A recombinant vector, an engineered bacterium, a transgenic duckweed and an application for high expression of astaxanthin in duckweed

Abstract

The present application relates to the technical field of genetic engineering, in particular to a recombinant vector for high expression of astaxanthin in duckweed, an engineered bacterium, transgenic duckweed and application. The recombinant vector provided by the present application can improve the expression activity of astaxanthin by mutating part of the amino acid sequences in the astaxanthin synthesis pathway gene, further improve the astaxanthin yield, and use the recombinant vector constructed by the present application to construct astaxanthin duckweed, which can improve the quality and economic value of duckweed as feed, and is a new way of efficient, safe, low-cost and sustainable astaxanthin production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a recombinant vector for high expression of astaxanthin in duckweed, engineered bacteria, transgenic duckweed, and its applications. Background Technology

[0002] duckweed( Lemna minor Duckweed (Lycoperdon perlatum) is a monocotyledonous aquatic plant widely distributed in freshwater areas. Among them, *Lycoperdon perlatum* and *Lycoperdon davidiana* are currently the most extensively studied and have the widest applications. Duckweed has a simple morphological structure, with tiny individuals, mostly thallus, and no true roots. It can rapidly accumulate biomass through asexual reproduction, with an extremely short growth cycle, averaging one generation every 2 to 3 days. It can be cultivated in industrial settings without occupying arable land, thus its growth rate is much higher than that of traditional crops. Furthermore, duckweed is rich in protein and starch, and low in crude fiber, making it easily digestible and absorbable. It is an ideal source of animal feed and shows broad application prospects in replacing traditional feeds.

[0003] Astaxanthin (3,3'-dihydroxy-4,4'-diketo-β,β'-carotene) is a carotenoid derivative with extremely strong antioxidant activity, more than 10 times that of β-carotene. As a natural antioxidant and colorant, astaxanthin can scavenge intracellular free radicals and enhance cell regeneration. It has significant effects in improving immunity, anti-aging, anti-tumor activity, prevention of cardiovascular and cerebrovascular diseases, and vision improvement, and is therefore widely used in cosmetics and pharmaceuticals. Adding astaxanthin to animal feed can improve disease resistance, and adding appropriate amounts to some aquatic and poultry feeds can also enhance their commercial appearance. Currently, astaxanthin is mainly obtained through chemical synthesis and biological sources. Chemically synthesized astaxanthin is not only expensive, but its biological activity is also significantly lower than that of natural astaxanthin. Biologically derived astaxanthin mainly includes extraction from aquatic waste, microalgae extraction, and synthesis from *Pseudomonas aeruginosa* yeast, which suffers from unstable raw material supply, limited yield, and high cultivation costs. Therefore, developing a new, efficient, safe, low-cost, and sustainable astaxanthin production method is of great significance for meeting the growing market demand for aquaculture and livestock farming. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a recombinant vector for high expression of astaxanthin in duckweed, engineered bacteria, transgenic duckweed, and its applications. The recombinant vector provided by this invention, by mutating certain amino acid sequences in the astaxanthin synthesis pathway genes, can enhance their expression activity, further increasing astaxanthin yield. Constructing astaxanthin-rich duckweed using the recombinant vector constructed by this invention can improve the quality and economic value of duckweed as feed, representing a new, efficient, safe, low-cost, and sustainable approach to astaxanthin production.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a recombinant vector for high expression of astaxanthin in duckweed, comprising a plant expression vector and four mutant target genes; the four mutant target genes are CrBKT-3 gene, HpCrtZ-1 gene, PaCrtl-4 gene and ZmPSY1-2 gene; The amino acid sequence of the protein encoded by the CrBKT-3 gene is relative to the amino acid sequence of the protein encoded by the wild-type CrBKT gene, with F at position 36 replaced by G and P at position 79 replaced by V; the amino acid sequence of the protein encoded by the wild-type CrBKT gene is shown in SEQ ID NO.2. The amino acid sequence of the protein encoded by the HpCrtZ-1 gene, compared to the amino acid sequence of the protein encoded by the wild-type HpCrtZ gene, has four amino acids (MITT) inserted at the N-terminus: T at position 129 is replaced with P, A at position 137 is replaced with Q, N at position 183 is replaced with K, and H at position 228 is replaced with S. The amino acid sequence of the protein encoded by the wild-type HpCrtZ gene is shown in SEQ ID NO.4. The amino acid sequence of the protein encoded by the PaCrtl-4 gene is relative to the amino acid sequence of the protein encoded by the wild-type PaCrtl gene, with H at position 278 replaced by K and G at position 312 replaced by W; the amino acid sequence of the protein encoded by the wild-type PaCrtl gene is shown in SEQ ID NO.6. The amino acid sequence of the protein encoded by the ZmPSY1-2 gene is such that, compared to the amino acid sequence of the protein encoded by the wild-type ZmPSY1 gene, position 67 is replaced with A; the amino acid sequence of the protein encoded by the wild-type ZmPSY1 gene is shown in SEQ ID NO. 8.

