CN108254569B - Method for detecting cold resistance of mulberry variety - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to a method for detecting cold resistance of mulberry varieties, which comprises the steps of preparing an antibody through a specific AKR2A protein sequence, and coating an arabidopsis AKR2A antibody on a 96-hole polystyrene reaction plate. After specific mulberry leaf tissue is treated, AKR2A protein antigen is obtained, the protein antigen is added to a reaction plate coated by an antibody according to the number, the immunoreaction is carried out with AKR2A antibody, the immunoreaction is further carried out with horseradish peroxidase-labeled secondary antibody, and the specific AKR2A protein in mulberry is detected through substrate color development. Through detecting the protein AKR2A in the mulberry, the contrast analysis shows that the low temperature stress resistance of the mulberry is in positive correlation with the content of AKR2A gene or protein, and the cold resistance of the mulberry is indicated through the detection of the content of AKR2A protein in 1-2 leaves at the top of the mulberry. The invention establishes a direct, rapid and reliable method for detecting the cold resistance of the mulberry, is used for screening and molecular breeding of cold-resistant mulberry varieties and remote and ultra-remote introduction of mulberry germplasm (varieties), and has great academic and economic values.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for detecting cold resistance of mulberry varieties.
Background
The natural distribution area of the mulberry is very wide, the mulberry is found in Asia, North America, Europe and Africa, and the mulberry resource is distributed in tropical zone, subtropical zone, warm zone to cold-warm zone. Due to the long-term growth in different natural environments, extremely rich genetic diversity is formed. Some mulberries can grow in arid and semi-arid desert regions with precipitation of less than 150mm, some mulberries can resist low temperature of minus 30 ℃ and can also bear high temperature of 40 ℃, and some mulberries have strong adaptability to the pH value of soil, can grow under the condition of pH value of 4.5-8.5, and can also grow normally when the salt content of the soil is 0.2%. More than 7000 portions of mulberry germplasm are preserved in a main mulberry germplasm resource preservation place around the world. To date, more than 3000 mulberry germplasm resources of various types have been collected and organized in China, and the mulberry resources guarantee the diversified development requirements of the silkworm industry in China in the future. At present, the silkworm industry faces transformation development, and the mulberry can not only be used for silkworm breeding, but also play a greater role in ecological environment protection. Therefore, the cultivation of mulberry varieties integrating the high-quality characteristics of the mulberries needs to be accelerated, and the germplasm range of high-quality germplasm resources is expanded, which is also very critical for the smooth introduction of mulberry varieties in areas with poor natural conditions (such as cold areas).
During the cold process, many of the intracellular enzymatic activities are inactivated and toxic Reactive Oxygen Species (ROS) accumulate, causing reduced fluidity of membrane lipids and increased membrane permeability, which are the direct causes of cell damage (Gombos et al, 1994). Under normal conditions, plants can effectively use ROS as a signal molecule to start the expression of corresponding genes so as to eliminate the toxicity of ROS, and the ROS comprises superoxide dismutase (SOD), Catalase (CAT), Peroxidase (POD), Ascorbate Peroxidase (APX), Glutathione Reductase (GR) and the like. Chaperones are a class of conserved proteins in cells that recognize unnatural conformations of specific peptide chains, help the nascent protein to fold correctly, and maintain this folded state. Without chaperones, many proteins cannot be targeted to target areas to perform their functions.
