CN113848202B

CN113848202B - Soybean lipoxygenase and screening and identifying method of 7S and 11S globulin subunit deletion hybrid offspring

Info

Publication number: CN113848202B
Application number: CN202111034644.9A
Authority: CN
Inventors: 王绍东; 王遂; 姜妍; 王晓云
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2021-09-03
Filing date: 2021-09-03
Publication date: 2024-03-01
Anticipated expiration: 2041-09-03
Also published as: CN113848202A

Abstract

The invention discloses a soybean fat oxidase and a screening and identifying method of 7S and 11S globulin subunit deletion hybrid offspring. According to the invention, through changing the operation flow and reagent components and proportions, three isoenzymes of lipoxygenase Lox-1, lox-2 and Lox-3 exist simultaneously and are simultaneously deleted, lox-3 is deleted, and the accurate identification of 4 characters possibly appearing in the filial generation such as Lox-1, lox-2 deletion and the like is realized through one trace sampling, the defect that the characters of lipoxygenase deletion cannot be identified through color difference because beta-carotene is extremely easy to oxidize is overcome, and the defect that whether a certain strip is deleted or not can not be judged in the existing detection process of whether three target characters of Lox-1,2 and 3 are deleted is overcome; the residue after lipoxygenase identification is utilized, and the synchronous identification of whether each subunit of the 7S globulin and the 11S globulin is deleted or not is realized without increasing the sampling times.

Description

Soybean lipoxygenase and screening and identifying method of 7S and 11S globulin subunit deletion hybrid offspring

Technical Field

The invention relates to a screening and identifying method of soybean seeds, in particular to a screening and identifying method of soybean lipoxygenase and 7S and 11S globulin subunit deletion filial generation, belonging to the field of screening and identifying soybean seeds.

Background

Lipoxygenase deletion trait is controlled by double recessive genes, 11S globulin 3 subunits deletion belongs to pure recessive trait, 7S deletion belongs to single dominant gene control. Therefore, in the soybean breeding process, if lipoxygenase, 7S and 11S globulin subunits are deleted, the character separation occurs in the F2 generation, namely, in theory, the seeds containing the deletion character can be screened out in the F2 generation, thus the target seeds can be obtained in the early generation, the detection workload and the detection cost are reduced, and the increase of the geometric progression of the workload and the detection cost caused by the screening in the high generation is prevented from being impossible. Therefore, if a very small number of seeds containing a plurality of target traits can be accurately identified in F2 generation with a small seed quantity, the method is an economical, effective, simple, convenient and feasible optimal screening and identification period.

In contrast, the soybean seed component improvement breeding which can meet the market demand and is used for emphasizing functional nutrition and health and high added value as breeding targets is not paid attention to by the masses of breeders, the original traditional conventional breeding technology which is used for breeding targets with high yield is basically realized by a ruler and a steelyard, the seed component improvement breeding which is used for breeding targets with functional nutrition and health cannot be met, and the F2 separation generation seed detection and identification technology which is suitable for the soybean seed component improvement breeding application has become the neck problem which restricts soybean germplasm resource innovation and new variety cultivation.

The existing method for analyzing the components of the soybean seeds is mainly a food-grade detection method, and is characterized in that the method has large sampling quantity, at least about 100mg, and the weight of one soybean seed is not more than 200mg, and the weight is only enough to analyze the content of one component, even if the seed contains the required target property, if the seed is to be continuously propagated, the seed can be excessively heavy due to injury, and the seed cannot normally emerge in the ground. Not to mention that more than two components are analyzed on the seed, one sample is taken on the seed for analyzing one component, two samples are not taken enough, and the seed can not be subjected to the secondary propagation. Therefore, the conventional soybean breeding component identification is carried out when the characters of the seeds are stable and the quantity is enough after the F6 generation, and the defect is that the quantity of the seeds is enough to identify the seeds when the seeds reach the F6 generation, but the seeds containing the target components are often only some tens of seeds, and from the seeds with the quantity of tens of thousands of seeds, a certain seed is accurately identified, so that a great amount of people, wealth, things and precious agricultural hours are wasted, like a sea fishing needle, and even so, the target seeds can not be screened.

The existing main methods for detecting the deletion of the 7S and 11S globulin subunits or lipoxygenase deletion include: polyacrylamide gel electrophoresis SDS-PAGE identification method: in the seed sampling mode: cutting 8-10mg of powder from a position far from the hilum, extracting protein, running gel, staining, decolorizing, and observing whether the protein bands of the three isozymes of 7S and 11S globulin subunits and lipoxygenase are deleted or not. The advantages are that: the method is simple and easy to operate and grasp, can be used for detecting and identifying various protein components of foods without being limited by the sampling amount, and cannot be used for screening and identifying the multi-component composite character deletion of the F2 hybrid offspring needing single grain analysis due to the overlarge sampling amount. In addition, this method has the following drawbacks: 1. the identification of lipoxygenase deletion is difficult for the distinction of protein subunits with close molecular weights, and has the problems that the lipoxygenase is composed of three isozymes, the molecular weight of the lipoxygenase is 94-97Kd, the lipoxygenase is reflected on SDS-PAGE gel, the bands of the lipoxygenase are very close to be almost dropped together, the identification of the three isozymes is easy when one of the three isozymes is deleted, the identification of one of the three isozymes is difficult when the identification of one of the three isozymes is needed, and the misjudgment rate is high; 2. the detection period is long, and more than 12 hours are needed for one period to come down. 3. The sampling amount is large, the seeds are seriously damaged, mildew is easy to cause in the soil germination process, the seeds containing the target genes cannot be ensured to safely emerge, and the offspring are propagated.

