Application of AUREO1c gene in improving organisms' resistance to high light stress

Abstract

The present invention discloses the role of the AUREO1c gene in improving the high light stress resistance of organisms. The present invention discloses for the first time a new function of the AUREO1c gene, which can improve the high light stress resistance of Pseudomonas triangularis. The present invention uses bacterial conjugation technology to mediate the transfer of exogenous plasmids into Pseudomonas triangularis, and uses gene editing technology to knock out the AUREO1c gene. The present invention uses electroporation to transfer DNA fragments into Pseudomonas triangularis cells, and uses AUREO1c driven by a strong promoter to complement mutant algae strains, and clarifies the function of the AUREO1c gene in high light stress response from a molecular mechanism. In addition, the present invention is also expected to improve photosynthesis under high light stress conditions in other photosynthetic organisms (algae, photosynthetic bacteria, plants), which is beneficial to photosynthesis efficiency and biomass accumulation, and is conducive to application in biotechnology.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of a Phaeodactylum tricornutum AUREO1c gene in high light stress resistance. Background Art

[0002] Land plants and algae face a complex and changeable light environment. High light levels can cause the energy absorbed by the photosynthetic system to exceed the energy limit required for carbon assimilation, inducing the production of reactive oxygen species (ROS), which in turn damages the photosynthetic system and reduces biomass accumulation. In order to cope with high light stress and excess energy, plants and algae have evolved a series of photoprotection mechanisms, the most common of which is the non-photochemical quenching (NPQ) mechanism that dissipates excess energy in the form of heat.

[0003] Diatoms dominate marine algae, contributing 20% ​​of the Earth's productivity and playing an important role in the Earth's ecology and the sustainable development of human society. Due to the movement of seawater and other reasons, marine algae face more severe light stress than terrestrial plants, and can face thousands of times the light intensity change at the minute level. The strong light protection ability of diatoms gives them a competitive advantage in harsh light environments.

[0004] The NPQ mechanism in diatoms mainly depends on the conversion of diatomaxanthin and diatomaxanthin in the xanthophyll cycle and the LHCXs proteins in the large family of fucoxanthin-chlorophyll complexes. At present, most of the research on the NPQ mechanism focuses on the downstream energy dissipation mechanism. However, the identification and mechanism of action of the upstream signaling molecules need to be further analyzed.

[0005] Photoreceptors are the primary factors in sensing light signals and light intensity. The blue light band is the main band in marine water, which shows the important function of blue light signals in the marine environment. The number of blue light receptors in P. triangularis photoreceptors is the largest, but how it specifically regulates high light stress resistance is unknown, and exploring genes related to high light stress is of great research significance. Summary of the invention

[0006] One of the purposes of the present invention is to provide an application of the AUREO1c gene of Phaeodactylum tricornutum in improving the high light stress of organisms, in order to expand the application scope of the AUREO1c gene.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is summarized as follows:

[0008] The AUREO1c gene, whose nucleotide sequence is shown in SEQ ID NO.1, consists of 981 bases and encodes a blue light receptor protein consisting of a bZIP domain at the amino terminal and a LOV domain at the carboxyl terminal, with a total of 327 amino acids. The amino acid sequence is shown in SEQ ID NO.2.

[0009] The protein encoded by the above-mentioned AUREO1c gene may also include a protein derived from (1) which is formed by replacing, deleting or adding one or more (such as 1-30; preferably 1-20; more preferably 1-10, such as 5, 3) amino acid residues of the amino acid sequence of SEQ ID NO.2, and has the protein function of (1); or a protein derived from (1) which has 80% (preferably more than 90%, such as 95%, 98%, 99% or higher) homology with the protein sequence defined in (1) and has the protein function of (1).

[0010] Transgenic organisms and gene editing vectors containing the AUREO1c gene all fall within the protection scope of the present invention, wherein the transgenic organisms include algae, photosynthetic bacteria or terrestrial plants.