When plants are damaged by low temperature, the biological membrane firstly undergoes membrane lipid phase change from liquid crystal lamellar phase to hard gel phase. The membrane tissue of the cell is mainly a double-layer membrane composed of fatty acid, fatty acid molecules are like tadpoles and are regularly arranged on the membrane, the head of the fatty acid molecules faces the outside of the membrane, and the tail of the fatty acid molecules is hidden in the double-layer membrane. When cells are subjected to low temperatures, the tails are in close proximity to reduce membrane fluidity and damage to the cells (Vijayan and Browse, 2002). Those "straight" fatty acid tails generally consist of saturated fatty acids, while twisted fatty acid tails consist of unsaturated fatty acids (Beck et al, 2004). Unsaturated fatty acid radicals are classified into monounsaturated fatty acids and polyunsaturated fatty acids according to the difference in the number of double bonds. The monounsaturated fatty acid is oleic acid (C18: 1), and the polyunsaturated fatty acid is linoleic acid (C18: 2), linolenic acid (C18: 3), arachidonic acid (C20: 4), eicosapentaenoic acid (C20: 5), docosahexaenoic acid (C22: 6). Fatty acids with a carbon number above 18C are called (super) long chain fatty acids. The synthesis of the fatty acid is to generate fatty acid with different chain lengths by the fatty acyl-CoA elongase catalyzing the elongation of 16C/18C fatty acid. 18C fatty acids are therefore an important extension point. Fatty acyl-CoA elongases are a multienzyme complex in which ketoacyl-CoA synthase (3-ketoacyl-CoA synthsase, KCS) is a key subunit of the multienzyme complex. KCS1 catalyzes the first condensation reaction in the synthesis of VLCFAs, the rate-limiting enzyme in the fatty acid elongation reaction (Lassner et al, 1996). The contents of various C20-C30 fatty acids are increased and the resistance is increased to different degrees after the heterologous expression of KCS1, KCS2 and the like in yeast (Trenkamp et al, 2004). Whereas the long-chain lipid content in kcs1 Arabidopsis mutant was significantly reduced (Todd et al, 1999). The gene of the gene engineering transformed fatty acid can also increase the synthesis of unsaturated fatty acid of plants, and the introduction of the Arabidopsis thaliana fatty acid desaturase gene Fad7 in tobacco can increase the content of octadecatriene fatty acid in the transformed plants and obviously improve the cold resistance of the transformed plants (Kodama et al, 1994). The interaction protein of the arabidopsis library and AKR2A is verified by yeast two-hybrid, and the obvious interaction relationship between AKR2A and KCS1 is found. The interaction relation between AKR2A and KCS1 suggests that AKR2A is at the upstream of long-chain fatty acid synthetase KCS1 to regulate the synthesis of fatty acid, and then regulates the cold resistance of plants by mediating the synthesis of fatty acid.
Disclosure of Invention
The invention provides a method for detecting cold resistance of mulberry varieties by detecting the content of AKR2A protein in mulberry in order to overcome the defects of the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting cold resistance of mulberry varieties comprises the following steps:
(1) antibody preparation: searching cDNA sequence of AKR2A in Tair Database of Arabidopsis thaliana, removing 1-198 bases from N end, matching with a sequence of starting base ATG connecting 199 th base of AKR2A to termination code; producing arabidopsis AKR2A target fragment protein by taking PET21 as a plasmid and BL21 as a strain; preparing an arabidopsis AKR2A target antibody;
(1) antibody preparation: searching cDNA sequence of AKR2A in Tair Database of Arabidopsis thaliana, removing 1-198 bases from N end, matching with a sequence of starting base ATG connecting 199 th base of AKR2A to termination code; producing arabidopsis AKR2A target fragment protein by taking PET21 as a plasmid and BL21 as a strain; preparing an arabidopsis AKR2A target antibody;
(2) antibody coating: diluting the arabidopsis AKR2A target antibody, coating a 96-hole polystyrene ELISA reaction plate with the concentration of 100 mu L/hole to obtain an antibody matrix for ELISA reaction, and placing the antibody matrix at 4 ℃ for later use;
(3) coating the plate with 5% skimmed milk powder blocking antibody, incubating at room temperature for 1h, wherein each well contains 200 μ L of the solution;
(4) preparation of mulberry AKR2A antigen: grinding 0.2g of mulberry sample in a mortar under the liquid nitrogen freezing state, then oscillating for 30s with 0.2mL of first extracting solution, mixing uniformly, and standing for 0.5h on ice; taking out, centrifuging at 4 ℃, transferring the supernatant into another test tube, and suspending the precipitate with 0.2mL of second extracting solution to obtain a suspension; boiling the suspension for 10min, cooling on ice, centrifuging at 4 deg.C, and collecting supernatant; mixing the above 2 supernatants to obtain mulberry AKR2A antigen;
(5) diluting a mulberry AKR2A antigen and a negative and positive control by PBST solution 1:1, adding the diluted antigen into reaction holes of a 96-hole polystyrene reaction plate coated with the antigen according to the number, adding 100 mu L of the antigen into each reaction hole, repeating two holes for negative and positive reactions, reacting for 2h at the saturation humidity of 22-28 ℃, and washing for later use;
(6) diluting a goat anti-rabbit IgG-HRP antibody marked by horseradish peroxidase with a PBST solution 1:1000, sequentially adding the diluted antibody into reaction holes of a 96-hole polystyrene reaction plate, adding 100 mu L of the antibody into each reaction hole, carrying out light-shielding reaction at the saturation humidity of 22-28 ℃ for 2h, and washing the antibody with PBST for later use;
(7) adding an alkaline phosphatase substrate TMB into reaction holes of a 96-hole polystyrene reaction plate in sequence, adding 100 mu L of alkaline phosphatase substrate TMB into each reaction hole, reacting for 20min in a dark place, adding 50 mu L of 2M concentrated sulfuric acid into each reaction hole, and stopping reaction;
(8) and (3) placing the 96-hole polystyrene reaction plate with the reaction stopped into a microplate reader, reading OD650 data, and judging the result.
