Evaluation method for target character of gene editing high oleic acid soybean

Abstract

The invention discloses a gene editing high oleic acid soybean target character evaluation method, and relates to an environmental safety evaluation technology in the field of agricultural biological breeding. The method comprises the following steps: respectively preparing fat extract samples of gene-edited soybeans and control varieties, sequentially carrying out a fat saponification reaction and a fatty acid methyl esterification reaction to obtain a to-be-detected sample, and carrying out gas chromatographic analysis; respectively calculating the content of corresponding monounsaturated fatty acid according to the content of single fatty acid methyl ester in the sample to be detected, and further calculating the content of the monounsaturated fatty acid in the sample; and analyzing the difference significance of the monounsaturated fatty acid content in the gene edited high oleic acid soybean and the control variety soybean by adopting a variance analysis method, so as to judge whether the target character of the gene edited high oleic acid soybean meets the requirement or not. The method provides a specific method for evaluating the functional efficiency of the target character of the gene editing high oleic acid soybean, and is helpful for promoting the progress of safety evaluation of gene editing crops in China.

Description

Technical Field

[0001] The present invention relates to environmental safety assessment technology in the field of agricultural biological breeding, and in particular to an evaluation method for the functional efficiency of target traits of gene-edited high-oleic soybeans. Background Art

[0002] Genome editing is one of the most important tools in biobreeding technology. It can accurately introduce genome modifications into plants to obtain desired traits. It is a technological breakthrough comparable to molecular cloning, polymerase chain reaction and other technologies. It has very significant application value and potential, and has broad application prospects in plant genetic improvement, livestock and poultry variety improvement, biomedicine research and development, disease treatment and other aspects.

[0003] Since the commercial application of genetically modified organisms, the public has been anxious and questioning the safety of genetically modified products. As a new biological technology, gene editing, whether the new materials after gene editing have potential safety risks, is an important part of the safety evaluation that must be conducted before the commercialization of biological breeding products. In order to standardize the safety evaluation of gene-edited plants for agricultural use, the Ministry of Agriculture and Rural Affairs has formulated the "Guidelines for Safety Evaluation of Gene-Edited Plants for Agricultural Use (Trial)" in accordance with the "Regulations on the Safety Management of Agricultural Genetically Modified Organisms" and the "Administrative Measures for Safety Evaluation of Agricultural Genetically Modified Organisms". This guideline is mainly aimed at gene-edited plants that have not introduced exogenous genes. For gene-edited plants that have introduced exogenous genes, safety evaluation should be reported in accordance with the requirements of the "Guidelines for Safety Evaluation of Genetically Modified Plants".

[0004] The Guidelines for Safety Assessment of Genetically Modified Plants clearly state that the contents of environmental safety assessment include survival competitiveness, environmental impact of gene drift, functional efficiency assessment, impact of pest-resistant transgenic plants on non-target organisms, impact on ecosystem community structure and evolution of pest status, and resistance risk of target organisms. The Guidelines for Safety Assessment of Genetically Edited Plants for Agricultural Use (Trial) divides gene-edited plants into four categories, namely: gene-edited plants whose target traits do not increase environmental safety and food safety risks, gene-edited plants whose target traits may increase food safety risks, gene-edited plants whose target traits may increase environmental safety risks, and gene-edited plants whose target traits may increase environmental safety and food safety risks. The above four types of gene-edited plants should provide stability data of target trait performance when applying for production and application safety certificates, and the test data provided should be no less than 3 generations. Therefore, regardless of whether exogenous genes are introduced into gene-edited plants, target trait evaluation, i.e. functional efficiency evaluation, should be carried out.

