CN118421588A

CN118421588A - Phytoene synthase NyPSY protein, encoding gene and application thereof

Info

Publication number: CN118421588A
Application number: CN202410657511.4A
Authority: CN
Inventors: 杨立恩; 李琤泠; 普加秋; 周伟; 胡传明; 邓银银
Original assignee: JIANGSU MARINE FISHERIES RESEARCH INSTITUTE
Current assignee: JIANGSU MARINE FISHERIES RESEARCH INSTITUTE
Priority date: 2024-05-25
Filing date: 2024-05-25
Publication date: 2024-08-02

Abstract

The invention discloses a phytoene synthase NyPSY protein, a coding gene and application thereof. The invention discovers phytoene synthase in the porphyra species involved in carotenoid anabolic pathway for the first time, and confirms that the enzyme is involved in carotenoid metabolism in the porphyra. The invention obtains the gene sequence for coding the enzyme, and provides a basis for improving the carotenoid metabolic pathway in the laver by utilizing the genetic engineering technology so as to improve the carotenoid content in the laver. By means of NyPSY protein and its coding gene, the present invention makes gene engineering improvement to Laver genus, and increases the content of carotenoid in Laver to raise the nutritive value of Laver and thus the economic value of Laver.

Description

Phytoene synthase NyPSY protein, encoding gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, relates to phytoene synthase NyPSY protein, and a coding gene and application thereof, and in particular relates to porphyra yezoensis phytoene synthase NyPSY protein and application of the coding gene thereof in metabolism of carotenoid in porphyra.

Background

Carotenoids are an important class of compounds with biological activity that are critical to human nutrition and health. Research has shown that beta-carotene is a precursor of vitamin a, which can be converted to retinol, and has an important role in human vision. The research also finds that the strong antioxidant effect of the carotenoid and the derivatives thereof, such as beta-carotene, lutein, fucoxanthin, astaxanthin and the like, has a plurality of benefits on the aspects of cardiovascular health, anti-inflammatory treatment, anticancer, tumor marker diagnosis, neuroprotection, diabetes treatment and the like of human beings.

Carotenoids are widely found as tetraterpenoids in bacteria, fungi, algae, and land plants, and other light and organisms, and can be used as auxiliary light harvesting pigments to absorb light energy and transfer this energy to chlorophyll for photosynthesis by singlet-singlet excitation. In case of excessive absorption of light energy, the photosynthetic system can be protected from oxidative damage caused by Reactive Oxygen Species (ROS) by lutein cycle (xanthophyll cycle). Meanwhile, the carotenoid can stabilize the structure of the reaction center of the optical system. In addition, carotenoids are precursors to certain plant hormones (such as strigolactone and abscisic acid), and are key regulatory molecules in plant architecture and in the process of growth and development. Recent studies have shown that carotenoids and their derivatives can also themselves act as signalling molecules in response to plant development and environmental signals.

The biosynthesis of carotenoids starts from geranylgeranyl diphosphate (GGPP), and Phytoene Synthase (PSY) catalyzes the condensation of two GGPP to colorless phytoene, a major rate-limiting step common to all organisms in the carotenoid metabolic pathway. Overexpression of the PSY gene results in significant downstream accumulation of phytoene, lycopene, beta-carotene and beta-cryptoxanthin, while mutation or knockout of the PSY gene significantly delays the accumulation of lycopene and beta-carotene even without accumulation. Because PSY plays a key role in controlling the flux of synthetic carotenoids, it is also subject to complex regulation by a variety of mechanisms and factors. The enzymatic activity such as PSY has been shown to require Mn ²⁺ as a cofactor for catalysis. Studies in Arabidopsis have shown that the orange protein OR protein is the primary posttranscriptional regulator of PSY, and can interact directly with PSY in plastids. The over-expression of OR gene can raise the enzyme activity of PSY and raise the accumulation of carotenoid obviously. It has also been reported that the STAY-GREEN protein (SlSGR 1) in tomato can down regulate SlPSY1 activity by inhibiting SlPSY1 transcription.