Beta-carotene fading method: according to the principle that beta-carotene can fade Lox-3 and methylene blue can fade Lox-1, according to strong linkage of lx and 1x2, lx3 and lx1x2 accord with independent separation rules, when three isozymes exist in seeds, the blue of methylene blue and the yellow of beta-carotene can be removed, and the detection liquid becomes colorless. However, when the lipid oxidase is missing, the color of the detection solution is that the mixture of the blue of methylene blue and the yellow of beta-carotene becomes green, and the lipid oxidase is identified as missing or not according to the principle. The method mainly has the following defects: beta-carotene is unstable, and is easily oxidized when encountering air in the operation process, so that color judgment errors are caused; the sampling amount is large, and the detection can be carried out only by 8-10mg, so that the damage to seeds is excessive and the emergence of hybrid offspring is affected.

The two detection methods have the problems of large sampling amount, influence on seedling emergence, incapability of accurate identification, unnecessary workload increase for the breeding process, inaccurate identification, waste of a large amount of human property, consumption of a large amount of reagents and easy environmental pollution.

The isoelectric focusing electrophoresis of polyacrylamide gel is a special polyacrylamide gel electrophoresis technology, and is characterized in that an ampholyte carrier is added into a gel column: ampholine, thereby creating a pH gradient across the gel column. When an electric field is applied to the amphoteric carrier gel, a pH gradient is formed, in which the order of pH increases gradually from anode to cathode. Because the protein is an ampholyte, the nature and amount of charge carried by the protein varies with the pH of the environment in which the protein is located, when the protein is electrophoresed in an isoelectric focusing gel column, charged protein ions migrate on the gel column, and when a protein sample migrates to a site, the net charge of the protein is zero and does not move when the pH value of the site corresponds to the isoelectric point of the protein, and the protein is focused into a protein band. The isoelectric focusing electrophoresis method is used for focusing at a certain corresponding position of the pH gradient according to the isoelectric point. Its advantages are high accuracy and less possibility of erroneous judgement. But suffer from several drawbacks: 1. the operation procedure is complicated, difficult to master, and suitable for analysis and detection with small sample amount. .2. The method has the problems of long detection period and low working efficiency, and is not suitable for detecting a large amount of F2 seeds with agricultural restrictions. 3. There is the sampling detection before seedling, hurts the seed, can't guarantee the safe emergence of seed containing target gene, breeds the problem of offspring.

In the breeding process of cultivating new soybean lipoxygenase, 7S and 11S globulin subunit composite character deletion gene polymerization varieties, how to separate the deletion of lipoxygenase, 7S and 11S globulin subunits of each seed of the filial generation F2, and accurately identify the deletion of lipoxygenase, 7S and 11S globulin subunits by one trace sample analysis, ensure that identified individuals can normally finish generation proliferation and breeding, and provide technical support for cultivating new soybean varieties with lipoxygenase, 7S and 11S globulin subunit composite character deletion.

Therefore, a method for simultaneously monitoring and identifying the content of various soybean seeds by one trace sampling without influencing the period of emergence and propagation of offspring has been developed, and has become a bottleneck problem of success of improved breeding of special high-added-value functional soybeans for food processing.

Disclosure of Invention

The main purpose of the invention is to provide a screening and identifying method for soybean lipoxygenase and filial generation of 7S and 11S globulin subunit deletion by only performing trace sampling on soybean seeds;

in order to achieve the above purpose, the main technical scheme provided by the invention comprises:

a method for screening and identifying soybean lipoxygenase and filial generation of 7S and 11S globulin subunit deletion, comprising:

peeling off the seed coats of the F2 generation soybean seeds on the opposite sides of the umbilicus, longitudinally cutting small samples, and filling the small samples into a test tube;

(II) adding Lox-3 detection liquid into a test tube, gently shaking, standing for observation and recording the color of the reaction liquid; adding Lox-1 detection liquid, gently shaking, standing, and observing and recording the color of the final reaction liquid; when the final reaction color is green, judging that the soybean seed sample is completely deleted by Lox-1,2 and 3 isozymes; when the final reaction color is colorless, judging that the soybean seed sample is Lox-1,2 and 3 isoenzymes are not deleted; when the final reaction color is yellow, judging that the soybean seed sample is Lox-3 missing; when the final reaction color is blue, judging that the soybean seed sample is Lox-1,2 missing;

the preparation method of the Lox-1 detection liquid comprises the following steps: sequentially adding 0.2M sodium borate buffer solution, 10mM sodium linoleate solution, deionized water and 100 mu M methylene blue solution in sequence, and mixing by light shaking to obtain the final product; wherein, the pH value of the sodium borate buffer solution is preferably 9.0; calculated as volume ratio, sodium borate buffer to sodium linoleate solution to deionized water to methylene blue solution=5:1:1:1.

The preparation method of the Lox-3 detection liquid comprises the following steps: sequentially adding 0.2M sodium phosphate buffer solution, 10mM sodium linoleate solution, deionized water and beta-carotene saturated solution in sequence, and mixing by light shaking to obtain the final product; wherein, the pH value of the sodium phosphate buffer solution is preferably 6.6; calculated as volume ratio, sodium phosphate buffer solution, sodium linoleate solution, deionized water, β -carotene saturated solution=5:1:1:1.

(iii) identification of deletion detection of 7S and 11S globulin subunits:

(a) Adding 1M hydrochloric acid into the test tube with isozyme identification in the step (II), adjusting the pH value of the detection liquid mixed with Lox-3 and Lox-1 to be acidic, standing, centrifuging, removing supernatant, and leaving precipitate for later use;

(b) Adding protein extract into the obtained precipitate to obtain crude protein;

(c) Performing ISDS-PAGE modified polyacrylamide gel electrophoresis on the obtained crude protein; wherein, the concentration of the separating gel is polyacrylamide gel of 12.0 percent, and the concentration of the concentrated gel is polyacrylamide gel of 7.5 percent; and (3) after the separation gel is subjected to corner cutting marking, the separation gel is taken off from the gel plate, and after dyeing and decoloring treatment are sequentially carried out, whether 3 subunits of 7S globulin and 3 subunits of 11S globulin are deleted or not is observed and identified on a common fluorescent lamp box or through a gel imaging system.

