Method for constructing a phaecoamabarium calidum calreticulin gene overexpression mutant strain

Abstract

This invention provides a method for constructing a mutant strain of *Phaeodactylum tricornutum* overexpressing the caloric reticulin gene. The specific steps include: 1) constructing an overexpression plasmid: obtaining the *Phaeodactylum tricornutum* caloric reticulin fragment, fluorescent label, and plasmid backbone, and ligating them using a recombinant cloning reagent; 2) transformation verification: transforming the constructed overexpression plasmid into competent cells, and verifying the correctness of the recombinant sequence after large-scale culture; 3) constructing a mutant strain: transforming the verified correct overexpression plasmid into *Phaeodactylum tricornutum*. The constructed *Phaeodactylum tricornutum* caloric reticulin gene overexpression mutant strain can cope with stressful environments, especially maintaining a good growth rate of *Phaeodactylum tricornutum* under nitrogen restriction conditions. Such a mutant strain can be used in conjunction with nitrogen restriction conditions to improve oil production efficiency, and experiments predict an increase of 23% in neutral lipid yield.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a method for constructing a mutant strain of *Phaeodactylum tricornutum* with overexpression of the calcified reticulin gene. Background Technology

[0002] Brown finger algae (Triangular brown finger algae) Phaeodactylum tricornutum Taxonomically belonging to the diatom phylum, *Phaeodactylum tricornutum* has an oil content ranging from 15.6% to 31.5% of its dry weight. Rich in polyunsaturated fatty acids (PUFAs), it is widely used in aquatic feed and is a high-potential, economically valuable algae. Studies on the growth conditions and nutrient composition of culture media affecting *Phaeodactylum tricornutum* have primarily focused on altering the composition of polyunsaturated fatty acids in its oil and regulating its growth to increase the proportion of PUFAs and thus improve its nutritional value. However, research on simultaneously increasing the biomass and oil content of *Phaeodactylum tricornutum* by adjusting the culture medium is still relatively rare.

[0003] Limited data exist to explore different Fe... 3+ The effects of concentration and light conditions on the growth and total lipid content of *Phaeodactylum tricornutum* were investigated. These results show that lipid accumulation in *Phaeodactylum tricornutum* can be regulated by altering growth conditions. Other studies have shown that nitrogen limitation can induce faster accumulation of neutral lipids in *Phaeodactylum tricornutum*, but nitrogen limitation also inhibits algal growth; therefore, it does not ultimately achieve the goal of improving oil production efficiency. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing a mutant strain of *Phaeodactylum tricornutum* overexpressing the calcified reticulin gene, in order to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0005] Calretin (CRT) is a type of calcium reticulin. 2+ These proteins bind to chaperones and play important roles both inside and outside the endoplasmic reticulum lumen, including responses to biotic and abiotic stresses. They also participate in the production of lectin-like chaperones and calcium-binding proteins. 2+ Processes such as storage, signal transduction, gene expression regulation, nuclear transport, cell adhesion, immunity, apoptosis, and N-glycosylation modification are mainly located in the endoplasmic reticulum and Golgi apparatus.

[0006] Surprisingly, the study found that overexpression of PtCRT (Phaeodactylum tricornutum calcnetin) enabled Phaeodactylum tricornutum to cope with nitrogen deficiency stress.

[0007] One aspect of this invention is to provide a method for constructing a mutant strain of *Phaeodactylum tricornutum* overexpressing the calcified reticulin gene, the specific steps of which include:

[0008] 1) Construction of overexpression plasmid: Obtain the gene fragment encoding the calcification protein of *Phaeodactylum tricornutum* and the plasmid backbone, and ligate them using recombinant cloning reagent;

[0009] 2) Transformation verification: The constructed overexpression plasmid was transformed into competent cells, and after large-scale culture, the correctness of the recombinant sequence of the constructed overexpression plasmid was detected and verified.

[0010] 3) Constructing mutant strains: Transform the validated overexpression plasmid into *Phaeodactylum tricornutum*.

