Fatty acid desaturase-modified genes and uses thereof

Abstract

The application discloses a comfrey delta(12)-fatty acid desaturase improved gene and a pistacia lentiscus fatty acid desaturase improved gene, and an expression cassette and an expression vector containing the improved genes. The comfrey delta(12)-fatty acid desaturase improved gene and the pistacia lentiscus fatty acid desaturase improved gene are respectively subjected to codon optimization and amino acid substitution, and are expressed in corn embryos by using a corn embryo specific promoter pZmESP and pZmMT, so that the content of alpha-linolenic acid in the corn embryos can be improved, and the yield of corn can be improved.

Description

Technical Field

[0001] This application relates to the fields of genetic engineering and protein engineering, and more specifically, to a modified gene for fatty acid desaturase and its applications. Background Technology

[0002] Alpha-linolenic acid (ALA, octadecanoic acid-9,12,15-trienoic acid) is a polyunsaturated fatty acid with three double bonds (C 18 H 30 Alpha-linolenic acid (ALA), also known as omega-3 fatty acid, is a fundamental component of cell membranes and biological enzymes, playing a crucial role in human health. ALA is more potent and safer than DHA and other fatty acids, and it can be converted into DHA, DPA, EPA, etc., in the body.

[0003] Fatty acid desaturase 2 (FAD2) and fatty acid desaturase 3 (FAD3) are the two most basic rate-limiting enzymes in the biosynthesis of polyunsaturated fatty acids (PUFAs). FAD2 catalyzes the formation of linoleic acid (LA) from oleic acid (OA), and FAD3 catalyzes the formation of alpha-linolenic acid (ALA) from linoleic acid (LA). Patent application CN109837290A discloses the application of the Chia ShFAD2 and ShFAD3 gene families in creating transgenic plants that produce high-yield ALA. The ultimate goal of bio-breeding is to introduce exogenous genes to acquire new traits or enhance existing traits and ensure stable inheritance. However, patent application CN109837290A suggests that using a single promoter to initiate the expression of a fusion gene during transgenic processes may lead to weakened gene expression or unstable inheritance in offspring, severely affecting the acquisition of transgenic plants and the stability of homozygous offspring. Furthermore, using the same promoter to initiate two genes separately may also cause gene silencing.

[0004] CN102277375A discloses a method for introducing an expression cassette of an endosperm-specific ω-3 fatty acid dehydrogenase gene into rice to increase the α-linolenic acid content in transgenic rice seeds. Corn germ oil, extracted from corn germ, is not only rich in unsaturated fatty acids needed by the human body but also contains various beneficial components. Other oil crops include soybeans, rapeseed, flax, and palm. Patent application CN102277375A uses rice as the substrate plant, but rice itself is not an oil crop and has low oleic and linoleic acid content, making it unsuitable as a substrate plant for producing high α-linolenic acid. CN110066812A discloses the plant linolenic acid synthase gene CsFAD2-2, which can catalyze the conversion of oleic and linoleic acids in plants into linolenic acid. The most important function of the Δ12-fatty acid dehydrogenase gene (FAD2) is to catalyze the conversion of oleic acid to linoleic acid during the biosynthesis and metabolism of plant fatty acids. The CN110066812A patent application only transformed one CsFAD2-2 gene, which has the function of catalyzing the conversion of linoleic acid to linolenic acid, but the conversion efficiency cannot reach the highest level.

[0005] Previous studies have shown that using a single promoter to initiate the expression of a fusion gene during transgenic processes may lead to weakened gene expression or unstable inheritance in offspring, severely impacting the acquisition of transgenic plants and the stability of homozygous offspring. Furthermore, using the same promoter to initiate two genes separately may also cause gene silencing.

[0006] With rapid social development and improved living standards, the market demand for vegetable oils is shifting towards high-quality and health-promoting products. Therefore, it is necessary to explore methods to increase the content of α-linolenic acid in plants. Summary of the Invention

[0007] This invention provides an improved gene for a flaxseed δ(12)-fatty acid desaturase (CsFAD2). The coding gene for CsFAD2 is derived from flaxseed, and its coding region sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. Based on codon preference in maize, CsFAD2 is codon optimized and amino acid substitutions are performed to improve its activity in maize, resulting in the improved flaxseed δ(12)-fatty acid desaturase gene, named CsFAD2-Ma. The nucleotide sequence of CsFAD2-Ma is shown in SEQ ID NO.3, or the amino acid sequence of the protein encoded by the improved flaxseed δ(12)-fatty acid desaturase gene is shown in SEQ ID NO.4.

