enzymolysis of the toxin by the digestive enzyme of the insect intestinal tract is as follows: excessive enzymatic hydrolysis of Cry toxins by proteases has been addressed by removing the protease cleavage site on the toxin (Bah et al, 2004), but the toxins engineered using this approach still have limitations. First, in the current studies, the selection of trypsin and chymotrypsin cleavage sites on toxins was mainly determined by two methods, one is to select mutation sites based on the specific recognition preferences of trypsin and chymotrypsin established by Keil, but no consideration was given to whether they are specific recognition sites for target insect gut proteases (Keil, 1992). However, the efficiency of amino acids recognized by trypsin and chymotrypsin in the gut is different for different insects (Zhang et al, 2011; Lightwood et al, 2000; Bah et al, 2004). Secondly, the enzymatic activity of insect midgut protease is analyzed and determined by a substrate colorimetry, so as to screen mutation sites (Walters et al, 2008). Due to the lack of advanced methods for detecting protease cleavage sites, these mutant sites are amino acids recognized by insect gut proteases, but these sites are not all capable of efficient cleavage by proteases. In addition, the existing research mainly focuses on mutation of a single site on the toxin, and can only solve the problem of excessive enzymolysis at the mutation site, but has no influence on the cleavage condition of other protease recognition sites on the toxin (Zhang et al, 2011; Lightwood et al, 2000; Bah et al, 2004), so that the toxin with mutation of the single site is limited in the capability of overcoming the enzymolysis of the intestinal proteases of the insect, and the toxicity of the mutant toxin to the corresponding target insect in the existing research is only improved by 2-4 times (Bah et al, 2004). Therefore, in order to overcome the excessive enzymolysis of the insect intestinal protease on the toxin, on one hand, the specific cleavage site of the target insect intestinal protease can be accurately determined, and on the other hand, mutation is carried out simultaneously aiming at a plurality of specific efficient recognition sites on the toxin. Aiming at the above ideas, the invention aims to provide a toxin modification method for solving the problem that the insecticidal toxin is excessively enzymolyzed by the intestinal digestive enzymes of insects, and provides a new idea and way for biological control of target insects by using the toxin.

Disclosure of Invention

The invention aims to provide a toxin modification method for solving the problem that insecticidal toxin is excessively enzymolyzed by digestive enzyme in insect intestinal tract. The invention provides a toxin modification method for solving the problem of excessive enzymolysis of insect intestinal tract digestive enzymes on toxins and improving the insecticidal activity of the toxins, and the toxin modification method is characterized in that PeptideCutter or fluorescent polypeptide enzyme activity determination or a multiple substrate mass spectrometry (MSP-MS) is used for determining specific cutting sites of the insect intestinal tract digestive enzymes; and then, screening the transformation sites positioned in the non-functional region by combining Swiss Model and PyMOL software, and finally mutating the screened multiple sites into unidentified amino acids.

In order to realize the purpose, the following technical scheme is adopted:

the insects of the invention comprise coleoptera, lepidoptera, diptera, hymenoptera, homoptera, hemiptera and orthoptera, as well as nematodes and mites. Preferably, the insect is monochamus alternatus.

The insecticidal toxin is parasporal crystal proteins Cry and Cyt toxoid generated by Bacillus thuringiensis (Bt) in a spore stage.

The method mainly comprises the following steps: 1) recognition of insect gut protease cleavage sites; 2) screening protease recognition sites on toxins; 3) molecular modification of the toxin.

The recognition of the insect intestinal protease cleavage site is determined by PeptideCutter or fluorescent polypeptide enzyme activity determination or multiple-substrate mass spectrometry (MSP-MS).

The screening of the protease recognition sites on the toxin adopts a method combining Swiss Model and PyMOL software prediction to screen out protease cleavage sites which are positioned on the surface of the toxin protein and in non-functional areas.

The molecular modification of the toxin comprises the step of carrying out molecular modification on the screened sites through a biosynthesis or point mutation method.

The PeptideCutter software was used to obtain all potential protease cleavage sites on the toxin protein; the enzyme activity determination of the fluorescent polypeptide substrate is used for determining the species and the activity of the insect intestinal protease; multiple-substrate mass spectrometry is used to identify specific cleavage sites for insect gut proteases.

The invention utilizes Swiss Model software and PyMOL software to screen out protease cleavage sites distributed on the surface of toxin protein and in non-functional areas. The toxin protein surface and non-functional regions are referred to as loop1, loop2, loop3, and loop α -8, excluding domain II, and other non-functional regions of β 16 of domain III.

The PeptideCutter software prediction method specifically comprises the following steps: and (3) predicting various protease cleavage sites on the toxin by using PeptideCutter software so as to obtain corresponding cleavage sites.

