Method for creating melatonin-rich soybean by using synthetic regulatory element and application of melatonin-rich soybean

Abstract

The invention discloses a method for creating melatonin-rich soybeans by utilizing synthetic regulatory elements and application of the melatonin-rich soybeans. According to the invention, a soybean standard line Williams 82 (W82) is used as a receptor, melatonin pathway genes COMT and SNAT are optimized through codons to synthesize regulation elements to efficiently and accurately activate expression of COMT and SNAT, and specific accumulation in soybean seeds is realized, so that a new soybean material rich in melatonin in the seeds is created. Compared with a control receptor, the melatonin content of the modified soybean is increased by more than 30 times. The method for efficiently producing the melatonin by synthesizing the regulatory element has an important value for increasing the content of the melatonin, provides a new thought and tool for improving the quality of soybeans and crops, is beneficial for supplementing high-value active substances in daily diet, and has a wide application prospect. The method has strong application potential in the aspect of assisting biosynthesis of plant-derived natural products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology applications and relates to a method for creating soybeans rich in melatonin by using synthetic regulatory elements and application thereof. Background Art

[0002] Melatonin (N-acetyl-5-methoxytryptamine) is a natural compound with multiple biological activities and is widely found in bacteria, fungi, plants and animals. In animals and humans, melatonin has been shown to regulate circadian rhythms, improve sleep disorders, delay aging, relieve allergic symptoms, improve diabetes, reduce inflammatory damage, and protect lymphocytes from DNA damage caused by ionizing radiation. It can be used as a medicinal ingredient and daily health product. In recent years, with the increase in the number of people suffering from insomnia, the demand for melatonin products in the consumer market has also increased. In the past, melatonin was mainly derived from chemical synthesis. Although the price was relatively low, it may be accompanied by by-products and cause adverse effects. Therefore, plant synthesis shows great potential. While ensuring that the source of melatonin is green, healthy and sustainable, it also improves the nutritional value and additional attributes of agricultural products and meets people's expectations for high-quality life.

[0003] Synthetic regulatory elements are artificially synthesized and computationally optimized promoter transcription regulatory factors. Compared with traditional promoters, these elements include core components (including TATA box, TF-IIB recognition element, Y element and downstream promoter element) and proximal regions (including cis elements that bind to activator and repressor proteins), omitting distal regions (such as enhancers, silencers and insulators), making the overall structure more efficient. Synthetic regulatory elements can precisely control the expression of target genes by controlling the copy number of DNA sites that bind to transcriptional activator proteins. In addition, since they do not exist in natural DNA sequences, these elements can minimize background noise and unnecessary crosstalk between synthetic elements and the underlying metabolic network, thereby creating precisely desired features.

[0004] Soybean is an important food crop and an important source of human diet and processing industry. Soybean is rich in nutrients such as protein, unsaturated fatty acids, isoflavones, etc., and also plays an important role in meeting the nutritional and health needs of residents. Creating soybean materials rich in melatonin and achieving biofortification not only meets the development needs of efficient resource utilization and environmental friendliness, but also meets people's high expectations for nutritional quality, and has great application potential. Summary of the invention

[0005] The purpose of the present invention is to provide a method and application of creating soybeans rich in melatonin by using synthetic regulatory elements in view of the deficiencies of the prior art.

[0006] To achieve the above object, the present invention is specifically implemented through the following technical solutions:

[0007] As a first aspect, the present invention provides a synthetic regulatory element, comprising a transcriptional activation element AmtR-ERF2-NLS and a DNA binding site; the transcriptional activation element AmtR-ERF2-NLS sequence is shown in SEQ ID NO.1; the DNA binding site sequence is shown in SEQ ID NO.2.

[0008] As a second aspect, a method for creating melatonin-rich soybeans using the above-mentioned synthetic regulatory elements is provided, specifically: the expression levels of the overexpressed melatonin pathway genes COMT and SNAT genes are affected by different synthetic regulatory element copy numbers to increase the melatonin content in soybean seeds.

[0009] Furthermore, it also includes overexpression of codon-optimized melatonin pathway genes COMT and SNAT genes to further increase the melatonin content in soybean seeds. The COMT codon-optimized sequence is shown in SEQ ID NO.3; the SNAT codon-optimized sequence is shown in SEQ ID NO.4.

[0010] Furthermore, the synthetic regulatory element and melatonin pathway gene were subcloned into the vector pCambia2300 and transformed into soybean Williams 82, successfully creating a new soybean material with a melatonin content in seeds increased by more than 30 times.

[0011] Furthermore, the primer sequences used for qRT-PCR expression measurement of the COMT gene are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0012] As a third aspect, there is provided an application of the synthetic regulatory element in the biosynthesis of plant-derived natural products.