[0011] One of the most important inventions of the present invention is to disclose a new function of the AUREO1c gene. The present invention obtains aureo1c mutants by gene editing. By comparing the wild-type triangular brown finger algae with the aureo1c mutant, the high light resistance and survival rate of the aureo1c mutant are lower than those of the wild type, and the NPQ induction and LHCX2 / 3 expression are significantly lower than those of the wild type. In addition, the results of the in vitro gel migration experiment found that the AUREO1c protein can bind to the promoter region of the effector gene LHCX2 / 3. The mutant algae strain is complemented by AUREO1c driven by a strong promoter, and the complemented (overexpressed) algae strain has a higher LHCX2 expression level and stronger NPQ induction ability than the wild type. It can be seen that the AUREO1c gene or protein is involved in the high light stress response of triangular brown finger algae, and it is mainly achieved through the induction of NPQ mediated by LHCX2 / 3. It can be inferred that the AUREO1c gene has the function of improving the high light stress resistance of P. tricornutum, and can also be used to improve the high light resistance of other photosynthetic organisms (algae, bacteria, plants), thereby improving photosynthetic efficiency, which is beneficial to biomass accumulation and the production of beneficial additional products in biofactories. The above method can be achieved through transgenic methods.

[0012] It should be noted here that the functions of the genes protected by the present invention include not only the above-mentioned AUREO1c gene, but also homologous genes with higher homology to SEQ ID NO.1 (e.g., the homology is higher than 40%; preferably higher than 50%; preferably higher than 60%; more preferably higher than 70%; more preferably higher than 80%; more preferably higher than 90%; more preferably higher than 95%; more preferably higher than 98%).

[0013] In addition, the present invention also discloses a method for obtaining a transgenic organism, wherein the method comprises transferring the AUREO1c gene into a target organism to obtain a transgenic organism, wherein the transgenic organism has a higher resistance to high light stress than the target organism, and preferably, the target organism is Phaeodactylum tricornutum.

[0014] Advantages of the present invention:

[0015] In P. triangularis, we first disclosed a new function of the AUREO1c gene, which can improve the high light stress resistance of P. triangularis. Moreover, through transgenic technology, it can be used to improve the high light stress resistance in other photosynthetic organisms (other algae, photosynthetic bacteria, plants), thereby improving the high light adaptability of photosynthetic organisms, improving photosynthetic efficiency, and being beneficial to biomass accumulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is evidence that the AUREO1c gene has been knocked out;

[0017] Figure 2 This is evidence that the strong promoter drives AUREO1c-GFP to complement the aureo1c mutant. The figure shows the Western blot results;

[0018] Figure 3 Evidence that the survival rate of aureo1c mutants is reduced under high light stress, showing 1200 μmol photonsm -2 s -1 Cell bleaching (death) after 2 days of light intensity treatment;

[0019] Figure 4 The wild type, aureo1c mutant and complemented strain were analyzed at 1200 μmol photons m -2 s -1 NPQ induction after 4 h of light intensity treatment;

[0020] Figure 5 The wild type, aureo1c mutant and complemented strain were detected at 550 μmol photons m -2 s -1 LHCX2 / 3 gene expression after 1 h of light intensity treatment;

[0021] Figure 6 There is evidence that AUREO1c protein binds to the promoter of effector gene LHCX2 / 3. DETAILED DESCRIPTION

[0022] The present invention will be described in detail below through specific embodiments. These embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0023] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained through commercial channels.

[0024] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0025] Unless otherwise specified, the implementation of the present invention will use conventional botanical techniques, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination and bioinformatics techniques that are obvious to those skilled in the art. These techniques are fully explained in the published literature. In addition, the methods of DNA extraction, construction of phylogenetic trees, gene editing methods, construction of gene editing vectors, and obtaining gene-edited plants used in the present invention can be achieved by using methods already disclosed in existing literature, except for the methods used in the following examples.

