Method for improving cold stress induction of nostoc flagelliforme to synthesize pseudocladodin by knocking out Ferritin-like protein gene

Abstract

The invention discloses a method for improving cold stress induction of nostoc flagelliforme to synthesize pseudocladodin by knocking out a Ferritin-like protein gene, and belongs to the technical field of gene engineering. The method comprises the following steps: firstly, cloning a Ferritin-like regulatory protein gene, designing a target sequence (TargetS) for knocking out the gene and homologous sequences HR at two ends of the target sequence, and respectively recombining the TargetS and the HR in a pCpf1b vector by applying seamless cloning and T4 connection technologies to obtain pCpf1b-TargetS-HR; then, pCpf1b-TargetS-HR is transformed into nostoc flagelliforme by adopting a conjugational transfer method, and a nostoc flagelliforme genetic engineering algal strain is obtained through resistance screening; and finally, inducing synthesis of cladosporin in a low-temperature environment. The method can promote the synthesis of the cladosporin under a non-ultraviolet induction condition, and has an important application value in the fields of sunscreen supplies and medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a method for improving the cold stress-induced synthesis of pseudocladactyl by Nostoc fusca by knocking out a Ferritin-like protein gene. Background Art

[0002] Scytonemin is a fat-soluble yellow-brown dimer compound, which is composed of two indole-phenol subunits symmetrically connected by carbon-carbon bonds, with a molecular weight of 544Da and a maximum absorption peak at 384nm. Scytonemin also has three derivative compounds, namely dimethoxyscytonemin, tetramethoxyscytonemin and scytonemin, but they are relatively rare in nature. Scytonemin has strong resistance to ultraviolet radiation and has strong antioxidant and free radical scavenging abilities. In addition, scytonemin also has the effects of anti-skin inflammation and inhibiting the proliferation of human fibroblasts, endothelial cells and tumor cells. Therefore, as a natural small molecule compound, scytonemin can be used in the cosmetics industry to develop new cosmeceuticals and in the pharmaceutical industry as a drug raw material, but its large-scale production and low-cost production still face huge challenges.

[0003] The synthesis of pseudocladonia in cyanobacteria is mainly induced by ultraviolet radiation (UV-A / B), and other factors such as high light, drought and salt stress also have a certain promoting effect on its biosynthesis. Current research reports mainly focus on improving the culture conditions of cyanobacteria to synthesize pseudocladonia. Although ultraviolet radiation can efficiently induce the synthesis of pseudocladonia, it also seriously inhibits cell growth. The decisive factor limiting the high yield of pseudocladonia is not entirely the improvement of production technology or production process. Its core technology or bottleneck lies in the screening of excellent algae species or the genetic improvement of existing algae species. At present, more than 300 species of cyanobacteria have been found to be able to synthesize pseudocladonia, but most of the culture technology is still immature and the genome sequence is unknown. At present, Nostocflagelliforme (commonly known as hair vegetable) is the algae strain with the greatest potential for the synthesis of pseudocladonia. At present, its genome has been sequenced and the genetic operation system has been established, which can be developed and utilized as an engineered algae strain.

[0004] At present, the molecular pathway of pseudocladactyl biosynthesis has been explored, and its direct synthesis gene cluster contains 17 genes located on an operon. The latest research shows that its biosynthesis process can be divided into four modules: UV-induced regulation module, Scytonemin direct synthesis module, tryptophan (as substrate) synthesis module and extracellular transport module, among which the synthesis of pseudocladactyl monomer is completed intracellularly, and the synthesis of pseudocladactyl (i.e. dimer) is completed in the extracellular periplasmic space. At present, the pseudocladactyl synthesis gene cluster has been successfully heterologously expressed in Escherichia coli, but it can only synthesize pseudocladactyl monomers, and the synthesis efficiency is poor. Therefore, a method that can efficiently and highly produce pseudocladactyl is needed. Studies have found that the regulatory protein gene containing ferritin-like domain (COO91_04051, NCBI) plays an important role in the resistance of Nostoc to low temperature stress, but whether it will affect the synthesis of pseudocladactyl is still unclear. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the object of the present invention is to provide a method for improving the cold stress-induced synthesis of pseudocladoxine by Nostoc fusca by knocking out the Ferritin-like protein gene, so as to solve the technical problems of low efficiency and low yield of the existing pseudocladoxine synthesis method and the cyanobacteria engineering strain.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention discloses the use of knocking out the Ferritin-like regulatory protein gene of Nostoc tricholoma in improving the production of pseudocladactylin, the nucleotide sequence of the Ferritin-like regulatory protein gene is shown in SEQ ID NO.1, and the amino acid sequence of the Ferritin-like regulatory protein is shown in SEQ ID NO.2.