[0006] Preferably, the recombinant vector includes a plant expression vector and four expression cassettes; The first expression cassette includes, in sequence, the promoter prFMV, the enhancer iZmHsp70, the signal peptide ssu1 encoding molecule, and the CrBKT-3 gene; The second expression cassette includes the promoter prZmUbi1, the enhancer iZmHsp70, the signal peptide ssu1 encoding molecule, and the HpCrtZ-1 gene, which are sequentially linked. The third expression cassette includes, in sequence, the promoter pr35s, the enhancer iZmHsp70, the signal peptide ssu1 encoding molecule, and the PaCrtl-4 gene; The fourth expression cassette includes the promoter prOsAct1 and the ZmPSY1-2 gene connected in sequence; The nucleotide sequence of the promoter prFMV is shown in SEQ ID NO.9; The nucleotide sequence of the enhancer iZmHsp70 is shown in SEQ ID NO.10; The nucleotide sequence of the signal peptide ssu1 encoding molecule is shown in SEQ ID NO.11; The nucleotide sequence of the promoter prZmUbi1 is shown in SEQ ID NO.13; The nucleotide sequence of the promoter pr35s is shown in SEQ ID NO.14; The nucleotide sequence of the promoter prOsAct1 is shown in SEQ ID NO.15; The nucleotide sequence of the promoter prFMV is shown in SEQ ID NO.9; The nucleotide sequence of the enhancer iZmHsp70 is shown in SEQ ID NO.10; The nucleotide sequence of the signal peptide ssu1 encoding molecule is shown in SEQ ID NO.11; The nucleotide sequence of the promoter prZmUbi1 is shown in SEQ ID NO.13; The nucleotide sequence of the promoter pr35s is shown in SEQ ID NO.14; The nucleotide sequence of the promoter prOsAct1 is shown in SEQ ID NO.15.

[0007] Preferably, all four expression cassettes further contain a transcription terminator tNos, the nucleotide sequence of which is shown in SEQ ID NO.12.

[0008] Preferably, the backbone vector of the recombinant vector includes the p2DBEN-CP-BZ vector.

[0009] Preferably, the nucleotide sequence of the CrBKT-3 gene is shown in SEQ ID NO.30, the nucleotide sequence of the HpCrtZ-1 gene is shown in SEQ ID NO.32, the nucleotide sequence of the PaCrtl-4 gene is shown in SEQ ID NO.34, and the nucleotide sequence of the ZmPSY1-2 gene is shown in SEQ ID NO.36.

[0010] The present invention provides an engineered bacterium, including Agrobacterium and the recombinant vector described in the above-mentioned technical solution, which is transferred into the Agrobacterium.

[0011] Preferably, the Agrobacterium includes Agrobacterium EHA105.

[0012] This invention provides a transgenic duckweed, which, relative to wild-type duckweed, is incorporating the recombinant vector described in the above-mentioned technical solution; the duckweed includes duckweed and purple duckweed.

[0013] This invention provides the application of the recombinant vector, engineered bacteria, or transgenic duckweed described in the above-mentioned technical solutions in the preparation of astaxanthin.

[0014] This invention provides the application of the recombinant vector, engineered bacteria, or transgenic duckweed described in the above-mentioned technical solutions in the preparation of products containing astaxanthin, including feed, cosmetics, or pharmaceuticals.

[0015] Beneficial effects: The recombinant vector provided by this invention contains modified astaxanthin synthesis pathway genes, such as the β-carotene 4-ketotransferase gene CrBKT-3, the β-carotene hydroxylase gene HpCrtZ-1, the phytoene dehydrogenase gene PaCrtl-4, and the phytoene synthase gene ZmPSY1-2. This recombinant vector can enhance the expression activity of astaxanthin synthesis pathway genes, further increasing astaxanthin yield. Constructing astaxanthin-rich duckweed using the recombinant vector developed in this invention can improve the quality and economic value of duckweed as feed, representing a new, efficient, safe, low-cost, and sustainable approach to astaxanthin production. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 A schematic diagram of the astaxanthin recombinant cloning vector JK7005; Figure 2 A schematic diagram of the astaxanthin recombinant cloning vector JK7005-5; Figure 3 The image shows a liquid chromatogram of astaxanthin-expressing duckweed; where A is the astaxanthin standard, B is wild-type duckweed, and C is duckweed expressing astaxanthin. Figure 4 The astaxanthin content of *Peperomia stenoptera* after integrating different astaxanthin recombinant expression vectors; Figure 5 To determine the astaxanthin content of *Lysimachia christinae* after integrating different astaxanthin recombinant expression vectors; Figure 4 and Figure 5 Among the groups with different lowercase letters, there were significant differences in astaxanthin content. P <0.05, there was no significant difference between groups with the same lowercase letter ( P >0.05). Detailed Implementation