[0008] This invention also provides a modified gene for *PvFAD3* fatty acid desaturase, the coding gene of which is derived from *PvFAD3*, and its coding region sequence is shown in SEQ ID NO. 5, and its amino acid sequence is shown in SEQ ID NO. 6. Based on codon preference in maize, codon optimization and amino acid substitution were performed on PvFAD3 to improve its activity in maize, resulting in the modified gene for *PvFAD3* fatty acid desaturase, named PvFAD3-Ma. The nucleotide sequence of PvFAD3-Ma is shown in SEQ ID NO. 7, or the amino acid sequence of the protein encoded by the modified gene for *PvFAD3* fatty acid desaturase is shown in SEQ ID NO. 8.

[0009] The present invention also provides an expression cassette containing the modified gene of flaxseed δ(12)-fatty acid desaturase (CsFAD2-Ma), wherein the promoter mediating the overexpression of the modified gene of flaxseed δ(12)-fatty acid desaturase is the maize pZmESP promoter.

[0010] The present invention also provides an expression cassette for a modified gene of fatty acid desaturase (PvFAD3-Ma) of *Pterocarya stenoptera*, wherein the promoter mediating the overexpression of the modified gene of fatty acid desaturase (PvFAD3-Ma) of *Pterocarya stenoptera* is the maize pZmMT promoter.

[0011] The embryo-specific expression promoters that mediate CsFAD2-Ma and PvFAD3-Ma overexpression are derived from maize or can be obtained through artificial synthesis.

[0012] Furthermore, the terminator for the overexpression of the modified gene for δ(12)-fatty acid desaturase of *Capsella bursa-pastoris* (CsFAD2-Ma) and the modified gene for stachys chinensis (PvFAD3-Ma) in the expression cassette is Nos. The terminator for overexpression of CsFAD2-Ma and PvFAD3-Ma can be derived from eukaryotes or prokaryotes, or can be obtained through artificial synthesis.

[0013] Furthermore, the expression cassette sequentially includes the maize pZmESP promoter, the modified gene of flaxseed δ(12)-fatty acid desaturase, the Nos terminator, the maize pZmMT promoter, the modified gene of star vine fatty acid desaturase, and the Nos terminator.

[0014] The present invention also provides an expression vector comprising the aforementioned expression cassette.

[0015] Furthermore, the aforementioned expression cassette is ligated into a T-DNA vector to obtain the expression vector.

[0016] The expression vector can be constructed by transforming a maize T-DNA vector containing the glyphosate resistance gene EPSPS, and then linking it with an overexpression cassette of the CsFAD2-Ma gene and an overexpression cassette of the PvFAD3-Ma gene, respectively. In this invention, the overexpression cassettes of the CsFAD2-Ma gene and the PvFAD3-Ma gene are constructed on the same T-DNA vector using molecular polymerization.

[0017] Alternatively, the pZmESP-CsFAD2-Ma-Nos-pZmMT-PvFAD3-Ma-Nos gene overexpression cassette can be constructed and then ligated into a T-DNA vector.

[0018] The present invention also provides the application of the aforementioned improved gene, expression cassette, or expression vector in increasing the α-linolenic acid content in maize embryo.

[0019] Furthermore, the application specifically involves overexpressing the modified gene for *Capsella bursa-pastoris* δ(12)-fatty acid desaturase and the modified gene for *Aristolochia debilis* fatty acid desaturase in maize. Specifically, the constructed recombinant expression vector can be transferred into the recipient plant genome via transgenic methods, thereby obtaining transgenic plants in the target plant that simultaneously express both the modified gene for *Capsella bursa-pastoris* δ(12)-fatty acid desaturase and the modified gene for *Aristolochia debilis* fatty acid desaturase.

[0020] The beneficial effects of this invention include:

[0021] This invention utilizes the maize embryo-specific promoters pZmESP and pZmMT to promote the expression of codon-optimized and amino acid-substituted flaxseed δ(12)-fatty acid desaturase (CsFAD2-Ma) and star vine fatty acid desaturase (PvFAD3-Ma) in maize embryos. This ensures high-level expression of the two genes and allows for the expression of multiple genes using different promoters, resulting in transgenic plants with stable offspring inheritance.

