Unexpected effect evaluation method of gene editing soybean phenotypic character

Abstract

The invention discloses an unexpected effect evaluation method of gene editing soybean phenotypic characters, and belongs to the technical field of biology. The method comprises the following steps: (1) preparing and planting soybean materials; (2) counting soybean germination time; (3) counting the germination rate; (4) counting the plant height in each period; (5) recording the early flowering stage time; (6) counting the width of a compound leaf; (7) counting the node number and the internode length of the main stem; and (8) counting the yield in the mature period. The method can be used for investigating main macroscopic agronomic traits such as related morphological characteristics of the gene edited soybeans under indoor controllable and outdoor field natural conditions, and compared with field investigation, the method has the advantages that the method is not easily influenced by the external environment, the investigation and identification period is short, the investigation method is simple, convenient, efficient and intuitive, the result is accurate and reliable, and the method is suitable for large-scale popularization and application. Meanwhile, the method has the advantages of saving time and cost and the like, and is of great significance to unexpected effect evaluation of gene editing soybeans and development of related research work.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for evaluating unexpected effects of gene-edited soybean phenotypic traits. Background Art

[0002] Soybeans are native to China and are widely grown around the world. They are important economic and oil crops. With the development of the economy and the improvement of people's living standards, my country's demand for soybeans is growing, but my country's total soybean production is low and mainly relies on foreign imports. In 2023, my country imported 99.41 million tons of genetically modified soybeans, with a high degree of foreign dependence. In order to improve the comprehensive development of my country's soybean planting industry and ensure food security, increasing soybean production capacity through genetic modification and gene editing technology and accelerating the pace of biological breeding soybeans in my country are one of the important means to solve the current soybean dilemma in my country.

[0003] Gene editing is an emerging technology that modifies specific target sequences in the genome. Gene editing can efficiently and accurately edit gene sequences to improve plant traits. However, while improving target plant traits through gene editing technology, important agronomic traits such as morphology may change due to changes in genome sequences and off-target effects, which may lead to unexpected effects. Summary of the invention

[0004] The present invention found that in addition to changes in the growth period, changes in agronomic traits such as plant height, compound leaf width, and soybean yield were observed during the planting process of a gene-edited long-juvenile soybean. Currently, there is no relevant method to evaluate the unexpected effects of gene-edited soybeans. The present invention establishes a simple, efficient, and intuitive identification method for the unexpected effects of gene-edited soybeans. It is of great significance to evaluate the unexpected effects of gene-edited soybeans, and provide suggestions and references for the application prospects of new gene-edited soybean materials through evaluation.

[0005] The purpose of the present invention is to fill the gap in the evaluation method of the unexpected effects of gene-edited soybeans, and to establish a method for evaluating the unexpected effects of a new gene-edited variety of long-juvenile soybeans. The experimental results can provide a reference for the evaluation of the unexpected effects caused by gene-edited soybeans.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for evaluating unexpected effects of gene-edited soybean phenotypic traits, the evaluation method comprising the following steps:

[0008] (1) Preparation and planting of soybean materials: setting up gene-edited soybeans and using corresponding receptor materials as controls; selecting seeds for sowing;

[0009] (2) Count the germination time after the soybean cotyledons emerge 1-2 cm from the soil layer;

[0010] (3) Count the soybean germination rate after the soybean cotyledons are fully expanded;

[0011] (4) Investigate the plant height of gene-edited soybean and recipient materials at the V3, R1, R3, and R5 stages;

[0012] (5) When a floret blooms at any node of the main stem, record the flowering time;

[0013] (6) Investigate the width of the compound leaves at the third-to-last node of the main stem at the R6 stage;

[0014] (7) Investigate the number of main stem nodes and internode length at R7 stage;

[0015] (8) Yield statistics of gene-edited soybeans and their control recipient materials at maturity.

[0016] The sowing method in step (1) is: selecting seeds for sowing, wherein the sowing depth is 1.5-4.5 cm, the culture temperature is 20-30° C., the culture relative humidity is 40-60%, the culture light cycle is divided into a long day period and a short day period, and the light intensity is 200-400 μM.

[0017] The seed sowing depth is 2-4 cm, the culture temperature is 23-27° C., the culture relative humidity is 45-55%, the light cycle is divided into a long-day period and a short-day period, and the light intensity is 250-350 μM.

[0018] Furthermore, the seed sowing depth is 3 cm.

[0019] Furthermore, the culture temperature is 25°C.

[0020] Furthermore, the culture relative humidity is 50%.

[0021] The culture light cycle is 10-20 hours of light and 4-14 hours of darkness during the long day period, and 10-14 hours of light and 10-14 hours of darkness during the short day period.