As a preferred embodiment of the invention, the opposite side of the soybean umbilicus is peeled off from the small pieces of seed coat in step (I) with a blade, and 3-4mg of the sample is cut longitudinally into 2.0ml test tubes for use.

As a preferred embodiment of the invention, in the step (II), 3-4mg of the sample to be detected is put into a 2.0ml test tube, 250 μl of Lox-3 detection liquid is firstly added, the mixture is gently shaken for 10s, and after standing for 3min, the color of the reaction liquid is observed and recorded; then 250. Mu.l of Lox-1 detection solution was added thereto, the mixture was gently shaken for 10s, allowed to stand for 1min, and then the color of the final reaction solution was observed and recorded.

As a preferred embodiment of the present invention, in step (a), 17.3. Mu.L of 1M hydrochloric acid is added to the test tube in which the isozymes discrimination of step (II) is completed; the standing time is 5min, the centrifugation is 12-000 rpm, and the centrifugation is 3-5min;

as a preferred embodiment of the present invention, the protein extract in step (b) has a composition of 0.05M Tris-HCl solution, 2% bromophenol blue, 4% glycerol, pH8.0,2% beta-mercaptoethanol; the protein extraction method of step (b) comprises: adding protein extract into the obtained precipitate, oscillating with vortex mixer for 30S, extracting in ultrasonic cleaner for 10min, standing for 20-30min at 6000rpm, centrifuging for 5min, and collecting the upper layer to obtain protein extract.

The principle of lipoxygenase deletion detection of the present invention: lox-3 can fade the yellow color of beta-carotene into colorless, lox-1 can fade the blue color of methylene blue into colorless, and the genetic rule of the lipoxygenase deletion character is adopted: the gene lx1 controlling Lox-1 and the gene lx2 controlling Lox-2 are in a pre-linkage relationship, lx1lx2 and the gene lx3 controlling Lox-3 are in an independent genetic relationship, and according to the principle, in the separation generation of the lipoxygenase deletion hybridization combination F2, only 4 genotype traits can appear: (1) Lox-1, lox-2, lox-3 is not deleted at all; (2) Lox-1 and Lox-2 deletions; (3) Lox-3 deletion; (4) Lox-1, lox-2 and Lox-3 are completely deleted, the separation ratio is 1:2:3:4=9:3:3:1, therefore, in the detection process, when the final reaction color of the detected seeds is colorless, the lipid oxidase Lox-1 and Lox-3 are not deleted, and because Lox-2 and Lox-1 are strongly linked, lox-2 is necessarily present in Lox-1, the detected seeds can be judged to be lipid oxidase-free seeds; when the final reaction color of the detected seed is colorless, changing the color into the color after the yellow and the green of the original detection liquid are mixed, which shows that the detected seed does not contain Lox-1 and Lox-3, and because Lox-2 and Lox-1 belong to strong linkage, the detected seed is judged to not contain Lox-1, lox-2 and Lox-3; when the final reaction color of the seed to be detected is yellow, the seed is proved to contain Lox-1 and does not contain Lox-3, because Lox-1 can fade methylene blue to be colorless, the yellow of beta-carotene is remained, and because Lox-2 and Lox-1 belong to a strong linkage relationship, the sample is judged to exist between Lox-2 and Lox-1, and Lox-3 is absent; similarly, when the final reaction color of the seed to be tested is blue, lox-3 exists, and Lox-1 and Lox-2 are absent.

Detection principle of 7S globulin subunit deletion and 11S globulin subunit deletion of the present invention: according to the polyacrylamide gel with a net structure, the molecular sieve effect is achieved, and as long as ionic detergent and strong reducing agent (SDS or sodium dodecyl sulfate) are added into a sample medium and the acrylamide gel, the protein subunit electrophoresis mobility is different along with the difference of subunit molecular weights, the migration rate of the protein subunit with small molecular weight is fast, the migration rate of the protein subunit with large molecular weight is slow, the protein subunit with large molecular weight is reflected on a rubber plate, the protein subunit with large molecular weight runs slowly and appears above the rubber, and conversely, the protein subunit with small molecular weight runs fast and appears in the current formula with large molecular weight of the protein, so that the proteins with different molecular weights can be distinguished.

The content of 7S and 11S globulin in soybean seeds is high, and the detection is easy, but the alpha', alpha and beta molecular weights of 7S globulin are 77kDa, 72K Da and 56kDa, and the molecular weights of 11S globulin IIb, IIa and I subunit are 40kDa,35kDa and 17kDa, respectively. According to the invention, the final determination is carried out through experiments, and the polyacrylamide gel with the concentration of the separating gel of 12.0% can be used for checking protein bands of macromolecules and micromolecules simultaneously.

Because the deletion of three isozymes, 7S and 11S globulins of lipoxygenase can not be clearly identified on the same gel, and secondly, the identification of lipoxygenase and the identification of 7S and 11S globulins can be separated, at least two samples must be taken, and the safety emergence of F2 seeds after sampling can not be ensured. Therefore, three isozymes of lipoxygenase Lox1, lox2 and Lox3 in the three categories can be simultaneously existed and simultaneously deleted, lox-1, lox-2 exists Lox-3 lacks, lox-3 exists Lox-1, 4 characters of Lox-2 deficiency and alpha', alpha, beta three subunits and IIb, IIa and I subunits are synchronously analyzed and identified, so that the method is a great technical innovation, and an irreplaceable technical support is provided for apparent identification of soybean polygenic polymerization breeding.