[0022] Meanwhile, this invention increases the α-linolenic acid content in maize embryos and improves maize yield by overexpressing the modified gene of *Capsella bursa-pastoris* δ(12)-fatty acid desaturase and the modified gene of *Aristolochia debilis* fatty acid desaturase in maize plants. Specifically, the CsFAD2-Ma protein catalyzes the formation of double bonds in oleic acid fatty acids to synthesize linoleic acid, and the PvFAD3-Ma protein catalyzes the formation of double bonds in linoleic acid fatty acids to synthesize α-linolenic acid. Overexpression of CsFAD2-Ma and PvFAD3-Ma fatty acid desaturases in maize embryos resulted in transgenic plants, OEFAD-Ma, where the content of the three unsaturated fatty acids increased from 1.66% to 8.98%–9.89%, and the content of linoleic acid increased from 28.36% to 49.14%–50.33%, leading to a 3.6%–5.1% increase in yield per maize plant compared to the non-transgenic control. Compared to the transgenic OEFAD plant, the linoleic acid content increased by 7.8%–11.6%, and the linolenic acid content increased by 31%–48%.

[0023] Corn itself contains over 84% unsaturated fatty acids, making it a suitable substrate for the production of alpha-linolenic acid (ALA). The transgenic corn obtained in this invention has an ALA content as high as 9.89%. Furthermore, the ratio of linoleic acid to ALA is 5:1, which better conforms to the recommended ratio in modern human nutrition. Additionally, the corn germ oil is extracted at low temperatures, which more effectively protects the ALA from oxidation. Attached Figure Description

[0024] Figure 1 A schematic diagram of the construction of the expression vector OEFAD-Ma;

[0025] Figure 2 This is a graph showing the yield analysis of OEFAD-Ma genetically modified maize. Detailed Implementation

[0026] The present invention will be further described and illustrated below with reference to embodiments. However, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the present invention and the embodiments, all other inventions and embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] Example 1: Carrier Construction

[0030] CsFAD2-Ma and PvFAD3-Ma gene overexpression cassettes were constructed. The pZmESP-CsFAD2-Ma-Nos-pZmMT-PvFAD3-Ma-Nos gene overexpression cassette was artificially synthesized, containing the pZmESP promoter, the CsFAD2-Ma coding gene, the Nos terminator, the pZmMT promoter, the PvFAD3-Ma coding gene, and the Nos terminator, respectively. To improve the activity of CsFAD2 in maize, codon optimization and amino acid substitution were performed on CsFAD2 according to the codon preference in maize, resulting in CsFAD2-Ma, whose coding sequence is shown in SEQ ID NO.3 and whose amino acid sequence is shown in SEQ ID NO.4. To improve the activity of PvFAD3 in maize, codon optimization and amino acid substitution were performed on PvFAD3 according to the codon preference in maize, resulting in PvFAD3-Ma, whose coding sequence is shown in SEQ ID NO.7 and whose amino acid sequence is shown in SEQ ID NO.8. The maize pZmESP promoter has the nucleotide sequence shown in SEQ ID NO. 9, and the maize pZmMT promoter has the nucleotide sequence shown in SEQ ID NO. 10. The terminator Nos has the nucleotide sequence shown in SEQ ID NO. 11.

[0031] The overexpression cassette pZmESP-CsFAD2-Ma-Nos-pZmMT-PvFAD3-Ma-Nos contains BamHI and KpnI sites at its 5' and 3' ends, respectively. This overexpression cassette is ligated into a T-DNA vector, and the final vector EPSPS-pZmESP-CsFAD2-Ma-Nos-pZmMT-PvFAD3-Ma-Nos is constructed using EPSPS selection markers. The vector is named OEFAD-Ma( Figure 1 ).

[0032] As a transgenic control, the gene overexpression vector EPSPS-pZmESP-CsFAD2-Nos-pZmMT-PvFAD3-Nos, named OEFAD, was constructed using the same method for CsFAD2 and PvFAD3 gene overexpression cassettes. The CsFAD2 gene is derived from *Capsella bursa-pastoris*, and its coding region sequence is shown in SEQ ID NO.1, while its amino acid sequence is shown in SEQ ID NO.2. The PvFAD3 gene is derived from *Ipomoea quamoclit*, and its coding region sequence is shown in SEQ ID NO.5, while its amino acid sequence is shown in SEQ ID NO.6.