PSY is a family of small genes, the first gene encoding PSY of the terrestrial plant was isolated from tomato 38. The gene encoding PSY has been isolated and identified in many species such as Arabidopsis, rice, wheat, etc. There is only one PSY gene in most algae and bacteria, but there are exceptions, such as two PSY's found in green alga Ostreococcus, micromonas and Dunaliella SALINA CCAP. PSY1 of Dunaliella SALINA CCAP has high catalytic activity, while PSY2 has little activity. OR promotes the accumulation of carotenoids, particularly beta-carotene, by interacting with PSY1/2 and modulating plastid development. In most photosynthetic plants PSY is multicopy and functional differentiation occurs. The advent of these subfunctions enabled carotenoids to accumulate in non-photosynthetic tissues and respond to environmental stresses, providing different regulatory mechanisms for carotenoid production. PSY differentiates up to three copies of genes with different functions during evolution. For example 3 specifically expressed PSY found in tomato. Wherein PSY1 mediates biosynthesis of fruit-specific carotenoids, PSY2 mediates biosynthesis of vane-specific carotenoids, and PSY3 expression is limited to roots under stress conditions.

Analysis of carotenoid metabolic pathway genes shows that early evolution of carotenoids originates from prokaryotes, evolved from a common ancestor of archaebacteria and bacteria, then co-evolved with photosynthesis, and then transferred from cyanobacteria to other algae and plant plastids through long-term endosymgenesis. There is evidence that the gene of the C40 pathway remains intact in archaea and bacteria, indicating that a common ancestor exists. Studies of the PSY structure also corroborate this insight, and find that the structure of the active site region is identical to that of the ancestral PSY. The subfunctionalization of PSY is therefore based on the results of common ancestral gene replication and gene loss. Arabidopsis experiences four gene duplication events, but only one PSY gene. The believable explanation is that the 5' -UTR of arabidopsis PSY has alternative splicing to allow the coding of multiple PSY isozymes to adapt to changing environments.

Porphyra yezoensis (Neopyropia yezoensis) belongs to the original red algae (Rhodophyta), is one of the important cultivated algae in the world, and is mainly produced in China, japan and Korea, and the total yield value of the purple laver in three countries in 2022 is about 21.9 hundred million dollars. The annual output value of Porphyra yezoensis in China exceeds 100 hundred million yuan. The thallus Porphyrae is used as edible seaweed, and has high nutritive value. However, the research on the metabolism of the carotenoid of the original red seaweed laver has been slow. Genes related to carotenoid metabolism such as GGPS, LCYs and CHYs have only been cloned and identified from Porphyra umbilicifolia, porphyra yezoensis and Porphyra tenera in the last decade. However, for several PSY encoding genes in the original red seaweed Porphyra yezoensis, the possible control mechanism and the evolution process are unknown. The discovery and the functional identification of NyPSY in the laver species represented by Porphyra yezoensis are beneficial to improving the nutritional value of the laver by improving the content of carotenoid and other nutritional ingredients in the laver in the future by using a genetic engineering technology, and improving the taste and the flavor of the laver by improving the content of terpenoid substances, thereby improving the economic value of the laver as a whole.

Disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing a phytoene synthase NyPSY protein, and a coding gene and application thereof, wherein the coding gene codes the phytoene synthase, so that carotenoid can be synthesized and accumulated in laver.

The technical scheme is as follows: in order to solve the above technical problems, the present invention provides a phytoene synthase NyPSY protein, the phytoene synthase NyPSY protein comprising:

(a) The amino acid sequence is shown as SEQ ID NO. 2; or (b)

(B) The amino acid sequence in (a) is an amino acid sequence which is substituted and/or deleted and/or added with one or more amino acids and has the same activity as the amino acid residue described in SEQ ID NO. 2.

Wherein the phytoene synthase NyPSY protein is derived from Porphyra yezoensis (Neopyropia yezoensis).

The invention also provides a nucleic acid molecule which codes for the phytoene synthase NyPSY protein.

Wherein said nucleic acid molecule comprises:

(i) Which encodes the protein of claim 1; or (b)

(Ii) A nucleotide sequence which hybridizes under stringent conditions to a nucleotide sequence of a nucleic acid or gene as defined in (i) and which encodes a protein having phytoene synthase activity; or has more than 80% homology with the nucleotide sequence of a sequence table SEQ ID NO. 1; or (b)

(Iii) The nucleotide sequence is shown as SEQ ID NO. 1.

