CN117106819B - Phaeodactylum tricornutum CHLC gene and application of encoded protein in chlorophyll c synthesis - Google Patents

Phaeodactylum tricornutum CHLC gene and application of encoded protein in chlorophyll c synthesis Download PDF

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CN117106819B
CN117106819B CN202311096732.0A CN202311096732A CN117106819B CN 117106819 B CN117106819 B CN 117106819B CN 202311096732 A CN202311096732 A CN 202311096732A CN 117106819 B CN117106819 B CN 117106819B
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李小波
蒋彦酉
曹天骏
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Westlake University
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Abstract

The invention discloses a CHLC gene of Phaeodactylum tricornutum and application of the coded protein in synthesis of chlorophyll c, and the invention discloses a novel function of the CHLC gene for the first time, which can improve the content of chlorophyll c in Phaeodactylum tricornutum; and, it can be used in photosynthetic organisms (plants, algae, photosynthetic bacteria) to increase chlorophyll c content in the photosynthetic organisms, and then increase light capturing efficiency, and is beneficial to light efficiency and biomass accumulation, and the protein encoded by the gene can be used for chlorophyll c synthesis in vitro.

Description

Phaeodactylum tricornutum CHLC gene and application of encoded protein in chlorophyll c synthesis
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a Phaeodactylum tricornutum CHLC gene, a Phaeodactylum tricornutum CHLC protein and application thereof in chlorophyll c synthesis.
Background
Chlorophyll is widely used in photosynthetic organisms, has the characteristic of capturing light energy, belongs to an important antenna molecule in a photosynthetic organism light capturing complex, and has important application in the aspect of capturing photosynthetic light energy. Due to the diversity of structures, different chlorophyll has different absorption spectra. Blue-green light has strong penetrability in seawater, so that most marine algae contain chlorophyll c and are used for capturing blue-green band visible light. Chlorophyll c, if transferred to other photosynthetic organisms, can be used to broaden the light-harvesting spectrum and increase the photosynthetic efficiency.
At present, the synthetic pathway of chlorophyll c has not been resolved. There is no report on the synthesis of chlorophyll c gene.
Disclosure of Invention
The invention aims at providing a Phaeodactylum tricornutum (Phaeodactylum tricornutum) CHLC gene and application of protein coded by the gene in chlorophyll c synthesis.
In order to achieve the above purpose, the technical scheme adopted by the invention is summarized as follows:
a CHLC gene whose nucleotide sequence is shown in SEQ ID NO.1 is composed of 1230 bases or DNA molecule hybridized with the DNA sequence defined by SEQ ID NO.1 under strict condition.
The amino acid sequence of the protein (1) coded by the gene is shown as SEQ ID NO. 2. The sequence consists of 409 amino acid residues.
The protein encoded by the CHLC gene may also include a protein derived from (1) having the function of (1) and formed by substitution, deletion or addition of one or more (e.g., 1 to 30, preferably 1 to 20, more preferably 1 to 10, e.g., 5, 3) amino acid residues of the amino acid sequence of SEQ ID NO. 2; or a protein derived from (1) having homology of 80% (preferably 90% or more, such as 95%,98%,99% or more) or more to the protein sequence defined in (1) and having the function of the protein of (1).
Transgenic organisms containing the CHLC gene, including algae, photosynthetic bacteria or land plants, are all within the scope of the present invention.
The most important invention is that a new function of CHLC gene is disclosed, CHLC knockout mutant is obtained through gene editing, compared with wild Phaeodactylum tricornutum, the mutant lacks chlorophyll c 1、c2, and the intermediate compounds of chlorophyll biosynthesis pathway, namely divinyl protochlorophyll lactone (DVPCHLIDE A, DVP) and monovinyl protochlorophyll lactone (MV PCHLIDE A, MVP) are accumulated. It can be seen that the CHLC gene or protein participates in the synthesis of chlorophyll c, that is, the chlorophyll c biosynthesis gene has the function of increasing the content of chlorophyll c in Phaeodactylum tricornutum, and can be used for increasing the yield of chlorophyll c produced by microorganisms (algae, fungi, bacteria); and, for increasing the chlorophyll c content in photosynthetic organisms (plants, algae, photosynthetic bacteria) and consequently increasing the light harvesting efficiency, the efficiency of photosynthesis and biomass accumulation are advantageous, which can be achieved by means of transgenesis.
In addition, through the identification of the enzymatic functions of the CHLC protein, the result shows that the divinylprotochlorophyll lactone and the monovinylprotochlorophyll lactone can be respectively converted into chlorophyll c 2、c1 only in the presence of the CHLC protein, so that the protein encoded by the CHLC gene can be used for in vitro synthesis of chlorophyll c.
It should be noted that the function of the gene protected by the present invention includes not only the CHLC gene described above, but also homologous genes having a high homology (e.g., homology of more than 40%, preferably more than 50%, preferably more than 60%, more preferably more than 70%, more preferably more than 80%, more preferably more than 90%, more preferably more than 95%, more preferably more than 98%) with SEQ ID NO. 1.