[0033] Finally, the T-DNA plasmid was transferred into Agrobacterium LB4404 by electroporation. Positive clones were screened using YEP solid medium containing 15 μg / mL tetracycline and 50 μg / mL kanamycin, and the bacteria were preserved for subsequent plant transformation.

[0034] Example 2: Corn Conversion

[0035] 1. Preparation of corn embryos

[0036] The company's internal maize inbred line AX808 was planted in the field or in a greenhouse, and maize embryos were collected 8-10 days (summer) or 10-13 days (autumn) after artificial pollination.

[0037] 2. Preparation of Agrobacterium

[0038] (1) Take the transformed and identified Agrobacterium glycerol bacteria and streak them on YEP solid medium supplemented with 100 mg / L kan and 12 mg / L tet, and incubate in the dark at 28°C for 2-3 days;

[0039] (2) Add 1 ml of infection culture medium to a sterile 2 ml centrifuge tube, take the Agrobacterium from step 1 and put it into the infection culture medium, and mix it thoroughly with a pipette.

[0040] (3) Take another sterile 2ml centrifuge tube and adjust the bacterial concentration with infection culture medium to make the OD 660 to 0.5-0.7.

[0041] 3. Co-culture of maize immature embryos and Agrobacterium

[0042] (1) Remove the infection medium from the centrifuge tube containing the embryos and add 1.5 ml of fresh infection medium to wash the embryos once.

[0043] (2) Remove the infecting culture medium and add the prepared Agrobacterium tumefaciens solution;

[0044] (3) Vibrate at maximum speed for 30 seconds, then let stand at room temperature for 5 minutes;

[0045] (4) Pour the embryos onto the co-culture medium and blot out the liquid;

[0046] (5) Place the embryo with the flat side facing up and the shield side facing up;

[0047] (6) Place the embryo in the dark at 22℃ for 2-3 days.

[0048] 4. Callus induction and selection

[0049] (1) After co-culture, the embryos were transferred to callus induction medium and cultured in the dark at 28°C for 7-10 days.

[0050] (2) Transfer the induced callus to the selection medium for selection culture. The selection pressure is 5.0 mM glyphosate. Incubate in the dark at 28℃ for 2-3 weeks.

[0051] (3) Take the callus that survived the first screening and perform a second screening. The screening pressure is 2.0 mM glyphosate.

[0052] 5. Regeneration and culture of transformed lines

[0053] (1) Take the embryogenic callus that has grown after screening and place it on the predifferentiation medium. Incubate in the dark at 28°C for 10-14 days.

[0054] (2) Take embryogenic callus onto differentiation medium and culture at 28℃ for 10-14 days until seedlings differentiate;

[0055] (3) Transfer the well-differentiated seedlings to the rooting medium and culture them at 28°C until the roots are fully developed;

[0056] (4) Transplant the healthy seedlings into the greenhouse substrate.

[0057] After the transgenic plants flower and bear fruit, the seeds are harvested. The harvested seeds are sown in a greenhouse, and when the plants grow to the 4-6 leaf stage, expression analysis is performed using PCR technology.

[0058] Example 3: Identification of genetically modified maize

[0059] The fatty acid composition of maize embryo tissue was analyzed using gas chromatography. 300 mg of maize leaves or seeds were crushed and mixed with 2.0 mL of 2.5% (v / v) sulfuric acid-methanol solution. The mixture was heated in a 70°C water bath for 30 min. After cooling, 5 mL of 1% NaCl (w / v) was added, and the mixture was shaken and allowed to stand at room temperature for 5 min. 3 mL of n-hexane was added, mixed, and allowed to stand at room temperature for 10 min to extract the methyl esterification product. The mixture was centrifuged at 9000 rpm for 10 min at room temperature, and the uppermost layer was collected in a centrifuge tube. After filtration, the sample was loaded onto a GC column for fatty acid separation and identification. In this embodiment, an Agilent 6890N gas chromatograph equipped with an FID detector and an Agilent HP-88 capillary column (100.0 m × 0.25 mm × 0.2 μm) were used. The initial column temperature was 120 °C, held for 10 min, then increased to 230 °C at a rate of 3.2 °C / min and held for 35 min. The carrier gas was high-purity nitrogen at a flow rate of 0.70 mL / min; the hydrogen flow rate was 30 mL / min; and the air flow rate was 450 mL / min. Split injection was used with a split ratio of 20:1. The vaporization chamber temperature was 250 °C, the detector temperature was 250 °C, and the injection volume was 2 μL.