[0018] This invention provides a recombinant vector for high expression of astaxanthin in duckweed, comprising a plant expression vector and four mutant target genes; the four mutant target genes are CrBKT-3 gene, HpCrtZ-1 gene, PaCrtl-4 gene and ZmPSY1-2 gene; The amino acid sequence of the protein encoded by the CrBKT-3 gene is relative to the amino acid sequence of the protein encoded by the wild-type CrBKT gene, with F at position 36 replaced by G and P at position 79 replaced by V; the amino acid sequence of the protein encoded by the wild-type CrBKT gene is shown in SEQ ID NO.2. The amino acid sequence of the protein encoded by the HpCrtZ-1 gene, compared to the amino acid sequence of the protein encoded by the wild-type HpCrtZ gene, has four amino acids (MITT) inserted at the N-terminus: T at position 129 is replaced with P, A at position 137 is replaced with Q, N at position 183 is replaced with K, and H at position 228 is replaced with S. The amino acid sequence of the protein encoded by the wild-type HpCrtZ gene is shown in SEQ ID NO.4. The amino acid sequence of the protein encoded by the PaCrtl-4 gene is relative to the amino acid sequence of the protein encoded by the wild-type PaCrtl gene, with H at position 278 replaced by K and G at position 312 replaced by W; the amino acid sequence of the protein encoded by the wild-type PaCrtl gene is shown in SEQ ID NO.6. The amino acid sequence of the protein encoded by the ZmPSY1-2 gene is such that, compared to the amino acid sequence of the protein encoded by the wild-type ZmPSY1 gene, position 67 is replaced with A; the amino acid sequence of the protein encoded by the wild-type ZmPSY1 gene is shown in SEQ ID NO. 8.

[0019] In one embodiment, the recombinant vector includes a plant expression vector and four expression cassettes; The first expression cassette includes, in sequence, the promoter prFMV, the enhancer iZmHsp70, the signal peptide ssu1 encoding molecule, and the CrBKT-3 gene; The second expression cassette includes the promoter prZmUbi1, the enhancer iZmHsp70, the signal peptide ssu1 encoding molecule, and the HpCrtZ-1 gene, which are sequentially linked. The third expression cassette includes, in sequence, the promoter pr35s, the enhancer iZmHsp70, the signal peptide ssu1 encoding molecule, and the PaCrtl-4 gene; The fourth expression cassette includes the promoter prOsAct1 and the ZmPSY1-2 gene connected in sequence; The nucleotide sequence of the promoter prFMV is shown in SEQ ID NO.9; The nucleotide sequence of the enhancer iZmHsp70 is shown in SEQ ID NO.10; The nucleotide sequence of the signal peptide ssu1 encoding molecule is shown in SEQ ID NO.11; The nucleotide sequence of the promoter prZmUbi1 is shown in SEQ ID NO.13; The nucleotide sequence of the promoter pr35s is shown in SEQ ID NO.14; The nucleotide sequence of the promoter prOsAct1 is shown in SEQ ID NO.15.

[0020] In one implementation, the 5' end of the promoter prFMV is connected to the 5' end of the promoter prZmUbi1, the 5' end of the promoter pr35s is connected to the 5' end of the promoter prOsAct1, and the 3' end of the transcription terminator tNos on the second expression cassette is connected to the 3' end of the transcription terminator tNos on the third expression cassette (see reference). Figure 2 ).

[0021] As one implementation scheme, the plant expression vector of the recombinant vector includes the pCambia1300 vector. The four expression cassettes of this invention are seamlessly cloned with the plant expression vector to obtain the recombinant vector. As another implementation scheme, the four expression cassettes are seamlessly cloned and ligated with the linearized pCambia1300 vector digested with Kpn I to obtain the recombinant vector.

[0022] As one embodiment, the nucleotide sequence of the CrBKT-3 gene is shown in SEQ ID NO.30, the nucleotide sequence of the HpCrtZ-1 gene is shown in SEQ ID NO.32, the nucleotide sequence of the PaCrtl-4 gene is shown in SEQ ID NO.34, and the nucleotide sequence of the ZmPSY1-2 gene is shown in SEQ ID NO.36.

[0023] As one embodiment, the amino acid sequence of the protein encoded by the CrBKT-3 gene is shown in SEQ ID NO.31, the amino acid sequence of the protein encoded by the HpCrtZ-1 gene is shown in SEQ ID NO.33, the amino acid sequence of the protein encoded by the PaCrtl-4 gene is shown in SEQ ID NO.35, and the amino acid sequence of the protein encoded by the ZmPSY1-2 gene is shown in SEQ ID NO.37.

[0024] The recombinant vector provided by this invention enables heterologous expression of multiple genes in the astaxanthin synthesis pathway. Modified phytoene synthase genes ZmPSY1-2, PaCrtI-4, CrBKT-3, and HpCrtZ-1 are introduced, respectively. The astaxanthin synthesis pathway is driven by constitutive promoters: cauliflower virus promoters prFMV and pr35s, maize ubiquitin promoter prZmUbi1, and rice actin promoter prOsAct1. Gene expression was enhanced by using the maize Hsp70 enhancer iZmHsp70 to increase the transcriptional activity of the target gene. Finally, the maize ribulose-bisphosphate carboxylase signal peptide ssu1 was linked to the target gene, guiding the target protein to be directed to the chloroplast, which greatly improved the substrate catalytic efficiency. This successfully reconstructed a highly efficient biosynthetic pathway for astaxanthin in duckweed, resulting in a significantly higher astaxanthin yield in transgenic duckweed containing this recombinant vector compared to the control group. Quantitative analysis by high-performance liquid chromatography showed that the highest astaxanthin content in duckweed was 426.0 μg / g in *Lemna minor* and 337.5 μg / g in *Lemna minor*, marking the first time that highly efficient astaxanthin synthesis has been achieved in duckweed. The astaxanthin-producing duckweed modified using this invention can improve the quality and economic value of duckweed as feed, and also provides a simple and efficient industrial production route for astaxanthin.

