Tomato slap2 gene for increasing content of five long-chain fatty acids in plants and application thereof

Abstract

The application discloses a tomato SlAP2 gene for increasing the content of five long-chain fatty acids in plants and application thereof, and belongs to the technical field of genetic engineering.The four SlAP2 transcription factors are overexpressed in Nicotiana benthamiana driven by a constitutive promoter 35S, and through comparison, it is found that overexpression of the tomato Sl03gAP2 and Sl06gAP2 genes can increase the content of five long-chain fatty acids, i.e., hexadecanoic acid (C16:0), hexadecatrienoic acid (C16:3), octadecanoic acid (C18:0), octadecatrienoic acid (C18:3) and eicosanoic acid (C20:0) in tobacco leaves.Through exploration of the action of the tomato Sl03gAP2 and homologous genes in regulating the synthesis of fatty acids, it is determined that the tomato Sl03gAP2 and Sl06gAP2 can be applied to the synthetic biology research of fatty acids.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a tomato SlAP2 gene that increases the content of five long-chain fatty acids in plants and its application. Background Technology

[0002] Long-chain fatty acids (LCFAs) play a variety of important functions in organisms, including energy storage, cell membrane structural components, and participation in signal transduction. In oil crops, LCFAs are the main components of oils and are of great significance for improving oil content and quality.

[0003] Long-chain fatty acids generally contain 18 to 22 carbons or more. When oxidized in the body, they produce large amounts of adenosine triphosphate (ATP), which is used for glucose and fatty acid metabolism, maintaining the body's basal metabolic rate. In addition, some long-chain fatty acids also play important roles in human health, such as alpha-linolenic acid (ALA). 18 H 30 Alpha-linolenic acid (ALA) is an omega-3 fatty acid that not only provides nutritional benefits but also helps regulate blood lipids, regulate blood pressure, and suppress allergic reactions. However, the human body cannot synthesize ALA and must obtain it from food.

[0004] The synthesis of long-chain fatty acids mainly relies on traditional chemical methods and bio-fermentation. However, these methods suffer from drawbacks such as high cost, low efficiency, and significant environmental impact. In recent years, with the rapid development of genetic engineering technology, optimizing biosynthetic pathways through the regulation of transcription factors has become a research hotspot.

[0005] The function of the tomato Sl03gAP2 transcription factor in regulating fatty acid synthesis remains to be determined. At the same time, there are multiple homologous genes of Sl03gAP2 in tomatoes, which may play similar functions and jointly regulate plant fatty acid synthesis and metabolism. Whether it can achieve the synthesis of long-chain fatty acids needs further verification. Summary of the Invention

[0006] To address the problems existing in the prior art, the purpose of this invention is to design and provide a technical solution for the tomato SlAP2 gene and its application to increase the content of five long-chain fatty acids in plants.

[0007] The present invention is specifically implemented using the following technical solutions:

[0008] The first aspect of this invention provides a tomato SlAP2 gene for increasing the content of five long-chain fatty acids in plants. The tomato SlAP2 gene includes the tomato Sl03gAP2 and Sl06gAP2 genes. The nucleotide sequence of the tomato Sl03gAP2 gene is shown in SEQ ID NO.3, and the nucleotide sequence of the tomato Sl06gAP2 gene is shown in SEQ ID NO.4. The five long-chain fatty acids include hexadecanoic acid (C16:0), hexadecanoic acid (C16:3), octadecanoic acid (C18:0), octadecanoic acid (C18:3), and eicosanoic acid (C20:0).

[0009] The second aspect of this invention provides the application of the above-mentioned tomato SlAP2 gene in increasing the content of five long-chain fatty acids in plants.

[0010] Furthermore, the plant described in this application is Nicotiana benthamiana.

[0011] The third aspect of this invention provides a method for increasing the content of five long-chain fatty acids in plants by using a transient conversion method to overexpress the Sl03gAP2 or Sl06gAP2 gene in plants.

[0012] Furthermore, the specific steps of this method include: amplifying the tomato SlAP2 gene, ligating the gene into a plant transient expression vector, using a transient transformation method to transfer the transient expression vector plasmid containing the SlAP2 gene into Agrobacterium, and simultaneously expressing the transient expression vector in the plant.

