CN113848202A

CN113848202A - Screening and identifying method for soybean lipoxygenase and 7S and 11S globulin subunit deletion hybrid progeny

Info

Publication number: CN113848202A
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: 2021-12-28
Anticipated expiration: 2041-09-03
Also published as: CN113848202B

Abstract

The invention discloses a screening and identifying method of soybean lipoxygenase and 7S and 11S globulin subunit deletion hybrid progeny. The method realizes the accurate identification of 4 characters possibly appearing in filial generations of lipoxygenase three isozymes Lox-1, Lox-2 and Lox-3, such as simultaneous existence, simultaneous deletion, Lox-3 deletion, Lox-1 and Lox-2 deletion and the like by changing the operation flow and the components and the proportion of reagents and sampling in a trace manner, overcomes the defect that each character of lipoxygenase deletion cannot be identified through color difference because beta-carotene is easily oxidized, and overcomes the defect that whether a certain strip is deleted or not cannot be determined due to the fact that the distances of the Lox-1,2 and 3 strips existing in the existing process of detecting the deletion or not of the three target characters of Lox-1,2 and 3 are too close; the residue after lipoxygenase identification is utilized, and synchronous identification of deletion of each subunit of 7S and 11S globulin is realized without increasing sampling times.

Description

Screening and identifying method for soybean lipoxygenase and 7S and 11S globulin subunit deletion hybrid progeny

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 hybrid progeny, belonging to the field of screening and identifying of soybean seeds.

Background

Lipoxygenase deletion traits are controlled by double recessive genes, deletion of 3 subunits of 11S globulin belongs to pure recessive traits, and deletion of 7S globulin belongs to control of single dominant genes. Therefore, in the soybean breeding process, if lipoxygenase, 7S and 11S globulin subunits are deleted, character separation can occur in F2 generation, namely theoretically, seeds containing the deletion character can be screened out in F2 generation, so that target seeds can be obtained in early generation, the detection workload and the detection cost are reduced, and the situation that the geometric progression increase of the workload and the detection cost caused by screening in high generation cannot be carried out is prevented. Therefore, if a very small number of seeds having a plurality of desired traits can be accurately identified in the F2 generation having a small number of seeds, it is an optimum screening and identification period that is economically efficient, simple and feasible.

In contrast, the improved breeding of soybean seed components which can meet the market demand and take emphasis on functional nutrition and health and high added value as the breeding targets is not taken into consideration by the broad breeders, while the original traditional conventional breeding technology taking high yield as the breeding target basically needs one ruler and one steelyard, so that the improved breeding of the seed components which take functional nutrition and health as the breeding targets cannot be met, and the seed detection and identification technology of F2 segregation generation which is suitable for the improved breeding of the soybean seed components is a calorie neck problem which restricts the innovation of soybean germplasm resources and the cultivation of new varieties.

Most of the existing methods for analyzing the components of soybean seeds are food-grade detection methods, and are characterized in that the method has large sampling amount, at least about 100mg is needed, the weight of one soybean seed is not more than 200mg, the weight is only enough for analyzing the content of one component, even if the seed is confirmed to contain the needed target character, if the seed is continuously propagated, the seed is too heavy due to damage and planted in the ground, and the seedling can not normally emerge. Needless to say, the content of more than two components is analyzed for the seeds, and the seeds are sampled once when one component is analyzed, so that the two samples are not sufficiently sampled, and the subculture of the seeds cannot be completed. Therefore, the conventional ingredient identification of soybean breeding is to identify related ingredients only when the seeds have stable properties and sufficient quantity after the F6 generation, and has the disadvantage that the seeds containing target ingredients are only some dozens of seeds even though the quantity of the seeds is sufficiently identified when the F6 generation is reached, and some seeds are accurately identified from hundreds of thousands of seeds, so that a large amount of people, money, materials and precious farming season are wasted, like a needle fishing from the sea, and even then the target seeds cannot be necessarily screened.

The existing main methods for detecting and detecting 7S and 11S globulin subunit deletion or lipoxygenase deletion comprise the following steps: polyacrylamide gel electrophoresis SDS-PAGE identification: in the seed sampling mode: cutting 8-10mg of powder from a position far away from the hilum, extracting protein, running glue, dyeing, decoloring, and observing whether protein bands of three isozyme of 7S and 11S globulin subunit deletion and lipoxygenase are deleted. The advantages are that: the method is simple and easy to operate, is easy to master, can be used for detecting and identifying various protein components of food which are not limited by sampling amount, and cannot be used for multi-component composite character deletion screening and identification of F2 hybrid progeny which need single-grain analysis due to overlarge sampling amount. In addition, the method has the following defects: 1. the lipoxygenase deletion identification has the problems that lipoxygenase deletion identification is difficult for distinguishing protein subunits with approximate molecular weights, because the lipoxygenase consists of three isozymes, the molecular weights of the lipoxygenase are between 94 and 97Kd and are reflected on SDS-PAGE gel, the bands of the lipoxygenase are very close to almost fall together, the identification of the three isozymes is easy when the three isozymes are deleted simultaneously, and the lipoxygenase deletion identification is difficult when one of the enzymes is deleted, so that the error rate is high; 2. the detection period is long, one period is less, and at least more than 12 hours are needed. 3. The method has the advantages of large sampling amount, serious damage to seeds, easy mildew in the soil germination process, and incapability of ensuring the safe seedling emergence and progeny propagation of seeds containing target genes.