The invention has the advantages that:
In Phaeodactylum tricornutum, we find a CHLC gene for the first time, the protein coded by the gene is responsible for biosynthesis of chlorophyll c, that is, the invention discloses a novel function of the CHLC gene for the first time, which can improve the chlorophyll c content in Phaeodactylum tricornutum. And, it can be used in photosynthetic organisms (plants, algae, photosynthetic bacteria) to increase chlorophyll c content in the photosynthetic organisms, and then increase light capturing efficiency, and is beneficial to light efficiency and biomass accumulation, and the protein encoded by the gene can be used for chlorophyll c synthesis in vitro.
Drawings
FIG. 1 is evidence that the CHLC gene was edited in CHLC knockout mutants;
FIG. 2 is evidence of reduced chlorophyll c content of chlc knockout mutants;
in the figure, the right image is a photograph of the appearance of the cell, and the left image is a High Performance Liquid Chromatography (HPLC) pigment analysis;
FIG. 3 is a chlorophyll precursor accumulated in chlc knockout mutants;
FIG. 4 is a vector used for producing CHLC protein in E.coli;
FIG. 5 shows the results of in vitro synthesis of chlorophyll c using CHLC protein in the examples.
Detailed Description
The present invention will be described in detail with reference to specific examples. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The test methods in the following examples are conventional methods unless otherwise specified. The reagents and materials employed, unless otherwise indicated, are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botanicals, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination, and bioinformatics, which will be apparent to one of skill in the art. These techniques are fully explained in the published literature, and the methods of DNA extraction, phylogenetic tree construction, gene editing method, gene editing vector construction, gene editing plant acquisition, etc. used in the present invention can be realized by the methods disclosed in the prior art except the methods used in the examples described below.
The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-or double-stranded structures. Such nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences of non-coding regions. These terms include a gene. "Gene" or "gene sequence" is used broadly to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons in genomic sequences, and/or coding sequences in cDNA, and/or cDNA and regulatory sequences thereof. In particular embodiments, for example in relation to isolated nucleic acid sequences, it is preferred that they are cDNA.
Examples
1. Acquisition of the CHLC Gene
Screening by using a plurality of technical means such as transcriptome and metabolome to obtain a Phaeodactylum tricornutum CHLC gene, wherein the length of the full-length coding frame nucleotide sequence of the gene is 1230bp, the gene consists of 409 amino acids, the nucleotide sequence is shown as a sequence SEQ ID NO.1, and the protein sequence is shown as a sequence SEQ ID NO. 2.
Phenotypic analysis of chlc mutants
Heretofore, no report has been made on the function of the CHLC gene in Phaeodactylum tricornutum, and the applicant has conducted the following studies, and has found for the first time that the CHLC gene plays an important role in the synthesis of chlorophyll c from Phaeodactylum tricornutum.
Construction of CHLC Gene editing vector
The coding guide sequence (sgRNA) for CHLC gene editing was selected using CRISPOR website (http:// crispor. Tefor. Net /), two primers were designed based on the selected sgRNA (Table 1), after annealing, double-stranded DNA containing cohesive ends was obtained, cloned into the target vector containing blasticidin-S deaminase gene, and the Phaeodactylum tricornutum U6 promoter was responsible for expression of the sgRNA. The Cas9 gene is controlled by the gamma tubulin promoter.
TABLE 1 CHLC sgRNA primers
2. Acquisition of Phaeodactylum tricornutum conjugal transformation and chlc knockout mutant
Coli containing the above vector and pTA-Mob vector required for conjugal transformation was mixed with wild type Phaeodactylum tricornutum at 1000:1, and after concentration, the f/2 medium plates are coated. Two days later, cells were scraped and plated on f/2 medium plates containing 5. Mu.g/mL blasticidin.
Two weeks later, the clones were picked, resuspended in liquid medium and plated again to obtain subclones. Multiple subclones were picked and colony PCR and sequenced. The resulting CHLC knockout mutant lacks the CHLC gene by 4 base pairs compared to the wild type (FIG. 1).
Chlorophyll c phenotyping of the chlc knockout mutant and identification of accumulated chlorophyll precursor structure
Wild type and mutant were cultured under constant light (80. Mu. Mol. M -2·s-1). 10 7 cells were collected by centrifugation, 250. Mu.L of 90% acetone was added and mixed well by sonication in dark conditions. After centrifugation, the supernatant was collected and subjected to a pigment analysis by High Performance Liquid Chromatography (HPLC).
HPLC parameters were as follows:
instrument: thermo Ultimate 3000 UHPLC;
chromatographic column: the C18 column (5 μm, 250×4.6mm，waters，USA)；
Temperature: 20 ℃;
Flow rate: 1mL per minute;