[0008] The second aspect of the present invention discloses a vector for genetically modifying Nostoc fusca to increase the production of pseudocladactin, wherein the vector contains a base sequence that causes the knockout of the Ferritin-like regulatory protein gene in the genome of Nostoc fusca;

[0009] Among them, the nucleotide sequence of the Ferritin-like regulatory protein gene is shown in SEQ ID NO.1, and the amino acid sequence of the Ferritin-like regulatory protein is shown in SEQ ID NO.2.

[0010] Preferably, the vector uses pCpf1b vector as a template, and the recognition sequence of the Ferritin-like regulatory protein gene and homologous sequences at both ends thereof are recombined in the pCpf1b vector.

[0011] The third aspect of the present invention discloses a genetically engineered strain of Nostoc for improving the production of pseudocladonia, wherein the Ferritin-like regulatory protein gene on the genome of the genetically engineered strain of Nostoc for increasing the production of pseudocladonia, has been knocked out; the nucleotide sequence of the Ferritin-like regulatory protein gene is shown in SEQ ID NO.1, and the amino acid sequence of the Ferritin-like regulatory protein is shown in SEQ ID NO.2.

[0012] The fourth aspect of the present invention discloses a method for constructing the genetically engineered strain of Nostoc for increasing the production of pseudocladina. The vector for increasing the production of pseudocladina is introduced into Nostoc cells in the logarithmic phase for homologous recombination, and a genetically engineered strain of Nostoc for producing pseudocladina is obtained through resistance screening and verification.

[0013] Preferably, the vector for increasing the production of pseudocladonia is introduced into Nostoc fulgens cells in the logarithmic phase by conjugation transfer, cultured under light, and subjected to resistance verification and screening to obtain a genetically engineered Nostoc fulgens strain producing pseudocladonia.

[0014] The fifth aspect of the present invention discloses the use of the above-mentioned recombinant vector for increasing the production of pseudocladonia or the genetically engineered algae strain of Nostoc fusca in increasing the production of pseudocladonia.

[0015] The sixth aspect of the present invention discloses a method for improving the cold stress-induced synthesis of pseudocladinocampus by Nostoc fusus by knocking out the Ferritin-like protein gene, wherein the genetically engineered strain of Nostoc fusus for improving the production of pseudocladinocampus is inoculated into a BG11 liquid culture medium and induced at 10°C to 20°C to induce the synthesis of pseudocladinocampus;

[0016] Among them, the nucleotide sequence of the Ferritin-like regulatory protein gene is shown in SEQ ID NO.1, and the amino acid sequence of the Ferritin-like regulatory protein is shown in SEQ ID NO.2.

[0017] Preferably, the light intensity during induction is 40 μmol photos m -2 s -1 .

[0018] Preferably, the induction time is 8 to 16 days.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The invention provides an application of knocking out a Ferritin-like regulatory protein gene in Nostoc tricholoma in improving the yield of pseudocladoxine. The synthesis of pseudocladoxine is induced by knocking out the Ferritin-like regulatory protein gene in Nostoc tricholoma, and it is found that compared with Nostoc tricholoma in which the Ferritin-like regulatory protein gene is not knocked out, the yield and production efficiency of pseudocladoxine in which the gene is knocked out are significantly improved, indicating that knocking out the Ferritin-like regulatory protein gene in Nostoc tricholoma can be used for efficiently producing pseudocladoxine.

[0021] The present invention provides a method for improving the cold stress-induced synthesis of pseudocladina by Nostoc spp. by knocking out the Ferritin-like protein gene. It is found that the Ferritin-like regulatory protein gene can promote the induction expression of pseudocladina synthesis-related genes under low temperature environment, synthesize more pseudocladina, and can be used to develop engineered cyanobacteria for non-ultraviolet induced synthesis of pseudocladina. After knocking out the Ferritin-like regulatory protein gene in Nostoc spp., it is found that the content of pseudocladina is significantly accumulated, proving that the Ferritin-like regulatory protein is a key regulatory factor for the synthesis of pseudocladina by Nostoc spp., and can increase the content of pseudocladina synthesized by Nostoc spp., so the method has important application value in the field of algae genetic engineering, new cosmeceuticals and raw material development in the pharmaceutical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure is the result of constructing the vector for producing pseudocladactyl; A, 800bp upstream homology arm (L-HR) and 800bp downstream homology arm (R-HR) of Ferritin-like regulatory protein gene; B, upstream homology arm and downstream homology arm fusion fragment (HR) for knocking out Ferritin-like regulatory protein gene; C, pCpf1b plasmid extraction; D, pCpf1b plasmid digested with BamH I; E, pCpf1b-HR plasmid construction; F, pCpf1b-HR plasmid digested with Aar I; G, blue-white screening; H, colony PCR identification;

[0023] Figure 2 This is a photograph of a genetically engineered strain of Nostoc fusca (Δcsrnf1) that produces pseudocladactyl;

[0024] Figure 3 This is a statistical chart showing the changes in chlorophyll a (Chl a) content in the genetically engineered Nostoc strain (Δcsrnf1) producing pseudocladactyls under normal temperature of 25°C.