Fading method of beta-carotene: according to the method, by utilizing the principle that beta-carotene can enable Lox-3 to fade and methylene blue can enable Lox-1 to fade, according to the fact that lx is strongly linked with 1x2 and lx3 and lx1x2 accord with an independent separation rule, when three isoenzymes exist in seeds, the blue color of the methylene blue and the yellow color of the beta-carotene fade, and the detected liquid becomes colorless. When the two are lacked, the color of the detected liquid is blue of methylene blue and the color of the yellow of beta-carotene are mixed to turn into green, and whether the lipoxygenase is lacked or not is identified according to the principle. The method mainly has the following defects: beta-carotene is very unstable, and is very easy to be oxidized when encountering air in the operation process, so that color judgment errors are caused; the sampling amount is large, the detection can be carried out only by 8-10mg, the damage to seeds is excessive, and the seedling emergence of filial generation is influenced.

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

Polyacrylamide gel isoelectric focusing electrophoresis is a special polyacrylamide gel electrophoresis technology and is characterized in that an ampholyte carrier is added into a gel column: amphiline, thereby generating a pH gradient on the gel column. When an electric field is applied to the amphoteric carrier gel, a pH gradient is formed in the order of increasing pH from anode to cathode. Because proteins are ampholytes and the nature and amount of charge vary with the pH of the environment, when proteins are electrophoresed in an isoelectric focusing gel column, charged protein ions are electrophoresed on the gel column, and when a protein sample is electrophoresed to a site whose pH value is exactly equivalent to the isoelectric point of the protein, the net charge of the protein is zero and does not move, and the protein is focused into a protein band. The method of focusing at a corresponding position of the pH gradient according to the size of the isoelectric point is an isoelectric focusing electrophoresis method. Its advantages are high correctness and not misjudgement. But has the following defects: 1. the operation procedure is complicated, is not easy to master, and is suitable for analysis and detection with small sample amount. .2. The method also has the problems of long detection period and low working efficiency, and is not suitable for detecting a large number of F2 seeds with farming time limitation. 3. The method has the problems that the sampling detection before the seedling is carried out, the seeds are damaged, the safe seedling emergence of the seeds containing the target genes cannot be ensured, and the offspring is bred.

In the breeding process of breeding new soybean lipoxygenase, 7S and 11S globulin subunit composite character deletion gene polymer varieties, how to accurately identify whether lipoxygenase, 7S and 11S globulin subunits of each seed of an isolated generation of filial generation F2 are deleted or not can be carried out through one-time trace sampling analysis, and the identified individuals can normally complete generation propagation and breeding, so that technical support is provided for breeding new soybean varieties with lipoxygenase, 7S and 11S globulin subunit composite character deletion genes.

Therefore, the development of a method for simultaneously monitoring and identifying the content of various soybean seed components by trace sampling at one time without influencing the seedling emergence and the propagation of offspring at the stage has become a bottleneck problem of successful realization of the improvement and breeding of the high value-added functional soybean special for food processing.

Disclosure of Invention

The invention mainly aims to provide a method for screening and identifying soybean lipoxygenase and 7S and 11S globulin subunit deletion hybrid offspring by only carrying out trace sampling once on soybean seeds;

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

a method for screening and identifying soybean lipoxygenase and 7S and 11S globulin subunit deletion hybrid progeny comprises the following steps:

peeling off small seed coats on the opposite side of an F2 generation soybean seed hilum, longitudinally cutting off small samples, and putting the small samples into a test tube;

(II) adding a Lox-3 detection solution into a test tube, slightly shaking, standing for observation and recording the color of the reaction solution; adding a Lox-1 detection solution, slightly shaking, standing, observing and recording the color of the final reaction solution; when the final reaction color is green, judging that the soybean seed sample is completely deficient in Lox-1,2 and 3 isozymes; when the final reaction color is colorless, judging that the soybean seed sample is not deficient in the three isozymes Lox-1,2 and 3; 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 solution 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 shaking gently to mix uniformly to obtain the sodium borate-containing aqueous solution; wherein the pH value of the sodium borate buffer solution is preferably 9.0; according to the volume ratio, the sodium borate buffer solution, the sodium linoleate solution, the deionized water and the methylene blue solution are 5: 1.

The preparation method of the Lox-3 detection solution comprises the following steps: sequentially adding 0.2M sodium phosphate buffer solution, 10mM sodium linoleate solution, deionized water and beta-carotene saturated solution, and uniformly mixing by gentle shaking to obtain the composition; wherein the pH value of the sodium phosphate buffer solution is preferably 6.6; according to the volume ratio, the sodium phosphate buffer solution, the sodium linoleate solution, the deionized water and the beta-carotene saturated solution are 5: 1.

(III) 7S and 11S globulin subunit deletion detection identification:

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

(b) adding a protein extracting solution into the obtained precipitate to extract crude protein;

(c) performing ISDS-PAGE (inverse denaturing gradient electrophoresis-polyacrylamide gel electrophoresis) on the obtained crude protein; wherein, the concentration of the separation gel is 12.0 percent of polyacrylamide gel, and the concentration of the concentration gel is 7.5 percent of polyacrylamide gel; and (3) taking the separation gel down from the gel plate after the separation gel is subjected to corner cutting marking, and observing and identifying whether 3 subunits of 7S globulin and 3 subunits of 11S globulin are lacked or not on a common fluorescent lamp box or through a gel imaging system after dyeing and decoloring treatment are sequentially carried out.