[0013] The beneficial effects of the present invention are as follows:

[0014] The present invention utilizes synthetic regulatory elements to efficiently and accurately activate the expression of melatonin pathway genes COMT and SNAT, thereby increasing the melatonin content in soybeans. Compared with the control receptor, the melatonin content of the modified soybeans is increased by more than 30 times. It can be used for daily dietary supplements to meet the consumption needs of people with relevant needs.

[0015] The method for synthesizing regulatory elements provided by the present invention can accurately regulate the expression of target genes, provide new ideas for improving the quality of crops, and improve breeding efficiency.

[0016] The method for synthesizing regulatory elements provided by the present invention can be applied to the biosynthesis of plant-derived natural products to increase the yield of target products, and has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram of the melatonin synthesis pathway in plants;

[0018] Figure 2 This is a schematic diagram of the core module structure of the transformation vector; the arrow indicates the promoter, AmtR-ERF2-NLS is the transcriptional activation element, 3X binding+35S is 3 copies of the DNA binding site fused to 35S, SNAT-COMT* is the codon-optimized melatonin pathway gene, and E9 and ADH are terminators;

[0019] Figure 3 This is a graph showing the expression of COMT genes in various tissues of melatonin-rich soybeans; the horizontal axis represents the root, stem, leaf, and seeds at 3 weeks (seed-1), 5 weeks (seed-2), 6 weeks (seed-3), 8 weeks (seed-4), and 10 weeks (seed-5), and the vertical axis represents the expression level; 2#, 14#, and 15# are three transgenic soybean clones;

[0020] Figure 4 is a graph of melatonin content in soybean seeds; W82 is the receptor control, 2#, 14#, and 15# are three transgenic soybean clones; the statistical significance between W82 and transgenic lines was evaluated using a two-tailed Student t test (ns=no significance, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001);

[0021] Figure 5 It is a graph of crude fat and crude protein content in soybean seeds; wherein, (a) is a graph of crude fat content in soybean seeds, and (b) is a graph of crude protein content in soybean seeds; W82 is a receptor control, and 2#, 14#, and 15# are three transgenic soybean clones; the two-tailed Student t test was used to evaluate the statistical significance between W82 and the transgenic line (ns=no significance, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001). DETAILED DESCRIPTION

[0022] The present invention will be described in detail below through specific embodiments. These embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0023] As used throughout the specification and claims, "including" or "comprising" is an open term and should be interpreted as "including but not limited to". The specification subsequently describes the preferred embodiments of the present invention, but the description is for the purpose of the general principles of the specification and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be determined by the attached claims.

[0024] like Figure 1 The diagram shows the melatonin synthesis pathway in plants. Melatonin in plants is synthesized from tryptophan through four enzymatic steps. Tryptophan decarboxylase (TDC) and tryptophan 5-hydroxylase (T5H) are the key enzymes in the first two steps of melatonin synthesis, catalyzing the production of serotonin in the cytoplasm. Subsequently, serotonin is synthesized into melatonin through serotonin N-acetyltransferase (SNAT) and N-acetylserotonin O-methyltransferase (ASMT) / caffeic acid O-methyltransferase (COMT). Among them, COMT and SNAT have been shown to be the key rate-limiting enzymes in melatonin biosynthesis.

[0025] Example 1: Method for creating melatonin-rich soybeans using synthetic regulatory elements

[0026] (1) Construction of pCambia2300-COMT-SNAT vector

[0027] First, the transcriptional activation element AmtR-ERF2-NLS and DNA binding site sequences, as well as codon-optimized melatonin pathway genes COMT and SNAT were obtained by gene synthesis. Among them, the transcriptional activation element AmtR-ERF2-NLS sequence is shown in SEQ ID NO.1; the DNA binding site sequence is shown in SEQ ID NO.2; the codon-optimized COMT sequence is shown in SEQ ID NO.3, and the codon-optimized SNAT sequence is shown in SEQ ID NO.4. The transcriptional activation element AmtR-ERF2-NLS is driven by the seed-specific promoter napin and terminated by E9, and COMT and SNAT are driven by 3 copies of the DNA binding site fusion (CaMV) 35S promoter and terminated by ADH. Using homologous recombination ABclonal 2XMultiF Seamless Assembly Mix, the above elements were subcloned into pCambia2300 to form the pCambia2300-COMT-SNAT vector.