According to the invention, an antibody is prepared through a specific AKR2A protein sequence, and an arabidopsis AKR2A antibody is coated on a 96-hole polystyrene reaction plate. After specific mulberry leaf tissue is treated, AKR2A protein antigen is obtained, the protein antigen is added to a reaction plate coated by an antibody according to the number, the immunoreaction is carried out with AKR2A antibody, the immunoreaction is further carried out with horseradish peroxidase-labeled secondary antibody, and the specific AKR2A protein in mulberry is detected through substrate color development. Through detecting the protein AKR2A in the mulberry, the contrast analysis shows that the low temperature stress resistance of the mulberry is in positive correlation with the content of AKR2A gene or protein, and the cold resistance of the mulberry is indicated through the detection of the content of AKR2A protein in 1-2 leaves at the top of the mulberry. Under cold conditions, the expression level of AKR2A protein in mulberry leaves is in a highly positive correlation with the cold resistance of mulberries. Therefore, the cold resistance of the mulberry varieties can be judged only by applying the expression quantity of AKR2A (namely the content of AKR2A antigen in the mulberry) in 1-2 leaf tissues at the top of the mulberry to be detected in the seedling stage by using an ELISA method, and important operation methods and theoretical bases are provided for flux screening of drought-resistant mulberry germplasm resources, ultra-long distance mulberry introduction and screening of cold-resistant mulberry varieties in large-area planting.
Preferably, in step (2), the Arabidopsis AKR2A target antibody is diluted at 1:1000 using a carbonate buffer solution of pH 9.6.
Preferably, in the step (4), the concentration of each component in the first extracting solution is 50mmol/L sodium phosphate and 1mmol/L EDTA; 2% (v/v) Triton X-100 and 2% (m/v) SDS.
Preferably, in the step (4), the first extract liquid and the second extract liquid have a pH of 6.8 to 7.2.
Preferably, in the step (4), the centrifugal rotating speed is 12000-15000 r/min, and the centrifugal time is 8-10 min.
Preferably, in the step (4), the mulberry sample is 1-2 leaf tissue samples at the top of the mulberry seedling to be detected for cuttage in 30 d.
The protein expression quantity of AKR2A in 1-2 leaf samples at the top of the mulberry is in a significant positive correlation with the cold resistance of mulberry varieties, and the mode is specific to the mulberry. Under cold conditions, the expression level of AKR2A protein in mulberry leaves is in a highly positive correlation with the cold resistance of mulberries.
Preferably, in step (8), OD650 data is read, and the result is determined according to the following formula: defining the ratio of the OD value of the mulberry sample to be detected to the known positive sample as S/P, the OD value of the known negative sample is less than or equal to 0.20, the OD value of the positive sample is greater than or equal to 0.8, and under the condition that the conditions are met:
when the S/P is less than 0.5, the mulberry is judged to be negative, namely the cold resistance of the corresponding mulberry is poor;
when the S/P is more than or equal to 0.5, the test result is positive;
when S/P is more than or equal to 0.5 and less than or equal to 0.75, the cold resistance of the corresponding mulberry is moderate;
when S/P is more than 0.75, the cold resistance of the corresponding mulberry is stronger.