[0011] Furthermore, the method for obtaining the gene fragment encoding the calecne protein of *Phaeodactylum tricornutum* uses *Phaeodactylum tricornutum* as a template and performs PCR amplification using two artificially synthesized primers. The primers include: CRT-insert1-F: 3'-cacttgtgcgaacggaattcATGAAGTTTACCTTGCTGACCG-5' (SEQ ID NO.1); CRT-insert1-R: 3'-gcccttgctcaccatGTAAGAAAGAGTCTCGCCACTT-5' (SEQ ID NO.2).

[0012] Furthermore, the sequence of the gene fragment encoding the triangular brown finger algae calcification protein is shown in SEQ ID NO.3.

[0013] Furthermore, the plasmid backbone contains a fluorescent marker selected from eGFP, mRFP, or mCherry. By inserting the fluorescent marker, the endoplasmic reticulum of the mutant strain can be observed under a laser scanning confocal microscope, thereby locating the PtCRT.

[0014] Furthermore, the plasmid backbone is obtained by double digestion of the pPha-T1 plasmid. Some commercially available pPha-T1 plasmids directly contain eGFP fluorescent markers, which can reduce the workload of constructing overexpression plasmids. Furthermore, the double digestion sites are EcoR I and Hind III.

[0015] Furthermore, the competent cells are Escherichia coli DH5α competent cells.

[0016] Furthermore, the overexpression plasmid was transformed into *Phaeodactylum tricornutum* using a gene gun.

[0017] The beneficial effect of this invention is that the constructed mutant strain of *Phaeodactylum tricornutum* overexpressing the calcification protein gene can cope with stressful environments, especially maintaining a good growth rate of *Phaeodactylum tricornutum* under nitrogen restriction conditions. This mutant strain can be used in conjunction with nitrogen restriction conditions to improve oil production efficiency, and experiments predict an increase of 23% in neutral lipid yield. Attached Figure Description

[0018] Figure 1 This is a subcellular fluorescence image of a mutant strain of *Phaeodactylum tricornutum*.

[0019] Figure 2 This is a fold change comparison of gene expression levels along the N-glycosylation modification pathway between wild-type and mutant strains;

[0020] Figure 3 This is a fold change comparison of gene expression levels between wild-type and mutant strains, reflecting endoplasmic reticulum stress (ER stress) status.

[0021] Figure 4 This is a fold change comparison of expression levels of endoplasmic reticulum-associated degradation pathway (ERAD) genes between wild-type and mutant strains;

[0022] Figure 5 This is a fold change comparison of gene expression levels in the unfolded protein response pathway (UPR) between wild-type and mutant strains;

[0023] Figure 6 This is the growth curve diagram from Example 2;

[0024] Figure 7 This is a bar chart of the maximum photosynthetic efficiency (Fv / Fm) in Example 2;

[0025] Figure 8 This is a bar chart of chlorophyll a content in Example 2;

[0026] Figure 9 This is a bar chart of the soluble sugar content in Example 2;

[0027] Figure 10 This is a bar chart of the soluble protein content in Example 2. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0030] Example 1: Construction of a mutant strain overexpressing the caloric reticulin gene from *Phaeodactylum tricornutum*

[0031] The steps for constructing mutant strains are as follows:

[0032] 1) DNA was extracted from *Phaeodactylum tricornutum* using the Plant Genomic DNA Extraction Kit from TIAN GEN. The extracted genome was used as a template for PCR amplification to obtain the PtCRT gene fragment. The sequences of the primers used for PCR amplification are shown in Table 1.

[0033] Table 1. List of PCR primers for the PtCRT gene

[0034]

[0035] PCR amplification was performed using the Q5® Hot Start High-Fidelity 2×Master Mix kit from New England Biolabs. The reaction volume was 50 μL, and the specific reaction volume is shown in Table 2.

[0036] Table 2 PCR reaction system for PtCRT gene

[0037]

[0038] The PCR reaction procedure is shown in Table 3.

