Soybean transformation vector and application thereof

Abstract

The invention relates to a soybean transformation vector and application thereof, and belongs to the field of plant genetic engineering. According to the soybean transformation vector provided by the invention, transgenic positive seeds can be accurately distinguished by naked eyes.

Description

Technical Field

[0001] The present invention relates to a soybean transformation vector and its application, belonging to the field of plant genetic engineering. Background Art

[0002] The identification of transgenic lines relies on molecular detection methods, which are time-consuming, laborious, and costly in terms of reagents and consumables. Therefore, establishing a simple and effective visualization marker system is crucial for solving this technical problem.

[0003] The red fluorescent protein (DsRed2) is a commonly used visual marker gene applied in the positive identification of transgenic plants such as maize, achieving rapid visualization of transgenic plants. However, its application effect in soybeans, especially the effect of direct visual discrimination by the naked eye, is still uncertain.

[0004] To solve the above problems, the present invention tested expression vectors constructed by connecting different combinations of promoters and enhancers to DsRed2, and directly discriminated the transgenic individuals by the naked eye in the seeds after transforming soybeans. Finally, a vector capable of accurately discriminating transgenic positive seeds by the naked eye was screened. Summary of the Invention

[0005] The purpose of the present invention is to provide a soybean transformation vector to accurately discriminate transgenic positive plants by the naked eye.

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

[0007] The present invention provides an application of an expression cassette in the identification and / or screening of transgenic soybean plants, characterized in that the expression cassette is composed of an enhancer shown by SEQ ID NO.4, an enhancer shown by SEQ ID NO.5, a promoter shown by SEQ ID NO.1, a coding gene shown by SEQ ID NO.6, and a terminator shown by SEQ ID NO.7 connected in sequence.

[0008] The present invention also provides an application of an expression vector in the identification and / or screening of transgenic soybean plants, characterized in that the expression vector contains the above-mentioned expression cassette.

[0009] The present invention also provides an application of a host cell in the identification and / or screening of transgenic soybean plants, characterized in that the host cell contains the above-mentioned expression cassette or the above-mentioned expression vector, and the host cell is a microbial cell or a non-renewable plant cell.

[0010] In some embodiments, the above-mentioned identification and / or screening of transgenic plants is carried out by directly observing the seeds with the naked eye for identification and / or screening.

[0011] The present invention also provides a method for producing transgenic soybean seeds, which is characterized by comprising the following steps:

[0012] (1) Transforming the above-mentioned expression cassette or the above-mentioned expression vector or the above-mentioned host cell into soybeans;

[0013] (2) Harvesting soybean seeds;

[0014] (3) Selecting red soybean seeds by the method of direct visual observation.

[0015] The present invention also provides an application of an expression cassette in soybean gene editing, which is characterized in that the expression cassette is composed of an enhancer shown by SEQ ID NO.4, an enhancer shown by SEQ ID NO.5, a promoter shown by SEQ ID NO.3, a coding gene shown by SEQ ID NO.6, and a terminator shown by SEQ ID NO.7 connected in sequence.

[0016] The present invention also provides a soybean gene editing method, which is characterized by comprising the following steps:

[0017] (1) Transforming a gene editing vector containing the above-mentioned expression cassette into soybeans;

[0018] (2) Selecting red soybean plants to harvest seeds or red seeds harvested from transformed soybean plants by the method of direct visual observation;

[0019] (3) Planting the seeds harvested in step (2), and selecting soybean plants in which the target site is successfully edited and does not contain the above-mentioned expression cassette.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The present invention tested expression vectors constructed by connecting different combinations of promoters and enhancers to DsRed2, and the effect of directly identifying transgenic individuals by the naked eye after transforming soybeans. Finally, a vector that can accurately identify transgenic positive seeds by the naked eye and does not affect the normal growth of soybeans was screened. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 VSES vector diagram. A: VSES2; B: VSES4. Elements are marked on the figure.

[0023] Figure 2 Seed display of different vectors under natural light and fluorescent light.

[0024] Figure 3 Representative comparison diagrams of wild-type and VSES4 transgenic plants at the seedling stage, flowering stage and maturity stage.

[0025] Figure 4 Sequencing results of the gene editing target site of GmEID1. Specific implementation manners

[0026] The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise specified, the terms are understood in accordance with the conventional usage of those of ordinary skill in the relevant art. All patent documents, academic papers, industry standards, and other publicly published materials cited herein are hereby incorporated by reference in their entirety.

