CN108489785B - Method for analyzing formation process and proteome change of mulberry silk - Google Patents
Method for analyzing formation process and proteome change of mulberry silk Download PDFInfo
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Abstract
The invention discloses a method for analyzing the formation process and proteome change of mulberry silk, which relates to the field of proteomics, marks the expression conditions of proteins of different silk length sections produced by the same silkworm under the same feeding condition, and analyzes the change of the silk formation process; the invention has the beneficial effects that: the invention13The relationship between the time for taking mulberry leaves and cocoon silk can be obtained by tracing and analyzing the glycine marked by C, and13c is a stable isotope without radioactivity, and has no influence on the growth and development of the silkworm; the invention adopts a flat plate spinning technology, changes the physical habit of silkworm cocooning, and ensures the accuracy of sectional collection of silk; the invention collects the silk in sections, which can realize the research on different expressions of protein in the process of silking of the silkworm; compared with the traditional high-concentration neutral salt solution, the invention adopts a low-sodium salt system to dissolve the cocoon shell, can reduce the dosage of chemicals, reduce the introduction of impurity ions, shorten the purification treatment time, reduce the cost and has no toxicity and harm.
Description
Technical Field
The invention relates to the field of proteomics, in particular to a method for analyzing a mulberry silk forming process and proteome change.
Background
Since ancient times, silk is used as a carrier of Chinese culture, silkworms are bred and reeled by the generations of Chinese people all the time along the way of silk, and with the research on the spinning mechanism of the silkworms, people find that a pair of semitransparent and multi-bent tubular organs, namely, spun silk glands exist in the bodies of the silkworms, are special organs for synthesizing and secreting silk substances, and can be divided into spinning tubes, anterior silk glands, middle silk glands and posterior silk glands in form and function; the main components of the silk, namely sericin and fibroin, are secreted by a middle silk gland and a rear silk gland respectively; jianan-ying Li and ZHaoming Dong et al have demonstrated this recognition through studies on silk glands of silkworms.
However, due to the physiological habit of silkworm cocoon self-binding, people are difficult to analyze the specific formation process of the silkworm cocoon, and meanwhile, the protein expression in the mulberry silk formation process is also difficult to analyze by adopting the traditional detection technologies such as infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, amino acid analysis and the like. Therefore, a more effective means is needed, which can carry out tracer analysis on silk formation and obtain the differential expression condition of proteome in the silk formation process, and is of great importance for the study on silk quality control and proteome.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for analyzing the formation process and the proteome change of mulberry silk.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for analyzing the formation process and proteome change of mulberry silk comprises the following steps:
1) a, B, C, D, E silkworm eggs of the same batch are taken and fed to the 3 rd day of 5 years old under the same external condition.
2) Feeding A, B, C, D, E groups in step 1) on 4 th, 5 th, 6 th, 7 th and 8 th days of 5 th age, respectively, and spraying13C marking the same mulberry leaves with glycine.
3) When the silkworm maturity rate of the 5 groups of the silkworms in the step 2) is 90-95%, laying the matured silkworms on a flat-plate spinning bed with the laying density of 0.5-1 kg/m2。
4) Collecting silkworms of 0-100 m, 200-400 m, 400-700 m and 700-1200 m in the 5 groups of silkworms in the step 3) respectively, wherein the silkworms are numbered as A, B, C and D.
5) Washing the silks with different groups and different lengths obtained in the step 4) with deionized water for 3-5 times, drying at 60 ℃, and then adding Na2CO3Dissolving the solution by water and heat; after cooling the solution, the incompletely dissolved fractions were removed to give a crude product of fibroin solution.
6) Dialyzing the crude product of the fibroin solution obtained in the step 5) for 24h through a membrane with the molecular weight cutoff of 1000, and carrying out vacuum freeze drying to obtain fibroin powder.
7) Analyzing the fibroin powder obtained in the step 6) by electrophoresis: dissolving fibroin powder in carbonate buffer solution with pH of 9.6 to obtain 20 μ g/μ L solution; taking 5-10 mu L of dissolving solution, performing high-temperature denaturation and centrifugation to obtain a protein sample, adding supernatant and a protein Marker of the sample into 10-15% polyacrylamide gel, and performing an electrophoresis experiment; after being stained with Coomassie brilliant blue G-250, the molecular weight distribution was analyzed.