[0039] Table 3 PCR reaction procedure for the PtCRT gene

[0040]

[0041] 2) Using a purchased plasmid containing the eGFP gene fragment as a template, the eGFP gene fragment was amplified by PCR.

[0042] 3) The pPha-T1 plasmid was prepared by double digestion with EcoR I and Hind III. The digestion system is shown in Table 4. The plasmid was incubated at 37 °C for 1 h using a PCR instrument.

[0043] Table 4. Preparation of linearized pPha-T1 plasmid digestion system

[0044]

[0045] 4) The PtCRT gene fragment, eGFP gene fragment, and pPha-T1 linearized plasmid were recovered using TIANGEN's standard agarose gel DNA recovery kit. The pPha-T1 linearized plasmid, PtCRT gene fragment, and eGFP gene fragment were ligated using TIANGEN EasyGeno rapid recombination cloning kit, and incubated at 50 °C for 15 min using a PCR instrument. The reaction system for constructing the recombinant pPha-T1-PtCRT-eGFP overexpression plasmid is shown in Table 5.

[0046] Table 5. Reaction system for constructing recombinant pPha-T1-PtCRT-eGFP plasmid

[0047]

[0048] 5) Transform the pPha-T1-PtCRT-eGFP overexpression plasmid into *E. coli* DH5α competent cells and plate them. Randomly select 5 colonies from the plate and inoculate them into 1 mL of LB broth containing Amp antibiotic. Incubate at 37 °C until the culture becomes turbid, then transfer to 5 mL of LB broth containing Amp and continue culturing overnight. Take 4 mL of the bacterial culture and extract plasmid using TIANGEN's Rapid Plasmid Mini-Prep Kit (DP05). Measure the DNA concentration using NanoDrop and send the obtained sample to the company for sequencing verification. *E. coli* cells with correct sequencing results are inoculated into 150 mL of LB broth containing Amp resistance and cultured overnight. Extract plasmid using TIANGEN's Endotoxin-Free Plasmid Mini-Prep Kit (DP117). Transfer the extracted pPha-T1-PtCRT-eGFP overexpression plasmid into a 1.5 mL centrifuge tube and store at -20 °C for later use.

[0049] 6) The extracted pPha-T1-PtCRT-eGFP overexpression plasmid was transformed into *Phaeodactylum tricornutum* using a gene gun.

[0050] Transformants grown on plates containing Amp were picked and transferred to liquid f / 2-zeocin medium in 48-well plates for further culture, with the medium changed every 2 weeks for subculturing. The resulting *Phaeodactylum tricornutum* transformants were subjected to PCR to verify successful gene sequence transfer into the genome. The transformant genome was crudely extracted using a Phire Plant Direct PCR MasterMix, and PCR was performed using the genome as a template. The PCR products were detected by gel electrophoresis. Additionally, the fluorescence of the *Phaeodactylum tricornutum* PtCRT overexpression mutant was observed using a laser confocal microscope. Transformants were spread onto f / 2 solid medium plates containing sodium nitrate as the nitrogen source and induced for 48 hours. Plastid fluorescence and eGFP green fluorescence were observed using a Zeiss laser confocal microscope. The results are as follows: Figure 1 As shown. Using 488 nm excitation light, red fluorescence of the plastids of *Phaeodactylum tricornutum* was detected at wavelengths of 625–720 nm, and green fluorescence of eGFP was detected at wavelengths of 500–520 nm. The resulting transformants were verified to be mutant strains of *Phaeodactylum tricornutum* overexpressing the caloric reticulin gene.

[0051] Example 2: Determination of the overexpression mutant strain of the caloric reticulin gene in *Phaeodactylum tricornutum*

[0052] Artificial seawater was prepared for culturing *Phaeodactylum tricornutum*. The formula is shown in Table 6.