As a preferred embodiment of the present invention, in step (I), a small piece of seed coat on the side opposite to the navel of soybean is peeled off with a blade, and 3 to 4mg of the sample is longitudinally cut out into a 2.0ml test tube for use.

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

As a preferred embodiment of the present invention, 17.3. mu.L of 1M hydrochloric acid was added to the test tube in step (a) in which the isozyme discrimination in step (II) was completed; the standing time is 5min, the centrifugation is 12000 rpm, and the centrifugation is 3-5 min;

as a preferred embodiment of the present invention, the composition of the protein extract in step (b) is 0.05M Tris-HCl solution, 2% bromophenol blue, 4% glycerol, pH8.0, 2% beta-mercaptoethanol; the protein extraction method described in step (b) comprises: adding protein extractive solution 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 extractive solution.

The principle of lipoxygenase deficiency detection of the invention is as follows: the yellow color of the beta-carotene fades to be colorless by Lox-3, the blue color of the methylene blue fades to be colorless by Lox-1, and the color of the blue-yellow pigment fades to be colorless according to the genetic rule of lipoxygenase deletion traits: according to the principle that the gene lx1 for controlling Lox-1 and the gene lx2 for controlling Lox-2 are in pre-linkage relationship, and lx1lx2 and the gene lx3 for controlling Lox-3 belong to independent genetic relationship, only 4 genotype traits can be shown in the F2 segregating generation of the lipoxygenase deletion hybrid combination: (1) lox-1, Lox-2, Lox-3 are not deleted at all; (2) lox-1 and Lox-2 are deleted; (3) a Lox-3 deletion; (4) the four properties of Lox-1, Lox-2 and Lox-3 are completely deleted, the separation ratio is 1:2:3:4 to 9:3:3:1, therefore, in the detection process, when the final reaction color of the detected seed is colorless, lipoxygenase Lox-1 and Lox-3 are not deleted, because Lox-2 and Lox-1 belong to strong linkage, Lox-2 is always present in Lox-1, and the detected seed can be judged to be lipoxygenase non-deleted seed; when the final reaction color of the detected seeds is colorless, changing the color into the color of the original detected liquid after mixing yellow and green, indicating that the detected seeds do not contain Lox-1 and Lox-3, and judging that the detected seeds do not contain Lox-1, Lox-2 and Lox-3 because Lox-2 and Lox-1 belong to strong linkage; when the final reaction color of the detected seed is yellow, the detected seed contains Lox-1 and does not contain Lox-3, because Lox-1 can enable methylene blue to fade to be colorless, yellow of beta-carotene is remained, and because Lox-2 and Lox-1 belong to a strong linkage relationship, the sample is judged to be that Lox-2 and Lox-1 exist and Lox-3 is absent; similarly, when the final reaction color of the tested seeds is blue, Lox-3 exists, and Lox-1 and Lox-2 are absent.

The detection principle of the deletion of the 7S globulin subunit and the deletion of the 11S globulin subunit comprises the following steps: according to the fact that polyacrylamide gel is of a net structure and has a molecular sieve effect, as long as an ionic detergent and a strong reducing agent (SDS (sodium dodecyl sulfate)) are added into a sample medium and the acrylamide gel, the electrophoretic mobility of protein subunits can be different along with the difference of the molecular weights of the subunits, the migration rate of the protein subunits with small molecular weights is high, the migration rate of the protein subunits with large molecular weights is low, the protein subunits with large molecular weights are reflected on a rubber plate, the proteins with large molecular weights can run slowly above the rubber, and the proteins with small molecular weights and the proteins with large molecular weights can run quickly on the spot of the protein molecular weights, so that the proteins with different molecular weights can be distinguished.

7S and 11S globulin, because it is high in content in the soybean seed, it is easy to detect separately, but the three subunit molecular weights of alpha', alpha, beta of 7S globulin are 77KDa, 72 KDa and 56KDa, respectively, the molecular weights of 11S globulin IIb, IIa, I subunit are 40KDa,35KDa and 17KDa, it is known that SDS-PAGE glue is used to detect large molecular weight protein, high concentration glue is used to detect small molecular weight protein, only two times of glue is needed to know the 7S or 11S deletion condition of the same seed, which results in the waste of human and financial materials, and when the large molecular and small molecular protein bands are checked on the same concentration glue, it is necessary to find an optimal separating glue concentration ratio. The invention finally determines through tests that the polyacrylamide gel with the separation gel concentration of 12.0% can be used for simultaneously checking protein bands of macromolecules and micromolecules.

Because the same glue cannot clearly identify whether the three isozymes of lipoxygenase, 7S and 11S globulin are missing or not, and the second step is required, the lipoxygenase identification and the 7S and 11S globulin identification can be separated, at least two times of sampling is required, and the F2 seeds cannot guarantee the safe emergence of the sampled F2 seeds. Therefore, one sampling can simultaneously exist and delete the three isozymes of lipoxygenase Lox1, Lox2 and Lox3 in the three categories, the Lox-3 deletion exists in Lox-1 and Lox-2 deletion 4 traits and the synchronous analysis and identification of ten traits of three subunits of alpha', alpha and beta and subunit IIb, IIa and I are important technical innovation, and irreplaceable technical support is necessarily provided for the apparent identification of soybean polygene polymerization breeding.