[0022] Furthermore, the culture light cycle is 15 hours of light and 9 hours of darkness per day during the long day period, and 12 hours of light and 12 hours of darkness per day during the short day period.

[0023] Furthermore, the illumination intensity is 300 μM.

[0024] Furthermore, in step (2), the germination time is counted after the soybean cotyledons emerge 1 cm from the soil layer.

[0025] Gene-edited soybeans are new materials for biological breeding. Currently, there is no evaluation method for the unexpected effects of gene-edited soybeans. The present invention is an evaluation method for the unexpected effects of gene-edited soybeans. Compared with the prior art, the present invention has the following beneficial effects:

[0026] The test materials are planted in an artificial climate chamber, with a high degree of environmental controllability, good repeatability, and multiple materials can be identified at the same time. Compared with field post-planting investigations, field identification is time-consuming and labor-intensive, and planting and growth conditions are easily affected by the environment, resulting in inconsistent soybean growth and interfering with the accuracy of the investigation results.

[0027] The present invention evaluates the unexpected effects of gene-edited soybeans by investigating the main agronomic traits of soybeans during growth and related phenotypes caused by prolonged juvenile period. The investigation method is simple and efficient to operate, the investigation results are intuitive, accurate and reliable, and the experimental results have good stability and usability, providing an efficient and stable method for the evaluation and research of the unexpected effects of gene-edited soybeans. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the plant phenotype survey of the early flowering period of the long-juvenile gene-edited soybeans in Example 1 (wherein the left picture is the wild-type soybean control group, and the right picture is the gene-edited soybean experimental group);

[0029] Figure 2 This is the plant phenotype survey of long-juvenile gene-edited soybeans at the grain filling stage in Example 1 (wherein the left picture is a wild-type soybean control group, and the right picture is a gene-edited soybean experimental group); DETAILED DESCRIPTION

[0030] In order to better illustrate the present invention, the following embodiments are listed. Obviously, the described embodiments are only a part of the present invention, not all embodiments. Based on the embodiments in the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0031] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0032] Example 1

[0033] Using "Huachun No. 1" soybean as the receptor and the CRISPR-cas9 gene editing vector, the GmFT of soybean was gene edited through Agrobacterium-mediated genetic transformation, and multiple mutants were obtained. Among them, the strains with significantly delayed growth period (long juvenile period) were selected as materials to study the unexpected effect evaluation method of the present invention.

[0034] (1) Preparation and planting of soybean materials:

[0035] a) Setting gene-edited soybeans and corresponding recipient materials as controls;

[0036] b) Before sowing, the gene-edited soybeans and the corresponding receptor materials were selected, and the shriveled, damaged, and moldy seeds were removed, and the plump and round seeds were selected;

[0037] c) The seeds were sown in pots filled with uniform soil (the soil was ordinary field soil taken from the field) at a sowing depth of 3 cm. The seeds were cultured under controllable conditions such as an artificial climate chamber. The culture temperature was set to 25°C, the relative humidity was 50%, the culture light cycle was long day (15 h light and 9 h dark), short day (12 h light and 12 h dark), and the light intensity was 300uM.

[0038] (2) Count the germination time after the soybean cotyledons emerge 1 cm from the soil layer.

[0039] (3) After the soybean cotyledons are fully expanded, the germination rate of soybeans is calculated.

[0040] (4) The plant height of gene-edited soybean and recipient materials was investigated at the V3, R1, R3, and R5 stages, respectively.

[0041] (5) When a small flower blooms at any node of the main stem of the plant, record the flowering time.

[0042] (6) Investigate the width of the compound leaves at the third-to-last node of the main stem at the R6 stage.

[0043] (7) Investigate the number of main stem nodes at R7 stage.

[0044] (8) Yield statistics of gene-edited soybeans and their control recipient materials at maturity.

[0045] In this experiment, 20 individual plants were set in each group to measure the germination time, germination rate, plant height, flowering time, compound leaf width, and main stem node number of the soybeans in the experimental group and the control group, and 30 individual plants were set to count the soybean yields in the experimental group and the control group.

[0046] The above statistical results are summarized and tabulated. The specific statistical results are shown in Table 1 and Table 2 below.

[0047] Table 1 Plant height statistics (cm)

[0048] Material V3 R1 R3 R5 Receptor Control 19.00±0.47a 38.9±1.60a 56.78±0.65a 66.94±0.85a Gene-edited soybeans 18.28±0.70a 37.72±1.04a 60.76±1.13b 81.36±1.09b

[0049] Note: The data in the table are mean ± standard error. The letters after the values ​​indicate the significant difference between treatments at the α=0.05 level. The same lowercase letters are not significantly different, while different lowercase letters are significantly different.