[0025] Based on the above advantages, the present invention provides an engineered bacterium, including Agrobacterium and the recombinant vector described in the above technical solution, which is transferred into the Agrobacterium.

[0026] As one implementation, the Agrobacterium includes Agrobacterium EHA105.

[0027] Based on the above advantages, the present invention provides a transgenic duckweed, which, relative to wild-type duckweed, is transfected with the recombinant vector described in the above technical solution; the duckweed includes duckweed and purple duckweed.

[0028] The transgenic duckweed provided by this invention integrates the phytoene dehydrogenase gene PaCrtl and the phytoene synthase gene ZmPSY1 into duckweed, which can effectively increase the accumulation of β-carotene, a precursor of astaxanthin. On this basis, the β-carotene 4-ketotransferase gene CrBKT and the β-carotene hydroxylase gene HpCrtZ are introduced to further convert β-carotene into astaxanthin, thereby transforming duckweed into a substrate plant for astaxanthin synthesis. This improves its nutritional value and quality as feed without the need for additional astaxanthin addition. At the same time, it can be rapidly industrialized based on existing research on duckweed feed applications.

[0029] Based on the aforementioned technical advantages, the high-yield astaxanthin-producing duckweed obtained by this invention exhibits broad application value and industrialization prospects. Because four tandem expression cassettes are integrated into a single vector and transferred into the duckweed genome, stable inheritance of traits in offspring is ensured, effectively overcoming the core pain points of long transformation cycles and low biomass in higher plants, thus meeting the core needs of large-scale industrial production. Through the technical modifications of this invention, duckweed can directly synthesize high-value-added astaxanthin. After harvesting, astaxanthin-rich duckweed can be directly used as a functional feed additive for aquaculture and poultry farming, utilizing the powerful antioxidant capacity of astaxanthin to enhance animal immunity, improve growth performance, and enhance product quality. Furthermore, because duckweed is easy to harvest, it can be used directly after harvesting or as a high-quality raw material for extracting natural astaxanthin, providing a successful example of using higher plants to produce high-value terpenoid compounds.

[0030] Based on the above advantages, the present invention provides the application of the recombinant vector, engineered bacteria, or transgenic duckweed described in the above technical solution in the preparation of astaxanthin.

[0031] Based on the above advantages, the present invention provides the application of the recombinant vector, engineered bacteria, or transgenic duckweed described in the above technical solution in the preparation of products containing astaxanthin, the products including feed, cosmetics, or pharmaceuticals.

[0032] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes a recombinant vector for high expression of astaxanthin in duckweed, engineered bacteria, transgenic duckweed, and applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1 1. Construction of wild-type recombinant vectors: The astaxanthin synthesis pathway genes include the maize-codon-optimized Chlamydomonas reinhardtii β-carotene 4-ketotransferase gene CrBKT (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2), the maize-codon-optimized Haematococcus pluvialis β-carotene hydroxylase gene HpCrtZ (nucleotide sequence as shown in SEQ ID NO.3, amino acid sequence as shown in SEQ ID NO.4), the maize-codon-optimized Erwinia phytoene dehydrogenase gene PaCrtl (nucleotide sequence as shown in SEQ ID NO.5, amino acid sequence as shown in SEQ ID NO.6), and the maize genome-optimized phytoene synthase gene ZmPSY1 (nucleotide sequence as shown in SEQ ID NO.7, amino acid sequence as shown in SEQ ID NO.8).

[0034] The recombinant expression vector JK7005, containing four tandem gene expression cassettes, was constructed using standard seamless cloning techniques. Figure 1 As shown, KanR represents the kanamycin resistance gene; RB represents the left boundary; and LB represents the right boundary.

[0035] The cauliflower virus promoter prFMV (SEQ ID NO. 9) is linked to the Hsp70 enhancer iZmHsp70 (SEQ ID NO. 10), which drives the expression of the β-carotene 4-ketotransferase gene CrBKT, which is linked to the signal peptide ssu1 (SEQ ID NO. 11) of the maize genome ribulose-bisphosphate carboxylase small chain. Finally, it is linked to the transcription terminator tNos (SEQ ID NO. 12) of annattoline synthase, forming the first expression cassette.

[0036] The maize ubiquitin promoter prZmUbi1 (SEQ ID NO.13) is linked to the Hsp70 enhancer iZmHsp70, which drives the expression of the β-carotene 4-ketotransferase gene HpCrtZ, which is linked to the signal peptide ssu1 of the maize genome ribulose-bisphosphate carboxylase chain. Finally, it is linked to the transcription terminator tNos of carmine synthase, forming a second expression cassette.