The sample treatment for the beta-carotene fading method and the pre-treatment of the 7S and 11S globulin samples are completely different, i.e. the samples identified by the beta-carotene fading method are not available for SDS-PAGE analysis of the 7S and 11S globulins. However, the invention solves the problem and realizes the synchronous analysis and identification of three isozymes of lipoxygenase Lox1, Lox2 and Lox3 which exist and are simultaneously deleted, Lox-1 and Lox-2 which exist and 4 properties of Lox-1 and Lox-2 which are deleted and ten properties of alpha', alpha and beta subunits, IIb, IIa and I subunits which are deleted at the same time by one trace sampling.

In the existing detection method, if 3 components of soybean seeds are analyzed, cotyledon powder of the seeds needs to be cut 3 times, the amount of cut seeds exceeds 40mg (waste in the cutting process) after 3 times of cutting about 10mg each time, and even if the seeds with target characters are obtained, the seeds are planted in the ground and cannot normally emerge and reproduce offspring due to excessive injury. The invention only needs to sample 3-4mg samples once, and can carry out three isoenzymes of lipoxygenase possibly appearing in F2 filial generation: the method is characterized in that the method comprises the steps of performing accurate identification on four separated characters of Lox-1, Lox-2 and Lox-3 deletion, performing accurate identification on the four separated characters of Lox-1, Lox-2 and Lox-3 deletion, performing ISDS-PAGE (inverse synthetic Aperture DS-PAGE) after the identified 3-4mg sample is processed, performing accurate identification on whether 7S and 11S globulin subunits are deleted, and simultaneously ensuring safe seedling emergence of target seeds, namely simultaneously performing tracking and accurate identification on 10 target characters of four possible characters of lipoxygenase deletion, three subunits of 7S globulin, three subunits of 11S globulin and the like by a trace sampling method, and providing technical support for breeding of polygene set soybeans.

The invention provides a method for screening and identifying F2 filial generation which can simultaneously detect and identify existence of Lox1,2 and 3, loss of Lox1,2 and 3, loss of Lox1 and 2, loss of Lox3, loss of 7S globulin a', three subunits of a and b and three subunits of 11S globulin I, IIa and IIb by aiming at F2 seeds by one trace sampling, and can complete identification of F2 filial generation with lipoxygenase loss, 7S globulin subunit loss and 11S globulin subunit loss and germplasm resources by sowing the previous trace sampling. Compared with the prior art, the method of the invention comprises the following steps: the method is characterized in that when three components of lipoxygenase, 7S and 11S globulin are analyzed in the prior art, the results are 1, the respective independent sampling and independent analysis are carried out, the sampling amount is more than 5 times of that of the method, after 1 seed is sampled for three times, the rest seeds are damaged and overweight, the seedling emergence and multiplication progeny cannot be guaranteed after the seeds are sowed in soil, the seedling emergence and breeding research cannot be finished until F2 generations, and new varieties cannot be bred. The three components are analyzed by adopting the prior art, after sampling is mostly adopted, an experiment process is completed by performing an SDS-PAGE identification method, an IEF-PAGE identification method and the like, at least 12 hours are needed, more than 36 hours are needed for completing the detection of the three characters, and the invention can be completed only by 1/3 of the total time. The invention is only below 1/3 in the prior art in terms of reagent and labor cost.

The main beneficial effects of the invention include:

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

2. Precisely identifying four separated characters of lipoxygenase three isoenzymes Lox-1, Lox-2 and Lox-3, such as total deletion, total absence, deletion of Lox-1 and Lox-2, deletion of Lox-3 and the like; 3. under the condition of not increasing sampling amount and sampling times, the deletion identification of 3 subunits of 7S and 11S globulin is carried out by using lipoxygenase identification used samples.

And increasing the detection target character species by changing the sampling amount. 10mg is sampled each time and more than 30mg is sampled for three times by the traditional method, each sampling can only analyze whether one property of lipoxygenase or 7S or 11S globulin subunit is lost or not, and is changed into 3-4mg sampling, and the trace sampling is realized, so that the full loss of 'lipoxygenase three isoenzymes Lox-1, Lox-2 and Lox-3' can be synchronously carried out; lox-1, Lox-2, Lox-3 are not deleted at all; lox-1 and Lox-2 are deleted; and precisely identifying 4 properties such as Lox-3 deletion and the like, and identifying 10 properties of the deletion of 3 subunits of 7S globulin and 3 subunits of 11S globulin in total by using lipoxygenase to identify used samples under the condition of not increasing the sampling amount and the sampling times.

After the detected seeds are slightly sampled, the water absorption speed of the seeds is accelerated, the seedling emergence time is shortened, the incidence rate of mildew of the seeds in soil is reduced, the seedling emergence rate of the detected seeds can be obviously improved compared with the original detection method, the death rate of individuals containing target gene characters is greatly reduced, and the survival rate of the individuals containing the target characters is improved to more than 99 percent from about 50 percent of that of the traditional sampling method.

3. The detection time is shortened, conditions are created for sowing in due time, accelerating the breeding process and creating high yield, the detection cost of chemical reagents, labor, electricity, water and the like is reduced, and the detection efficiency is improved by more than 3 times.