[0026] 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-stranded or double-stranded structures. These nucleic acids or polynucleotides include gene coding sequences, antisense sequences and regulatory sequences of non-coding regions, but are not limited to these. These terms include a gene. "Gene" or "gene sequence" is widely used to refer to a functional DNA nucleic acid sequence. Therefore, a gene may include introns and exons in a genomic sequence, and / or include a coding sequence in a cDNA, and / or include a cDNA and its regulatory sequences. In specific embodiments, such as with respect to an isolated nucleic acid sequence, it is preferably assumed to be cDNA.

[0027] Example 1 Acquisition of AUREO1c gene and acquisition of mutants

[0028] According to previous studies, the sequences of aureochrome family members were obtained, among which the nucleotide sequence of the AUREO1c gene coding frame is 981 bp long and consists of 327 amino acid residues. Its nucleotide sequence is shown in SEQ ID NO.1, and its protein sequence is shown in SEQ ID NO.2.

[0029] Previously, there was no report on the function of the AUREO1c gene in P. triangularis under high light stress. The applicant conducted the following research and discovered for the first time that the AUREO1c gene plays an important role in high light stress in P. triangularis. The specific research process is described as follows.

[0030] 1. Construction of AUREO1c gene editing vector

[0031] The guide RNA (sgRNA) design website CRISPOR (http: / / crispor.tefor.net / ) was used to design sgRNA, and CRISPR / Cas9-mediated gene editing technology was used for gene knockout. Primers were designed according to the selected sgRNA (Table 1), and double-stranded DNA with sticky ends was obtained after annealing. It was ligated into the conjugated plasmid using T4 ligase, and the sgRNA was expressed by the U6 promoter, and the Cas9 protein was expressed by the γ-tubulin promoter. The antibiotic screened was blasticidin.

[0032] Table 1. Aureo1c mutant knockout primers

[0033]

[0034] 2. Conjugative transformation of Phaeodactylum tricornutum and generation of aureo1c mutant

[0035] The conjugated plasmid was transferred into the E. coli containing epi300 of pTA_Mob. E. coli and Phaeodactylum triangularis were incubated at 1 mL of bacteria (OD600 = 0.8-1.0): 5 × 10 6 The cells were mixed evenly in a ratio of 1:1 and 2:1, and after co-culture on an f / 2 plate containing 5% LB for two days, the plates were transferred to an f / 2 screening plate containing 5 μg / mL blasticidin for screening.

[0036] About 2 weeks later, single clones were selected for PCR and sequencing verification. The edited clones were cultured in liquid medium containing blasticidin. Two weeks after plating, single clones were grown and re-pick plaques were sequenced for verification. The mutants finally obtained lacked 1 base pair compared to the wild type, resulting in a frameshift mutation ( Figure 1 ), the AUREO1c gene was knocked out and the aureo1c mutant was obtained.

[0037] Example 2 Obtaining aureo1c mutant complement strain

[0038] 1. Construction of complementation vector

[0039] RNA was extracted using the RNAsimple total RNA extraction kit, and cDNA was obtained by reverse transcription using the HiScript III 1st StrandcDNA Synthesis Kit (+gDNA wiper). AUREO1c cDNA (with synonymous mutations at the sgRNA) was constructed into the complementation vector by homologous recombination, and the primers are shown in Table 2. The transformation method was electroporation, the strong promoter EF1a drove the expression of AUROE1c-GFP, and the screening antibiotic was bleomycin.

[0040] Table 2. Primers for constructing complementation vectors

[0041]

[0042] Among them, one electroporation transformation system contains 2×10 8 After transformation, the cells were placed on f / 2 screening plates containing 75 μg / mL bleomycin. After about 4 weeks, single clones were picked for Western blot identification.

[0043] 3 × 10 7 The algae cells were resuspended in RIPA lysis buffer, disrupted by ultrasound in a bioruptor, centrifuged at low temperature to obtain the supernatant, and an equal volume of 2×SDS loading buffer was added. The protein was denatured at 95°C for 5 minutes, and then SDS-PAGE was run, transferred to a membrane, and antibody was applied for analysis. Figure 2 As shown, AUREO1c-GFP protein was expressed correctly and was not expressed in the wild type (negative control), indicating that the complementation was successful and the complemented strain was obtained.