8) Cutting the glue and decoloring: and cutting the protein band from the separation gel, placing the protein band in a clean centrifugal tube, washing the protein band for 2-3 times by using deionized water, carrying out reductive alkylation and further decoloration treatment, and freeze-drying.
9) And (3) enzymolysis in glue: the gel strip was immersed in 25mM NH4HCO3Adding 2-10 mu g of sequencing-grade and modified trypsin, and incubating at 37 ℃ for 12-16 h; after the extracting solution is collected, adding 30-50 mu L of extracting solution mixed by 5% formic acid solution and acetonitrile according to the volume ratio of 1:1, and repeatedly extracting polypeptide fragments in the adhesive tape; the extract was lyophilized and redissolved in 0.1% formic acid solution.
10) Performing mass spectrometry on the solution obtained in step 9): LC-MS test is carried out to obtain different lengths of different groups of mulberry silk13C isotope distribution and protein expression.
Preferably, in said step 2)13C configuration of labeled glycine solution: at 25 deg.C, 20g13And dissolving the C-labeled glycine in 100mL of deionized water to obtain a glycine spray solution.
Preferably, said step 2)13C, when spraying the glycine to the mulberry leaves, spraying the glycine while turning, repeatedly spraying for 3-5 times, and spraying 10kg of mulberry leaves with 2-3 kg of glycine spraying liquid.
Preferably, in the step 3), when 90-95% of the silkworms are ripe, putting the ripe silkworms on A1 paper, laying plant ash under A1 paper, and uniformly putting the ripe silkworms on a flat spinning bed within 12 hours; the density is sparse and uniform.
Preferably, the step 3) adopts a screen window type flat plate silking bed, the upper layer of which is screen window cloth with through holes, and the lower layer of which is a plastic film; and (5) shading the silkworm room.
Preferably, in the step 4), the length of the flat yarn is measured every 4h, the silk length is recorded, and the collected silk is cut.
Preferably, in the step 5), the silk is placed in 0.5 wt% Na at a bath ratio of 1:502CO3In the solution, the pH is adjusted to 11-12, and the solution is dissolved in water at 110-130 ℃ for 3-5 h.
Preferably, in the step 6), dialysis is performed at 4-6 ℃.
Preferably, in the step 7), 5-10 μ L of sample dissolving solution is taken and put into a small centrifuge tube, the sample loading buffer solution is added according to the volume ratio of 1:1, the sample is bathed for 8-10 min at 95-100 ℃, and the sample is centrifuged for 2-3 min at 8000r/min after being cooled, so as to obtain the protein sample.
Preferably, during the electrophoresis operation in step 7): the voltage is first constant at 80V, and is adjusted to 120V after 10 min. The invention has the beneficial effects that: the invention13The relationship between the time for taking mulberry leaves and cocoon silk can be obtained by tracing and analyzing the glycine marked by C, and13c is a stable isotope without radioactivity, and has no influence on the growth and development of the silkworm; the invention adopts the flat plate spinning technology, changes the physical habit of silkworm cocooning, forms flat plate silk and naked pupa, and ensures the accuracy of silk sectional collection; the invention collects the silk in sections, which can realize the research on different expressions of protein in the process of silking of the silkworm; the invention adopts a low sodium salt system to dissolve the cocoon shell, compared with the traditional high-concentration neutral salt solution, the invention can reduce the dosage of chemicals, reduce the introduction of impurity ions, shorten the purification treatment time, reduce the cost and has no toxicity and harm; according to the silk dissolving method, the pH value of a low-sodium salt system is controlled to be 11-12, so that silk can be dissolved, and damage to the structure of the silk is reduced; the invention is an analysis technology based on proteomics, and can rapidly, efficiently and accurately detect and quantitatively analyze proteins.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the embodiments of the present invention are not limited to the following examples.