Therefore, the invention has the following beneficial effects: the method for detecting the cold resistance of a certain variety (strain or germplasm) is judged and predicted in the early stage (seedling stage), reduces the blindness of mulberry seedling growers and breeders in variety breeding and ultra-remote introduction of mulberry germplasm resources in actual production in selection of cold-resistant varieties, and provides an important theoretical method for researchers to research the cold resistance mechanism. The invention establishes a direct, rapid and reliable method for detecting the cold resistance of the mulberry, is used for screening and molecular breeding of cold-resistant mulberry varieties and remote and ultra-remote introduction of mulberry germplasm (varieties), and has great academic and economic values.
Drawings
FIG. 1 is a graph comparing the germination rates of different mulberry samples after cold treatment according to the present invention.
FIG. 2 shows the level of AKR2A and SOD expression by the fluorescent quantitative PCR analysis of the present invention.
FIG. 3 shows the expression level of AKR2A and SOD in Westernblot analysis of the present invention: control N; cold treatment C.
Detailed Description
The technical solution of the present invention is further specifically described below by using specific embodiments and with reference to the accompanying drawings.
In the present invention, all the equipment and materials are commercially available or commonly used in the art, and the methods in the following examples are conventional in the art unless otherwise specified.
The invention uses the same AKR2A protein detection method to detect the cold resistance of Xinjiang white mulberry, Hu mulberry 32 and Yunnan Changsu mulberry. White mulberry in Xinjiang (Morus alba L., white mulberry for short), Hu mulberry 32(Morus alba Linn. var. multicaulis (Perrot) Loud. Hu mulberry for short) and Yunan Changsu mulberry (Morus wittiorum hand. -Mazz. Changsu mulberry for short). The original white mulberry in Xinjiang is a mulberry variety with strong cold resistance in northern China. The main planting area of the Hu mulberry 32 is a general control mulberry variety in the silkworm area of the Yangtze river basin in China, the Yunan Changsu mulberry is a variety with weak cold resistance, and the germination rate of the mulberry in the next year is still less than 20 percent even if the Yunan Changsu mulberry is planted in the middle and lower reaches of the Yangtze river.
Mulberry cold experiment:
annual potted Xinjiang white mulberry, agricultural mulberry 12 and Yunnan long-ear mulberry with the diameter of about 1cm are cultured in dark for 30 days at the temperature of-20 ℃ and 2 ℃ respectively at the beginning of 12 months. Subsequently, the mulberry trees are transplanted to a climatic chamber, and the culture conditions are as follows: humidity is 70%; the temperature is 25 ℃; photoperiod 16h light/8 h dark; culturing under 20,000lx for 15 days, and recording to count the germination condition of mulberry.
Taking mulberry twigs with the diameter of about 1cm at the beginning of 12 months, ensuring that each mulberry twig has about 6 active teeth, and carrying out cuttage in a climatic chamber. The culture conditions are as follows: humidity is 70%; the temperature is 25 ℃; photoperiod 16h light/8 h dark; the illumination intensity was 20,000lx cultured until the second new leaf was fully extended, followed by chilling. And (4) cold treatment, namely transferring the normally grown mulberry twigs into an incubator at 4 ℃ for 8 hours. The leaves were then removed and frozen at-80 ℃.
The cold treatment has influence on the mulberry germination rate and the AKR2A and SOD expression level, the plant is placed at 2 ℃, the germination rates of three mulberry varieties are not obviously different, the germination rates of the three mulberry varieties are obviously different after the cold treatment at-20 ℃, and the comparison result of the germination rates of different mulberry samples after the cold treatment is shown in figure 1. As can be seen from the fluorescent quantitative PCR results of FIG. 2 and the Westernblot results of FIG. 3, the gene levels of mAKR2A and SOD of the three mulberry varieties were reduced after cold treatment, but the gene and protein expression levels of mAKR2A and SOD of white mulberry were still higher than those of Morus longicornus and Morus laevigata.