[0044] Example 3 Analysis of the survival of aureo1c mutants under high light conditions

[0045] Under constant illumination (40 μrnol photons m -2 s -1 ) were used to culture wild-type and mutant cells at 21°C until 3×10 6 cells / mL, 1200 μmol photons m -2 s -1 The cells were treated with high light stress under illumination, and the pigments were measured after 1 day of treatment. The cell whitening (death) phenotype was observed after 2 days of treatment. The results showed that 1200 μmol photons m -2 s -1 After 2 days of light treatment, the mutant cells completely turned white (died) ( Figure 2), but the wild-type cells were still in a relatively normal brown state, indicating that compared with the wild-type, the high-light survival rate of the aureo1c mutant was significantly reduced, and the complemented strain could completely recover to the level of the wild-type.

[0046] The results of pigment determination showed that the pigment of the mutant could not be detected after 2 days of treatment, so the pigment component analysis was carried out after 1 day of treatment. The results are shown in Table 3. The pigment of the aureo1c mutant was about one-tenth of the wild type, and the complemented strain was equivalent to the wild type.

[0047] Table 3. 1200 μmol photons m -2 s -1 Pigment determination after 2 days of light intensity treatment

[0048]

[0049] Example 4 AUREO1c gene is involved in the induction of NPQ by high light

[0050] Under constant light (40 μmol photons m -2 s -1 ) were used to culture wild-type and mutant cells at 21°C until 3×10 6 cells / mL, 1200 μmol photons m -2 s -1 High light stress was carried out under light, and NPQ ( Figure 4 ).

[0051] NPQ determination method: After 15 mL of diatom cells were concentrated to 2 mL and dark-adapted for 40 minutes, Dual-PAM-100 (Walz) was used for determination. The NPQ calculation formula was NPQ = (Fm-Fm′) / Fm′, where Fm was the maximum chlorophyll fluorescence in the dark-adapted state and Fm′ was the maximum chlorophyll fluorescence at each illumination time point.

[0052] The results of NPQ test showed that ( Figure 4 ), high light treatment can significantly induce NPQ in the wild type, initiate photoprotection, and use excess energy for heat dissipation, but the induction of NPQ is basically lost in the aureo1c mutant. The NPQ induction intensity of the complemented strain is higher than that of the wild type, indicating that the AUREO1c gene is overexpressed in the aureo1c mutant, that is, the overexpression of the AUREO1c gene can improve the photoprotection ability of P. tricornutum.

[0053] Example 5 AUREO1c regulates LHCX2 / 3 expression at the transcriptional level

[0054] Under constant light (40 μmol photons m -2 s -1 ) were used to culture wild-type and mutant cells at 21°C until 3×10 6 cells / mL, 550 μmol photons m -2 s -1 High light stress treatment was carried out under light, and the LHCX2 / 3 gene expression was measured after 1 h of treatment.

[0055] RNA was extracted using the RNAsimple total RNA extraction kit, and reverse transcription was performed using the HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper). The real-time fluorescence quantitative PCR (qPCR) reaction system and reaction procedure are shown in Table 4.

[0056] Table 4. qPCR reaction system and reaction procedure

[0057]

[0058] qPCR experimental results ( Figure 5 ) showed that high light could significantly induce the expression of effector protein encoding genes LHCX2 / 3 in the wild type, but there was basically no induction in the aureo1c mutant. The expression of LHCX2 in the complemented strain was significantly higher than that in the wild type. It can be seen that AUREO1c mediated the high-light-induced expression of effector protein encoding genes LHCX2 / 3.

[0059] Example 6 AUREO1c protein binds to the LHCX2 / 3 promoter region

[0060] 1. Construction of AUREO1c exogenous protein expression vector

[0061] RNA was extracted using the RNAsimple total RNA extraction kit, and reverse transcribed using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper). The obtained cDNA was used as a template for PCR to obtain the full-length sequence of AUREO1c. The PCR related parameters are shown in Table 5.