[0037] The cauliflower mosaic virus promoter pr35s (SEQ ID NO.14) is linked to the Hsp70 enhancer iZmHsp70, which drives the expression of the phytoene dehydrogenase gene PaCrtl, which is linked to the signal peptide ssu1 of the maize genome ribulose-bisphosphate carboxylase small chain. Finally, it is linked to the transcription terminator tNos of annattoline synthase, forming a third expression cassette.

[0038] The rice promoter prOsAct1 (SEQ ID NO.15) drives the expression of the phytoene synthase gene ZmPSY1, which is then linked to the transcription terminator tNos of cadherin synthase to form a fourth expression cassette.

[0039] The construction steps of the recombinant expression vector JK7005 are as follows: The above four astaxanthin synthesis pathway gene expression cassette sequences were synthesized by Nanjing Genscript Biotech Co., Ltd. The backbone vector pCambia1300 (Shanghai Weidi Biotechnology) was digested with Kpn I enzyme, the linearized plasmid was recovered by gel, and the four astaxanthin synthesis pathway gene expression cassettes and hygromycin selection marker expression cassettes were integrated into the linearized plasmid according to the seamless cloning kit operation steps to obtain the recombinant vector JK7005.

[0040] 2. Construction of the mutant gene recombination vector: Recombinant expression vectors JK7005-CrBKT-1, JK7005-CrBKT-2, and JK7005-CrBKT-3 were constructed using the above-mentioned method for constructing wild-type recombinant vectors. These vectors contain the modified sequences CrBKT-1, CrBKT-2, and CrBKT-3 of the wild-type CrBKT gene, respectively. The amino acid mutations involved in the modification, their locations, and their sequences are shown in Table 1. The remaining sequences are consistent with those of the JK7005 vector.

[0041] Recombinant expression vectors JK7005-HpCrtZ-1 and JK7005HpCrtZ-2 were constructed using the above-mentioned method for constructing wild-type recombinant vectors. They contain the modified sequences HpCrtZ-1 and HpCrtZ-2 of the wild-type HpCrtZ gene, respectively. The amino acid mutations involved in the modification, the mutation locations and mutation sequences are shown in Table 1. The remaining sequences are consistent with those of the JK7005 vector.

[0042] Recombinant expression vectors JK7005-PaCrtl-1, JK7005-PaCrtl-2, JK7005-PaCrtl-3, and JK7005-PaCrtl-4 were constructed using the above-mentioned method for constructing wild-type recombinant vectors. These vectors contain the modified sequences of the wild-type PaCrtl gene, namely PaCrtl-1, PaCrtl-2, PaCrtl-3, and PaCrtl-4, respectively. The amino acid mutations involved in the modification, their locations, and their sequences are shown in Table 1. The remaining sequences are consistent with those of the JK7005 vector.

[0043] Recombinant expression vectors JK7005-ZmPSY1-1 and JK7005-ZmPSY1-2 were constructed using the above-mentioned method for constructing wild-type recombinant vectors. They contain the modified sequences ZmPSY1-1 and ZmPSY1-2 of the wild-type ZmPSY1 gene, respectively. The amino acid mutations involved in the modification, the mutation locations and mutation sequences are shown in Table 1. The remaining sequences are consistent with those of the JK7005 vector.

[0044] The recombinant expression vector JK7005-5 was constructed using the method described above for constructing wild-type recombinant vectors (e.g., Figure 2 As shown in Table 1, CrBKT-3, HpCrtZ-1, PaCrtl-4, and ZmPSY1-2 are the modified sequences of the wild-type CrBKT, HpCrtZ, PaCrtl, and ZmPSY1 genes, respectively. The amino acid mutations involved in the modification, the mutation locations, and the mutation sequences are shown in Table 1. The remaining sequences are consistent with the JK7005 vector.

[0045] Table 1. Related genes and sequence mutations.

[0046] 3. E. coli transformation: The recombinant vectors were transformed into *E. coli* Trans1-T1 competent cells using a heat shock method. The transformation process was as follows: 50 μl of *E. coli* competent cells and 10 μl of recombinant vector were mixed and incubated at 42 °C for 30 s, then at 37 °C for 45 min, followed by shaking at 200 rpm for 1 h. The mixture was then plated on LB agar plates containing the corresponding antibiotics and incubated overnight. White colonies were picked and cultured overnight in LB liquid medium containing the corresponding antibiotics at 37 °C on a shaker. The plasmid was extracted using the alkaline method, with the following steps: Centrifuge the bacterial culture at 12000 rpm for 1 min, discard the supernatant, and resuspend the precipitated bacterial cells in 100 μl of pre-chilled solution I; add 150 μl of freshly prepared solution II, invert the centrifuge tube 4 times to mix, and place on ice for 3-5 min; add 150 μl of ice-cold solution III, mix thoroughly immediately, and place on ice for 5-10 min; centrifuge at 4 ℃ and 12000 rpm for 5 min, add 2 volumes of anhydrous ethanol to the supernatant, mix well, and place at room temperature for 5 min; centrifuge at 4 ℃ and 12000 rpm for 5 min, discard the supernatant, wash the precipitate with 70% ethanol and air dry; dissolve the precipitate in 30 μl of TE containing 20 μg / ml ribonuclease; and digest the RNA by incubating in a water bath at 37 ℃ for 30 min. The extracted plasmids were identified by enzyme digestion and verified by sequencing, and then stored in a -20 ℃ freezer for later use.