4. By changing the operation flow and the reagent proportion of the original method, the defects that the beta-carotene is easy to oxidize, the color changes too fast and the lipoxygenase deficiency characters can not be accurately identified through color difference in the original method are effectively overcome. The defect that whether a certain band is deleted or not can not be accurately judged due to too close distance of the existing Lox-1,2 and 3 bands in the process of detecting whether the three target characters of Lox-1,2 and 3 are deleted or not by an SDS-PAGE technology is avoided, so that the efficiency and the accuracy of detecting the lipoxygenase deletion are fundamentally improved.

The invention realizes that only one time of sampling is carried out, the sampling amount is more than 30mg compared with the original method, the sampling amount is reduced to 3-4mg, only 1/10 of the original sampling amount, after the lipoxygenase deficiency is identified by the fading method on the same seed, when 7S and 11S globulin are analyzed next, the sampling is not carried out for 2 times and 3 times, but the waste residual liquid identified by the fading method is treated by the technology of the invention to be used as the sample for identifying the 7S and 11S globulin, and whether the 7S and 11S globulin subunits are deficient or not is identified by the improved polyacrylamide electrophoresis technology of the invention. Because reagent type solution proportioning systems used by the fading method and the like are completely different from the preparation method of the protein extracting solution required by the SDS-PAGE polyacrylamide gel electrophoresis technology, residual liquid precipitates after the fading method experiment cannot be directly used for SDS-PAGE polyacrylamide gel electrophoresis loading. However, the method realizes synchronous detection and identification of various components such as lipoxygenase, 7S, 11S globulin and the like of the same seed by one trace sampling, thoroughly solves the bottleneck problem that multi-component tracking identification cannot be carried out on precious F2 character segregation generation seeds in soybean polygenic polymerization breeding, and provides technical support for soybean polygenic polymerization breeding.

Definition of terms to which the invention relates

Lipoxygenase (Lox), also called lipoxygenase for short, comprises three isoenzymes Lox-1, Lox-2 and Lox-3. When soybean seeds are broken and exposed to oxygen, unsaturated fatty acids such as linoleic acid and linolenic acid contained in the seeds can be oxidized into micromolecular substances such as hexenal, alcohol and ketone under the action of lipoxygenase, and the soybean seeds are a main source for generating beany flavor of soybeans. Therefore, by means of genetic improvement breeding, as long as a variety with complete deficiency of lipoxygenase is bred, the soybean protein has the characteristic of no fishy smell, and the problem of removing the beany smell of soybean deep processing enterprises can be effectively solved.

Soy storage protein: the protein content of soybean seeds is about 40 percent generally, the soybean storage protein can be divided into four globulins of 2S, 7S, 11S and 15S according to a sedimentation coefficient classification method, wherein 2S cannot be utilized in the practical application process because the molecular weight of the 2S is too small, 15S is too large because the molecular weight of the 15S, 7S and 11S globulins become one of the main components of the two storage proteins, the two globulins form a significant negative correlation relationship, the globulins account for about 75 percent of the total protein content of the soybean, and the specific content is different according to varieties.

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

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

F2 soybean seed characteristics: in order to cultivate a new soybean variety, proper parents are selected for hybridization, harvested seeds which are hybridized successfully are called F1 seeds, 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 by screening in the F2 generation, and can be stably inherited to the next generation; the 7S globulin subunit deletion belongs to dominant characters controlled by a single gene, and homozygous individuals can be identified by screening through continuous tracking selection identification of F2 and F3 generations, and can be stably inherited to the next generation.

ISDS-PAGE (improvement Sodium didecylsulfopate polyacrylamide gel electrophoresis): modified polyacrylamide gel electrophoresis.

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

Drawings

FIG. 1 is a flow chart of detection and identification of deletion of lipoxygenase and 7S, 11S globulin subunits provided by the present invention.

FIG. 2 uses the detection method of the present invention for F₂The soybean seed lipoxygenase isozyme lacks color reaction results; a: green, full loss of Lox-1,2, 3; b: colorless, Lox-1,2,3 is not missing; c: yellow, Lox-3 deleted; d: blue, Lox-1,2 deleted.

FIG. 3 uses the prior art for F₂Detecting the result of the color reaction of the soybean seed lipoxygenase isozyme deletion; b: colorless, Lox-1,2,3 is not missing; c: yellow, Lox-3 deleted; d: blue, Lox-1,2 deleted.

FIG. 4ISDS-PAGE modified polyacrylamide gel electrophoresis pattern.

Detailed Description

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Test example 1F2 Rapid Trace detection test for Soy seed lipoxygenase isozyme

1 test Material

1.1 seed material examined from soybean seeds of F2.

1.2 main reagents: trisodium phosphate (Trisodium phosphate anhydrous Na)₃PO₄·12H₂Otianjin Tianli chemical reagent Co., Ltd.), Sodium tetraborate (Sodium borate Na)₂B₄O₇·10H₂O, Tianjin Tianli chemical reagent, ltd.), β -carotene (SIGMA corporation), Methylene blue (methyl blue C16H18N3ClSIUPAC SIGMA corporation), and the like.

2 test method

2.1 pretreatment of the sample Small pieces of seed coats on the opposite side of the soybean hilum were peeled off with a blade, and 3 to 4mg of the sample was longitudinally cut into a 2.0ml test tube for use.

2.2 preparation of reagents

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

2.2.2 preparation of β -carotene saturated solution 10mg of β -carotene are weighed and dissolved in 10ml of acetone and centrifuged at 12000 rpm for 10min, the orange supernatant is β -carotene saturated acetone solution, which is preferably prepared before use, protected from light and stored by short-term refrigeration.