[0013] Furthermore, the plant described in this method is Nicotiana benthamiana.

[0014] Furthermore, the plant transient expression vector used in this method is pEAQ.

[0015] Furthermore, the nucleotide sequence of the Sl03gAP2 gene described in this method is shown in SEQ ID NO.3, and the nucleotide sequence of the Sl06gAP2 gene is shown in SEQ ID NO.4.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] This invention compares the transient expression effects of four homologous genes (Sl01gAP2, Sl02gAP2, Sl03gAP2, and Sl06gAP2) from the tomato SlAP2 family in tobacco. It was found that Sl03gAP2 and Sl06gAP2 have a more significant effect on the synthesis of five long-chain fatty acids compared to the other two. Therefore, tomato Sl03gAP2 and Sl06gAP2 can be used to synthesize five long-chain fatty acids. Based on experimental data and using the method provided in this invention, significant changes in hexadecanoic acid (C16:0) content were observed in tobacco leaves by overexpressing different AP2 transcription factors. Specifically, in tobacco leaves overexpressing Sl01gAP2, the hexadecanoic acid content increased to 146% of the control; while overexpression of Sl02gAP2 had no significant effect on the hexadecanoic acid content. In contrast, the hexadecanoic acid content in tobacco leaves overexpressing Sl03gAP2 and Sl06gAP2 increased to 155% and 183% of the control, respectively, showing a more significant improvement. Furthermore, we analyzed the effects of overexpressing different AP2 transcription factors on the hexadecanoic acid (C16:3) content in tobacco leaves. Compared with the empty vector control pEAQ, the hexadecanoic acid content in tobacco leaves overexpressing Sl01gAP2 increased by 26%; overexpressing Sl02gAP2 by 23%; overexpressing Sl03gAP2 by 32%; and overexpressing Sl06gAP2 by a significant 47%. Furthermore, it is worth noting that overexpression of Sl03gAP2 and Sl06gAP2 in tobacco leaves, compared with overexpression of Sl01gAP2 and Sl02gAP2, not only significantly increased the content of hexadecanoic acid, but also had a more significant effect on increasing the content of octadecanoic acid (C18:0), octadecadienoic acid (C18:3) and eicosanoic acid (C20:0).

[0018] This invention enables the synthesis of large amounts of long-chain saturated fatty acids in plants by overexpressing the tomato Sl03gAP2 and Sl06gAP2 genes: hexadecanoic acid (C16:0), hexadecyltrienoic acid (C16:3), octadecanoic acid (C18:0), octadecyltrienoic acid (C18:3), and eicosanoic acid (C20:0). Attached Figure Description

[0019] Figure 1 The images show PCR amplification diagrams of the tomato Sl01gAP2, Sl02gAP2, Sl03gAP2, and Sl06gAP2 gene fragments, where (a) is the tomato Sl01gAP2 gene fragment, (b) is the tomato Sl02gAP2 gene fragment, (c) is the tomato Sl03gAP2 gene fragment, and (d) is the tomato Sl06gAP2 gene fragment.

[0020] Figure 2The molecular formulas and chromatograms of five long-chain fatty acids (in methyl ester form) and two internal standards after extraction are shown. Among them, ①-⑦ are methyl pentadecanoate, methyl hexadecyltrienoate, methyl hexadecanoate, methyl heptadecanoate, methyl octadecyltrienoate, methyl octadecanoate, and methyl eicosanoate, respectively.

[0021] Figure 3 The image shows a comparison of the contents of five long-chain fatty acids in tobacco leaves that transiently overexpress Sl01gAP2, Sl02gAP2, Sl03gAP2, and Sl06gAP2, where (a) to (e) represent hexadecanoic acid (C16:0), hexadecanetrienoic acid (C16:3), octadecanoic acid (C18:0), octadecanetrienoic acid (C18:3), and eicosanoic acid (C20:0), respectively. Detailed Implementation

[0022] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.

[0023] Example 1: A method for increasing the content of five long-chain fatty acids in plants

[0024] 1. Acquisition of tomato Sl01gAP2, Sl02gAP2, Sl03gAP2 and Sl06gAP2 gene sequences and primer design.