Example 1
Mulberry AKR2A antigen ELISA detection:
(1) antibody preparation: searching cDNA sequence of AKR2A in Tair Database of Arabidopsis thaliana, removing 1-198 bases from N end, matching with a sequence of starting base ATG connecting 199 th base of AKR2A to termination code; producing arabidopsis AKR2A target fragment protein by taking PET21 as a plasmid and BL21 as a strain; preparing an arabidopsis AKR2A target antibody;
(2) antibody coating: diluting the arabidopsis AKR2A target antibody, coating a 96-hole polystyrene ELISA reaction plate with the concentration of 100 mu L/hole to obtain an antibody matrix for ELISA reaction, and placing the antibody matrix at 4 ℃ for later use;
(3) coating the plate with 5% skimmed milk powder blocking antibody, incubating at room temperature for 1h, wherein each well contains 200 μ L of the solution;
(4) preparation of mulberry AKR2A antigen: taking 30d leaf tissue samples of 1-2 leaves at the top end of a mulberry seedling to be detected in cuttage of Sinkiang white mulberry and 17 subsequent different mulberry varieties to be detected, grinding the samples in a mortar in a liquid nitrogen freezing state, oscillating for 30s with 0.2mL of first extracting solution containing 50mmol/L sodium phosphate and 1mmol/L EDTA and having the pH value of 7.0, uniformly mixing, and standing for 0.5h on ice; taking out, centrifuging at 13000r/min for 10min at 4 ℃, transferring the supernatant into another test tube, and suspending the precipitate with 0.2mL of a second extract containing 50mmol/L phosphate buffer, 2% (v/v) Triton X-100 and 2% (m/v) SDS and having a pH of 7.0 to obtain a suspension; boiling the suspension for 10min, cooling on ice, centrifuging at 13000r/min at 4 deg.C for 10min, and collecting supernatant; mixing the above 2 supernatants to obtain mulberry AKR2A antigen;
(5) diluting a mulberry AKR2A antigen and a negative and positive control by PBST solution 1:1, adding the diluted antigen into reaction holes of a 96-hole polystyrene reaction plate coated with the antigen according to the number, adding 100 mu L of the antigen into each reaction hole, repeating two holes for negative and positive reactions, reacting for 2h at the saturation humidity of 22-28 ℃, and washing for later use;
(6) diluting a goat anti-rabbit IgG-HRP antibody marked by horseradish peroxidase with a PBST solution 1:1000, sequentially adding the diluted antibody into reaction holes of a 96-hole polystyrene reaction plate, adding 100 mu L of the antibody into each reaction hole, carrying out light-shielding reaction at the saturation humidity of 22-28 ℃ for 2h, and washing the antibody with PBST for later use;
(7) adding an alkaline phosphatase substrate TMB into reaction holes of a 96-hole polystyrene reaction plate in sequence, adding 100 mu L of alkaline phosphatase substrate TMB into each reaction hole, reacting for 20min in a dark place, adding 50 mu L of 2M concentrated sulfuric acid into each reaction hole, and stopping reaction;
(8) placing the 96-hole polystyrene reaction plate for terminating the reaction into a microplate reader, reading OD650 data, and judging the result according to the following formula: defining the ratio of the OD value of the mulberry sample to be detected to the known positive sample as S/P, the OD value of the known negative sample is less than or equal to 0.20, the OD value of the positive sample is greater than or equal to 0.8, and under the condition that the conditions are met:
when the S/P is less than 0.5, the mulberry is judged to be negative, namely the cold resistance of the corresponding mulberry is poor;
when the S/P is more than or equal to 0.5, the test result is positive;
when S/P is more than or equal to 0.5 and less than or equal to 0.75, the cold resistance of the corresponding mulberry is moderate;
when S/P is more than 0.75, the cold resistance of the corresponding mulberry is stronger.
Example 2
Example 2 differs from example 1 in step (4):
taking a sample of 1-2 leaf tissues at the top end of 30d of a mulberry seedling to be detected and subjected to cuttage in a 0.2g mode, grinding the sample in a mortar in a liquid nitrogen freezing state, oscillating the sample for 30s by using 0.2mL of a first extracting solution containing 50mmol/L sodium phosphate and 1mmol/L EDTA and having the pH value of 6.8, uniformly mixing, and standing the sample on ice for 0.5 h; taking out, centrifuging at 12000r/min at 4 deg.C for 8min, transferring supernatant to another tube, and suspending precipitate with 0.2mL of second extractive solution containing 50mmol/L phosphate buffer, 2% (v/v) Triton X-100 and 2% (m/v) SDS, and pH 6.8 to obtain suspension; boiling the suspension for 10min, cooling on ice, centrifuging at 12000r/min at 4 deg.C for 8min, and collecting supernatant; mixing the above 2 supernatants to obtain mulberry AKR2A antigen;
the rest of the procedure was exactly the same as in example 1.