[0005] In soybean oil, the content of polyunsaturated fatty acids accounts for the largest proportion, while the content of oleic acid is only about 20%, accounting for only 1 / 4 of the unsaturated fatty acid content. Oleic acid is a monounsaturated fatty acid with strong antioxidant capacity and good stability, which is beneficial to health. High-oleic soybean varieties have the characteristics of high oxidative stability and long shelf life. Therefore, reducing the content of polyunsaturated fatty acids, increasing the content of oleic acid, and cultivating high-oleic soybean varieties are important goals of soybean quality breeding. Gene editing technology can accurately edit endogenous genes, achieve rapid improvement of crop traits, shorten breeding time, and reduce costs. At present, scientists have used CRISPR / Cas9 gene editing technology to achieve site-directed mutation of the soybean fatty acid desaturase encoding gene that controls the conversion of oleic acid to linoleic acid in soybeans, thereby achieving an increase in oleic acid content. However, there is a lack of target trait evaluation methods for gene-edited high-oleic soybeans. Summary of the invention

[0006] In order to overcome the current situation described in the background technology that there is no unified and standard method for evaluating target traits of gene-edited high oleic soybeans, the present invention provides a method for evaluating target traits of gene-edited high oleic soybeans.

[0007] To achieve the above purpose, the present invention proposes the following technical scheme: taking the recipient soybean of gene-edited soybean as the control variety, preparing fat extract samples of gene-edited soybean and control variety respectively, then performing saponification of fat and methylation of fatty acid, and then taking the sample to be tested, single fatty acid methyl ester standard solution and mixed fatty acid methyl ester standard solution respectively and injecting them into gas chromatograph for analysis. The corresponding monounsaturated fatty acid content is calculated by the content of single fatty acid methyl ester in the sample, and then the monounsaturated fatty acid content in gene-edited soybean and control variety is calculated. The variance analysis method is used to analyze the significance of the difference in the content of monounsaturated fatty acids between gene-edited high oleic acid soybean and control variety soybean. If the monounsaturated fatty acid content of gene-edited high oleic acid soybean is significantly higher than that of the control variety, the target traits of gene-edited high oleic acid soybean meet the requirements.

[0008] The beneficial effects of the present invention are as follows: a method for evaluating target traits of gene-edited high-oleic soybeans provided by the present invention clarifies that a mixed fatty acid methyl ester standard contains 37 components, and uses 9 monounsaturated fatty acid standards to determine the oleic acid content in the sample, and uses the variance analysis method to determine whether the monounsaturated fatty acid content of the gene-edited high-oleic soybeans is significantly higher than that of the control variety, thereby evaluating whether the target traits of the gene-edited soybeans meet the requirements of industrialization. DETAILED DESCRIPTION

[0009] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments.

[0010] [Example 1]

[0011] Undecaned triglyceride internal standard solution (5.00 mg / mL): Accurately weigh 2.5 g (accurate to 0.1 mg) of undecaned triglyceride into a beaker, add methanol solution, transfer to a 500 mL volumetric flask and make up to volume with methanol. Refrigerate in a refrigerator and store for 1 month; Mixed fatty acid methyl ester standard solution: Take out an appropriate amount of fatty acid methyl ester mixed standard and transfer it to a 10 mL volumetric flask, dilute it with n-heptane to make up the volume, and store it in a refrigerator below -10°C. The validity period is 3 months; the mixed fatty acid methyl ester is a standard product containing 37 kinds of fatty acid methyl esters; Standard solutions of individual fatty acid methyl esters: Take out individual fatty acid methyl esters from the ampoules and transfer them to 10 mL volumetric flasks. Rinse the ampoules with n-heptane and then make up to volume with n-heptane to obtain single standard solutions of different fatty acid methyl esters. Store in a refrigerator below -10°C with a shelf life of 3 months. The molecular formula and CAS number of the individual fatty acid methyl ester standards are shown in Table 1 in the attached table.

[0012] Serial number Fatty acid methyl esters Fatty acid abbreviation Molecular formula CAS Number 1 Cis-9-Tetradecenoic acid methyl ester C14:1 <![CDATA[C 15 H 28 O2]]> 56219-06-8 2 Cis-10-pentadecanedenoic acid methyl ester C15:1 <![CDATA[C 12 H 24 O2]]> 90176-52-6 3 Cis-9-hexadecenoic acid methyl ester C16:1 <![CDATA[C 16 H 30 O2]]> 1120-25-8 4 Cis-10-heptadecanedioic acid methyl ester C17:1 <![CDATA[C 17 H 32 O2]]> 75190-82-8 5 Methyl trans-9-octadecenoate C18:1n9t <![CDATA[C 19 H 36 O2]]> 1937-62-8 6 Cis-9-octadecenoic acid methyl ester C18:1n9c <![CDATA[C 19 H 36 O2]]> 112-62-9 7 Cis-11-eicosenoic acid methyl ester C20:1 <![CDATA[C 21 H 40 O2]]> 2390-09-2 8 Cis-13-docosaenoic acid methyl ester C22:1n9 <![CDATA[C 23 H 44 O2]]> 1120-34-9 9 Cis-15-tetradecenoic acid methyl ester C24:1 <![CDATA[C 25 H 48 O2]]> 2733-88-2