[0025] The gene number of the tomato Sl01gAP2 gene is Solyc01g096865, and its gene sequence is shown in SEQ ID NO.1; the gene number of the tomato Sl02gAP2 gene is Solyc02g030210, and its gene sequence is shown in SEQ ID NO.2; the gene number of the tomato Sl03gAP2 gene is Solyc03g117720, and its gene sequence is shown in SEQ ID NO.3; the gene number of the tomato Sl06gAP2 gene is Solyc06g068570, and its gene sequence is shown in SEQ ID NO.4, as shown in Table 1.

[0026] The pEAQ vector fragment was introduced into primers Sl01gAP2-Q-1F, Sl01gAP2-Q-1R, Sl02gAP2-Q-1F, Sl02gAP2-Q-1R, Sl03gAP2-Q-1F, Sl03gAP2-Q-1R, and Sl06gAP2-Q-1F, Sl06gAP2-Q-1R. This resulted in the introduction of the pEAQ vector fragment into primers Sl01gAP2, Sl02gAP2, and Sl06gAP2-Q-1R, which stimulated the development of tomato Sl01gAP2, Sl02gAP2, and Sl06gAP2-Q-1F primers. PCR amplification was performed on the sequences l03gAP2 and Sl06gAP2. The nucleotide sequences of Sl01gAP2-Q-1F, Sl01gAP2-Q-1R, Sl02gAP2-Q-1F, Sl02gAP2-Q-1R, Sl03gAP2-Q-1F, Sl03gAP2-Q-1R, and Sl06gAP2-Q-1F and Sl06gAP2-Q-1R are shown in SEQ ID NO. As shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12; during the PCR amplification process, using the designed primers and cDNA of cultivated tomato M82 as a template, PCR amplification was performed using the high-fidelity enzyme KOD, followed by recovery and purification. The amplified products were analyzed by gel electrophoresis to obtain purified Sl01gAP2, Sl02gAP2, Sl03gAP2, and Sl06gAP2 gene fragments. Figure 1 The images show PCR amplification diagrams of the tomato Sl01gAP2, Sl02gAP2, Sl03gAP2, and Sl06gAP2 gene fragments, where (a) is the tomato Sl01gAP2 gene fragment, (b) is the tomato Sl02gAP2 gene fragment, (c) is the tomato Sl03gAP2 gene fragment, and (d) is the tomato Sl06gAP2 gene fragment.

[0027] 2. Constructing transient expression vectors for the Sl01gAP2, Sl02gAP2, Sl03gAP2, and Sl06gAP2 genes in tobacco.

[0028] (1) Using homologous recombination, the tomato SlAP2 fragment and the pEAQ vector fragment were recombinated into the transient expression vector pEAQ-SlAP2. The specific method is as follows:

[0029] In this embodiment, using the designed primers and cDNA of cultivated tomato M82 as a template, PCR amplification was performed using the high-fidelity enzyme KOD, followed by recovery and purification. The amplification products were analyzed by gel electrophoresis to obtain purified Sl01gAP2, Sl02gAP2, Sl03gAP2, and Sl06gAP2 gene fragments. The specific method for obtaining the vector fragment using the double enzyme digestion method is as follows: the pEAQ empty vector plasmid was digested with XhoI and XmaI enzymes, and the digestion products were recovered and purified to obtain linearized pEAQ vector fragments. The specific method for homologous recombination ligation is as follows: the purified Sl01gAP2, Sl02gAP2, Sl03gAP2, and Sl06gAP2 gene fragments were ligated with the pEAQ vector fragments to obtain recombinant plasmids.

[0030] (2) The recombinant plasmid was transformed into different E. coli DH5α competent cells, and the specific method is as follows:

[0031] The recombinant plasmids were transformed into different E. coli DH5α competent cells and cultured in LB medium containing kanamycin. The grown colonies were then identified by PCR. The PCR-positive colonies were shaken and cultured before the plasmids were extracted and sequenced to obtain transient expression vector plasmids pEAQ-Sl01gAP2, pEAQ-Sl02gAP2, pEAQ-Sl03gAP2, and pEAQ-Sl06gAP2 with the correct mutant sequences.