Example 3
Example 3 differs from example 1 in step (4):
taking 1-2 leaf tissue samples of 30d of Yunnan Changsui mulberry to be detected at the top end of a mulberry seedling to be cut, grinding the samples in a mortar in a liquid nitrogen freezing state, oscillating the samples for 30s by using 0.2mL of first extracting solution containing 50mmol/L sodium phosphate and 1mmol/L EDTA and having the pH value of 7.2, uniformly mixing, and standing the samples for 0.5h on ice; centrifuging at 15000r/min at 4 deg.C for 9min, transferring the supernatant to another tube, and suspending the precipitate with 0.2mL of a second extract containing 50mmol/L phosphate buffer, 2% (v/v) Triton X-100 and 2% (m/v) SDS at pH 7.2 to obtain a suspension; boiling the suspension for 10min, cooling on ice, centrifuging at 15000r/min at 4 deg.C for 9min, and collecting supernatant; mixing the above 2 supernatants to obtain mulberry AKR2A antigen;
the rest of the procedure was exactly the same as in example 1.
Example 4 example 20
Example 4-example 20 differs from example 1 in that: in the step (4), 30d leaf tissue samples of 1-2 leaves at the top of the mulberry seedlings to be detected are selected from 17 different mulberry varieties to obtain mulberry AKR2A antigen, and the rest steps are completely the same as in the example 1.
The results of testing different varieties of mulberry trees in examples 1 to 20 for cold resistance are summarized in Table 1:
TABLE 1 Mulberry AKR2A protein ELISA test result AKR2A coating
| Sample (I) | OD650 | Mean | S/P value | Determination of negative or positive | Judgment of Cold resistance |
| Positive for | 1.163 | 1.0605 | |||
| Positive for | 0.958 | ||||
| Negative of | 0.198 | 0.188 | |||
| Negative of | 0.178 | ||||
| White mulberry in Xinjiang | 1.55 | 1.461575 | + | High strength | |
| Rhizoma picrorhizae 32 | 0.55 | 0.518623 | + | Medium and high grade | |
| Yunnan Changsui mulberry | 0.16 | 0.150872 | - | Weak (weak) | |
| Mulberry variety 1 | 1.038 | 0.978784 | + | High strength | |
| Mulberry variety 2 | 0.208 | 0.196134 | - | Weak (weak) | |
| Mulberry variety 3 | 0.382 | 0.360207 | - | Weak (weak) | |
| Mulberry variety 4 | 1.084 | 1.022159 | + | High strength | |
| Mulberry variety 5 | 0.331 | 0.312117 | - | Weak (weak) | |
| Mulberry variety 6 | 0.87 | 0.820368 | + | High strength | |
| Mulberry variety 7 | 0.56 | 0.528053 | + | Medium and high grade | |
| Mulberry variety 8 | 0.79 | 0.744932 | + | Medium and high grade | |
| Mulberry variety 9 | 1.355 | 1.277699 | + | High strength | |
| Mulberry variety 10 | 1.458 | 1.374823 | + | High strength | |
| Mulberry variety 11 | 0.872 | 0.822254 | + | High strength | |
| Mulberry variety 12 | 1.071 | 1.009901 | + | High strength | |
| Mulberry variety 13 | 0.449 | 0.423385 | - | Weak (weak) | |
| Mulberry variety 14 | 0.732 | 0.69024 | + | Medium and high grade | |
| Mulberry variety 15 | 1.241 | 1.170203 | + | High strength | |
| Mulberry variety 16 | 0.694 | 0.654408 | + | Medium and high grade | |
| Mulberry variety 17 | 1.357 | 1.279585 | + | High strength |
As can be seen from Table 1, the S/P value of Sinkiang white mulberry is 1.46, and the cold resistance is strong. The S/P value of Waxgourd 32 is 0.51, and the cold resistance is moderate. The S/P value of the Yunnan Changsu mulberry is 0.15, and the cold resistance is weaker. In contrast, white mulberry in Xinjiang has the strongest cold resistance, 32 times of white mulberry, and the worst Yunnan long-ear mulberry.