[0013] Prepare gene-edited soybean and control variety powders separately. Avoid sample contamination during sampling and preparation. Use a tissue crusher or grinder to crush the solid sample, freeze it below -18°C, and thaw it before analysis;

[0014] Weigh 0.1 g to 10 g of a uniform sample (accurate to 0.1 mg, containing about 100 mg to 200 mg of fat) into a 250 mL flat-bottom flask, and accurately add 2.0 mL of 11-carbonic acid triglyceride internal standard solution. Add about 100 mg of pyrogallic acid, a few zeolites, 2 mL of 95% ethanol and 4 mL of water, and mix well.

[0015] Add 8 mL of 2% sodium hydroxide methanol solution to the fat extract, connect a reflux condenser, and reflux in a 80℃±1℃ water bath until the oil droplets disappear. Add 7 mL of 15% boron trifluoride methanol solution from the top of the reflux condenser and continue to reflux in a 80℃±1℃ water bath for 2 min. Rinse the reflux condenser with a small amount of water. Stop heating, remove the flask from the water bath, and quickly cool to room temperature.

[0016] Accurately add 10 mL~30 mL of n-heptane, shake for 2 min, then add saturated sodium chloride aqueous solution, and let stand to separate. Pipette about 5 mL of the upper n-heptane extraction solution into a 25 mL test tube, add about 3 g~5 g of anhydrous sodium sulfate, shake for 1 min, let stand for 5 min, and pipette the upper solution into a sample injection bottle for determination.

[0017] Take a single fatty acid methyl ester standard solution and a mixed fatty acid methyl ester standard solution and inject them into the gas chromatograph to qualitatively analyze the chromatographic peaks. The retention time and relative retention time of fatty acid methyl esters are shown in Table 2 in the attached table.

[0018] a) Capillary column: Poly(dicyandiamide) siloxane strong polar stationary phase column with a length of 100 m, an inner diameter of 0.25 mm and a film thickness of 0.2 μm.

[0019] b) Injector temperature: 270°C; c) Detector temperature: 280°C; d) Temperature program: initial temperature 100 °C, lasting 13 min; 100℃~180℃, heating rate 10℃ / min, hold for 6 min; 180℃~200℃, heating rate 1℃ / min, hold for 20 min; 200℃~230℃, heating rate 4℃ / min, hold for 10.5 min.

[0020] e) Carrier gas: ammonia.

[0021] f) Split ratio: 100:1.