[0047] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, pH adjusted to 7.5 with sodium hydroxide.

[0048] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH adjusted to 7.5 with NaOH.

[0049] Solution I: 25 mM Tris-HCl, 10 mM EDTA, 50 mM glucose, pH adjusted to 8.0.

[0050] Solution II: 0.2 M NaOH, 1% sodium dodecyl sulfate (SDS).

[0051] Solution III: 4 M potassium acetate, 2 M acetic acid.

[0052] TE: 10 mM Tris-HCl, 1 mM EDTA, pH adjusted to 8.0.

[0053] Example 2: Agrobacterium-mediated genetic transformation and molecular detection of duckweed (I) Genetic transformation and molecular detection of *Peperomia stenoptera* 1. Callus induction Prepare the induction medium: MS 4.43 g / L, 2,4-D 1.0 mg / L, TDZ 0.1 mg / L, sucrose 30 g / L, agar 7.9 g / L, adjust the pH to 5.8 and sterilize at 121℃ for 30 min; Take leaves of *Peperomia stenoptera* and place them on an induction medium. Incubate at 25°C in the dark, and change the induction medium regularly until callus tissue grows.

[0054] 2. Preparation of Agrobacterium Agrobacterium EHA105 competent cells were thawed on ice, and the recombinant expression plasmid from Example 1 was added. The cells were then incubated on ice for 5 min, followed by sequential treatments of liquid nitrogen flash freezing for 5 min, water bath at 37°C for 5 min, and ice bath for 5 min. 600 μl of LB liquid medium was added, and the cells were incubated at 28°C with shaking for 2 h. The cells were then plated onto LB solid medium with the corresponding antibiotic and incubated upside down at 28°C. Single-clone transformants were picked for PCR detection and sequencing. Successfully sequenced Agrobacterium transformants were inoculated into 100 mL of LB liquid medium with the corresponding antibiotic and incubated with shaking until OD (out of 100°C). 600 When the absorbance reaches 0.3-0.4, add 100 μL of acetylsuccinone (AS) and continue shaking and incubating until the OD value reaches 0.5. 600 When the absorbance reaches 0.6-0.8, take an appropriate amount of bacterial culture, centrifuge, collect the bacterial cells, resuspend them in 30 mL of conversion buffer, and add 30 μL of AS for later use.

[0055] Conversion solution: MS 4.43 g / L, 2,4-D 1.0 mg / L, thiabendazole (TDZ) 0.1 mg / L, sucrose 30 g / L, pH adjusted to 5.8 and then sterilized at 121℃ for 30 min.

[0056] 3. Genetic transformation of *Peperomia stenoptera* Place the duckweed callus tissue into the suspended bacterial solution, and sequentially vacuum for 10 min, sonicate for 5 min, and vacuum for 10 min. Then transfer the callus tissue to a petri dish lined with three layers of sterile filter paper and dry it appropriately in a clean bench with the lights off. Place the callus tissue in a petri dish lined with two layers of sterile filter paper and treat it in the dark for 3 days. After three days, transfer the callus tissue to differentiation medium and continue to treat it in the dark for 2-3 weeks before transferring it to light. Change the differentiation medium every 2-3 weeks according to the growth status of the callus tissue. Finally, transfer the regenerated leaves differentiated from the callus tissue to SH solid medium and culture them until they are in a suitable condition before taking samples for identification.

[0057] Differentiation medium: B5 medium salt 3.21 g / L, kinetin (KT) 1 mg / L, sucrose 10 g / L, adjust pH to 5.8, add agar 7.9 g / L, termethin (TMT) 400 mg / L, hygromycin B 10 mg / L, indoleacetic acid (IAA) 4.5 mg / L; SH solid medium: SH base salts 3.2 g / L, sucrose 10 g / L, agar 8 g / L, pH adjusted to 5.8, sterilized at 121℃ for 15 min.

[0058] 4. Molecular detection of genetically modified Aquatic Plants Transgenic *Peperomia stenoptera* PCR identification: Molecular detection was performed on the offspring of all T0 plants that were stably inherited. The steps were as follows: approximately 100 mg of transgenic and wild-type *Peperomia stenoptera* samples were placed in liquid nitrogen for quick freezing, ground and broken up using a tissue homogenizer, and genomic DNA was extracted using Qiagen's DNeasy Plant Maxi Kit. The extracted genomic DNA was used as a template for PCR amplification and sequencing to screen for positive lines.