Example 1
1): feeding the same silkworm eggs to the 3 rd day of 5 years old under the same external condition, and regularly disinfecting the ground and the air of a silkworm rearing room by using medicines in the silkworm feeding process; the temperature and humidity of the silkworm rearing room are strictly controlled to ensure the consistent growth of the silkworm; well ventilating the silkworm rearing room; dividing the silkworms into 5 groups, wherein the numbers of the silkworms are A, B, C, D, E;
2): at 25 deg.C, 20g13Dissolving glycine marked C in 100mL of deionized water to obtain glycine spray liquid, and spraying folium Mori while turning over for 3 times, wherein 2kg of glycine spray liquid can spray 10kg of folium Mori. A. B, C, D, E groups of silkworm are fed on 4 th, 5 th, 6 th, 7 th and 8 th days of 5 th age13C, marking glycine mulberry leaves, and feeding the same common mulberry leaves in the rest time;
3): when 5 groups of silkworm reach 90% maturity, placing the mature silkworm on A1 paper, laying plant ash on the lower surface, and uniformly placing the mature silkworm on a flat plate spinning bed within 12h after excretion is clean; the distribution density is 0.5kg/m2Sparse and uniform;
4): measuring the length of the flat yarn every 4h, recording the length of the silk, and respectively collecting the silks 100m, 200-400 m, 400-700 m and 700-1200 m before 5 groups of silkworms, wherein the silks are numbered as A, B, C and D;
5): washing silk of different groups and lengths with deionized water for 3 times, drying at 60 deg.C, and placing in 0.5 wt% Na at a bath ratio of 1:502CO3In the solution, the pH was adjusted to 11 and the mixture was dissolved hydrothermally at 110 ℃ for 5 hours. After the solution is cooled, centrifuging to remove the part which is not completely dissolved to obtain a crude product of the fibroin solution;
6): dialyzing the fibroin solution at 4 deg.C (molecular weight cut-off of 1000) for 24 hr, removing small molecular impurities in the solution, and vacuum freeze drying to obtain fibroin powder;
7): electrophoretic analysis: fibroin powder dissolved in CB 9.6 (Na)2CO3/NaHCO3Buffer solution) to obtain 20 mu g/mu L of solution, taking 5 mu L of sample solution to put in a small centrifugal tube, adding the sample buffer solution according to the volume ratio of 1:1, carrying out water bath at 95 ℃ for 10min, cooling, centrifuging at 8000r/min for 2min to obtain a protein sample, adding the supernatant and the protein Marker of the protein sample into 10% polyacrylamide gel (including separation gel and concentrated gel), and firstly keeping the constant pressure at 80V to ensure that silk is kept at constant pressureConcentrating the protein in the concentrated gel, adjusting the voltage to 120V after 10min, when the bromophenol blue indicator in the sample runs to the bottom of the gel, indicating that the electrophoresis is finished, taking out the gel, and analyzing the molecular weight distribution of the gel after passing through Coomassie brilliant blue G-250 staining solution.
8): cutting the glue and decoloring: cutting off protein band from separation gel, placing in clean centrifuge tube, washing with deionized water for 2 times, performing reductive alkylation and further decolorizing treatment, and freeze drying.
9): and (3) enzymolysis in glue: the gel strips were placed in 25mM ammonium bicarbonate (solution submerged gel strips), 2. mu.g sequencing grade and modified trypsin was added and incubated at 37 ℃ for 12 h. After the extract was collected, 30. mu.L of an extract (5% formic acid solution and acetonitrile were mixed at a volume ratio of 1: 1) was added, and the polypeptide fragments in the gel strip were repeatedly extracted. The extract was lyophilized and redissolved in 0.1% formic acid solution.
10): performing mass spectrometry on the solution obtained in step 9): LC-MS/MS tests are carried out to obtain different lengths of different groups of mulberry silk13C isotope distribution and protein expression.