[0022] g) Injection volume: 1.0 μL

[0023] Serial number Fatty acid methyl esters Fatty acid abbreviation Retention time / min Relative retention time / (C11:0) 1 Methyl butyrate C4:0 12.56 0.47 2 Methyl hexanoate C6:0 15.54 0.59 3 Methyl octanoate C8:0 19.83 0.75 4 Methyl decanoate C10:0 24.32 0.92 5 Methyl undecanoate C11:0 26.46 1.00 6 Methyl dodecanoate C12:0 28.49 1.08 7 Methyl tridecanoate C13:0 30.46 1.15 8 Methyl tetradecanoate C14:0 32.45 1.23 9 Cis-9-Tetradecenoic acid methyl ester C14:1 34.31 1.30 10 Methyl pentadecanoate C15:0 34.56 1.31 11 Cis-10-pentadecanedenoic acid methyl ester C15:1 36.62 1.38 12 Methyl hexacarbonate C16:0 36.87 1.39 13 Cis-9-hexadecenoic acid methyl ester C16:1 38.81 1.47 14 Methyl heptadecanoate C17:0 39.42 1.49 15 Cis-10-heptadecanedioic acid methyl ester C17:1 41.50 1.57 16 Methyl octadecanoate C18:0 42.27 1.60 17 Methyl trans-9-octadecenoate C18:1n9t 43.73 1.65 18 Cis-9-octadecenoic acid methyl ester C18:1n9c 44.38 1.68 19 trans, trans-9,12-octadecadienoic acid methyl ester C18:2n6t 46.16 1.74 20 Cis, cis-9,12-octadecadienoic acid methyl ester C18:2n6c 47.73 1.80 21 Methyl icosacarbonate C20:0 48.90 1.85 22 Cis, cis, cis-6,9,12-octadecatrienoic acid methyl ester C18:3n6 50.50 1.91 23 Cis-11-eicosenoic acid methyl ester C20:1 51.51 1.95 24 Cis, cis, cis-9,12,15-octadecatrienoic acid methyl ester C18:3n3 52.15 1.97 25 Methyl hexonecarbonate C21:0 52.95 2.00 26 Cis, cis-11,14-eicosadienoic acid methyl ester C20:2 55.99 2.12 27 Methyl dodecanoate C22:0 57.75 2.18 28 Cis, cis, cis-8,11,14-eicosatrienoic acid methyl ester C20:3n6 59.78 2.26 29 Cis-13-docosaenoic acid methyl ester C22:1n9 61.35 2.32 30 Cis-11,14,17-eicosatrienoic acid methyl ester C20:3n3 62.12 2.35 31 Cis-5,8,11,14-eicosatetraenoic acid methyl ester C20:4n6 63.04 2.38 32 Methyl triscarbonate C23:0 63.53 2.40 33 Cis-13,16-docosadienoic acid methyl ester C22:2 67.68 2.56 34 Methyl tetracosanoate C24:0 69.99 2.64 35 Cis-5,8,11,14,17-eicosapentaenoic acid methyl ester C20:5n3 70.36 2.66 36 Cis-15-tetradecenoic acid methyl ester C24:1 72.98 2.76 37 Cis-4,7,10,13,16,19-Docosahexaenoic acid methyl ester C22:6n3 81.72 3.09

[0024] Under the above chromatographic conditions, the fatty acid standard solution and the sample solution were respectively injected into the gas chromatograph and quantified by the chromatographic peak area.

[0025] The content of individual fatty acid methyl ester in the sample is calculated according to formula (1): X i = F i ×( A i ÷ A C11 )×( ρ C11 × V C11 ×1.0067÷ m ) × 100......(1) Where: X i ——Fatty acid methyl esters in the sample i Content, in grams per hundred grams (g / 100 g); F i ——Fatty acid methyl esteri The response factor of A i ——Fatty acid methyl esters in the sample i The peak area of A C11 ——Peak area of ​​methyl undecanoate, the internal standard substance added to the sample; ρ C11 ——undecanoic acid triglyceride concentration, in milligrams per milliliter (mg / mL); V C11 ——The volume of undecaned triglyceride added to the sample, in milliliters (mL); 1.0067 – conversion factor of triglyceride undecanoate to methyl undecanoate; m ——The mass of the sample, in milligrams (mg); 100——The coefficient for converting the content into the content per 100 g of sample.

[0026] Fatty acid methyl esters i The response factor F i Calculate according to formula (2): F i =( ρ Si ×A11)÷(Asi× ρ 11 )......(2) Where: F i ——Fatty acid methyl ester i The response factor of ρ Si ——Fat acid methyl esters in mixed standard i The concentration is expressed in milligrams per milliliter (mg / mL); A11——methyl undecanoate peak area; Asi - fatty acid methyl ester i The peak area of ρ 11 ——Concentration of methyl undecanoate in the standard mixture, in milligrams per milliliter (mg / mL).

[0027] Step 8: Calculation of monounsaturated fatty acid content in the sample Monounsaturated fatty acid content in the sample ( X Mono-Unsaturated Fat) is calculated according to formula (3), and the content of each monounsaturated fatty acid methyl ester in the sample is calculated according to formula (4): X Mono Unsaturated Fat =∑X MUFAᵢ ...... (3) X MUFAᵢ =X FAMEᵢ ×F FAMEᵢ-FAᵢ ...... (4) Where: X Mono-Unsaturated Fat ——Monounsaturated fatty acid content in the sample, in grams per 100 grams (g / 100 g); X MUFAᵢ ——Content of each monounsaturated fatty acid in the sample, in grams per 100 grams (g / 100 g); X FAMEᵢ ——Content of each monounsaturated fatty acid methyl ester, in grams per 100 grams (g / 100 g); F FAMEᵢ-FAᵢ ——Fatty acid methyl ester i Conversion coefficient to fatty acids.