[0059] Real-time quantitative PCR identification: Genomic DNA was extracted using Qiagen's DNeasy Plant Maxi Kit. The copy numbers of CrBKT, HpCrtZ, PaCrtI, and ZmPSY1 genes were detected by Taqman probe quantitative PCR. Wild-type Aquatic Plants served as a control. The fluorescent group (FAM) and quencher group (BHQ-1) of the Taqman probe were modified at the 5′ and 3′ ends, respectively. Each gene was repeated three times, and the average value was taken.

[0060] The primer and probe sequences for real-time PCR are as follows: The following primers and probes are used to detect the CrBKT gene sequence: Primer 1: TGGAGGACGGAGTAATCAAACC (SEQ ID NO.16); Primer 2: GAGGAATAGGAGGCTTAACGACTTC (SEQ ID NO. 17); Probe 1: TACAGGCTAATGGCAAGCTGCCCCA (SEQ ID NO.18); The following primers and probes are used to detect the HpCrtZ gene sequence: Primer 3: GCGGAGGAAAAGAGAACAACTG (SEQ ID NO.19); Primer 4: GCCGGACACGCCGATA (SEQ ID NO.20); Probe 2: CAAGCCGCCGCCATTGCC (SEQ ID NO.21); The following primers and probes are used to detect the PaCrtI gene sequence: Primer 5: TGATCATGCCTTGCACAAGAG (SEQ ID NO.22); Primer 6: TCTACACCCTCATCCACGCTTT (SEQ ID NO.23); Probe 3: CTTGGAAACCAAACTCCCCACTCCCTTT (SEQ ID NO. 24); The following primers and probes are used to detect the ZmPSY1 gene sequence: Primer 7: CTCGTCCGAGCAGAAGGTCTA (SEQ ID NO.25); Primer 8: TGCGCAGCTGGCGTTT (SEQ ID NO.26); Probe 4: TCGTGCTCAAGCAGGCCGCATT (SEQ ID NO.27); Data were analyzed using SDS2.3 (Applied Biosystems) software to obtain single-copy transgenic duckweed transformants.

[0061] (II) Genetic transformation and molecular detection of *Dysmena purpurea* Genetic transformation and molecular detection were performed using leaves of *Lycoris radiata* as experimental material. The genetic transformation and molecular detection methods were the same as those used for *Lycoris radiata*, resulting in *Lycoris radiata* plants transgenic with the astaxanthin gene.

[0062] It should be noted that the duckweed and water lily in this invention are two duckweed species, not specific strains. In this field, astaxanthin content and other plant physiological characteristics can be maintained between different strains by simply transforming them with Agrobacterium tumefaciens. That is, the transgenic duckweed constructed in this invention is a plant intermediate material rather than a plant variety.

[0063] Example 3: High Performance Liquid Chromatography Detection of Astaxanthin in Duckweed 1. Duckweed astaxanthin extraction: After freeze-drying and pulverizing the astaxanthin-containing duckweed, weigh 100 mg of duckweed powder, add 10 mL of dichloromethane-methanol solution and shake to extract for 5 min, then sonicate for 5 min. Centrifuge to collect the supernatant residue and repeat the extraction until the residue is white. Combine the supernatants and dilute to 50 mL with dichloromethane-methanol. Let stand for 15 min before use.

[0064] 2. Saponification: Accurately transfer 5 mL of the above extract into 0.7 mL of sodium hydroxide-methanol solution, vortex to mix, seal under nitrogen, and let stand in a refrigerator at 5 °C for 12–14 h. Then, add 0.4 mL of 2% phosphoric acid-methanol solution to the reaction solution to neutralize the remaining alkali, and bring the volume to 5 mL under nitrogen purging. After filtering through a 0.45 μm filter membrane, the sample is ready for detection.

[0065] 3. High-performance liquid chromatography (HPLC) for the detection of astaxanthin: Chromatographic conditions: HPLC analysis was performed using a Shimadzu LC-20AD liquid chromatograph. A C30 column (250 mm × 4.6 mm, 5 μm) was used. The injection volume was 20 μL, the flow rate was 1 ml / min, and the column temperature was 25℃. The characteristic absorption peak of cordycepin was detected at 474 nm. Gradient elution was used: phase A was methanol, phase B was tert-butyl methyl ether, and phase C was 1% phosphoric acid solution. The elution program is shown in Table 2 below. Each sample was repeated three times.

[0066] Table 2 Elution Procedure for High Performance Liquid Chromatography Mobile Phase

[0067] Preparation of standards: Astaxanthin standards were diluted with acetone to prepare working solutions with concentrations of 0.1 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 5.0 μg / mL, and 10.0 μg / mL, respectively, before being analyzed by liquid chromatography and a standard curve was plotted. The solutions were prepared and used immediately. Yeast fermentation broth was diluted 10-fold with 20% methanol, filtered through a 0.22 μm filter membrane, and then analyzed by liquid chromatography.

[0068] Astaxanthin determination results in duckweed liquid chromatography are as follows: Figure 3 As shown, the astaxanthin content in duckweed with different astaxanthin recombinant expression vectors was analyzed using SAS software, and the significance of differences between means was determined by one-way ANOVA.