[0028] The recombinant vector was transformed into competent E. coli DH5α: Add 5 μL of the recombinant product to be transformed to 50 μL of competent cells, tap to mix, and place on ice for 30 minutes. Place the centrifuge tube in a 42°C water bath for heat shock for 90 seconds. Add 700 μL of non-resistant LB liquid culture medium to the centrifuge tube and incubate at 37°C shaker at 220 rpm for 30 minutes. Centrifuge the incubated bacterial solution at 3000 rpm for 5 minutes, discard the supernatant, aspirate 100 μL of non-resistant LB liquid culture medium to resuspend the bacterial pellet, and transfer it to LB solid culture medium containing the corresponding antibiotics, and spread it evenly. After the bacterial solution on the surface of the plate is absorbed, seal the culture dish, invert it and culture it at 37°C for 12-16 hours. Use a sterilized toothpick to pick a single colony in 600 μL of resistant liquid LB culture medium, and culture it at 37°C shaker at 200 rpm for 8 hours. Then the turbid bacterial solution is sequenced by Sanger to ensure the accuracy of the vector construction ( Figure 2 ).

[0029] (2) Stable transformation of soybean

[0030] The vector was transferred into Agrobacterium EHA105 by electroporation, and the PCR identification was positive. Soybeans that had germinated for one day were used as materials, placed in a culture dish containing 50 ml of Agrobacterium suspension, and 150 explants were treated and placed at room temperature for 30 minutes for infection. After the infection, the Agrobacterium liquid was discarded, and the explants were placed in a co-culture medium and co-cultured in the dark at 23°C for 3 days. After co-culture, the explants were transferred to a resting culture medium, cultured at 25°C for 7 days, and then placed on a screening culture medium with corresponding resistance, screened and cultured for three weeks to induce resistant buds. Then they were transferred to an elongation culture medium with corresponding resistance and cultured in light for 6-9 weeks. The regenerated elongated seedlings were rooted to produce transgenic soybeans.

[0031] (3) Identification of COMT expression levels in soybean tissues

[0032] First, RNA was extracted from soybean tissues. Roots, stems, leaves, and seeds at 3, 5, 6, 8, and 10 weeks were ground with liquid nitrogen, and RNA was extracted according to the instructions of the EASYspin Plus Plant RNA kit, and then stored in a -80°C ultra-low temperature refrigerator for subsequent analysis.

[0033] Reverse transcription reaction uses ABclonal reverse transcriptase to obtain cDNA. Denaturation of RNA template helps to open the secondary structure, which can greatly increase the yield of first-strand cDNA. The reverse transcription reaction conditions are: 37℃ reaction for 2min, 55℃ reaction for 15min, and 85℃ termination reaction. The reverse transcription product can be used immediately for subsequent experiments or stored in a refrigerator at 20℃ and used within half a year to avoid repeated freezing and thawing.

[0034] Afterwards, the cDNA concentrations of all test samples were adjusted to be consistent, with a concentration difference of ±5ng / μL. RT-qPCR was performed using the fluorescent quantitative PCR enzyme AceQ qPCR SYBR Green Master Mix. Three technical replicates and three biological replicates were set for each sample. The sequence of the amplification primer F1 used is shown in SEQ ID NO.5, and the sequence of the amplification primer R1 is shown in SEQ ID NO.6. The reaction conditions are: the first step of pre-denaturation: 95°C for 5 minutes; the second step of cycle reaction: 95°C for 10s, 60°C for 30s; the third step: drawing a melting curve. The reaction procedure was performed on a Roche Light Cycler 4800II fluorescent quantitative PCR instrument. The quantitative data were standardized according to the minimum sample threshold cycle number (CT value) and the maximum fluorescence value, and 2 -ΔΔCT Methods The relative gene expression was calculated.

[0035] Figure 3 These are the results of COMT expression in various soybean tissues. The seed-specific promoter used has a significant effect, with COMT being dominantly expressed in seeds during the 6th to 8th week of development and hardly expressed in roots, stems, and leaves.

[0036] (4) Melatonin extraction and content determination in soybean seeds

[0037] Determination of melatonin content in soybean seeds was performed using HPLC-HPLC. Dried soybean seeds from three independent biological replicates were crushed into powder and analyzed using the Waters Acquity Premier Xevo TQAbsolute platform. 500 mg of soybean powder was ultrasonically extracted with 2.0 mL of 80% methanol in water at 4 °C for 5 min. Subsequently, the mixture was incubated in the dark at 4 °C for 12 h. After centrifugation at 1500 g and 4 °C for 20 min, the supernatant was transferred to a new test tube. The remaining residue was washed with 1 mL of 80% methanol in water and combined with the supernatant for analysis.

[0038] Figure 4 This is the result of the determination of melatonin content; W82 is the receptor used as the control; 2#, 14#, and 15# are three transgenic soybean clones; compared with the control, the melatonin content in the soybean seeds with overexpressed genes increased significantly, among which 14# had the most significant increase, which increased by 30 times.