The sample treatment for the beta-carotene fade method and the 7S and 11S globulin sample pretreatment methods are quite different, i.e., the samples identified by the beta-carotene fade method are not available for SDS-PAGE analysis for the analysis of 7S and 11S globulins. However, the invention solves the problem, and realizes the synchronous analysis and identification of ten characters of three subunits of alpha', alpha, beta and IIb, IIa and I of lipoxygenase Lox1, lox2 and Lox3 by one trace sampling, wherein three isozymes of lipoxygenase Lox1, lox2 and Lox3 exist and Lox-1 exists and Lox-2 exists and Lox-3 exists and Lox-1 and Lox-2 lacks.

In the existing detection method, if 3 components of soybean seeds are analyzed, cotyledon powder of the seeds needs to be cut off for 3 times, about 10mg of the seeds are cut off each time, the amount of the cut off seeds exceeds 40mg (waste exists in the cutting off process) for 3 times, and the respective analysis is carried out, so that even if the seeds with target characters are obtained, the seeds are in the ground, and the seeds cannot normally emerge and reproduce offspring due to excessive injury. The invention can sample 3-4mg of sample once, namely three isoenzymes of lipoxygenase which may appear in F2 hybridization offspring: the method has the advantages that four separated characters of Lox-1, lox-2, lox-3 complete deletion, lox-1 and Lox-2 deletion, lox-3 deletion and the like are accurately identified, 3-4mg of identified samples can be utilized to carry out accurate identification of whether 7S and 11S globulin subunits are deleted or not after being processed, meanwhile, safe emergence of target seeds can be ensured, namely, the method can simultaneously track and accurately identify 10 target characters of four possible characters of lipoxygenase deletion, three 7S globulin subunit deletions, three 11S globulin subunit deletions and the like through a trace sampling method while ensuring safe emergence of target seeds after sampling, and provides technical support for multi-gene collection soybean breeding.

The invention provides an F2 hybrid offspring screening and identifying method which can simultaneously detect and identify Lox1,2 and 3 existence, lox1,2 and 3 deletion, lox1,2 deletion, lox3 deletion, a' of 7S globulin, a, b three subunits, I, IIa and IIb of 11S globulin of F2 seeds by taking trace samples for one time, and can finish identification of F2 hybrid offspring and germplasm resources of lipoxygenase deletion, 7S globulin subunit deletion and 11S globulin subunit deletion by taking trace samples before sowing. Compared with the prior art, the method of the invention has the advantages that: the method is characterized in that the prior art shows 1. Each of the three components of lipoxygenase, 7S and 11S globulin is independently sampled and independently analyzed, the sampling amount is more than 5 times of that of the method, after three times of sampling of 1 seed, the residual seed can be excessively heavy due to injury, the seedling emergence and the reproduction of the seed cannot be ensured after the seed is sowed into soil, the seedling emergence and the breeding research cannot be finished until the F2 generation is finished, and a new variety cannot be bred. The three components are analyzed by adopting the prior art, and an experimental flow is finished by adopting a SDS-PAGE identification method, an IEF-PAGE identification method and the like after sampling, wherein at least 12 hours are needed, more than 36 hours are needed for finishing three character detection, and the invention can be finished only by 1/3 of the total time. The cost of the reagent and the labor is only less than 1/3 of that of the prior art.

The invention has the main beneficial effects that:

1. the minimum sample size is determined: each time 10mg is sampled by the traditional method, more than 30mg is sampled for three times, and the sampling is reduced to 3-4mg.

2. The three isoenzymes Lox-1, lox-2 and Lox-3 of lipoxygenase are completely deleted, and the four separated characters Lox-1, lox-2, lox-3 and the like are accurately identified; 3. the deletion identification of 3 subunits of each of 7S and 11S globulins was performed by using lipoxygenase-identified used samples without increasing the amount and number of samples.

By changing the sampling amount, the variety of the target trait to be detected is increased. Namely, 10mg of the lipoxygenase is sampled each time by the traditional method, more than 30mg of the lipoxygenase is sampled for three times, and whether one of the characteristics of the lipoxygenase or the 7S or 11S globulin subunit is missing or not can be analyzed only in each sampling, and the sampling is changed into 3-4mg, so that the purpose that the total loss of three isoenzymes Lox-1, lox-2 and Lox-3 of the lipoxygenase can be synchronously carried out by the trace sampling is realized; lox-1, lox-2 and Lox-3 are not deleted at all; lox-1 and Lox-2 deletions; the deletion of 10 traits in total of 3 subunits of 7S globulin and 3 subunits of 11S globulin was identified by using lipoxygenase to identify the used samples without increasing the sampling amount and the sampling times while precisely identifying 4 traits such as Lox-3 deletion.

After the detected seeds are slightly sampled, the water absorption speed of the seeds is increased, the seedling emergence time is shortened, the probability of mildew of the seeds in soil is reduced, the seedling emergence rate of the detected seeds can be remarkably improved compared with the original detection method, the death rate of individuals containing target genes is greatly reduced, and the survival rate of the individuals containing the target genes is improved to more than 99% from about 50% by the traditional sampling method.

3. Shortening the detection time, creating conditions for proper sowing, accelerating breeding process and high yield, reducing the detection cost of chemical reagents, manpower, electricity, water and the like, and improving the detection efficiency by more than 3 times.

4. By changing the operation flow and the reagent proportion of the original method, the defects that beta-carotene existing in the original method is easy to oxidize, the color change is too fast, and the characteristics of lipoxygenase deficiency cannot be accurately identified through the color difference are effectively overcome. The defect that whether one strip is missing or not can not be accurately judged due to the fact that the distances of the existing Lox-1,2 and 3 strips are too close in the process of detecting whether the three target characters of Lox-1,2 and 3 are missing or not through an SDS-PAGE technology is avoided, and the lipoxygenase missing detection efficiency and accuracy are improved fundamentally.