[0053] Table 6 Artificial seawater (f / 2) culture medium

[0054]

[0055] The artificial seawater (f / 2) culture medium prepared according to the formula shown in Table 6 was used as the standard seawater sample, the artificial seawater (f / 2) culture medium without NaNO3 was used as the nitrogen-limited seawater sample, and the artificial seawater (f / 2) culture medium prepared according to the formula shown in Table 6 with tunicamycin (abbreviated as TUN) was used as the tunicamycin seawater sample (the amount of tunicamycin added was 0.3 g / mL).

[0056] 2-1) Transcriptional level analysis of endoplasmic reticulum stress-related genes:

[0057] Wild-type strains of *Phaeodactylum tricornutum* and the *Phaeodactylum tricornutum* caloric reticulin gene overexpression mutant strain constructed in Example 1 were cultured separately in standard seawater samples. Algal strains in the logarithmic growth phase were collected by centrifugation, and transcriptome data were analyzed. The analysis results are as follows: Figures 2 to 5 As shown in the figure. By comparing the fold change between wild-type and mutant strains, the effect of PtCRT overexpression on the transcriptional levels of endoplasmic reticulum stress-related genes can be observed. Figure 2The study showed that the expression levels of genes related to the N-glycosylation modification pathway, such as ALG2, ALG8, GntⅠ, and α(1,3)-Fust, were increased in the mutant strain compared to the wild-type strain. Figure 3 , 4 The results showed that most genes related to endoplasmic reticulum stress (ER stress), degradation pathways, and unfolded protein response pathways were downregulated, suggesting that overexpression of calreticulin could reduce ER stress in *Phaeodactylum tricornutum*.

[0058] 2-2) Determination of growth curve:

[0059] Wild-type strains and the mutant strain constructed in Example 1 were cultured and tested using standard seawater samples, nitrogen-limited seawater samples, and tunicamycin-treated seawater samples, resulting in 6 groups of samples, with 3 parallel controls in each group, for a total of 18 samples. At culture times of 0 h, 12 h, 24 h, 48 h, and 72 h, 1 mL of algal culture was taken from each sample and placed in a 1.5 mL centrifuge tube. Before each sampling, the algal culture was thoroughly mixed, and the sample was diluted 20-fold with cell dilution buffer specifically for cell counters. The cell density of each of the 18 samples was measured using a cell counter. The measured growth curves are shown below. Figure 6 As shown, under nitrogen limitation conditions, the cell density of the wild-type strain at 72 h was 1,081,507 cells / ml, while that of the mutant strain at 72 h was 1,331,333 cells / ml, an increase of 23%, demonstrating that the mutant strain has a significant effect in coping with stress.

[0060] 2-3) Determination of maximum photosynthetic efficiency (Fv / Fm):

[0061] During growth curve determination, 2 mL of algal solution was taken from each of the 18 samples at 48 h and 72 h of culture time and placed in 2 mL centrifuge tubes. The tubes were then incubated in the dark for 15 minutes. A Water-PAM was connected, and the light frequency and intensity were set to a suitable range of PAM GAIN: 200-400. The dark-treated algal solution was then transferred to the Water-PAM's matching cuvette, and the maximum photosynthetic efficiency (Fv / Fm) was measured. The results are as follows: Figure 7 As shown, the overexpression plasmid containing eGFP did not adversely affect the photosynthesis of *Phaeodactylum tricornutum*.

[0062] 2-4) Determination of chlorophyll a:

[0063] During the growth curve determination, 5 mL of algal solution was taken from each of the 18 samples at 48 h and 72 h of culture time and placed into 10 mL centrifuge tubes. The algal solution was filtered using a vacuum filtration device, the filter membrane was recovered, and the filter membrane was placed into a 10 mL brown centrifuge tube containing 5 mL of HPLC-grade methanol. The tube was vortexed for 30 s, keeping the liquid surface completely submerged in the filter membrane, and then placed at 4 ℃ in the dark for 24 h. The samples were then removed and centrifuged at 10000 g for 15 min using a high-speed centrifuge. The supernatant was collected, and the absorbance was measured using a UV spectrophotometer. The absorbance was measured at 750 nm, 665 nm, and 652 nm using methanol as the zeroing agent.