[0028] Conversion coefficient of fatty acid methyl esters to fatty acids F FAMEᵢ-FAᵢ See Table 3 in the Appendix.

[0029] Table 3 Conversion coefficients of fatty acid methyl esters and fatty acids Serial number Fatty acid abbreviation <![CDATA[F FAME-FA ]]> 1 C14:1n5 0.9417 2 C15:1n5 0.9449 3 C16:1n7 0.9477 4 C17:1n7 0.9503 5 C18:1n9t 0.9527 6 C18:1n9c 0.9527 7 C20:1 0.9568 8 C22:1n9 0.9602 9 C24:1n9 0.9965

[0030] The variance analysis method was used to analyze the significance of the difference in monounsaturated fatty acid content between gene-edited high oleic acid soybeans and control soybean varieties. If the monounsaturated fatty acid content of gene-edited high oleic acid soybeans is significantly higher than that of the control variety, the target traits of gene-edited high oleic acid soybeans meet the requirements.

[0031] The target traits of gene-edited high oleic acid soybeans and control soybean varieties were measured by the method of the present invention. The results showed that the monounsaturated fatty acid content of the gene-edited high oleic acid soybeans was 10.2455 g / 100 g, and the monounsaturated fatty acid content of the control soybean variety was 2.4897 g / 100 g, and the difference reached a significant level, indicating that the target traits of the gene-edited high oleic acid soybeans met the requirements.

[0032] Those skilled in the art should understand that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for evaluating target traits of gene-edited high-oleic soybeans, characterized in that: Prepare fat extract samples of gene-edited soybeans and control varieties respectively, and then carry out fat saponification reaction and fatty acid methyl esterification reaction in sequence to obtain test samples, and then take the test samples, single fatty acid methyl ester standard solutions and mixed fatty acid methyl ester standard solutions and inject them into gas chromatograph for analysis respectively; calculate the corresponding monounsaturated fatty acid contents according to the single fatty acid methyl ester contents in the test samples, and then calculate the monounsaturated fatty acid contents in the gene-edited soybeans and the control varieties; use variance analysis to analyze the significance of the difference in monounsaturated fatty acid content between gene-edited high oleic acid soybeans and control varieties of soybeans; if the monounsaturated fatty acid content of gene-edited high oleic acid soybeans is significantly higher than that of the control varieties, then the target traits of the gene-edited high oleic acid soybeans meet the requirements.

2. The method for evaluating target traits of gene-edited high oleic soybeans according to claim 1, characterized in that: The control variety is the recipient variety of gene-edited soybeans.

3. The method for evaluating target traits of gene-edited high oleic soybeans according to claim 1, characterized in that: The fatty acid methyl esters in the single fatty acid methyl ester standard solution are respectively methyl undecanoate, methyl cis-9-tetradecenoate, methyl cis-10-pentadedecenoate, methyl cis-9-hexadecenoate, methyl cis-10-heptadedecenoate, methyl trans-9-octadecenoate, methyl cis-9-octadecenoate, methyl cis-11-eicosenoate, methyl cis-13-docosaenoate, and methyl cis-15-tetracosenoate; and the mixed fatty acid methyl ester standard solution is a mixed standard solution of different types of single fatty acid methyl esters.

4. The method for evaluating target traits of gene-edited high oleic soybeans according to claim 1, characterized in that: The monounsaturated fatty acid content in the gene-edited high-oleic acid soybeans or the control variety of soybeans is the sum of the contents of one or more of nine monounsaturated fatty acids, including cis-9-tetradecenoic acid, cis-10-pentadedecenoic acid, cis-9-hexadecenoic acid, cis-10-heptadedecenoic acid, trans-9-octadecenoic acid, cis-9-octadecenoic acid, cis-11-eicosadecenoic acid, cis-13-docosadecenoic acid and cis-15-tetracosenoic acid.