The invention realizes that the sample is sampled only once, the sampling amount is reduced to 3-4mg which is 1/10 of the original sampling amount compared with the original method, when the lipoxygenase deletion is identified by the same seed through the fading method, the 7S and 11S globulin is analyzed, the 2 times and 3 times of sampling are not carried out, the waste residual liquid identified by the fading method is used as the sample for identifying the 7S and 11S globulin after being treated by the technology of the invention, and the deletion of the 7S and 11S globulin subunits is identified by the modified polyacrylamide electrophoresis technology of the invention. The preparation method of the protein extract required by the SDS-PAGE polyacrylamide gel electrophoresis technology is completely different from the preparation method of the reagent type solution proportioning system and the like used by the fading method, so that the residual liquid precipitation after the experiment of the fading method cannot be directly used for the SDS-PAGE polyacrylamide gel electrophoresis loading. However, the invention realizes synchronous detection and identification of multiple components such as lipoxygenase, 7S, 11S globulin and the like of the same seed by one trace sampling, thoroughly solves the problem of neck blockage which restricts the multi-component tracking and identification of precious F2 character separation generation seeds in soybean polygene polymerization breeding, and provides technical support for soybean polygene polymerization breeding.

Definition of terms in connection with the invention

Lipoxygenase (abbreviated as Lox) is called lipoxygenase, which comprises three isoenzymes Lox-1, lox-2 and Lox-3. The soybean seeds are crushed and meet oxygen, unsaturated fatty acids such as linoleic acid, linolenic acid and the like contained in the seeds are oxidized into substances of small molecules such as hexenal, alcohol, ketone and the like under the action of lipoxygenase, and the substances are the main source spring for generating beany flavor of soybeans. Therefore, by means of genetic improvement and breeding, the soybean deep processing enterprises can effectively solve the problem of removing beany flavor by only breeding varieties with completely deleted lipoxygenase, which have the characteristic of no fishy smell.

Soybean storage protein: the soybean seed protein content is generally about 40%, and according to the sedimentation coefficient classification method, soybean storage proteins can be divided into four globulins of 2S, 7S, 11S and 15S, wherein the 2S has too small molecular weight, the 15S has too large molecular weight, the two globulins cannot be utilized in the practical application process, the 7S and 11S globulins become one of the main components forming the two large storage proteins, the two components form a remarkable negative correlation relationship, the two components account for about 75% of the total protein content of the soybean, and the specific content varies from variety to variety.

7S globulin: is a glycosyl-containing protein, which is composed of three subunits of alpha', alpha and beta. According to Japanese scholars' researches, the protein has obvious effects of reducing visceral fat, blood fat and blood sugar. Because 11S globulin and 7S globulin are in a remarkable negative correlation, a new variety of functional soybean with high 7S globulin content and three-high reducing property can be obtained by cultivating a variety with the 11S globulin subunit deleted. But it also has drawbacks: it is one of the main allergen proteins of soybean, and the use of the main allergen protein as a feed for feeding piglets or a bean-based infant powder for feeding infants can cause anaphylactic reaction.

11S globulin: is a sulfur-containing amino acid protein rich in high nutrition, is composed of three subunits of I, IIa and IIb, and can show the characteristics of high gel stability, good emulsifying property, good oil-retaining and water-retaining properties and the like in processing. Because 7S and 11S globulin are in a remarkable negative correlation, the content of the 11S globulin can be improved through the variety with 7S globulin deletion, and soybean varieties with high 11S globulin content are cultivated. It is also one of animal allergen proteins, and feeding piglets with it as a feed may cause allergic reactions.

F2 soybean seed characteristics: in order to cultivate new soybean varieties, proper parental stocks are selected for hybridization, seeds which are successfully hybridized are harvested and called F1 seeds, the F1 seeds are planted, the harvested seeds are called F2 seeds, the F2 generation can have character separation, each seed has different character differences, a plurality of characters in the soybean seeds such as lipoxygenase and 11S globulin subunit deletion characters belong to recessive characters in quality characters, homozygous individuals can be identified through screening in the F2 generation, and stable inheritance can be realized for the next generation; the deletion of the 7S globulin subunit belongs to the dominant character controlled by a single gene, and through continuous tracking selection identification of two generations of F2 and F3, homozygous individuals can be identified through screening, and can be stably inherited to the next generation.

ISDS-PAGE (Improvement Sodium dodecylsulphate polyacrylamide gel electrophoresis): modified polyacrylamide gel electrophoresis.

IEF-PAGE (Isoelectric focusing polyacrylamide gel electrophoresis): polyacrylamide gel isoelectric focusing electrophoresis.

Drawings

FIG. 1 shows a flow chart for detecting and identifying deletion of subunits of lipoxygenase and 7S and 11S globulins.

FIG. 2 pair F using the detection method of the present invention ₂ The color reaction result of the soybean seed fat oxidase isozyme is lost; a: green, lox-1,2,3 total loss; b: colorless, lox-1,2,3 is not missing; c: yellow, lox-3 deleted; d: blue, lox-1,2 is absent.

FIG. 3 prior art is a diagram of F ₂ The detection result of the soybean seed fat oxidase isozyme deficiency color reaction; b: colorless, lox-1,2,3 is not missing; c: yellow, lox-3 deleted; d: blue, lox-1,2 is absent.

FIG. 4is an ISDS-PAGE modified polyacrylamide gel electrophoresis.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.

Test example 1F2 Rapid detection test of Soy seed lipid oxidase isozymes trace

1 test materials

1.1 seed material examined F2 soybean seeds.

1.2 main reagents: trisodium phosphate (Trisodium phosphate anhydrous Na) ₃ PO ₄ ·12H ₂ Sodium tetraborate (Sodium tetra borate Na) from OTianjin Tianji chemical reagent Co., ltd ₂ B ₄ O ₇ ·10H ₂ O, tianjin Tianji chemical Co., ltd.), β -carbotene (SIGMA), methylene blue (methyl blue C16H18N3ClSIUPAC SIGMA), and the like.

