Rapid screening method for soybean germplasm and variety with high content of genistin and total flavonoids

Abstract

The invention relates to the technical field of soybean variety breeding, in particular to a rapid screening method for soybean germplasm and variety with high content of genistin and general flavone. The contents of genistin and total flavonoids of soybean varieties are in extremely significant positive correlation with the qualitative enzyme activity of lipoxygenase isoenzyme LOX-1 and are in extremely significant negative correlation with the quantitative reaction time of LOX-1 at a certain temperature, so that the qualitative enzyme activity of the lipoxygenase isoenzyme LOX-1 and the shorter quantitative reaction time at a certain temperature are utilized for preliminary screening; according to the method, a large number of resources or breeding materials can be primarily screened according to the qualitative activity of lipoxygenase isoenzyme LOX-1 and the quantitative reaction time at a certain temperature, and then the high performance liquid chromatography is combined to detect the variety and germplasm of the soybeans with the high content of genistin and total flavonoids, and a large number of resources or breeding materials can be screened out according to the qualitative activity of the lipoxygenase isoenzyme LOX-1 and the quantitative reaction time at a certain temperature; a material with high lipoxygenase isoenzyme LOX-1 activity and extremely short reaction time at a certain temperature is obtained, and then the material is subjected to high performance liquid chromatography detection on the content of genistin and the content of total flavonoids.

Description

Technical Field

[0001] The invention relates to the technical field of soybean variety breeding, in particular to a method for quickly screening soybean germplasms and varieties with high contents of genistein and total flavonoids. Background Art

[0002] Modern soybean science has discovered that soy isoflavones are a mixture of three isoflavone free aglycones and nine bound isoflavone glycosides, of which glycoside isoflavones account for about 98%. Glycoside isoflavones are mainly genistin and daidzin. Different types of soy isoflavones have different medical and health effects on the human body.

[0003] At present, the pilot-stage products of "high genistin content soybean isoflavones" (total isoflavones ≥ 90%, of which genistin ≥ 80%, daidzein ≤ 15%) have been exported to Japan. Although genistin and daidzein belong to the same family of soybean isoflavones, due to their different structures and medical and health effects, the international market has extremely strict requirements on the composition and content of genistin (G) and daidzein (D), and the price difference is huge. Meiji Pharmaceutical Co., Ltd. of Japan has put forward clear indicators for the content of soybean isoflavones and genistin as two different components: "At present, scholars from all over the world have confirmed that the efficacy of high-purity genistin far exceeds that of soybean isoflavones, and the side effects are lower than those of soybean isoflavones. Japan already has genistin with a content of 60%. Although the price of your company's genistin (US$1,200 / kg) is higher than that of soybean isoflavones, the genistin content is ≥ 80%. Our company finally decided to purchase your company's genistin." It is generally recognized both at home and abroad that the structure of genistin (G) in soy isoflavones is similar to the basic structure of estrogen in the human body. When the body's own estrogen level is low and a low dose of exogenous soy genistin is given, genistin (G) can occupy estrogen receptor sites, show estrogen activity, and improve female secondary sexual characteristics, prevent and treat menopausal syndrome, etc.; when the body's estrogen level is high and a high dose of exogenous genistin (G) is given, it can resist its own estrogen and reduce the level of autologous estrogen, thereby inhibiting tumor lesions caused by highly active estrogen. Summary of the invention

[0004] The present invention aims to establish a method for rapidly screening soybean germplasms and varieties with high genistein and total flavonoids based on the relevant characteristics of lipoxygenase isozyme LOX-1, and to use it in the field of screening and purifying materials and supplementary foods.

[0005] To achieve the above object, the present invention provides a rapid screening method for soybean germplasms and varieties with high contents of genistin and total flavonoids, which is characterized in that it is demonstrated that the contents of genistin and total flavonoids in soybean varieties are extremely significantly positively correlated with the qualitative enzyme activity of lipoxygenase isozyme LOX-1, and extremely significantly negatively correlated with the reaction time of lipoxygenase isozyme LOX-1 in the quantitative detection of LOX-1 enzyme activity;

[0006] By screening soybean varieties with high qualitative enzyme activity of lipoxygenase isozyme LOX-1 and short reaction time of lipoxygenase isozyme LOX-1 in the quantitative detection of LOX-1 enzyme activity, soybean germplasms and varieties with high contents of genistin and total flavonoids are screened out.