[0064] The chlorophyll a content Cb of *Phaeodactylum tricornutum* is calculated using the following formula:

[0065] Ca=16.29×(A665-A750)-8.54×(A652-A750)

[0066] Cb=(Ca×V 甲醇 ) / (Dcell×V 藻液 )×10 7

[0067] The Cb is the chlorophyll content (ug × 10). 7 cell -1 ); V 甲醇 V represents methanol volume; Dcell represents cell density; V 藻液 This represents the volume of the algal solution. The test results are as follows: Figure 8 As shown, the overexpression plasmid containing eGFP did not adversely affect the photosynthesis of *Phaeodactylum tricornutum*.

[0068] 2-5) Determination of soluble polysaccharides:

[0069] Simultaneously with the growth curve determination, 20 mL of algal solution was taken from each of the 18 samples and placed into 50 mL centrifuge tubes at 48 h and 72 h of culture time. The soluble polysaccharides of *Phaeodactylum tricornutum* were determined using the Plant Soluble Sugar Detection Kit (A145-1-1) sold by Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. The test results are as follows: Figure 9 As shown, under nitrogen-limited conditions, the soluble sugar content accumulated in both wild-type and mutant strains was significantly higher than that in the control group, and the soluble sugar content of the mutant strain was significantly higher than that of the wild-type strain at 24 h, at 5.41 ug × 10⁻⁶. 7 cell -1 5.91 ug × 10 7 cell -1 .

[0070] 3-6) Determination of soluble proteins:

[0071] During growth curve determination, 20 mL of algal culture was taken from each of the 18 samples at 48 h and 72 h of culture time and placed into 50 mL centrifuge tubes. The tubes were centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the algal sludge was collected in 2 mL centrifuge tubes with 1 mL of 1×PBS added. Cell disruption was performed using an ultrasonic cell disruptor, with sonication on for 5 s and off for 5 s, for a total of 10 min. The resulting product was centrifuged at 12000 rpm / min for 30 min at 4 ℃, and the supernatant was collected and kept on ice throughout. Soluble protein concentration was determined using the BCA protein concentration assay kit (enhanced version - P0010) sold by Beyotime Biotechnology. The results are as follows: Figure 10 As shown, in the nitrogen-deficient group samples after 72 h, the soluble protein content of the wild-type strain and the mutant strain was 3.02 ug × 10⁻⁶. 7 cell -1 and 3.43 ug × 10 7 cell -1 There are significant differences.

[0072] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for constructing a mutant strain of *Phaeodactylum tricornutum* overexpressing the calcification protein gene, characterized in that, Including the following steps: Construction of overexpression plasmid: The gene fragment encoding the calcification protein of *Phaeodactylum tricornutum* and the plasmid backbone were obtained and ligated using a recombinant cloning reagent. The sequence of the gene fragment is shown in SEQ ID NO.

3. Transformation verification: The constructed overexpression plasmid was transformed into competent cells, and the correctness of the recombinant sequence of the constructed overexpression plasmid was detected and verified after large-scale culture. Construction of mutant strains: The overexpression plasmid that was verified to be correct was transformed into *Phaeodactylum tricornutum*.

2. The construction method according to claim 1, characterized in that, The method for obtaining the gene fragment encoding the caloric reticulin of *Phaeodactylum tricornutum* is to use *Phaeodactylum tricornutum* as a template and perform PCR amplification using primers shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. The construction method according to claim 1, characterized in that, The plasmid backbone contains a fluorescent label, which is selected from eGFP, mRFP or mCherry.

4. The construction method according to claim 1 or 3, characterized in that, The plasmid backbone was obtained by double digestion of the pPha-T1 plasmid.

5. The construction method according to claim 4, characterized in that, The double enzyme digestion sites are EcoRⅠ and HindⅢ.

6. The construction method according to claim 1, characterized in that, The competent cells are Escherichia coli DH5α competent cells.

7. The construction method according to claim 1, characterized in that, The overexpression plasmid was transformed into *Phaeodactylum tricornutum* using a gene gun.

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