2 test method

2.1 sample pretreatment the opposite side of the soybean umbilicus was peeled off with a blade and 3-4mg of the sample was cut longitudinally into 2.0ml tubes for use.

2.2 preparation of reagents

2.2.1buffer preparation 0.2M sodium phosphate buffer (pH 6.6): 15.2048g of trisodium phosphate is weighed, dissolved in 200ml of deionized water, adjusted to pH6.6 with 1M HCl solution and stored at normal temperature. 0.2M sodium borate buffer (pH 9.0): 15.2548g of sodium tetraborate is weighed, dissolved in 200ml of deionized water, adjusted to pH9.0 with 1M HCl solution and stored at normal temperature.

2.2.2. 2. Beta. -carotene saturated solution is prepared by weighing 10mg of beta. -carotene, dissolving in 10ml of acetone, centrifuging at 12 rpm for 10min, and collecting orange supernatant as beta. -carotene saturated acetone solution.

2.2.3 preparation of methylene blue solution 3.20mg of methylene blue was weighed and dissolved in 100ml of deionized water to prepare 100. Mu.M methylene blue solution, and the brown reagent bottle was stored at room temperature in a dark place.

2.2.4 preparation of sodium linoleate solution proper quantity of deionized water is boiled and deaerated in a Erlenmeyer flask for use. 30.00mg of sodium linoleate was weighed and dissolved in 10ml of deaerated water to prepare a 10mM sodium linoleate solution, and the upper layer was kept in a refrigerator at-20℃with nitrogen being filled therein. The prepared sodium linoleate solution needs to be refrigerated, and when the sodium linoleate solution needs to be stored for more than one day after being used in a half day, the sodium linoleate solution needs to be stored in a refrigerator at the temperature of minus 20 ℃ after being filled with nitrogen at the upper layer.

2.2.5 preparation of Lox-1 detection solution 0.2M sodium borate buffer (pH9.0) 1. Mu.l, 10mM sodium linoleate solution 250. Mu.l, deionized water 250. Mu.l, 100. Mu.M methylene blue solution 250. Mu.l were added sequentially, and mixed gently to prepare Lox-1 detection solution for use.

2.2.6 preparation of Lox-3 detection solution 0.2M sodium phosphate buffer solution (pH 6.6) 625. Mu.l, 10mM sodium linoleate solution 250. Mu.l, deionized water 725. Mu.l, beta-carotenes saturated solution 400. Mu.l were added sequentially, and the mixture was gently shaken and mixed to prepare Lox-3 detection solution for later use.

2.3F2 soybean seed fat oxidase isozyme deletion detection and identification

In a 2.0ml test tube containing 3-4mg of the detected sample, 250 μl of Lox-3 detection liquid is firstly added, the mixture is gently shaken for 10s, and after standing for 3min, the color of the reaction liquid is observed and recorded; then 250. Mu.l of the Lox-1 detection solution was added, and after 10s and 1min, the color of the final reaction solution was observed and recorded, and as shown in FIG. 2 and Table 1, it was judged that three isoenzymes of Lox-1,2 and 3 were completely deleted (FIG. 2-A) when the final reaction color was green, that none of the three isoenzymes of Lox-1,2 and 3 was deleted (FIG. 2-B) when the final reaction color was colorless, that Lox-3 was deleted (FIG. 2-C) when the final reaction color was yellow, and that Lox-1,2 was deleted (FIG. 2-D) when the final reaction color was blue.

TABLE 1 lipoxygenase deletion type and final reaction solution color

Comparative test example 1 identification of F2 soybean seed lipid oxidase isoenzyme deletion detection Using the Prior Art

The test compares the detection method of the present invention (i.e., test example 1) with the detection method of the prior art (Suda I, hajika M, nishiba Y, et al sample and rapid method for the selective detection of individual lipoxygenase isozymes in soybean seeds [ J ]. J.Agr.food chem.,1995,43 (3): 742-747.).

TABLE 2 preparation of buffer for detection method of the present invention and detection method of the prior art

Note that: the old method is a detection method in the prior art; the novel method is the detection method provided by the invention.

TABLE 3 comparison of the detection method of the present invention with the composition of the detection solution, the detection sequence, the mass of the sample to be detected and the number of times of weighing in the detection method of the prior art

The detection result using the old method is shown in fig. 3: lox-1,2 represented by A before improvement cannot be detected, and Lox-1,2,3 represented by D is not green enough and is easy to confuse with Lox-1,2,3 represented by C.

Test examples 2 identification of deletion of the globulins 7S and 11S

1 test materials and test methods

1.1 materials to be tested: sample precipitation identified by lipoxygenase Lox-1,2,3 deletion detection was used for 2.3 in test example 1.

1.2 analytically pure reagents: hydrochloric acid (HCl), tris (Tris), sodium Dodecyl Sulfate (SDS), acrylamide (Acrylamide), bisacrylamide (Bisacrylamide), riboflavin, ammonium persulfate, bromophenol blue, glycerol, methanol (. Gtoreq.95%), glacial acetic acid, coomassie brilliant blue (G-250).

1.3 main reagent proportion required by the preparation of gum and protein extract

Through the improved method, the solution required by the experiment comprises separating gel, concentrated gel solution, protein extracting solution, electrophoresis buffer solution and dyeing and decolorizing solution; the main reagent ratio required by the ISDS-PAGE electrophoresis experiment is as follows:

and (3) solution A: 181.5g Tris,2g SDS pH was adjusted to 8.82 with HCl and volume was set to 500mL.

And (2) liquid B: 15g Tris,2g SDS, pH 6.78 with HCl, and volume to 500mL.

And C, liquid: 150g Acrylamide (Acrylamide), 4g Bisacrylamide (bisacryiamide), and the volume was set to 500mL.

E, liquid: 8mg of riboflavin, and the volume was set to 200mL.