[0007] Preferably, the soybean germplasm varieties with high contents of genistin and total flavonoids are: Nai Dou, Jiyu 3517, Dongnongdou 405, Jiyu 303, Qinong 7.

[0008] Preferably, this method can be applied to: screening suitable materials for the purification of genistin and total flavonoids in soybeans, and screening auxiliary foods for menopausal syndrome patients or cancer patients as health products.

[0009] Preferably, when detecting lipoxygenase isozyme LOX-1, the invention with the patent application number ZL00112539.7 is selected as the detection method for lipoxygenase isozyme LOX-1.

[0010] Preferably, when determining the contents of soybean isoflavone components, the contents of soybean isoflavone components of several varieties are required, and high performance liquid chromatography is selected as the detection method. The high performance liquid chromatography is referred to GB / T 26625-2011.

[0011] Preferably, in the quantitative detection of LOX-1 enzyme activity, the temperature is set at 18°C - 26°C.

[0012] Preferably, in the quantitative detection of LOX-1 enzyme activity, it needs to be carried out under the conditions of an artificial climate chamber, and the amount of the detection sample is between 10 mg and 20 mg.

[0013] Preferably, the reaction time of enzyme LOX-1 is defined as follows: in the quantitative detection of LOX-1 enzyme activity, the time from the start of the reaction when the detection solution and the indicator solution are mixed until the color of the reaction system changes from "present" to "absent" is defined as the reaction time of LOX-1 enzyme.

[0014] The beneficial effects of the present invention are as follows:

[0015] 1. The present invention demonstrates that there is a highly significant positive correlation between the contents of genistin and total flavonoids in soybean varieties and the qualitative enzyme activity of lipoxygenase isozyme LOX-1, and a highly significant negative correlation with the quantitative reaction time of LOX-1. By measuring the qualitative enzyme activity of lipoxygenase isozyme LOX-1 and the quantitative reaction time of LOX-1, the contents of genistin and total flavonoids in soybean varieties can be deduced, which can greatly reduce the time for variety screening and breeding.

[0016] 2. The present invention can use the qualitative activity and quantitative reaction time of lipoxygenase isozyme LOX-1 to preliminarily screen a large number of resources or breeding materials, and obtain materials with high lipoxygenase isozyme LOX-1 activity and extremely short reaction time. By then performing high-performance liquid chromatography detection on their genistin and total flavonoid contents, a large amount of manpower and material resources can be reduced, and the screening and breeding efficiency of high-genistin soybean materials can be greatly improved.

[0017] 3. Traditional soybean variety screening methods usually take a long time, while the rapid screening method of the present invention can evaluate a large number of soybean samples in a short time, greatly improving the screening efficiency. Through rapid screening, soybean varieties that do not meet the requirements can be excluded at an early stage, thereby reducing the cost of subsequent detailed analysis.

[0018] 4. The rapid screening method of the present invention is usually based on precise detection techniques such as high-performance liquid chromatography (HPLC), etc., which can accurately measure the contents of genistin and total flavonoids in soybeans, ensuring the accuracy of screening results.

[0019] 5. The rapid screening method of the present invention can provide strong technical support for the improvement of soybean germplasm and varieties, and promote the cultivation of new varieties. Detailed implementation mode

[0020] A rapid screening method for soybean germplasm and varieties with high contents of genistin and total flavonoids, which can be applied to: screening suitable materials for the purification of genistin and total flavonoids in soybeans, and screening auxiliary foods for menopausal syndrome patients or cancer patients as health products.