sample injection volume: 10. Mu.L;
Gradient mobile phase: consists of solvent A (acetonitrile: methanol: ethyl acetate=7:2:1) and solvent B (10 mmol/L ammonium acetate). 0 minutes: 40% A,60% B;4 minutes: 25% A,75% B;12 minutes: 0% A,100% B;15 minutes: 2%A;98% b;16 minutes: 40% A;60% B;20 minutes: 40% A;60% B.
HPLC results showed that the chlc knockout mutant had complete deletion of chlorophyll c. This suggests that the CHLC gene or protein is involved in the synthesis of chlorophyll c. Furthermore, the deletion of chlc knockout mutant chlorophyll c also resulted in a reduced carotenoid-fucoxanthin content in the light harvesting complexes as well. Thus, the ratio of total chlorophyll content to total carotenoid content increases, rendering it green. (FIG. 2).
The accumulated chlorophyll c precursor is collected during HPLC, and is subjected to structural analysis by mass spectrometry, the analyzed molecules are known compounds of divinylprotochlorophyll lactone and monovinylprotochlorophyll lactone (figure 3), and the molecules are used as reaction substrates of CHLC protein to further identify the activity function of CHLC protease in an in vitro system.
Construction of expression vector and protein acquisition
1. Cloning of Phaeodactylum tricornutum CHLC Gene cDNA (SEQ ID NO. 1)
Extracting wild Phaeodactylum tricornutum RNA by adopting RNEASY PLANT MINIKIT kit. Reverse transcription was performed using SuperScript TM IIIREVERSE TRANSCRIPTASE kit. The CHLC gene cDNA was cloned by PCR using the obtained total cDNA as a template.
PCR system (table 2):
TABLE 2.20. Mu.L amplification System
PCR cycle:
1)94℃：5min；
2)94℃：30s；
3)55℃：30s；
4)72℃：2min；
Steps 2) -4) are cycled 35 times;
6)72℃：5min。
the PCR primers (Table 3) include not only sequences that overlap with the CHLC-cDNA sequence (uppercase), but also sequences homologous to the target vector (lowercase).
TABLE 3 CHLC cDNA amplification primers
The PCR product was cloned into the pMAL-c5x vector by Information homologous recombination. In the vector, CHLC gene is fused with MBP at N end, polyhistidine tag (His-tag) is arranged at C end, and exogenous gene expression is induced by isopropyl-beta-D-thiogalactoside (IPTG). The constructed plasmid was transformed into the expression BL21 (DE 3) strain and positive clones were screened by PCR. The constructed successful vector is shown in FIG. 4.
2. Expression of Phaeodactylum tricornutum CHLC gene in E.coli
The strain was cultured at 37℃using LB medium containing 100mg/L of ampicillin until the OD600 was 0.6 to 0.8. Isopropyl- β -D-thiogalactoside (IPTG) was added to a final concentration of 0.4mM and the incubation was continued for 12 hours at 16 ℃.
3. Purification of Phaeodactylum tricornutum CHLC protein
After high pressure disruption, the mixture was centrifuged at 13 g for 15 minutes at 4 ℃. The obtained supernatant was subjected to protein purification using an AKTA system and an amylose resin affinity column. Protein concentration was performed using 30kDa MWCO ultrafiltration tube. Bovine Serum Albumin (BSA) was used as a configuration standard and protein concentration was determined by the bicinchoninic acid method. Split charging to 50 μg per tube, quick freezing with liquid nitrogen, and storing at-80deg.C.
4. Enzymatic functional identification of CHLC proteins
And adding the divinylorthochlorophyll lactone and the monovinylorthochlorophyll lactone which are precursors for synthesizing chlorophyll c and purifying CHLC protein into an in-vitro system, and verifying the generation of chlorophyll c. The method comprises the following steps:
1. establishment of enzymatic research system
50. Mu.L of buffer (20%Methanol,100mM Tris-HCl, pH 8.0) was added to the purified and dried divinylorthochlorophyll lactone and monovinylorthochlorophyll lactone, and the precursors were dissolved by ultrasonic homogenization.
Three components were then added to the final concentration in brackets (concentration in 50ul of the final reaction system):
l-ascorbic acid (L-ascorbate, 5 mM);
alpha-ketoglutarate (2-OG, 5 mM);
Ferrous sulfate (FeSO 4, 0.1 mM);
50 μg purified CHLC protein.
The CHLC protein was omitted in the control reaction. After two hours, 30. Mu.L of acetone and 60. Mu.L of ethyl acetate were added and mixed well to terminate the reaction.
Analysis of CHLC protein products
After termination of the reaction, the chlorophyll c product was extracted by centrifugation: the organic layer of the supernatant was aspirated, dried with nitrogen and dissolved in 50. Mu.L of aqueous methanol.
The dissolved substances were analyzed by High Performance Liquid Chromatography (HPLC) to analyze the generation of chlorophyll c and the residue of the precursor compound. HPLC parameters were as follows:
instrument: waters Acquity UPLC;
Chromatographic column: ACQUITY UPLC HSS T31.8.8 μm (3 μm, 2.1×150mm)；
Temperature: 45 ℃;
Flow rate: 0.3mL per minute;
Sample injection volume: 3 μL;
gradient mobile phase: consists of solvent A (acetonitrile: methanol: methyl tert-butyl ether=70:20:10) and solvent B (10 mM ammonium acetate). 0 minutes: 56% A,44% B;1 minute: 58% A,42% B;4 minutes: 58% A,42% B;6 minutes: 60% A,44% B.
As can be seen from fig. 5, the divinylprotochlorophyll lactone and the monovinylprotochlorophyll lactone are converted into chlorophyll c 2、c1, respectively, by adding the CHLC protein, compared with the control, that is, the CHLC protein has a function of synthesizing chlorophyll c in vitro.