[0032] In this embodiment, the specific PCR amplification method is as follows: 25 μL of KOD high-fidelity enzyme, 2 μL of forward primer, 2 μL of reverse primer, 2 μL of cDNA, and water added to 50 μL; the reaction program is: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 65℃ annealing for 10 s, 68℃ extension for 30 s, denaturation, annealing, and extension for 36 cycles, and 68℃ extension for 5 min.

[0033] In this embodiment, the specific XhoI and XmaI double digestion method is as follows: 1 μg pEAQ empty vector plasmid, 1 μL XhoI, 1 μL XmaI, and NE Buffer. TM Add 3.12 μL of r and water to a final volume of 20 μL; the reaction procedure is: incubate at 37°C for 15 min.

[0034] In this embodiment, the specific ligation reaction system for the recombinant plasmid is as follows: 5 μL of one-step seamless cloning premix (2x), 200 ng of purified gene fragment, 50 ng of purified pEAQ vector fragment, and water added to 10 μL.

[0035] In this embodiment, the primers for PCR identification are pEAQ-F with the sequence shown in SEQ ID NO.13 and pEAQ-R with the sequence shown in SEQ ID NO.14.

[0036] In the above process of this embodiment, the nucleotide sequences of all primers SEQ ID NO.5 to 14 used and their respective uses are summarized in Table 2.

[0037] Table 1 Gene Sequences

[0038]

[0039]

[0040]

[0041] Table 2 Primer sequences

[0042]

[0043]

[0044] 3. Instantaneous conversion method to Benzoic tobacco

[0045] (1) The specific method for expressing pEAQ-Sl01gAP2, pEAQ-Sl02gAP2, pEAQ-Sl03gAP2 and pEAQ-Sl06gAP2 vectors using the transient transformation method is as follows: The transient expression vector plasmids pEAQ-Sl01gAP2, pEAQ-Sl02gAP2, pEAQ-Sl03gAP2 and pEAQ-Sl06gAP2 are transformed into Agrobacterium GV3101 competent cells and cultured in LB medium containing kanamycin and rifampin resistance. The grown colonies are identified by PCR. The primers used for PCR identification are pEAQ-F with the sequence shown in SEQ ID NO.13 and pEAQ-R with the sequence shown in SEQ ID NO.14.

[0046] (2) The PCR-positive Agrobacterium was activated and amplified to obtain Agrobacterium bacterial suspensions containing transient expression vectors pEAQ-Sl01gAP2, pEAQ-Sl02gAP2, pEAQ-Sl03gAP2, and pEAQ-Sl06gAAP2; the required volume of each bacterial suspension was calculated according to the actual OD value of the bacterial suspension, and the final OD value of the bacterial suspension was measured. 600 Adjust the concentration to 1.0; collect Agrobacterium after centrifugation, then wash, resuspend, centrifuge, collect the precipitate and resuspend to obtain Agrobacterium infection solution; inject the Agrobacterium infection solution into plant leaves.

[0047] 4. Determining changes in fatty acid content in tobacco leaves using GC-MS.

[0048] (1) Collect infiltrated tobacco leaves and extract fatty acids. The specific method is as follows:

[0049] Four to five days after injection, infiltrated tobacco leaves were collected, freeze-dried, and stored at -80°C. 10 mg of the ground sample was weighed, and 1 mL of extraction buffer (2.5% sulfuric acid: methanol; v / v) was added. 25 μg of pentadecanoic acid (internal reference) was added, along with 300 μl of toluene (co-solvent). The mixture was heated at 80°C for 60 min, cooled to room temperature, and 1.5 mL of 0.9% NaCl was added to terminate the reaction. 25 μg of methyl heptadecanate was added, along with 2 mL of n-hexane. After vortexing, the mixture was centrifuged horizontally at 4000 rpm for 10 min. The supernatant was collected, and the process was repeated twice. The mixture was dried under nitrogen, dissolved in 400 μl of n-hexane, and transferred to a sample vial.

[0050] (2) The changes in fatty acid content in tobacco leaves were determined using GC-MS. The specific method is as follows:

[0051] Fatty acids were separated and determined using an Agilent GC-MS 7890B gas chromatograph-mass spectrometer. The chromatographic column was an Agilent 122-5532G DB-5MS with a length of 40 m, an inner diameter of 250 μm, and a film thickness of 0.25 μm. The carrier gas was nitrogen. The temperature program was as follows: initial temperature 50 °C, 50 °C–200 °C, 25 °C / min, hold for 1 min; 200 °C–300 °C, hold for 8 min; split ratio was 5:1; injection volume was 4 μl.