The invention also provides an expression cassette, a recombinant vector, a recombinant cell or a recombinant bacterium comprising said nucleic acid molecule.

The NyPSY gene of the present invention may be used as a target gene to construct a plant expression vector, and the cauliflower mosaic virus CAMV35S promoter, the ethanol-inducible promoter, etc., and may include enhancers as necessary.

The invention also provides application of the phytoene synthase NyPSY protein, the nucleic acid molecule, the expression cassette, the recombinant vector, the recombinant cell or the recombinant bacterium in improving carotenoid in plants.

Wherein the plants include, but are not limited to, laver, other similar plants such as rice, etc. are also suitable for the present invention.

The invention also provides a method for increasing the carotenoid content in plants, which comprises the following steps:

1) Allowing the plant to contain said nucleic acid molecule; or (b)

2) Allowing the plant to express the phytoene synthase NyPSY protein.

Wherein the method comprises the steps of transgene, crossing, backcrossing or asexual propagation.

To simplify the identification of transformed plants, selectable markers (e.g., antibiotics enzymes) may be used. As the expression vector, ti plasmid, ri plasmid, plant virus vector and the like can be used. Transformation methods Agrobacterium-mediated or other methods may be used to transform plants.

The beneficial effects are that: compared with the prior art, the invention discovers an enzyme in the porphyra species which participates in the anabolism pathway of carotenoid, namely phytoene synthase for the first time, and confirms that the enzyme participates in the metabolism of carotenoid in the porphyra. The invention obtains the gene sequence for coding the enzyme, and provides a basis for improving the carotenoid metabolic pathway in the laver by utilizing the genetic engineering technology so as to improve the carotenoid content in the laver. Through NyPSY genes and proteins provided by the invention, genetic engineering improvement is carried out on the porphyra species, and the nutritional value of the porphyra is improved by improving the carotenoid content in the porphyra, so that the economic value of the porphyra is improved.

Drawings

FIG. 1 is a nucleotide and deduced amino acid sequence of NyPSY; wherein "×" represents a stop codon.

FIG. 2 is a multiple sequence alignment of phytoene synthase protein sequences from different species showing conserved domains of PSY from different species.

FIG. 3 is a diagram of HPLC analysis of NyPSY experiments in E.coli; wherein A, B and C: positive control accumulated beta-carotene E.coli photographs, chromatograms and beta-carotene absorption spectrograms; D. e and F: the sample accumulates a beta-carotene escherichia coli photograph, a chromatogram and a beta-carotene absorption spectrum; g and H: pMAL-C5X and pAC-85b were co-transferred into E.coli as negative controls.

FIG. 4 is a phylogenetic analysis of phytoene synthase in different species.

Detailed Description

The present invention is not limited to the above examples, and recombinant expression vectors, transgenic cell lines and host bacteria containing the genes of the present invention are all within the scope of the present invention.

Gene acquisition of example 1NyPSY

1. Porphyra yezoensis NyPSY transcript identification

The Open Reading Frame (ORF) of the PSY gene encoding Arabidopsis thaliana was used to search for homologous sequences in the Porphyra yezoensis genome by tblastx, and the possible homologous sequences were screened by intercepting the e-value of 1X 10 ^-10 to find only one transcript. The ORF of RACE primer amplification NyPSY (shown in FIG. 1) was designed based on the transcript gene sequence.

2. Extraction of Total RNA

Porphyra yezoensis (Neopyropia yezoensis) (from national Porphyra yezoensis germplasm library of ocean aquatic institute of Jiangsu province) is selected, total RNA of Porphyra yezoensis (Yang et al 2013) is extracted by adopting the published technology of the team, and after the integrity, purity and concentration of RNA are confirmed by electrophoresis detection and ultraviolet spectrophotometer detection, the Porphyra yezoensis is preserved at-80 ℃.