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps, or conditions of the present invention shall fall within the scope of the present application. Unless otherwise specified, the examples are carried out under conventional experimental conditions, such as those described in Molecular Cloning: A Laboratory Manual by Sambrook et al. (Sambrook J & Russell D W, Molecular cloning: a laboratory manual, 2001), or according to the conditions recommended by the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are commercially available conventional reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0028] In order to facilitate the understanding of the technical solutions of the present invention, the present invention will be further described below with reference to specific examples.

[0029] Example 1 Design and construction of a soybean transformation vector

[0030] The inventors selected the red fluorescent protein DsRed2 as a color marker to achieve the technical effect of visually differentiating positive plants. Since the color intensity is a key factor affecting the visual differentiation effect, it is necessary to increase the expression level of DsRed2. At the same time, excessive expression of DsRed2 will also affect the normal growth of plants, so the expression of DsRed2 needs to be controlled at an appropriate level in soybeans.

[0031] The inventors selected several different promoters: two soybean seedcoat-specific promoters (SCSP1 and SCSP2, the sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively), and one soybean S-adenosylmethionine synthetase promoter (SAMS, the sequence is shown in SEQ ID NO.3). And enhancer 2xEnh composed of FMV enhancer (the sequence is shown in SEQ ID NO.4) and CaMV35S enhancer (the sequence is shown in SEQ ID NO.5) was selected. These promoters and enhancers were combined, and the DsRed2 coding gene (the sequence is shown in SEQ ID NO.6) and the tPinII terminator (the sequence is shown in SEQ ID NO.7) were ligated to construct a series of expression cassettes, which were respectively ligated to the soybean CRISPR / Cas9 gene editing backbone vector to obtain a series of soybean transformation vectors (see Table 1). Among them, taking VSES2 and VSES4 as examples, the vector diagrams are shown as Figure 1 shown below.

[0032] Table 1 Information of soybean transformation vectors

[0033]

[0034] Example 2 Obtaining of soybean transgenic plants

[0035] The vectors constructed above were respectively transferred into Agrobacterium tumefaciens GV3101 by heat shock method and identified by PCR, and then the Agrobacterium and glycerol were mixed in a volume ratio of 1:1 and stored at -80°C. The cotyledon node genetic transformation method mediated by Agrobacterium was used to transform soybean Dongnong 50. The specific transformation steps are as follows:

[0036] (1) Seed sterilization: First, select mature soybean seeds with complete grains and clean surfaces, and spread them evenly in a petri dish, about 100 seeds per dish. Place the petri dish in a desiccator, slowly add 5 mL of concentrated hydrochloric acid to a glass beaker containing 100 mL of sodium hypochlorite solution, and then place the beaker in the center of the desiccator. When sealing the desiccator, it is necessary to reinforce the joint between the lid and the base with petroleum jelly. After maintaining the airtight environment for 16 hours, take out the seeds, and collect the mixed waste liquid into a special waste liquid bottle. Then place the seeds in a laminar flow hood, blow them with sterile air flow for 5 minutes to thoroughly remove the residual chlorine, and finally seal and store the seeds in a clean petri dish.

[0037] (2) Agrobacterium activation culture: 200 μL of the preserved Agrobacterium was inoculated into YEP liquid medium containing 100 μg / mL rifampicin and 50 μg / mL kanamycin. The culture was activated in an incubator at 28°C and 100 rpm for 12 h. The shaken culture was spread onto a YEP solid plate containing the same antibiotic concentration and cultured overnight in a 28°C incubator.

[0038] (3) Seed germination: Soybean seeds were placed with the hilum facing downwards in a germination medium, with 20-25 seeds placed in each dish. The dish was sealed with breathable tape and incubated in a dark environment at 23°C for 24 hours to allow the seeds to fully absorb water and swell.

[0039] (4) Infection and co-cultivation: Scrape the Agrobacterium from the surface of the solid culture medium and suspend it in the infection medium. Use an ultraviolet spectrophotometer to detect the bacterial concentration and adjust it to OD600 = 0.5-0.6. Cut the pre-cultured soybeans longitudinally under sterile conditions, remove the growth point, and create 3-5 wound zones at the incision. Immerse the treated explants completely in the Agrobacterium infection solution and continue to shake and infect for 30 minutes. Transfer them to the co-culture medium covered with sterile filter paper and place them neatly with the incision side facing up, with 10-15 explants per dish. Then, seal with breathable tape and culture in a dark environment at 23°C for 5 days.