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 normal temperature in the dark.

2.2.4 preparation of sodium linoleate solution an appropriate amount of deionized water was boiled in a Erlenmeyer flask and degassed for further use. 30.00mg of sodium linoleate was weighed and dissolved in 10ml of degassed water to prepare a 10mM sodium linoleate solution, and the upper layer was sealed in nitrogen and stored in a refrigerator at-20 ℃. The prepared sodium linoleate solution needs to be refrigerated, is used within half a day, and is sealed with nitrogen at the upper layer and stored in a refrigerator at the temperature of-20 ℃ when the prepared sodium linoleate solution needs to be stored for more than one day.

2.2.5 preparation of Lox-1 detection solution 0.2M sodium borate buffer (pH9.0) 1250. mu.l, 10mM sodium linoleate solution 250. mu.l, deionized water 250. mu.l, 100. mu.M methylene blue solution 250. mu.l are added in sequence, and the mixture is shaken and mixed evenly to prepare the Lox-1 detection solution for standby.

2.2.6 preparation of Lox-3 detection solution 625. mu.l of 0.2M sodium phosphate buffer solution (pH6.6), 250. mu.l of 10mM sodium linoleate solution, 725. mu.l of deionized water and 400. mu.l of beta-carotene saturated solution are sequentially added, and the mixture is uniformly mixed by gentle shaking to prepare the Lox-3 detection solution for later use.

2.3F2 Soybean seed lipoxygenase isozyme deletion detection and identification

Adding 250 μ l Lox-3 detection solution into a 2.0ml test tube containing 3-4mg of sample, shaking gently for 10s, standing for 3min, observing and recording the color of reaction solution; then 250. mu.l of the LOX-1 test solution was added, the mixture was shaken gently for 10 seconds for 1min, and the color of the final reaction solution was observed and recorded, as shown in FIG. 2 and Table 1, when the final reaction color was green, it was judged that three isoenzymes Lox-1,2, and 3 were completely absent (FIG. 2-A), when the final reaction color was colorless, it was judged that none of the three isoenzymes Lox-1,2, and 3 were absent (FIG. 2-B), when the final reaction color was yellow, it was judged that Lox-3 was absent (FIG. 2-C), and when the final reaction color was blue, it was judged that Lox-1,2 was absent (FIG. 2-D).

TABLE 1 Lipoxygenase deficiency type and color of final reaction solution

Comparative test example 1 lipoxygenase isozyme deletion detection and identification of F2 soybean seeds by adopting prior art

This test compares the detection method of the present invention (i.e., test example 1) with the prior art detection method (Suda I, Hajika M, Nishiba Y, et al, simple and rapid method for the selective detection of the inductive enzymes in the sensory beans seeds [ J ]. J.Agr.food chem.,1995,43(3):742- & 747.).

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

Note: the old method is the detection method of the prior art; the new method is the detection method provided by the invention.

TABLE 3 comparison of the composition and detection order of the detection solutions of the detection method of the present invention and the detection method of the prior art, and the quality and weighing frequency of the sample to be detected

The detection results using the old method are shown in fig. 3: lox-1,2 represented by A before improvement could not be detected, and the missing green color of Lox-1,2,3 represented by D was not green enough and easily confused with the missing colorless color of Lox-1,2,3 represented by C.

Experimental example 27S and 11S globulin subunit deletion assay identification

Test Material and test method

1.1 materials to be detected: the sample precipitate identified by lipoxygenase Lox-1,2,3 deletion detection in 2.3 of test example 1 was used.

1.2 analytical pure reagents: hydrochloric acid (HCl), Tris (hydroxymethyl) aminomethane (Tris), Sodium Dodecyl Sulfate (SDS), Acrylamide (Acrylamide), Bisacrylamide (Bisacrylamide), riboflavin, ammonium persulfate, bromophenol blue, glycerol, methanol (not less than 95%), glacial acetic acid, and Coomassie brilliant blue (G-250).

1.3 Main reagent ratio for preparing glue and protein extract

The improved method is characterized in that the solution required by the experiment comprises separation gel, a solution for concentrating gel, protein extracting solution, electrophoresis buffer solution, and dyeing and decoloring solution; the ISDS-PAGE electrophoresis experiment requires the following main reagent proportions:

solution A: 181.5g Tris, 2g SDS, adjusted to pH 8.82 with HCl and made up to 500 mL.

And B, liquid B: 15g Tris, 2g SDS, pH adjusted to 6.78 with HCl and made up to 500 mL.

And C, liquid C: 150g of Acrylamide (Acrylamide), 4g of Bisacrylamide (Bisacrylamide), and a volume of 500 mL.

E, liquid E: 8mg of riboflavin, and the volume is up to 200 mL.

Liquid P: 0.78g ammonium persulfate to 50 mL.

Protein extracting solution: 6.057g Tris, 0.1g bromophenol blue, 50g glycerol, adjusted to pH8.0 with HCL and made up to 1000 mL.

10 × electrophoresis buffer: 30g Tris, 10g SDS, 144g Glycine, constant volume to 1000 mL.

Dyeing liquid: 65mL of glacial acetic acid, 310mL of methanol, and Coomassie brilliant blue G-2501.5G to a volume of 1000 mL.

Decoloring liquid: 60mL of glacial acetic acid and 210mL of methanol are added to the volume of 1000 mL.