The data show that the cold resistance of mulberry varieties can be identified and predicted by the protein expression quantity of the specific gene AKR2A in the mulberry seedlings, and an identification method is provided for accurately selecting excellent varieties for seedling promotion by seed selection personnel.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
Claims (5)
1. A method for detecting cold resistance of mulberry varieties is characterized by comprising the following steps:
(1) antibody preparation: searching cDNA sequence of AKR2A in Tair Database of Arabidopsis thaliana, removing 1-198 bases from N end, matching with a sequence of starting base ATG connecting 199 th base of AKR2A to termination code; producing arabidopsis AKR2A target fragment protein by taking PET21 as a plasmid and BL21 as a strain; preparing an arabidopsis AKR2A target fragment antibody;
(2) antibody coating: diluting an arabidopsis AKR2A target fragment antibody by using a carbonate buffer solution with the pH of 9.6 according to the ratio of 1:1000, coating a 96-hole polystyrene ELISA reaction plate with the concentration of 100 mu L/hole to obtain an antibody matrix for ELISA reaction, and placing the antibody matrix at 4 ℃ for later use;
(3) coating the plate with 5% skimmed milk powder blocking antibody, incubating at room temperature for 1h, wherein each well is 200 μ L;
(4) preparation of mulberry AKR2A antigen: grinding 0.2g of mulberry sample in a mortar under the liquid nitrogen freezing state, then oscillating for 30s with 0.2mL of first extracting solution, mixing uniformly, and standing for 0.5h on ice; taking out, centrifuging at 4 ℃, transferring the supernatant into another test tube, and suspending the precipitate with 0.2mL of second extracting solution to obtain a suspension; boiling the suspension for 10min, cooling on ice, centrifuging at 4 deg.C, and collecting supernatant; mixing the above 2 supernatants to obtain mulberry AKR2A antigen;
(5) diluting a mulberry AKR2A antigen and a negative and positive control by PBST solution 1:1, adding the diluted PBST solution into reaction holes of a 96-hole polystyrene reaction plate coated by an arabidopsis AKR2A target fragment antibody according to the number, adding 100 mu L of each reaction hole, repeating two holes in negative and positive, reacting for 2h at the saturated humidity of 22-28 ℃, and washing for later use;
(6) diluting a goat anti-rabbit IgG-HRP antibody marked by horseradish peroxidase with a PBST solution 1:1000, sequentially adding the diluted antibody into reaction holes of a 96-hole polystyrene reaction plate, adding 100 mu L of the antibody into each reaction hole, carrying out light-shielding reaction at the saturation humidity of 22-28 ℃ for 2h, and washing the antibody with PBST for later use;
(7) adding a peroxidase HRP substrate TMB into reaction holes of a 96-hole polystyrene reaction plate in sequence, adding 100 mu L of each reaction hole, reacting for 20min in a dark place, adding 50 mu L of 2M concentrated sulfuric acid into each reaction hole, and stopping reaction;
(8) and (3) placing the 96-hole polystyrene reaction plate with the reaction stopped into a microplate reader, reading OD650 data, and judging the result.
2. The method for detecting cold resistance of mulberry varieties according to claim 1, wherein in the step (4), the concentrations of the components in the first extracting solution are 50mmol/L sodium phosphate and 1mmol/L EDTA; the concentration of each component in the second extracting solution is 50mmol/L phosphate buffer solution, 2% (v/v) Triton X-100 and 2% (m/v) SDS.
3. The method for detecting cold resistance of mulberry varieties according to claim 1, wherein in the step (4), the pH of the first extracting solution and the pH of the second extracting solution are 6.8-7.2.
4. The method for detecting the cold resistance of mulberry varieties according to claim 1, wherein in the step (4), the centrifugal rotation speed is 12000-15000 r/min, and the centrifugal time is 8-10 min.
5. The method for detecting cold resistance of mulberry varieties according to any one of claims 1 to 4, wherein in the step (4), the mulberry sample is 1-2 leaf tissue samples at the top of a cuttage mulberry seedling to be detected for 30 d.
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