3. Cloning of Porphyra yezoensis phytoene synthase Gene (NyPSY)

Using 1. Mu.g of total RNA as a template for reverse transcription, the first strand of cDNA was synthesized by reverse transcription using SSRT-II reverse transcriptase, following the procedure of SMARTER RACE CDNA KIT (Clontech), and amplified by nested PCR to obtain NYPSY MRNA full length. Wherein,

The primers used for 5' -RACE were as follows:

NyPSY-ER1：GATTACGCCAAGCTCGCCGACGACTCGCAGGTGCATCGTTT

NyPSY-ER2：GATTACGCCAAGCTTGGCAAGGGAGCACCTGCAAACGCCAAGG

The primers used for 3' -RACE were as follows:

NyPSY-HF1：GATTACGCCAAGCTCCTGGACATGTACGCCGGCATCCTCGAGGT NyPSY-HF2：GATTACGCCAAGCTTACCCTGCCTGGCTCGTGGGCGCGTAT

the PCR product was ligated to pMD19-T (Takara Co.) vector, sequenced, and spliced to obtain the ORF sequence of Porphyra yezoensis phytoene synthase gene NyPSY having the complete coding region as shown in SEQ ID NO. 1.

The gene mRNA has the total length of 2066bp, including 332bp 5'-UTR,135bp 3' -UTR and 1599bp ORF, and the total code of 532 amino acid residue protein sequence and one stop codon, and the deduced protein sequence is compared with homologous phytoene synthase multiple sequences in other species (shown in figure 2), which shows that the obtained NyPSY is homologous to phytoene synthase in other species.

4. Construction of Porphyra yezoensis NyPSY expression vector

The first strand of cDNA was synthesized in reverse using 1. Mu.g of the total RNA obtained as a reverse transcription template using the Takara "PRIMESCRIPT ^TM 1st Strand cDNA Synthesis Kit" kit.

Based on the full-length mRNA sequence NyPSY obtained by RACE, primers with homology arms (the primers are synthesized by Genscript company) are designed, nyPSY and ORF are amplified, and an expression vector is constructed by using a homologous recombination method.

The primer sequences were as follows:

NyPSY-pF：GAAGGATTTCACATATGATGAGCCTCAATCCGCCG

NyPSY-pR：GTTTTATTTGAAGCTTCTACCCAGAACCAAACGACAC

After synthesis of the first strand of cDNA by reverse transcription using 1. Mu.g of the total RNA obtained as a template for reverse transcription, PCR was performed

Amplification, PCR procedure was as follows: pre-denaturation at 94℃for 2min; denaturation at 94℃for 30s, annealing at 63℃for 1min, extension at 72℃for 1min, after 35 cycles, at 72℃for 10min. After the reaction is finished, the PCR product is connected to a pMAL-C5X vector, and positive clones are screened after the connection product is transformed into competent cells of escherichia coli BL21 (DE 3), so as to obtain a recombinant vector carrying NyPSY, which is named pMAL-NyPSY.

Functional verification of example 2NyPSY

To identify NyPSY functions, we used an in vivo E.coli enzyme activity identification system, which was combined with HPLC to analyze the enzyme activity by color complementation. The system is used for a plasmid pAC-85b (purchased from Addgene), which carries the genes geranylgeranyl diphosphate synthase (CrtE, GGPS), phytoene desaturase (CrtI, PDS/ZDS) and lycopene beta-cyclase (CrtY, LCYB) related to the synthesis of E.coli carotene by the enzyme E.summer, and can synthesize and accumulate beta-carotene in E.coli cells in the presence of phytoene synthase, so that E.coli presents an orange yellow color; while E.coli is white without phytoene synthase, it is easy to distinguish whether or not there is enzymatic activity of phytoene synthase.

1. Heterologous expression of E.coli

The sequenced pMAL-NyPSY and pAC-85b were co-transferred into E.coli BL21 (DE 3) strain by heat shock. The empty vector pMAL-C5X (purchased from NEB) was cotransformed with pAC-85b as negative control, and the synthetic pMAL-AaPSY containing the Fushou PSY gene was cotransformed with pAC-85b as positive control. Co-transformed strains were cultured in LB medium containing chloramphenicol (50. Mu.g. ML ^-1) and carboxin (50. Mu.g. ML ^-1). Yellow colonies were selected and inoculated into 5mL of liquid LB medium with chloramphenicol and carbenicillin, shaking culture was performed at 37℃and 200rpm for overnight, 200. Mu.L was inoculated into 20mL of resistant LB medium, shaking culture was performed at 200rpm for 3d, and cells were collected by centrifugation, and it was found that in the sample containing Porphyra yezoensis NyPSY and the positive control E.coli was orange-yellow in color, while the negative control E.coli containing the empty vector pMAL-C5X was colorless, indicating that NyPSY had activity of catalyzing phytoene synthase, thereby allowing E.coli cells to accumulate beta-carotene.