1.4 Assembly of glass plates for electrophoresis

If detect 96 samples once, can assemble 4 sets of clean glass board that does not have the water mark, one has a pair of ground glass clearance strip at the both ends with one side in every set of glass board, another glass's top is fluted, there is the inclined plane incision along the recess, place the glass board of taking ground glass strip in the desktop (ground glass one side upwards), with U type silica gel strip on a left side, down, ground glass clearance strip is hugged closely to the three direction in the right side and is put straightly, prevent the hourglass and glue, cover the glass board of taking the recess (inclined plane incision one side inwards), then fix respectively four angular position in the upper and lower both sides of glass board with 4 long tail clamps, place perpendicularly on the horizontally laboratory bench, wait for the encapsulating.

1.5 preparation of the separation gel and the concentrated gel

Preparation of separation gel

The method needs to prepare polyacrylamide gel with 12.0% of separation gel concentration. Taking 4 polyacrylamide gels with a concentration of 12.0% as an example: and taking a 150mL conical flask, sequentially adding 18.0mL of A solution, 28.8mL of C solution, 3.6mL of P solution and 21.6mL of degassed distilled water, then shaking gently for mixing uniformly, finally adding 48 mu L of TEMED, shaking gently for mixing uniformly again, slowly pouring the prepared separation gel along a gap at one side of the mounted glass plate, and stopping pouring the gel until the liquid level is 3cm or so away from the lower part of the groove of the glass plate. Then, a pipette is used to gently sprinkle deionized water on the upper surface of the contact part of the glue and the air until the water overflows the groove. And then placing the filled rubber plate on a fluorescent lamp box, standing at room temperature for about 40min to fully condense the rubber, pouring out the distilled water above the rubber plate after an obvious boundary line is visible between the rubber and the deionized water above the rubber plate to indicate that the separation rubber is completely solidified, absorbing the residual water on the glass plate by qualitative filter paper, taking down the glass plate from the lamp box, inverting the glass plate and removing the clean residual water to prepare for adding the concentrated rubber.

Preparation of concentrated gum

The concentration of the concentrated gum in this test was 7.5%. Taking a 50mL conical flask, adding 6.4mL of liquid B, 4.0mL of liquid C, 0.8mL of liquid P, 5.2mL of liquid E and 7.6mL of distilled water in turn, shaking gently for mixing uniformly, finally adding 24 mu L of TEMED, shaking gently again for mixing uniformly, slowly pouring the prepared separation gel upper layer until the liquid level is equal to or slightly lower than the upper edge of the groove of the glass plate, inserting a 28-tooth lane comb (obliquely inserting from left to right to remove bubbles), standing on a sunlight lamp box for about 30min, after the gel is solidified, sequentially removing a long tail clamp fixed at four corners and a U-shaped silica gel strip, slowly pulling out the lane comb, washing off residual gel attached to the gap of the glass plate by deionized water, sealing by a preservative film, inverting the groove downwards, and refrigerating at 4 ℃ for later use.

Pretreatment method of sample for 1.6ISDS-PAGE run

Then, in the last step of the lipoxygenase isoenzyme deletion assay of soybean seeds F2 in 2.3 of test example 1 (i.e., adding Lox-1 test solution, recording the final reaction color, confirming the lipoxygenase deletion property of the sample), 17.3. mu.L of 1M hydrochloric acid was directly added to the test tube, the pH value of the mixed Lox-3 and Lox-1 test solution was adjusted to 4.6, the test tube was left to stand for 5min, the protein dissolved in the lipoxygenase deletion assay was subjected to acid precipitation and centrifugation at 12000 rpm for 3-5min, the protein in the sample was reprecipitated, and the supernatant was removed to leave the precipitate for further use.

1.7 extraction of crude protein

To the remaining precipitated sample was added 250. mu.L of a protein extract (0.05M Tris-HCl solution, 2% bromophenol blue, 4% glycerol, pH8.0, 2% beta-mercaptoethanol), shaken for 30S with a vortex mixer, followed by extraction in an ultrasonic cleaner for 10min, allowed to stand for 20-30min, at 6000rpm, centrifuged for 5min, and 10. mu.L of the supernatant protein extract was taken 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 putting the prepared rubber plate into the electrophoresis solution (paying attention to the inward side of the groove), paying attention to discharge gas in a gap between the two glass plates at the bottom of the rubber plate while putting the rubber plate into the electrophoresis tank, fixedly fixing the rubber plate on the electrophoresis tank, and continuously adding the electrophoresis solution into the upper tank to ensure that the liquid level is submerged at the top of the groove lane of the rubber plate. Injecting 10 μ L of protein extract into lane with injector or 12-channel gun, switching on electrophoresis apparatus and electrophoresis tank power supply, adjusting voltage to 100V, starting cooling system of electrophoresis tank, performing electrophoresis for about 75min, adjusting voltage to 150V when the indication strip moves to junction of concentrated gel and separation gel, and continuing electrophoresis for about 4.5 h. And (3) when the blue indicating strip is 2-3cm away from the bottom of the rubber plate, turning off a power supply, taking down the rubber plate, removing a concentrated rubber part, marking a corner cut of the separation rubber in order to distinguish the sequence of each piece of gel and the sequence of sample adding of the same rubber plate, carefully taking down the separation rubber from the rubber plate, putting a dyeing solution into the rubber plate, slowly shaking and dyeing the separation rubber on a shaking table for about 2 hours, carefully taking out the dyed separation rubber, simply washing the dyeing solution with distilled water, draining the water, putting the drained separation rubber into a decoloring solution, and placing the separation rubber in the shaking table and lightly shaking the shaking table at the lowest speed for more than 4 hours. And then taking out the gel, putting the gel into a flat-bottom plastic box filled with distilled water, and observing and identifying whether 3 subunits of 7S globulin and 3 subunits of 11S globulin are deleted or not on a common fluorescent lamp box or through a gel imaging system.