Example 2
1): feeding the same silkworm eggs to the 3 rd day of 5 years old under the same external condition, and regularly disinfecting the ground and the air of a silkworm rearing room by using medicines in the silkworm feeding process; the temperature and humidity of the silkworm rearing room are strictly controlled to ensure the consistent growth of the silkworm; well ventilating the silkworm rearing room; dividing the silkworms into 5 groups, wherein the numbers of the silkworms are A, B, C, D, E;
2): at 25 deg.C, 20g13Dissolving glycine marked C in 100mL of deionized water to obtain glycine spray liquid, and spraying folium Mori while turning, repeatedly spraying for 4 times, wherein 2kg of glycine spray liquid can spray 10kg of folium Mori. A. B, C, D, E groups of silkworm are fed on 4 th, 5 th, 6 th, 7 th and 8 th days of 5 th age13C, marking glycine mulberry leaves, and feeding the same common mulberry leaves in the rest time;
3): when 5 groups of silkworm reach 93% of maturity, placing the mature silkworms on A1 paper, laying plant ash on the lower surface of the paper, and uniformly placing the mature silkworms on a flat spinning bed within 12 hours after excretion is clean; is distributed at a density of0.7kg/m2Sparse and uniform;
4): measuring the length of the flat yarn every 4h, recording the length of the silk, and respectively collecting the silks 100m, 200-400 m, 400-700 m and 700-1200 m before 5 groups of silkworms, wherein the silks are numbered as A, B, C and D;
5): washing silk of different groups and lengths with deionized water for 3 times, drying at 60 deg.C, and placing in 0.5 wt% Na at a bath ratio of 1:502CO3In the solution, the pH was adjusted to 11.5 and the solution was dissolved hydrothermally at 120 ℃ for 4 h. After the solution is cooled, centrifuging to remove the part which is not completely dissolved to obtain a crude product of the fibroin solution;
6): dialyzing the fibroin solution at 4 deg.C (molecular weight cut-off of 1000) for 24 hr, removing small molecular impurities in the solution, and vacuum freeze drying to obtain fibroin powder;
7): electrophoretic analysis: fibroin powder dissolved in CB 9.6 (Na)2CO3/NaHCO3Buffer solution) to obtain 20 mug/muL of solution, taking 7 muL of sample solution to a small centrifugal tube, adding the sample solution into a sample loading buffer solution according to the volume ratio of 1:1, carrying out water bath for 9min at 98 ℃, cooling, centrifuging for 3min at 8000r/min to obtain a protein sample, adding a sample supernatant and a protein Marker into 12.5% polyacrylamide gel (including separation gel and concentrated gel), firstly keeping the constant voltage at 80V to concentrate fibroin in the concentrated gel, adjusting the voltage to 120V after 10min, when a bromophenol blue indicator in the sample runs to the bottom of the gel, indicating that electrophoresis is finished, taking out the gel, and analyzing the molecular weight distribution after Coomassie brilliant blue G-250 staining solution.
8): cutting the glue and decoloring: cutting off protein band from separation gel, placing in clean centrifuge tube, washing with deionized water for 3 times, performing reductive alkylation and further decolorizing treatment, and freeze drying.
9): and (3) enzymolysis in glue: the gel strips were placed in 25mM ammonium bicarbonate (solution submerged gel strips), 5. mu.g sequencing grade and modified trypsin was added, and incubated at 37 ℃ for 12 h. After the extract was collected, 40. mu.L of an extract (5% formic acid solution and acetonitrile mixed at a volume ratio of 1: 1) was added, and the extraction of the polypeptide fragments in the gel strip was repeated. The extract was lyophilized and redissolved in 0.1% formic acid solution.
10): performing mass spectrometry on the solution obtained in step 9): LC-MS/MS tests are carried out to obtain different lengths of different groups of mulberry silk13C isotope distribution and protein expression.