[0039] (5) Determination of crude fat and crude protein content of soybeans

[0040] Determination of crude fat content: Dry and grind the sample into powder, weigh 2.5g of the mixed sample, accurate to 0.001g, and transfer all of it into the filter paper tube. Place the filter paper tube into the extraction tube of the Soxhlet extractor, connect it to the receiving bottle that has been dried to constant weight, add anhydrous ether or petroleum ether from the upper end of the extractor condenser to two-thirds of the bottle volume, heat it on a water bath, and make the anhydrous ether or petroleum ether reflux and extract for 3 hours. At the end of the extraction, close the valve to recover the reagent. Remove the receiving bottle, recover the anhydrous petroleum ether, evaporate it on a water bath when there is 2.0mL of solvent remaining in the receiving bottle, and then dry it at 100℃±5℃ for 1h, cool it in a dryer for 0.5h, and weigh it. Repeat the above operation until constant weight.

[0041] The crude fat content X (g / 100g) in the sample is calculated according to the formula:

[0042]

[0043] in:

[0044] X—fat content in the sample, in grams per 100 grams (g / 100g);

[0045] m1—the content of the receiving bottle and fat after constant weight, in grams (g);

[0046] m0—the mass of the receiving bottle, in grams (g);

[0047] m2—mass of the sample, in grams (g);

[0048] 100—Conversion factor.

[0049] The calculation result shall be rounded to three significant figures.

[0050] Determination of crude protein content: weigh 1g of the mixed sample into a digestion tube, then add 0.4g of copper sulfate, 6g of potassium sulfate and 20mL of sulfuric acid to the digestion furnace for digestion. When the temperature of the digestion furnace reaches 420℃, continue digestion for 1h. At this time, the liquid in the digestion tube is green and transparent. Take it out and cool it down before adding 50mL of water. After the sample is digested, perform automatic nitrogen determination in a Kjeldahl nitrogen analyzer. The N in the sample is finally absorbed in the boric acid solution in the receiving bottle. Add a mixed indicator to the receiving bottle of the sample, titrate with sodium hydroxide and record the titration data.

[0051] The crude protein content X (g / 100g) in the sample is calculated according to the formula:

[0052]

[0053] in:

[0054] X—protein content in the sample, in grams per hundred grams (g / 100g);

[0055] V1—the volume of hydrochloric acid standard titrant consumed by the test solution, in milliliters (mL);

[0056] V2—volume of hydrochloric acid standard titrant consumed by reagent blank, in milliliters (mL);

[0057] c—concentration of hydrochloric acid standard titration solution, in moles per liter (mol / L);

[0058] The mass of nitrogen equivalent to 0.0140-1.0mL hydrochloric acid [c(HCl)=1.000mol / L] standard titration solution, in grams (g);

[0059] m—mass of the sample, in grams (g);

[0060] V3—the volume of digestive fluid absorbed, in milliliters (mL);

[0061] F—nitrogen conversion factor to protein;

[0062] 100—Conversion factor.

[0063] The result is rounded to three significant figures.

[0064] Figure 5 is the crude fat and crude protein content in soybean seeds; W82 is the recipient as the control; 2#, 14#, and 15# are three transgenic soybean clones; compared with the control, the crude fat content in the soybean seeds of the gene overexpression was significantly reduced, and the crude protein content was significantly increased.

[0065] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Without making creative work, any modification, equivalent replacement, improvement, etc., shall be included in the protection scope of the present invention.

Claims

1. A synthetic regulatory element, characterized in that The synthetic regulatory element includes a transcriptional activation element AmtR-ERF2-NLS and a DNA binding site; The transcriptional activation element AmtR-ERF2-NLS sequence is shown in SEQ ID NO.1; The DNA binding site sequence is shown in SEQ ID NO.

2.

2. A method for producing soybeans rich in melatonin using the synthetic regulatory element according to claim 1, characterized in that: The expression levels of the overexpressed melatonin pathway genes COMT and SNAT were affected by changing the copy number of different synthetic regulatory elements to increase the melatonin content in soybean seeds.

3. The method according to claim 2, characterized in that Also includes: Overexpression of codon-optimized COMT and SNAT genes to further increase the melatonin content in soybean seeds; The COMT codon-optimized sequence is shown in SEQ ID NO.3; The SNAT codon-optimized sequence is shown in SEQ ID NO.

4.

4. The method according to claim 2, characterized in that: The primer sequences used for qRT-PCR expression measurement of the COMT gene are shown in SEQ ID NO.5 and SEQ ID NO.

6.

5. Use of the synthetic regulatory element according to claim 1 in the biosynthesis of plant-derived natural products.

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