[0069] The results showed that transgenic duckweed with high astaxanthin expression was obtained by transforming recombinant expression vectors carrying astaxanthin synthesis pathway genes into *Lemna minor* and *Lemna minor*. Statistical analysis showed that although optimization of individual genes in the astaxanthin synthesis pathway did not result in a significant difference in astaxanthin content compared to duckweed lines integrating the JK7005 vector (…). P >0.05), but after optimizing the genes of four astaxanthin synthesis pathways simultaneously, the astaxanthin content in duckweed can be significantly increased ( P <0.05%, with the highest astaxanthin content in freeze-dried Aquatica reaching 426.0 μg / g ( Figure 4 As shown), the astaxanthin content in freeze-dried aquatica can reach up to 337.5 μg / g (as shown). Figure 5 (As shown).

[0070] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A recombinant vector for high expression of astaxanthin in duckweed, characterized in that, It includes a plant expression vector and four mutant target genes; the four mutant target genes are CrBKT-3, HpCrtZ-1, PaCrtl-4, and ZmPSY1-2 genes; relative to the amino acid sequence of the protein encoded by the wild-type target gene, the amino acid sequence of the protein encoded by the mutant target gene contains the following mutations: CrBKT-3 gene: F at position 36 is replaced with G, and P at position 79 is replaced with V; the amino acid sequence of the protein encoded by the wild-type CrBKT gene is shown in SEQ ID NO.2; HpCrtZ-1 gene: MITT, a 4-amino acid insertion at the first position of the N-terminus, T at position 129 is replaced with P, A at position 137 is replaced with Q, N at position 183 is replaced with K, and H at position 228 is replaced with S; The amino acid sequence of the protein encoded by the wild-type HpCrtZ gene is shown in SEQ ID NO.4; PaCrtl-4 gene: H at position 278 is replaced with K, and G at position 312 is replaced with W; the amino acid sequence of the protein encoded by the wild-type PaCrtl gene is shown in SEQ ID NO.6; ZmPSY1-2 gene: V at position 67 is replaced with A; the amino acid sequence of the protein encoded by the wild-type ZmPSY1 gene is shown in SEQ ID NO.

8.

2. The recombinant vector according to claim 1, characterized in that, The recombinant vector includes a plant expression vector and four expression cassettes; The first expression cassette includes, in sequence, the promoter prFMV, the enhancer iZmHsp70, the signal peptide ssu1 encoding molecule, and the CrBKT-3 gene; The second expression cassette includes the promoter prZmUbi1, the enhancer iZmHsp70, the signal peptide ssu1 encoding molecule, and the HpCrtZ-1 gene, which are sequentially linked. The third expression cassette includes, in sequence, the promoter pr35s, the enhancer iZmHsp70, the signal peptide ssu1 encoding molecule, and the PaCrtl-4 gene; The fourth expression cassette includes the promoter prOsAct1 and the ZmPSY1-2 gene connected in sequence; The nucleotide sequence of the promoter prFMV is shown in SEQ ID NO.9; The nucleotide sequence of the enhancer iZmHsp70 is shown in SEQ ID NO.10; The nucleotide sequence of the signal peptide ssu1 encoding molecule is shown in SEQ ID NO.11; The nucleotide sequence of the promoter prZmUbi1 is shown in SEQ ID NO.13; The nucleotide sequence of the promoter pr35s is shown in SEQ ID NO.14; The nucleotide sequence of the promoter prOsAct1 is shown in SEQ ID NO.

15.

3. The recombinant vector according to claim 2, characterized in that, All four expression cassettes also contain a transcription terminator tNos, the nucleotide sequence of which is shown in SEQ ID NO.

12.

4. The recombinant vector according to claim 1, characterized in that, The nucleotide sequence of the CrBKT-3 gene is shown in SEQ ID NO.30, the nucleotide sequence of the HpCrtZ-1 gene is shown in SEQ ID NO.32, the nucleotide sequence of the PaCrtl-4 gene is shown in SEQ ID NO.34, and the nucleotide sequence of the ZmPSY1-2 gene is shown in SEQ ID NO.

36.

5. An engineered bacterium, characterized in that, Includes Agrobacterium and the recombinant vector of any one of claims 1-4 inserted into said Agrobacterium.

6. The engineered bacteria according to claim 5, characterized in that, The Agrobacterium species mentioned include Agrobacterium EHA105.

7. A genetically modified duckweed, characterized in that, Compared to wild-type duckweed, the transgenic duckweed is infused with the recombinant vector as described in any one of claims 1-4; the duckweed includes duckweed and purple duckweed.

8. The use of the recombinant vector according to any one of claims 1-4, the engineered bacteria according to claim 5 or 6, or the transgenic duckweed according to claim 7 in the preparation of astaxanthin.

9. The use of the recombinant vector according to any one of claims 1-4, the engineered bacteria according to claim 5 or 6, or the transgenic duckweed according to claim 7 in the preparation of products containing astaxanthin, said products including feed, cosmetics, or pharmaceuticals.

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