And (3) P solution: 0.78g ammonium persulfate to a volume of 50mL.

Protein extract: 6.057g Tris,0.1g bromophenol blue, 50g glycerol, pH adjusted to 8.0 with HCl, and volume fixed to 1000mL.

10 Xrunning buffer: 30g Tris,10g SDS,144g Glycine to 1000mL.

Dyeing liquid: 65mL glacial acetic acid, 310mL methanol, coomassie Brilliant blue G-250.5G, and constant volume to 1000mL.

Decolorization liquid: 60mL glacial acetic acid, 210mL methanol, to 1000mL.

1.4 assembling of glass plates for electrophoresis

If 96 samples are detected at a time, 4 sets of clean glass plates without water marks can be assembled, a pair of frosted glass gap strips are arranged at two ends of the same side of one glass plate, grooves are formed in the top end of the other glass plate, bevel cuts are formed in the lower edge of the grooves, the glass plate with the frosted glass strips is placed on a tabletop (one side of the frosted glass is upwards), the U-shaped silica gel strips are tightly clung to the frosted glass gap strips in the left, lower and right directions and are vertically placed, glue leakage is prevented, the glass plate with the grooves is covered (one side of the bevel cuts is inwards), and then 4 long tail clamps are respectively fixed at four corner positions of the upper side and the lower side of the glass plate and are vertically placed on a horizontal experiment table to wait for glue filling.

1.5 preparation of separation gel and concentrated gel

Preparation of separation gel

The method needs to prepare the polyacrylamide gel with the concentration of the separation gel of 12.0 percent. Taking 4 polyacrylamide gels with a concentration of 12.0% as an example: taking a 150mL conical flask, sequentially adding 18.0mL of A liquid, 28.8mL of C liquid, 3.6mL of P liquid and 21.6mL of deaerated distilled water, immediately and slightly shaking and uniformly mixing, finally adding 48 mu L of TEMED, slightly shaking and uniformly mixing again, slowly pouring the prepared separating gel along a gap on one side of the glass plate, and stopping pouring the gel about 3cm away from the liquid surface of the separating gel from the lower edge of the groove of the glass plate. Then, the deionized water is lightly sprinkled on the upper surface of the contact part of the glue and the air by a liquid-transfering device until the water overflows out of the groove. And then placing the poured rubber plate on a fluorescent lamp box, standing for about 40min at room temperature to fully coagulate the rubber, pouring distilled water above the rubber plate after an obvious boundary line is visible between the rubber and deionized water above the rubber plate to indicate that the rubber is completely coagulated, sucking residual water on the glass plate by qualitative filter paper, taking down the rubber plate from the lamp box, and inverting the rubber plate to remove clean residual water to prepare for adding concentrated rubber.

Preparation of concentrated gel

The concentration of the concentrated gel in this test was 7.5%. Taking a 50mL conical flask, sequentially adding 6.4mL B liquid, 4.0mL C liquid, 0.8mL P liquid, 5.2mL E liquid, 7.6mL distilled water, immediately and gently mixing, finally adding 24 mu L TEMED, gently mixing again, slowly pouring into the prepared separation gel upper layer until the liquid level is level with or slightly lower than the upper edge of the groove of the glass plate, inserting a 28-tooth-specification lane comb (from left to right in an inclined manner to drive away bubbles), placing the separation gel upper layer on a sunlight lamp box, standing for about 30min, sequentially removing a long tail clamp fixed at four corners and a U-shaped silica gel strip after the colloid is solidified, slowly removing the lane comb, flushing residual glue suspended at a gap of the glass plate with deionized water, sealing with a preservative film, pouring the groove downwards into a refrigerator, and refrigerating at 4 ℃ for standby.

1.6 pretreatment method of sample for ISDS-PAGE running gel

Then, in the final step (namely, lox-1 detection liquid is added, final reaction color is recorded, and lipoxygenase deletion characteristics of a sample are confirmed) in the detection and identification process of F2 soybean seed lipoxygenase isoenzyme deletion in 2.3 in test example 1, then 17.3 mu L of 1M hydrochloric acid is directly added into the test tube, the pH value of the mixed Lox-3 and Lox-1 detection liquid is adjusted to 4.6, and the mixture is left stand for 5min, so that the dissolved protein in the lipoxygenase deletion identification is subjected to acid precipitation and centrifugation at 12 000rpm for 3-5min, the protein of the sample is reprecipitated, and then supernatant is removed, and precipitation is left for standby.

1.7 extraction of crude proteins

To the residual precipitated sample, 250. Mu.L of protein extract (0.05M Tris-HCl solution, 2% bromophenol blue, 4% glycerol, pH8.0,2% beta-mercaptoethanol) was added, followed by shaking with a vortex mixer for 30S, followed by extraction in an ultrasonic cleaner for 10min, standing for 20-30min,600 rpm, centrifuging for 5min, and taking 10. Mu.L of the upper protein extract for ISDS-PAGE modified polyacrylamide gel electrophoresis.