Claims (5)

1. The application of Phaeodactylum tricornutum CHLC gene or the coded protein thereof in chlorophyll c synthesis is characterized in that the nucleotide sequence of the Phaeodactylum tricornutum CHLC gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
2. The use according to claim 1, wherein the chlorophyll c comprises chlorophyll c 1、c2.
3. Use according to claim 1, characterized in that the synthetic precursors of chlorophyll c are divinylorthochlorophyll lactone and monovinylorthochlorophyll lactone.
4. The application of the protein coded by the Phaeodactylum tricornutum CHLC gene in synthesizing chlorophyll c in vitro is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.
5. A method for synthesizing chlorophyll c in vitro, characterized in that the method comprises the following steps: adding 50 mu L of buffer solution into the purified and dried precursors, namely divinyl protochlorophyll lactone and monovinyl protochlorophyll lactone, and uniformly mixing by ultrasonic to dissolve the precursors; then adding 5 mM L-ascorbic acid, 5 mM alpha-ketoglutaric acid, 0.1 mM ferrous sulfate and 50 mug purified CHLC protein, reacting for two hours, adding 30 mug of acetone and 60 mug of ethyl acetate, and uniformly mixing to terminate the reaction; after termination of the reaction, the chlorophyll c product was extracted by centrifugation: sucking the organic layer of the supernatant, drying with nitrogen, and dissolving with 50 μl of methanol aqueous solution to obtain the final product; the amino acid sequence of the CHLC protein is shown as SEQ ID NO. 2.
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光合作用抑制剂DCMU对三角褐指藻岩藻黄素含量和叶绿素荧光特征及关键基因表达的影响;郑小恽;龚一富;李申睿;方清姝;王何瑜;唐道军;;核农学报;20200617(第08期);第91-98页 *

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