[0040] (5) Recovery culture: After co-cultivation, the hypocotyls were cut 5 mm from the cotyledonary nodes in a clean bench and inserted obliquely into the recovery medium. Cultured at 23°C for 5-7 days until new shoots emerged from the medium.

[0041] (6) Screening culture: After the culture is resumed, the hypocotyl is cut 5 mm from the cotyledonary node in a clean bench and inserted obliquely into the screening medium for two weeks. During the screening process, the leaves on the large buds gradually turn yellow, while the cotyledonary node continues to swell and grow small green buds.

[0042] (7) Regeneration culture: After screening and culture, the yellow leaves of the explants are removed, the base of the cotyledonary nodes is cut off, and the explants are transferred to the regeneration medium for culture.

[0043] (8) Rooting culture: When the seedlings in the regeneration medium have grown to 3-4 cm, they are cut and transferred to the rooting medium. When there are more than two new roots, they are transplanted to nutrient soil.

[0044] The culture medium used in the transformation process is as follows:

[0045] Germination medium: B5 + 2% sucrose + 0.7% agar, pH 5.7;

[0046] Infection medium: 1 / 10 B5 + BAP 1.67 mg / L + GA3 250 mg / L + As 200 μM / L + MES 20 mM + 3% sucrose, pH 5.4;

[0047] Co - culture medium: 1 / 10 B5 + BAP 1.67 mg / L + GA3 250 mg / L + As 200 μM / L + Cys 400 mM + DTT 154 mg / L + MES 20 mM + 3% sucrose + 0.7% agar, pH 5.4;

[0048] Recovery medium: B5 + BAP 1.67 mg / L + Tim 50 mg / L + Cef 150 mg / L + Cb 100 mg / L + MES 3 mM + 3% sucrose + 0.7% agar, pH 5.7;

[0049] Selection medium: B5 + BAP 1.67 mg / L + Tim 50 mg / L + Cef 150 mg / L + Cb 100 mg / L + G 10 mg / L + MES 3 mM + 3% sucrose + 0.7% agar, pH 5.7;

[0050] Regeneration medium: MS + IAA 0.1 mg / L + GA3 500 mg / L + Zeatin - R 1 mg / L + ASP 50 mg / L + Glu 50 mg / L + Tim 50 mg / L + Cef 150 mg / L + Cb 100 mg / L + G 5 mg / L + MES 3 mM + 3% sucrose + 0.7% agar, pH 5.8;

[0051] Rooting medium: MS + MES 3 mM + 3% sucrose + 0.7% agar, pH 5.8.

[0052] Example 3: Effect test of soybean transformation vector

[0053] A total of 20 transgenic plants were obtained with the VSES1 vector, and the total number of harvested seeds was 454. At the fluorescence excitation wavelength, red - fluorescent seeds accounted for 98.7% of the total harvest ( Figure 2 , Table 2). However, under natural light conditions, all seeds showed yellow and could not be distinguished by the naked eye ( Figure 2 ). For transgenic plants expressing VSES2, the expression level of the fluorescent protein in the harvested seeds was significantly increased, enabling seeds carrying DsRed2 to be directly identified by the naked eye ( Figure 2)。The red fluorescent seeds accounted for 94.2% of the total harvested seeds, and 57.5% of the seeds showed red under natural light (Table 2). The performance of the harvested seeds of the VSES3 transgenic plants was lower than that of the transgenic plants expressing VSES2. The red fluorescent seeds only accounted for 71.4% of the total harvested seeds, and the proportion of visible seeds under natural light was 48.7% ( Figure 2 , Table 2). In the transgenic plants expressing VSES4, the proportion of red visible seeds under natural light was significantly increased to 81.3% (Table 2). In addition, the plants expressing VSES4 showed red stems and leaves at the seedling stage, flowering stage and maturity stage ( Figure 3 ).