1.8ISDS-PAGE modified Polyacrylamide gel electrophoresis

Slowly pouring electrophoresis buffer solution into the electrophoresis tank, slowly and obliquely placing the manufactured rubber plate into the electrophoresis solution (note that the groove is inward), simultaneously paying attention to discharge gas in a gap between two glass plates at the bottom of the rubber plate, fixing the rubber plate on the electrophoresis tank, and continuously adding the electrophoresis solution into the upper tank to enable the liquid level to overflow the top of the groove lane of the rubber plate. Injecting 10 mu L of protein extract into the lanes by using a syringe or a 12-channel gun, switching on the power supply of the electrophoresis apparatus and the electrophoresis tank, starting a cooling system of the electrophoresis tank after the voltage is regulated to 100V, running for about 75min, regulating the voltage to 150V when the strip is moved to the junction of the concentrated gel and the separation gel, and continuing electrophoresis for about 4.5 h. When the blue indicator strip is 2-3cm away from the bottom of the rubber plate, the power supply is turned off, the rubber plate is taken down, the concentrated rubber part is removed, in order to distinguish the sequence of each gel and the sequence of sample adding of the same rubber plate, the angle cutting mark is needed to be carried out on the separating rubber, then the separating rubber is carefully taken down from the rubber plate, the dyeing liquid is put into the separating rubber, the separating rubber is slowly shaken on a shaking table for dyeing for about 2 hours, then the dyed separating rubber is carefully taken out, the dyeing liquid is simply washed by distilled water, the separating rubber is put into a decoloring liquid after draining, and the separating rubber is placed into the shaking table for shaking at least 4 hours at the lowest speed. The gel was then removed and placed in a flat bottom plastic box with distilled water and observed on a common fluorescent light box or by gel imaging system to identify the absence or absence of 3 subunits of 7S globulin and 3 subunits of 11S globulin.

2 detection results

The results of lipoxygenase and 7S and 11S globulin subunit deletion detection and identification are shown in FIG. 4. Electrophoresis lanes 1-4 are electrophoresis pictures of precipitated samples identified by lipoxygenase, wherein lanes 1-4 correspond to four samples of FIG. 1, A, B, C, D, and lanes 5-6 are samples with 7S globulin subunit deletion and 11S subunit deletion, respectively.

As can be seen from FIG. 4, the protein after lipoxygenase deletion detection can be used to detect and identify the deletion and presence of the 7S and 11S subunits after reprocessing. On the premise of not affecting the emergence of the F2 seeds after detection, the four characters of Lox-1,2,3 deletion, lox-1,2,3 non-deletion, lox-1,2 deletion and Lox-3 deletion of the F2 seeds, and the three characters of a' -subunit, a-subunit and b-subunit of 7S, namely the I subunit, IIa subunit and IIb subunit of 11S globulin subunits, can be synchronously and rapidly tracked and identified by adding up 10 characters, so that the multi-gene polymerization breeding screening process is greatly shortened, the manpower, material resources, financial resources and screening time are reduced, the bottleneck problem that the same F2 seed is separated in the soybean seed component improvement breeding, and the necks of multiple components cannot be synchronously tracked and screened and detected is solved, and technical support is provided for the cultivation of special high-function soybean varieties for food processing and the identification of quality phenotype characters.

Claims

1. A screening and identifying method for soybean seed fat oxidase and 7S and 11S globulin subunit deletion hybrid offspring is characterized by comprising the following steps:

stripping off the small pieces of seed coats on the opposite sides of the seed umbilicus of the F2 generation soybean seeds, and longitudinally cutting a small sample into a test tube for standby;

(iii) identification of deletion detection of 7S and 11S globulin subunits:

(c) Performing ISDS-PAGE modified polyacrylamide gel electrophoresis on the obtained crude protein; the separation gel is taken down from the gel plate after being subjected to corner cutting marking, and after being subjected to dyeing and decoloring treatment in sequence, the deletion of 3 subunits of 7S globulin and 3 subunits of 11S globulin is observed and identified on a common fluorescent lamp box or through a gel imaging system;

the preparation method of the Lox-1 detection liquid comprises the following steps: sequentially adding 0.2M sodium borate buffer solution, 10mM sodium linoleate solution, deionized water and 100 mu M methylene blue solution, and mixing by light shaking to obtain the final product;

the preparation method of the Lox-3 detection liquid comprises the following steps: sequentially adding 0.2M sodium phosphate buffer solution, 10mM sodium linoleate solution, deionized water and beta-carotene saturated solution in sequence, and mixing by light shaking to obtain the final product;

in the preparation method of the Lox-1 detection liquid, the pH value of the sodium borate buffer solution is 9.0; calculated as volume ratio, sodium borate buffer to sodium linoleate solution to deionized water to methylene blue solution=5:1:1:1;

in the preparation method of the Lox-3 detection liquid, the pH value of the sodium phosphate buffer solution is 6.6; calculated by volume ratio, sodium phosphate buffer solution, sodium linoleate solution, deionized water, beta-carotene saturated solution=5:1:1:1;

the separating gel in the ISDS-PAGE modified polyacrylamide gel electrophoresis is polyacrylamide gel with the concentration of 12.0 percent;

the concentrated gel in the ISDS-PAGE modified polyacrylamide gel electrophoresis is a polyacrylamide gel with a concentration of 7.5%.

2. The method of claim 1, wherein in step (I) the seed coats of the soybean seed umbilicus are peeled off by a blade and 3-4mg of the sample is cut longitudinally into 2.0ml test tubes for use.

3. The screening and identifying method according to claim 1, wherein in the step (II), 3-4mg of the sample to be tested is put into a 2.0ml test tube, 250 μl of Lox-3 detection solution is added first, the mixture is gently shaken for 10s, and after standing for 3min, the color of the reaction solution is observed and recorded; then 250. Mu.l of Lox-1 detection solution was added thereto, the mixture was gently shaken for 10s, allowed to stand for 1min, and then the color of the final reaction solution was observed and recorded.

4. The screening assay of claim 1 wherein in step (a) 1M hydrochloric acid 17.3 μl is added to the test tube for isozyme identification in step (ii); the standing time is 5min, the centrifugation is 12-000 rpm, and the centrifugation is 3-5min.

5. The screening assay of claim 1 wherein the protein extract of step (b) comprises the following composition: 0.05M Tris-HCl solution, 2% bromophenol blue, 4% glycerol, pH8.0,2% beta-mercaptoethanol.

6. The screening assay of claim 1 wherein the protein extraction method of step (b) comprises: adding protein extract into the obtained precipitate, oscillating with vortex mixer for 30S, extracting in ultrasonic cleaner for 10min, standing for 20-30min at 6000rpm, centrifuging for 5min, and collecting the upper layer to obtain protein extract.