Example 3
1): feeding the same silkworm eggs to the 3 rd day of 5 years old under the same external condition, and regularly disinfecting the ground and the air of a silkworm rearing room by using medicines in the silkworm feeding process; the temperature and humidity of the silkworm rearing room are strictly controlled to ensure the consistent growth of the silkworm; well ventilating the silkworm rearing room; dividing the silkworms into 5 groups, wherein the numbers of the silkworms are A, B, C, D, E;
2): at 25 deg.C, 20g13Dissolving glycine marked C in 100mL of deionized water to obtain glycine spray liquid, and spraying folium Mori while turning, repeatedly spraying for 5 times, wherein 2kg of glycine spray liquid can spray 10kg of folium Mori. A. B, C, D, E groups of silkworm are fed on 4 th, 5 th, 6 th, 7 th and 8 th days of 5 th age13C, marking glycine mulberry leaves, and feeding the same common mulberry leaves in the rest time;
3): when 5 groups of silkworm reach 95% maturity, placing the mature silkworm on A1 paper, laying plant ash on the lower surface of the paper, and uniformly placing the mature silkworm on a flat spinning bed within 12 hours after excretion is clean; the distribution density is 1kg/m2Sparse and uniform;
4): measuring the length of the flat yarn every 4h, recording the length of the silk, and respectively collecting the silks 100m, 200-400 m, 400-700 m and 700-1200 m before 5 groups of silkworms, wherein the silks are numbered as A, B, C and D;
5): washing silk of different groups and lengths with deionized water for 5 times, drying at 60 deg.C, and placing in 0.5 wt% Na at a bath ratio of 1:502CO3In the solution, 12 was adjusted and dissolved in water at 130 ℃ for 3 hours. After the solution is cooled, centrifuging to remove the part which is not completely dissolved to obtain a crude product of the fibroin solution;
6): dialyzing the fibroin solution at 4 deg.C (molecular weight cut-off of 1000) for 24 hr, removing small molecular impurities in the solution, and vacuum freeze drying to obtain fibroin powder;
7): electrophoretic analysis: fibroin powder dissolved in CB 9.6 (Na)2CO3/NaHCO3Buffer solution) to obtain 20 mu g/mu L of solution, taking 10 mu L of sample solution to a small centrifugal tube, adding the sample solution into the sample buffer solution according to the volume ratio of 1:1, carrying out water bath at 100 ℃ for 8min, cooling, and centrifuging at 8000r/min for 3min to obtain a protein sample. Adding the sample supernatant and the protein Marker into 15% polyacrylamide gel (including separation gel and concentrated gel), firstly keeping the constant voltage at 80V to concentrate the fibroin in the concentrated gel, adjusting the voltage to 120V after 10min, indicating that the electrophoresis is finished when the bromophenol blue indicator in the sample runs to the bottom of the gel, taking out the gel, and analyzing the molecular weight distribution after passing through Coomassie brilliant blue G-250 staining solution.
8): cutting the glue and decoloring: cutting off protein band from separation gel, placing in clean centrifuge tube, washing with deionized water for 3 times, performing reductive alkylation and further decolorizing treatment, and freeze drying.
9): and (3) enzymolysis in glue: the gel strips were placed in 25mM ammonium bicarbonate (solution submerged gel strips), 10. mu.g sequencing grade and modified trypsin was added and incubated at 37 ℃ for 12 h. After the extract was collected, 50. mu.L of an extract (5% formic acid solution and acetonitrile were mixed at a volume ratio of 1: 1) was added, and the polypeptide fragments in the gel strip were repeatedly extracted. The extract was lyophilized and redissolved in 0.1% formic acid solution.