[0050] Table 2 Germination time, germination rate, compound leaf width and main stem node number of soybean

[0051] Material Germination time (d) Germination rate (%) Leaf width (cm) Main stem nodes (nodes) Yield per plant (g) Receptor Control 3.4±0.55a 95.2±3.27a 6.04±0.30a 11.2±0.45a 39.3±4.1a Gene-edited soybeans 4.6±0.55b 96.2±1.79b 7.65±0.45b 14.2±0.45b 46.2±3.5b

[0052] Note: The data in the table are mean ± standard error. The letters after the values ​​indicate the significant difference between treatments at the α=0.05 level. The same lowercase letters are not significantly different, while different lowercase letters are significantly different.

[0053] The material used in the present invention is a delayed growth period gene-edited soybean (long-juvenile soybean). According to observations during multiple growing seasons, the gene-edited soybean is consistent with our initial expectations except for the delayed growth period. In addition, there is no significant difference in plant height between the gene-edited soybean and the wild type in the V3 and R1 stages. However, the plant height of the gene-edited soybean in the R3 and R5 stages is significantly higher than that of the wild type. In terms of germination rate, compound leaf width and main stem node number, the gene-edited soybean is significantly higher than the wild type; and in terms of yield, which is of great concern in production, the single plant yield of the gene-edited soybean is significantly higher than that of the wild type. The above results all illustrate the significant changes and unexpected effects of gene-edited soybeans in agronomic traits. Therefore, the development of this project can conduct centralized evaluation and prediction of relevant materials before the promotion of new products, which has important guiding significance for production and breeding.

[0054] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for evaluating unexpected effects of gene-edited soybean phenotypic traits, characterized in that: The evaluation method comprises the following steps: (1) Preparation and planting of soybean materials: setting up gene-edited soybeans and using corresponding receptor materials as controls; selecting seeds for sowing; (2) Count the germination time after the soybean cotyledons emerge 1-2 cm from the soil layer; (3) Count the soybean germination rate after the soybean cotyledons are fully expanded; (4) Investigate the plant height of gene-edited soybean and recipient materials at the V3, R1, R3, and R5 stages; (5) When a floret blooms at any node of the main stem, record the flowering time; (6) Investigate the width of the compound leaves at the third-to-last node of the main stem at the R6 stage; (7) Investigate the number of main stem nodes and internode length at R7 stage; (8) Yield statistics of gene-edited soybeans and their control recipient materials at maturity.

2. A method for evaluating unexpected effects of gene-edited soybean phenotypic traits according to claim 1, characterized in that: The sowing method in step (1) is: selecting seeds for sowing, wherein the sowing depth is 1.5-4.5 cm, the culture temperature is 20-30° C., the culture relative humidity is 40-60%, the culture light cycle is divided into a long day period and a short day period, and the light intensity is 200-400 μM.

3. A method for evaluating unexpected effects of gene-edited soybean phenotypic traits according to claim 2, characterized in that: The seed sowing depth is 2-4 cm, the culture temperature is 23-27° C., the culture relative humidity is 45-55%, the light cycle is divided into a long-day period and a short-day period, and the light intensity is 250-350 μM.

4. A method for evaluating unexpected effects of gene-edited soybean phenotypic traits according to claim 3, characterized in that: The seed sowing depth is 3 cm.

5. A method for evaluating unexpected effects of gene-edited soybean phenotypic traits according to claim 3, characterized in that: The culture temperature is 25°C.

6. A method for evaluating unexpected effects of gene-edited soybean phenotypic traits according to claim 3, characterized in that: The relative humidity of the culture was 50%.

7. A method for evaluating unexpected effects of gene-edited soybean phenotypic traits according to claim 3, characterized in that: The culture light cycle is 10-20 hours of light and 4-14 hours of darkness during the long day period, and 10-14 hours of light and 10-14 hours of darkness during the short day period.

8. A method for evaluating unexpected effects of gene-edited soybean phenotypic traits according to claim 7, characterized in that: The culture light cycle is 15 hours of light and 9 hours of darkness during the long day period, and 12 hours of light and 12 hours of darkness during the short day period.

9. A method for evaluating unexpected effects of gene-edited soybean phenotypic traits according to claim 3, characterized in that: The illumination intensity is 300 μM.

10. A method for evaluating unexpected effects of gene-edited soybean phenotypic traits according to claim 1, characterized in that: In the step (2), the germination time is counted after the soybean cotyledons emerge 1 cm from the soil layer.

Images