2 results of detection

The detection and identification results of the deletion of the lipoxygenase, 7S and 11S globulin subunits are shown in figure 4. The electrophoresis lanes of lanes 1-4 are the electrophoresis pictures of the precipitates identified by lipoxygenase after running gel, wherein lanes 1-4 correspond to A, B, C, D samples of FIG. 1, and lanes 5-6 are the 7S globulin subunit and 11S subunit deletion samples.

As can be seen from FIG. 4, the lipoxygenase deletion-detected protein can be reprocessed to detect the deletion and presence of the 7S and 11S subunits. Only one trace sampling is carried out on the seeds of the F2 seeds after detection on the premise of not influencing the emergence of the seeds, namely four characters of the F2 seed, namely Lox-1,2,3 deletion, Lox-1,2 deletion and Lox-3 deletion, and 3 characters of a' -subunit, a-subunit and b-subunit of 7S, three characters of I subunit, IIa subunit and IIb subunit of 11S globulin subunit, 10 characters are synchronously and quickly tracked, detected and identified, the multi-gene polymerization breeding and screening process is greatly shortened, the manpower, material resources, financial resources and screening time are reduced, the problem of improving and breeding soybean seed components is solved, the bottleneck problem that the neck of a plurality of components of the same F2 segregating generation seed cannot be synchronously tracked, screened and detected is solved, and technical support is provided for cultivation of a new high-added-value functional soybean variety special for food processing and identification of quality phenotypic characters.

Claims

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

peeling off small seed coats on the opposite side of the F2 generation soybean seed hilum, and longitudinally cutting small samples into test tubes for later use;

(III) 7S and 11S globulin subunit deletion detection identification:

(c) performing ISDS-PAGE (inverse denaturing gradient electrophoresis-polyacrylamide gel electrophoresis) on the obtained crude protein; and (3) taking the separation gel down from the gel plate after the separation gel is subjected to corner cutting marking, and observing and identifying whether 3 subunits of 7S globulin and 3 subunits of 11S globulin are lacked or not on a common fluorescent lamp box or through a gel imaging system after dyeing and decoloring treatment are sequentially carried out.

2. The screening and identification method according to claim 1, wherein in the step (I), a small piece of seed coat on the side opposite to the navel of soybean is peeled off by a blade, and 3 to 4mg of the sample is longitudinally cut out into a 2.0ml test tube for use.

3. The screening and identification method of claim 1, wherein the Lox-1 detection reagent is formulated by a method comprising: sequentially adding 0.2M sodium borate buffer solution, 10mM sodium linoleate solution, deionized water and 100 mu M methylene blue solution, and shaking gently to mix uniformly to obtain the sodium borate-containing aqueous solution;

the preparation method of the Lox-3 detection solution comprises the following steps: sequentially adding 0.2M sodium phosphate buffer solution, 10mM sodium linoleate solution, deionized water and beta-carotene saturated solution, and uniformly mixing by gentle shaking to obtain the composition.

4. The screening and identification method of claim 1, wherein the Lox-1 detection solution is prepared by a method in which a sodium borate buffer solution has a pH of 9.0; according to the volume ratio, the sodium borate buffer solution, the sodium linoleate solution, the deionized water and the methylene blue solution are 5: 1;

in the preparation method of the Lox-3 detection solution, the pH value of a sodium phosphate buffer solution is 6.6; according to the volume ratio, the sodium phosphate buffer solution, the sodium linoleate solution, the deionized water and the beta-carotene saturated solution are 5: 1.

5. The screening and identifying method according to claim 1, wherein in step (ii), 3-4mg of the test sample is loaded into a 2.0ml test tube, 250 μ l of Lox-3 detection solution is added, 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 test solution was added, the mixture was shaken gently for 10 seconds, and after standing for 1min, the color of the final reaction solution was observed and recorded.

6. The screening and identifying method according to claim 1, wherein in the step (a), 17.3. mu.L of 1M hydrochloric acid is added to the test tube in which the isozyme identification in the step (II) is performed; the standing time is 5min, the centrifugation is 12000 rpm, and the centrifugation is 3-5 min.

7. The screening and identifying method according to claim 1, wherein the composition of the protein extract in step (b) is: 0.05M Tris-HCl solution, 2% bromophenol blue, 4% glycerol, pH8.0, 2% beta-mercaptoethanol.

8. The screening method according to claim 1, wherein the protein extraction method in step (b) comprises: adding protein extractive solution 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 extractive solution.

9. The screening and identifying method of claim 1, wherein the separation gel in the ISDS-PAGE modified polyacrylamide gel electrophoresis is a polyacrylamide gel with a concentration of 12.0%.

10. The screening method of claim 1, wherein the gel concentrate in the ISDS-PAGE modified polyacrylamide gel electrophoresis is 7.5% polyacrylamide gel.