The method for measuring the activity of the fluorescent polypeptide enzyme comprises the following specific steps: dissecting insect intestinal tract tissue under a microscope, suspending in double distilled water, grinding, homogenizing, centrifuging to obtain intestinal tract digestive enzyme solution, and determining protein concentration. Quenched fluorescent peptide substrate was selected, and an amount of insect intestinal enzyme solution and an equimolar amount of quenched fluorescent polypeptide substrate were added to Tris-HCl buffer (50 mM, pH 7.5; 100 mM NaCl, 2 mM DTT, and 0.01% Tween-20) for reaction. And setting an inhibitor experiment. The set excitation wavelength of 330 nm and emission wavelength of 400nm was run in a black round bottom microplate for 2 hours at room temperature. The change in fluorescence units per second was recorded. Thus, the types of the insect intestinal proteases are determined, and the cleavage sites of the insect intestinal proteases are obtained according to the created substrate model and the protease specific binding rules summarized by combining the substrate model.

The multiple-substrate mass spectrometry (MSP-MS) comprises the following specific steps: an amount of insect intestinal enzyme solution was added to equimolar amounts of tetradecapeptides (tetradecapeptide library) in Tris-HCl buffer (50 mM, pH 7.5; 100 mM NaCl, 2 mM DTT) for reaction. All enzymes were incubated at room temperature for 15, 60, 240 and 1200 minutes and stopped by acidification with concentrated formic acid to a final pH of 2.5. Desalted using C18tips and analyzed by LC-MS/MS polypeptide sequencing. And visualizing the frequency of the amino acids around the cleavage site by adopting IceLoco software and then analyzing. Thereby defining the specific cleavage sites with higher insect intestinal protease recognition efficiency.

The screening of protease recognition sites on the toxin comprises the following specific steps: modeling the toxin protein by using Swiss Model software, and screening the protease enzyme cutting sites of the non-functional region of the toxin protein by using PyMOL software to obtain the sites positioned on the protein surface.

The biosynthesis is carried out by a biological company directly according to the designed modified toxin nucleic acid sequence.

And the point mutation is to carry out point mutation on mutation sites on the toxin in sequence by using a point mutation kit, and finally obtain the modified toxin.

The invention has the following beneficial effects:

the two toxins modified by the method can be hydrolyzed by trypsin and intestinal digestive enzyme of Monochamus alternatus larvae in vitro to obtain effective active fragments. Compared with the in vitro enzymolysis of the original toxin protein, the modified toxin can more stably activate more active fragments. The phenomenon of excessive enzymolysis of the modified toxin protein is obviously improved, and the insecticidal activity of the modified toxin protein is also obviously improved, so that a favorable way is provided for biological control of coleoptera insect Monochamus alternatus.

Drawings

FIG. 1A shows the predicted trypsin cleavage site in the Cry3Aa protein by PeptideCutter software.

FIG. 1B shows the chymotrypsin cleavage site in the Cry3Aa protein predicted by PeptideCutter software.

FIG. 2 is a schematic model diagram of PeptideCutter software.

A schematic of the eight binding sites of the enzyme-substrate is shown, with the cleavage site between P1 and P1 'in the middle of Pn and Pm' in the substrate.

FIG. 3 is a schematic diagram of the trypsin and chymotrypsin cleavage sites in PeptideCutter software.

Where a and B are chymotrypsin cleavage efficiencies in the substrate model, when P1= Lys (a) and P1= Arg (B), the inhibition of trypsin cleavage by the amino acid sequences at the positions P2 (abscissa) and P1 ' (ordinate) is shown by the black regions indicating the percentage of inhibition corresponding to each of the P2-Lys-P1 ' and P2-Arg-P1 ' tripeptide sequences. C is the trypsin cleavage efficiency in the substrate model, with the ordinate being the amino acid at position P1 and the abscissa being the amino acid at position P1'. The black area represents the percentage of each dipeptide amino acid sequence cleaved by chymotrypsin.

FIG. 4 shows the determination of the activity of the fluorogenic substrate polypeptide enzyme of the intestinal digestive enzyme of Monochamus alternatus larvae.

Wherein AEBSF is a serine protease inhibitor; e-64 is a cysteine protease inhibitor; pepstatin-A is aspartic acid; EDTA is a metalloproteinase inhibitor.

FIG. 5 is a map of specific cleavage sites of intestinal digestive enzymes of Monochamus alternatus larvae.

Wherein the upper side of the horizontal axis represents amino acids that are easily cleaved by protease at each position, the lower side of the horizontal axis represents amino acids that are not easily cleaved by protease at each position, and p is less than 0.05.

FIG. 6 is a three-dimensional block diagram of the modified toxins of BRC1912 and BRC 1913.

A is a three-dimensional structural diagram of Cry3Aa protoxin, and A' is a three-dimensional structural diagram of BRC1912 modified toxin.

B is a three-dimensional structural diagram of Cry3Aa protoxin, and B' is a three-dimensional structural diagram of BRC1913 modified toxin.

FIG. 7 is a plasmid map of the universal vector pGEX-6P-1.

FIG. 8 is a SDS-PAGE pattern of BRC1912 and BRC1913 engineered toxins.

FIG. 9 is an in vitro enzymatic digestion of BRC1912 and BRC1913 engineered toxins by Monochamus alternatus larva gut digestive enzymes.

Detailed Description

In order to make the objects, technical solutions and advantageous technical effects of the present invention more clear, the present invention is further described in detail with reference to the following embodiments. It should be understood that the examples in this specification are for the purpose of illustration only and are not intended to limit the invention.