[0054] Table 2 Statistical analysis of red fluorescent seeds and naked-eye visible seeds of VSES1-4 transgenic plants

[0055]

[0056]

[0057] However, the results of continuous tracking surveys of transgenic plants of each vector showed that in the transgenic plants expressing VSES4, about 9.23% of the transgenic seedlings showed abnormal growth and development, resulting in complete lethality (Table 3).

[0058] Table 3 Statistical analysis of the number of planted and seedling lethal VSES4 transgenic plants

[0059]

[0060] Therefore, based on the above results, it can be seen that the 2xEnh-pSCSP1-DsRed2-tPinII expression cassette is more suitable for identifying or screening soybean transgenic plants by directly observing the seeds with the naked eye; the 2xEnh-pSCSP1-DsRed2-tPinII expression cassette can be constructed onto a vector together with another expression cassette, such as the expression cassette of a candidate gene that needs to be functionally identified, and after transforming soybeans, positive lines can be screened by directly observing the seed color with the naked eye, so as to study the function of the candidate gene by relevant means. Although the 2xEnh-pSAMS-DsRed2-tPinII expression cassette can make the plants show color before seed harvest, can identify transgenic plants earlier, and the proportion of seeds showing red to the naked eye is also higher, it is not suitable for continuous expression in soybean plants because it will cause abnormal growth and development of seedlings and complete lethality of some plants; the 2xEnh-pSAMS-DsRed2-tPinII expression cassette can be used as a color marker for gene editing vectors (such as VSES4), and the editing vector can be deleted after the target gene editing is completed, so as to avoid the influence of the 2xEnh-pSAMS-DsRed2-tPinII expression cassette on the growth and development of soybean plants.

[0061] Example 4 Effect Test of Soybean Gene Editing

[0062] The inventors tested the effect of using the VSES4 vector for soybean gene editing. The soybean GmEID1 gene was selected as the target gene for editing, and the gRNA sequence was designed to construct an editing vector with the VSES4 vector as the backbone, and the editing vector was transformed into soybeans. After obtaining the T0 generation plants, the T1 seeds were harvested, and the red seeds were selected by direct visual observation. Then the red T1 seeds were planted to obtain T1 plants. The editing status of the target sites of the T1 plants was identified. The results showed that all the obtained plants had various different edits ( Figure 4 ), indicating that the VSES4 vector can be used for soybean gene editing.

[0063] Although the present invention has been described in detail above with general descriptions and specific embodiments, on the basis of the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. Use of an expression cassette in the identification and / or screening of transgenic soybean plants, characterized in that, The expression cassette is formed by sequentially connecting an enhancer shown by SEQ ID NO.4, an enhancer shown by SEQ ID NO.5, a promoter shown by SEQ ID NO.1, a coding gene shown by SEQ ID NO.6, and a terminator shown by SEQ ID NO.

7.

2. Use of an expression vector in identifying and / or screening transgenic soybean plants, characterized in that: The expression vector contains the expression cassette described in claim 1.

3. Use of a host cell in the identification and / or screening of transgenic soybean plants, characterized in that, The host cell contains the expression cassette described in claim 1 or the expression vector described in claim 2, and the host cell is a microbial cell or a non-renewable plant cell.

4. The use according to any one of claims 1 to 3, characterized in that: The identification and / or screening of the transgenic plant is carried out by directly observing the seeds with the naked eye.

5. A method for producing transgenic soybean seeds, characterized in that, It includes the following steps: (1) Transforming soybean with the expression cassette described in claim 1, the expression vector described in claim 2, or the host cell described in claim 3; (2) Harvesting soybean seeds; (3) Selecting red soybean seeds by directly observing with the naked eye.

6. Use of the expression cassette in soybean gene editing, characterized in that, The expression cassette is formed by sequentially connecting an enhancer shown by SEQ ID NO.4, an enhancer shown by SEQ ID NO.5, a promoter shown by SEQ ID NO.3, a coding gene shown by SEQ ID NO.6, and a terminator shown by SEQ ID NO.

7.

7. A soybean gene editing method, characterized in that, It includes the following steps: (1) Transforming soybean with a gene editing vector containing the expression cassette described in claim 6; (2) Selecting red soybean plants harvested by directly observing with the naked eye or red seeds harvested from transformed soybean plants; (3) Planting the seeds harvested in step (2), and selecting soybean plants in which the target site is successfully edited and does not contain the expression cassette described in claim 6.

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