[0052] Peak areas were analyzed using Agilent MassHunter Workstation software. The peak areas of the target fatty acid were compared with those of the internal reference peaks in the sample to calculate the relative concentration of fatty acids.

[0053] Figure 2 The molecular formulas and chromatograms of five long-chain fatty acids (in methyl ester form) and two internal standards after extraction are shown. Among them, ①-⑦ are methyl pentadecanoate, methyl hexadecyltrienoate, methyl hexadecanoate, methyl heptadecanoate, methyl octadecyltrienoate, methyl octadecanoate, and methyl eicosanoate, respectively.

[0054] Comparative Example 1

[0055] In this comparative example, the pEAQ empty vector plasmid was transformed into Agrobacterium GV3101, and the changes in fatty acid content in tobacco leaves were determined by GC-MS. The results were compared with those of the example. The specific operating steps are as described in steps 3 and 4 of Example 1.

[0056] The experimental results show that (e.g.) Figure 3In tobacco leaves, overexpression of different AP2 transcription factors significantly altered the content of hexadecanoic acid (C16:0). Specifically, overexpression of Sl01gAP2 increased the hexadecanoic acid content to 146% of the control; while overexpression of Sl02gAP2 had no significant effect on hexadecanoic acid content. In contrast, overexpression of Sl03gAP2 and Sl06gAP2 increased the hexadecanoic acid content to 155% and 183% of the control, respectively, showing a more significant enhancement effect.

[0057] Furthermore, we analyzed the effects of overexpression of different AP2 transcription factors on the content of hexadecanoic acid (C16:3) in tobacco leaves. Compared with the empty vector control pEAQ, the content of hexadecanoic acid in tobacco leaves overexpressing Sl01gAP2 increased by 26%; overexpressing Sl02gAP2 by 23%; overexpressing Sl03gAP2 by 32%; and overexpressing Sl06gAP2 by a significant 47%.

[0058] Furthermore, it is worth noting that overexpression of Sl03gAP2 and Sl06gAP2 in tobacco leaves, compared with overexpression of Sl01gAP2 and Sl02gAP2, not only significantly increased the content of hexadecanoic acid, but also had a more significant effect on increasing the content of octadecanoic acid (C18:0), octadecadienoic acid (C18:3) and eicosanoic acid (C20:0).

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. Tomato Sl03gAP2 or Sl06gAP2 The application of genes in increasing the content of five long-chain fatty acids in plants, specifically tomatoes. Sl03gAP2 The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the tomato... Sl06gAP2 The nucleotide sequence of the gene is shown in SEQ ID NO.

4. The plant is Nicotiana benthamiana, and the five long-chain fatty acids are hexadecanoic acid (C16:0), hexadecanoic acid (C16:3), octadecanoic acid (C18:0), octadecanoic acid (C18:3), and eicosanoic acid (C20:0).

2. A method for increasing the content of five long-chain fatty acids in plants, characterized in that, Using the transient transformation method, tomato was overexpressed in plants. Sl03gAP2 or Sl06gAP2 The gene, the plant is Nicotiana benthamiana, the five long-chain fatty acids are hexadecanoic acid (C16:0), hexadecanoic acid (C16:3), octadecanoic acid (C18:0), octadecanoic acid (C18:3) and eicosanoic acid (C20:0), the tomato Sl03gAP2 The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the tomato... Sl06gAP2 The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

3. The method for increasing the content of five long-chain fatty acids in plants as described in claim 2, characterized in that, The specific steps include: expanding the tomato Sl03gAP2 or Sl06gAP2 The aforementioned gene was ligated into a plant transient expression vector, and a transient transformation method was used to transform the tomato-containing gene. Sl03gAP2 or Sl06gAP2 The transient expression vector plasmid of the gene is transferred into Agrobacterium and expressed in the plant.

4. The method for increasing the content of five long-chain fatty acids in plants as described in claim 3, characterized in that, The plant transient expression vector is pEAQ.

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