[0062] Table 5. PCR amplification system and amplification procedure

[0063]

[0064]

[0065] The PCR amplification primers are shown in Table 6. The primers include the cDNA homologous sequence (uppercase) and homology arm sequence (lowercase) of AUREO1c.

[0066] Table 6. Primers for constructing AUREO1c expression vector

[0067]

[0068] The obtained AUREO1c cDNA fragment was ligated into the pET28a vector by homologous recombinase and transformed into the expression strain Transetta (DE3).

[0069] 2. AUREO1c protein expression in vitro

[0070] The above strain was cultured in LB medium containing 50 μg / mL kanamycin to an OD600 of 0.6 to 0.8, and isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM. After induction at 16°C for 16 hours, the cells were collected by centrifugation.

[0071] 3. AUREO1c protein purification

[0072] After high-pressure crushing, centrifuge at 16000g and 4°C for 20 minutes. The obtained supernatant was purified by Ni-NTA agarose beads, and then purified by AKTA system for a new round, and then concentrated by 30kDa MWCO ultrafiltration tube, and the protein concentration was determined by BCA method, and stored at -80°C after aliquoting.

[0073] 4. AUREO1c protein binds to LHCX2 / 3 promoter sequence in vitro

[0074] 600 bp of the promoter region of LHCX2 / 3 was used for protein-DNA binding analysis. The primers were biotinylated and labeled as shown in Table 7. The labeled and non-labeled primers were PCR amplified, and the obtained fragments were used as protein-DNA binding analysis and competitive probes, respectively.

[0075] Table 7. LHCX2 / 3 promoter region amplification

[0076]

[0077] In the table: “*” represents biotin labeling.

[0078] Gel shift assay results ( Figure 6) showed that AUREO1c protein could bind to the promoter regions of LHCX2 and LHCX3. After adding excess (50-fold, 100-fold, 200-fold) non-biotinylated labeled competitive probes, the bound probes were competed off, indicating that the binding of AUREO1c protein to the promoter region of LHCX2 / 3 was specific.

Claims

1. The use of AUREO1c gene in improving the high light stress resistance of organisms, characterized in that: The nucleotide sequence of the AUREO1c gene is shown in SEQ ID NO.1, and the organism is Phaeodactylum tricornutum.

2. The use of the AUREO1c gene according to claim 1 in improving the resistance of organisms to high light stress, characterized in that: The AUREO1c gene is derived from Phaeochromis tricornutum.

3. The use of the AUREO1c gene according to claim 1 in improving the high light stress resistance of organisms, characterized in that: High light stress refers to the condition where the light intensity is between 550-1200 μmol photons m -2 s -1 Process under light.

4. The use of the AUREO1c gene according to claim 1 in improving the high light stress resistance of organisms, characterized in that: Compared with the wild type, the high-light survival rate of the aureo1c mutant was significantly reduced, while the complemented strain could completely restore it to the wild type level.

5. The use of the AUREO1c gene according to claim 1 in improving the resistance of organisms to high light stress, characterized in that: High light stress can significantly induce NPQ in the wild type, initiate photoprotection, and use excess energy for heat dissipation, but the induction of NPQ is basically lost in the mutant. The NPQ induction intensity of the complemented strain is higher than that of the wild type, indicating that overexpression of AUREO1c can improve the photoprotection ability of P. tricornutum.

6. The use of the AUREO1c gene according to claim 1 in improving the resistance of organisms to high light stress, characterized in that: The protein encoded by the AUREO1c gene can act as a transcription factor to regulate the expression of downstream genes LHCX2 / 3, and the amino acid sequence of the protein is shown in SEQ ID NO.

2.

7. The use according to claim 6, characterized in that: The protein binds to the promoter region of LHCX2 / 3.

8. A method for obtaining a transgenic organism, characterized in that: The method comprises transferring the AUREO1c gene into a target organism to obtain a transgenic organism, wherein the transgenic organism has a higher resistance to high light stress than the target organism, the target organism is Phaeodactylum tricornutum, and the nucleotide sequence of the AUREO1c gene is shown in SEQ ID NO.1.

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