[0021] This method requires demonstrating that there is a highly significant positive correlation between the contents of genistin and total flavonoids in soybean varieties and the qualitative enzyme activity of lipoxygenase isozyme LOX-1, and a highly significant negative correlation with the reaction time of lipoxygenase isozyme LOX-1 in the quantitative detection of LOX-1 enzyme activity, where the temperature is set between 18°C and 26°C and it needs to be carried out under the conditions of an artificial climate chamber, and the sample amount for detection is between 10 mg and 20 mg. When determining the content of soybean isoflavone components, the contents of soybean isoflavone components of several varieties are required, and high performance liquid chromatography is selected as the detection method. For the said high performance liquid chromatography, refer to GB / T 26625-2011. When detecting lipoxygenase isozyme LOX-1, the invention patent with the application number ZL00112539.7 is selected as the detection method for lipoxygenase isozyme LOX-1.

[0022] By screening soybean varieties with high qualitative enzyme activity of lipoxygenase isozyme LOX-1 and short reaction time of lipoxygenase isozyme LOX-1 in the quantitative detection of LOX-1 enzyme activity, soybean germplasms and varieties with high contents of genistin and total flavonoids are screened out. The finally screened soybean germplasm varieties with high contents of genistin and total flavonoids are: Nainai Dou, Jiyu 3517, Dongnongdou 405, Jiyu 303, Qinong 7.

[0023] Example:

[0024] 1. Experiment on determining the content of soybean isoflavone components by high performance liquid chromatography

[0025] The contents of soybean isoflavone components of 4 varieties, namely Wandou 37, Wandou 22467, Wandou 901, and Wandou 903, are determined by high performance liquid chromatography, as shown in Table 1. In Table 1, the contents of each isoflavone component and total isoflavones of the varieties Wandou 37 and Wandou 22467 are significantly higher than those of Wandou 901 and Wandou 903. Among them, for the content of genistin, Wandou 37 and Wandou 22467 are 9.9 times and 6 times that of Wandou 901 respectively, and 33.2 times and 20.2 times that of Wandou 903 respectively.

[0026]

[0027]

[0028]

[0029]

[0030]

[0031] Table 4 Qualitative detection of LOX isozyme activity for 62 samples

[0032]

[0033]

[0034] Note: "-" indicates the absence of LOX; "+" indicates normal LOX activity; "++" indicates high LOX activity; "+++" indicates extremely high LOX activity

[0035] 4. Study the correlation between the contents of genistin and total flavonoids and the quantitative reaction time of lipoxygenase isoenzyme LOX-1 in 61 soybean variety resources

[0036] The quantitative detection of LOX-1 enzyme activity was carried out under the conditions of an artificial climate chamber, with the temperature set at 21 °C and the sample amount at 10 mg. The reaction time (min) of LOX-1 enzyme was defined as the time from the start of the mixed reaction of the detection solution and the indicator solution to the change of the color of the reaction system from "present" to "absent" until the reaction ended. Refer to the invention patent application document with the patent application number ZL00112539.7, in which the detection solution composition is pH 9.0, 200 mM boric acid buffer, containing 20 mM CaCl 2 , and the composition of the indicator solution is pH 9.0, 25 ml of 200 mM boric acid buffer, 5 ml of 50 - 150 μM methylene blue, 5 ml of 10 mM sodium linoleate, and 5 ml of distilled water. The specific reaction time records are shown in Table 5

[0037] Since 1 of them lacked LOX-1, the Pearson correlation analysis of the contents of genistin and total flavonoids and the reaction time of lipoxygenase isoenzyme LOX-1 in the remaining 61 materials using SPSS software showed that the Pearson correlations between the reaction time of lipoxygenase isoenzyme LOX-1 and the contents of genistin and total flavonoids were r = -0.556 (**), p = 0.000 and r = -0.455 (**), p = 0.000 respectively, showing a highly significant negative correlation and the correlation was significantly higher than the qualitative detection result of LOX-1

[0038] Table 5 Quantitative determination reaction time of LOX-1 enzyme

[0039]

[0040]