[0060] Compared with the non-transgenic control AX808 (CK), the content of three unsaturated fatty acids in the germ of the transgenic plant OEFAD-Ma maize increased from 1.66% to 8.98%–9.89% for linolenic acid (C18:3) and from 28.36% to 49.14%–50.33% for linoleic acid (C18:2), while the content of oleic acid (C18:1) decreased (Table 1). These results indicate that the CsFAD2-Ma and PvFAD3-Ma genes function as desaturase enzymes in maize germ, ultimately leading to a significant increase in the content of linolenic acid and linoleic acid in the germ.

[0061] Table 1. Determination of fatty acid content

[0062]

[0063] Note: The data in Table 1 represent the percentage of different fatty acid components in the total fatty acid composition.

[0064] Compared with OEFAD transgenic plants, the OEFAD-Ma transgenic plants, formed through codon optimization and amino acid substitution, showed varying degrees of increase in linoleic acid and linolenic acid content. Linoleic acid content increased by 7.8%–11.6% compared to OEFAD, and linolenic acid content increased by 31%–48%. These results indicate that codon optimization and amino acid substitution significantly enhanced the activities of two fatty acid desaturases.

[0065] In addition, such as Figure 2 As shown, the yield of the transgenic maize OEFAD-Ma in this invention is improved to a certain extent. Compared with the control AX808, the yield per plant of OEFAD-Ma transgenic maize is increased by 3.6% to 5.1%. Compared with transgenic maize OEFAD, the yield per plant of OEFAD-Ma transgenic maize is also improved.

Claims

1. A modified gene for flaxseed δ(12)-fatty acid desaturase, characterized in that, The nucleotide sequence of the modified gene for δ(12)-fatty acid desaturase of *Capsella flaxensis* is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the modified gene for δ(12)-fatty acid desaturase of *Capsella flaxensis* is shown in SEQ ID NO.

4.

2. A modified gene for fatty acid desaturase in *Acer palmatum*, characterized in that, The nucleotide sequence of the modified gene for fatty acid desaturase of *Stellaria media* is shown in SEQ ID NO.7, and the amino acid sequence of the protein encoded by the modified gene for fatty acid desaturase of *Stellaria media* is shown in SEQ ID NO.

8.

3. An expression box, characterized in that, The expression cassette contains the modified gene for *Capsella bursa-pastoris* δ(12)-fatty acid desaturase as described in claim 1 and the modified gene for *Aristolochia debilis* fatty acid desaturase as described in claim 2. The promoter mediating the overexpression of the modified gene for δ(12)-fatty acid desaturase in *Capsella bursa-pastoris* is the maize pZmESP promoter; the nucleotide sequence of the maize pZmESP promoter is shown in SEQ ID NO.

9. The promoter mediating the overexpression of the modified gene for fatty acid desaturase in *Aristolochia debilis* is the maize pZmMT promoter; the nucleotide sequence of the maize pZmMT promoter is shown in SEQ ID NO.

10.

4. The expression box according to claim 3, characterized in that, The terminator mediating the overexpression of the modified gene of δ(12)-fatty acid desaturase from *Capsella bursa-pastoris* and the modified gene of fatty acid desaturase from *Aristolochia debilis* is Nos.

5. The expression box according to claim 4, characterized in that, The expression cassette sequentially includes the maize pZmESP promoter, the modified gene of flaxseed δ(12)-fatty acid desaturase, the Nos terminator, the maize pZmMT promoter, the modified gene of star vine fatty acid desaturase, and the Nos terminator.

6. An expression carrier, characterized in that, The expression vector comprises the expression cassette as described in any one of claims 3-5.

7. The expression vector according to claim 6, characterized in that, The expression vector is obtained by ligating the expression cassette according to any one of claims 3-5 into a T-DNA vector.

8. The use of the improved gene of claim 1 or 2, or the expression cassette of any one of claims 3-5, or the expression vector of claim 6 or 7 in increasing the α-linolenic acid content in maize embryo.

9. The application according to claim 8, characterized in that, The modified gene of flaxseed δ(12)-fatty acid desaturase and the modified gene of star oil vine fatty acid desaturase were overexpressed in maize plants.

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