10): performing mass spectrometry on the solution obtained in step 9): LC-MS/MS tests are carried out to obtain different lengths of different groups of mulberry silk13C isotope distribution and protein expression.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (10)
1. A method for analyzing the formation process and the proteome change of mulberry silk is characterized by comprising the following steps:
1) a, B, C, D, E feeding silkworm eggs of the same batch under the same external condition to the 3 rd day of 5 years old;
2) feeding A, B, C, D, E groups in step 1) on 4 th, 5 th, 6 th, 7 th and 8 th days of 5 th age, respectively, and spraying13C, marking the same mulberry leaves with glycine;
3) when the silkworm maturity of the 5 groups of the silkworms in the step 2) is 90-95%, laying the matured silkworms on a flat-plate silking bed with the laying density of 0.5-1 kg/m2；
4) Respectively collecting silkworms of 0-100 m, 200-400 m, 400-700 m and 700-1200 m in the 5 groups of silkworms in the step 3), wherein the silkworms are numbered as A, B, C and D;
5) washing silks of different groups and different lengths obtained in the step 4) with deionized water for 3-5 times, drying at 60 ℃, and then performing hydrothermal dissolution in a Na2CO3 solution; after the solution is cooled, removing the part which is not completely dissolved to obtain a crude product of the fibroin solution;
6) dialyzing the crude product of the fibroin solution obtained in the step 5) for 24 hours by a membrane with molecular weight cutoff of 1000, and carrying out vacuum freeze drying to obtain fibroin powder;
7) analyzing the fibroin powder obtained in the step 6) by electrophoresis: dissolving fibroin powder in carbonate buffer solution with pH of 9.6 to obtain 20 μ g/μ L solution; taking 5-10 mu L of dissolving solution, performing high-temperature denaturation and centrifugation to obtain a protein sample, adding supernatant and a protein Marker of the sample into 10-15% polyacrylamide gel, and performing an electrophoresis experiment; analyzing the molecular weight distribution of the sample after passing through Coomassie brilliant blue G-250 staining solution;
8) cutting the glue and decoloring: cutting a protein band from the separation gel, placing the protein band in a clean centrifugal tube, washing the protein band for 2-3 times by using deionized water, carrying out reductive alkylation and further decoloration treatment, and then carrying out freeze drying;
9) and (3) enzymolysis in glue: the gel strip was immersed in 25mM NH4HCO3Adding 2-10 mu g of sequencing-grade and modified trypsin, and incubating at 37 ℃ for 12-16 h; after the extracting solution is collected, adding 30-5 parts of the extracting solution0 mu L of extract liquid mixed by 5 percent formic acid solution and acetonitrile according to the volume ratio of 1:1 is used for repeatedly extracting the polypeptide fragments in the adhesive tape; freeze drying the extractive solution, and dissolving in 0.1% formic acid solution;
10) performing mass spectrometry on the solution obtained in step 9): LC-MS test is carried out to obtain different lengths of different groups of mulberry silk13C isotope distribution and protein expression.
2. The method of claim 1, wherein the method comprises the steps of: in the step 2)13C configuration of labeled glycine solution: at 25 deg.C, 20g13And dissolving the C-labeled glycine in 100mL of deionized water to obtain a glycine spray solution.
3. The method of claim 1, wherein the method comprises the steps of: said step 2)13C, when spraying the glycine to the mulberry leaves, spraying the glycine while turning, repeatedly spraying for 3-5 times, and spraying 10kg of mulberry leaves with 2-3 kg of glycine spraying liquid.
4. The method of claim 1, wherein the method comprises the steps of: in the step 3), when 90-95% of the silkworms are ripe, putting the ripe silkworms on A1 paper, laying plant ash under A1 paper, and uniformly putting the ripe silkworms on a flat-plate spinning bed within 12 hours; the density is sparse and uniform.
5. The method of claim 1, wherein the method comprises the steps of: the step 3) adopts a screen window type flat plate spinning bed with the upper layer being screen window cloth with through holes and the lower layer being a plastic film; and (5) shading the silkworm room.
6. The method of claim 1, wherein the method comprises the steps of: and in the step 4), measuring the length of the flat yarn every 4h, recording the length of the silk, and intercepting and collecting the silk.
7. The method of claim 1, wherein the method comprises the steps of: in the step 5), the silk is placed in 0.5 wt% of Na at a bath ratio of 1:502CO3And (3) adjusting the pH value of the solution to 11-12, and performing hydrothermal dissolution at the temperature of 110-130 ℃ for 3-5 hours.
8. The method of claim 1, wherein the method comprises the steps of: in the step 6), dialysis is performed at 4-6 ℃.
9. The method of claim 1, wherein the method comprises the steps of: preparation of the protein sample in the step 7): and (3) putting 5-10 mu L of sample solution into a small centrifugal tube, adding a sample loading buffer solution according to the volume ratio of 1:1, carrying out water bath for 8-10 min at 95-100 ℃, cooling, and centrifuging for 2-3 min at 8000r/min to obtain a protein sample.
10. The method of claim 1, wherein the method comprises the steps of: during the electrophoresis operation in the step 7): the voltage is first constant at 80V, and is adjusted to 120V after 10 min.
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