[0041] 5. Preliminary screening implementation effect

[0042] Among the above 62 varieties, preliminary screening was carried out based on the qualitative enzyme activity of LOX-1 and the quantitative reaction time, and combined with high performance liquid chromatography technology, 5 soybean varieties, namely Naindou, Jiyu 3517, Dongnongdou 405, Jiyu 303, and Qinong 7 (Table 6), were screened out. The qualitative enzyme activity of LOX-1 was "++" or "+++", and the LOX-1 enzyme reaction time was between 5 and 9 minutes. For the control, Wandou 38, the qualitative enzyme activity of LOX-1 was "-", and the LOX-1 enzyme reaction time was 18 minutes. The genistin content of the above 5 materials was all greater than 300 mg / kg, the total isoflavones were above 392.69 mg / kg, and the proportion (%) of genistin in the total flavonoids was all greater than 70%. For the control Wandou 38, the genistin content was 55.57 mg / kg, the total isoflavones were 114.28 mg / kg, and the proportion (%) of genistin in the total flavonoids was 48.63%, and the implementation effect was obvious.

[0043] Table 6 Preliminary screening effect of 62 varieties

[0044]

[0045] Note: "-" indicates LOX deficiency; "+" indicates normal LOX activity; "++" indicates high LOX activity; "+++" indicates ultra-high LOX activity

[0046] The data parameters in the present invention belong to the common knowledge in the art. The parameters are already known technologies, and the appropriate parameters are selected according to the actual use. Therefore, the data parameters of the present invention will not be further explained in detail.

[0047] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for rapid screening of soybean germplasms and varieties with high content of genistin and total flavonoids, characterized in that: The results showed that the genistin and total flavonoids content of soybean varieties was significantly positively correlated with the qualitative enzyme activity of lipoxygenase isozyme LOX-1, and significantly negatively correlated with the enzyme reaction time of lipoxygenase isozyme LOX-1 in the quantitative detection of LOX-1 enzyme activity. By screening soybean varieties with high qualitative enzyme activity of lipoxygenase isozyme LOX-1 and short enzyme reaction time of lipoxygenase isozyme LOX-1 in quantitative detection of LOX-1 enzyme activity, soybean germplasms and varieties with high genistein and total flavonoids contents were screened out.

2. The method for rapid screening of soybean germplasms and varieties with high contents of genistin and total flavonoids according to claim 1, characterized in that ,The soybean germplasm varieties with high contents of genistein and total flavonoids screened out are: milk bean, Jiyu 3517, Dongnong bean 405, Jiyu 303, and Qinong 7.

3. The use of soybean germplasm and varieties with high content of genistin and total flavonoids bred by the method according to claim 1, characterized in that ,This method can be applied to: screening suitable materials for the purification of soybean genistein and total flavonoids, and screening supplementary foods for menopausal syndrome patients or cancer patients as health products.

4. The method for rapid screening of soybean germplasms and varieties with high contents of genistin and total flavonoids according to claim 1, characterized in that: When detecting the lipoxygenase isozyme LOX-1, refer to the invention patent application document with patent application number ZL00112539.7 as a method for detecting the lipoxygenase isozyme LOX-1.

5. The method for rapid screening of soybean germplasms and varieties with high contents of genistin and total flavonoids according to claim 1, characterized in that: When determining the content of soybean isoflavone components, the content of soybean isoflavone components of several varieties is required, and high performance liquid chromatography is selected as the detection method. The high performance liquid chromatography method is described in GB / T 26625-2011.

6. The method for rapid screening of soybean germplasms and varieties with high contents of genistin and total flavonoids according to claim 1, characterized in that: In the quantitative detection of LOX-1 enzyme activity, the temperature was set at 18°C-26°C.

7. The method for rapid screening of soybean germplasms and varieties with high contents of genistin and total flavonoids according to claim 1, characterized in that: The quantitative detection of LOX-1 enzyme activity was carried out under artificial climate chamber conditions, and the detection sample amount was between 10 mg and 20 mg.

8. The LOX-1 enzyme reaction time according to claim 1, characterized in that: The enzyme reaction time of LOX-1 is the time it takes for the color of the reaction system to change from "presence" to "absence" from the start of the mixed reaction of the test solution and the indicator solution to the end of the reaction in the quantitative detection of LOX-1 enzyme activity.

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