2. Pigment extraction and HPLC analysis

Coli cells containing the dual vectors pAC-85b and pMAL-C5X (negative control), containing the dual vectors pAC-85b and pMAL-NyPSY (sample) and containing the dual vectors pMAL-AaPSY and pAC-85b (positive control) were collected by centrifugation at 10,000g for 1min, respectively, and then sonicated. 400 mu L of 80% acetone is added into the ultrasonic crushed material, the mixture is vigorously shaken for 30min to thoroughly extract pigment contained in the material, then 250 mu L of ethyl acetate and ddH ₂ O are sequentially added, the mixture is respectively and vigorously shaken for 15sec, and after standing for 5min, the mixture is centrifuged for 5min at 10,000g and 4 ℃. The supernatant was aspirated, dried with nitrogen and dissolved in 100. Mu.L of ethyl acetate. The extracted pigment was separated by reverse phase high performance liquid chromatography (reverse-PHASE HPLC) on a Sphermsorb ODS 2C 18 column (Waters) of 4.6X1250 mm with a mobile phase of linear ethyl acetate (0-100%) in acetonitrile/water/triethylamine (9:1:0.01). The development time was 45min, the flow rate was 1mL min ^-1, and the column temperature was 50 ℃. The scanning wavelength of the detector is 300-800 nm. Scan results of 296nm and 440nm were selected. The identification of carotenoids is determined from standard or reported retention times and absorption spectra. All chemical reagents were chromatographically pure. The analytical structure showed that the samples accumulated β -carotene as well as the positive control, whereas the negative control did not (fig. 3), further demonstrating that NyPSY could catalyze the β -cyclization of lycopene to β -carotene.

6) Systematic analysis

To determine the type of cloned gene NyPSY, we searched for homologous sequences in GenBank NyPSY and reported the phytoene synthase sequences from different plants and performed phylogenetic analyses. All sequences were aligned using ClustalX and phylogenetic tree was constructed using the adjacency method in MEGA5.1 (Tamura et al 2011). The bootstrap test was repeated 1,000 times for analysis of the reliability of each node. Analysis showed NyPSY to be indeed phytoene synthase in plants (shown in FIG. 4).

Claims

1. A phytoene synthase NyPSY protein, wherein the phytoene synthase NyPSY protein comprises:

(a) The amino acid sequence is shown as SEQ ID NO. 2; or (b)

2. The phytoene synthase NyPSY protein according to claim 1, wherein the phytoene synthase NyPSY protein is derived from Porphyra yezoensis.

3. A nucleic acid molecule encoding the phytoene synthase NyPSY protein of claim 1.

4. A nucleic acid molecule according to claim 3, comprising:

(i) Which encodes the protein of claim 1; or (b)

(Ii) A nucleotide sequence which hybridizes under stringent conditions to a nucleotide sequence of a nucleic acid or gene as defined in (i) and which encodes a protein having phytoene synthase activity; or (b)

(Iii) The nucleotide sequence is shown as SEQ ID NO. 1.

5. An expression cassette, recombinant vector, recombinant cell or recombinant bacterium, characterized in that it comprises a nucleic acid molecule according to claim 3 or 4.

6. Use of the phytoene synthase NyPSY protein of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, the expression cassette of claim 5, the recombinant vector, the recombinant cell or the recombinant bacterium for increasing carotenoids in plants.

7. The use according to claim 6, wherein the plant is laver.

8. A method for increasing carotenoid content in a plant comprising the steps of:

1) Allowing a plant to contain the nucleic acid molecule of claim 3 or 4; or (b)

2) Allowing a plant to express the phytoene synthase NyPSY protein according to claim 1 or 2.

9. The method of claim 8, comprising the step of transgenesis, crossing, backcrossing, or asexual propagation.