[0025] Figure 4This is a statistical chart showing the changes in chlorophyll a (Chl a) content in the genetically engineered Nostoc strain (Δcsrnf1) producing pseudocladactyls under low temperature treatment of 15°C;

[0026] Figure 5 This is a statistical chart showing the changes in chlorophyll a (Chl a) content in the genetically engineered Nostoc strain (Δcsrnf1) producing pseudocladactyls under low temperature treatment of 10°C;

[0027] Figure 6 This is a statistical graph showing the changes in scytonemin content in the genetically engineered Nostoc strain (Δcsrnf1) that produces scytonemin under normal temperature and different low temperature environments. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0029] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0030] Herein, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0031] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0032] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0033] Herein, the nucleotide sequence of the Ferritin-like regulatory protein gene is shown in SEQ ID NO.1 in Table 1, and the amino acid sequence of the Ferritin-like regulatory protein is shown in SEQ ID NO.2 in Table 1.

[0034] The invention provides a method for improving cold stress-induced synthesis of pseudocladinocampus by Nostoc fusus by knocking out a Ferritin-like protein gene. The method comprises the following steps: firstly, a recognition sequence (TargetS) of a Ferritin-like regulatory protein gene and homologous sequences (L-HR and R-HR) at both ends of the Ferritin-like regulatory protein gene are cloned, and the TargetS sequence and the HR sequence are respectively recombined in a pCpf1b vector by using a seamless cloning technology and a T4 connection technology to obtain a knockout vector of the Ferritin-like regulatory protein gene (i.e., pCpf1b-TargetS-HR); then, the pCpf1b-TargetS-HR is transformed into Nostoc fusus by using a conjugation transfer method, and a genetically engineered algae strain of Nostoc fusus (i.e., Δcsrnf1) is obtained through multiple rounds of resistance screening; and finally, the genetically engineered algae strain of Nostoc fusus is induced in a low temperature environment of 10°C to 20°C for 8 to 16 days to induce synthesis of pseudocladinocampus.

[0035] Table 1 Sequence Listing

[0036]

[0037]

[0038]

[0039]

[0040] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0041] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, are conventional commercial products, and their specifications are conventional specifications in the art.

[0042] The experimental methods for which specific conditions are not specified in the following examples are generally performed under conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer.

[0043] In the following examples, unless otherwise specified, "Spe" means "spectinomycin", "Cm" means "chloramphenicol" and "Amp" means "ampicillin".

[0044] Example 1 Construction of the TargetS sequence of the Ferritin-like regulatory protein gene of Nostoc fusca and homologous sequences at both ends of the knockout gene

[0045] 1. Clone the homology arms into the pCpf1b vector

[0046] 1. Target gene amplification: Using the genome of Nostoc hair-like as a template, PCR amplified the upstream and downstream homology arms HR of the Ferritin-like regulatory protein gene. The nucleotide sequences of the upstream and downstream homology arms are shown in SEQ ID NO.3 and SEQ ID NO.4 in Table 1, respectively. The PCR reaction system is shown in Table 2, and the reaction procedure is shown in Table 3. The size of the PCR product band is detected by gel electrophoresis, and the amplified fragments are recovered using a DNA fragment gel recovery kit. The operating steps refer to the instructions of the Qingke Biotechnology DNA Fragment Gel Recovery Kit.

[0047] Table 2 PCR reaction system of upstream and downstream homology arms of Ferritin-like regulatory protein gene

[0048]

[0049] Table 3 PCR reaction procedures for upstream and downstream homology arms of Ferritin-like regulatory protein genes

[0050]

[0051] Test results such as Figure 1 As shown in A, after PCR amplification, an upstream homology arm with a size of 800 bp was obtained, and a downstream homology arm with a size of 800 bp was obtained.

[0052] 2. Homology arm fusion: The upstream and downstream homology arms of the Ferritin-like regulatory protein gene were connected by fusion PCR. The homology arm fusion program of the Ferritin-like regulatory protein gene is shown in Table 4 to obtain the fused HR. The fused HR was then amplified by PCR. The PCR reaction system is shown in Table 5. The PCR reaction program is shown in Table 6. The band size of the PCR product was detected by gel electrophoresis. The amplified fragment was recovered by a DNA fragment gel recovery kit. The operating steps refer to the instructions of the Qingke Biotechnology DNA Fragment Gel Recovery Kit.

[0053] Table 4 Ferritin-like regulatory protein gene homology arm fusion program

[0054]

[0055] Table 5 Ferritin-like regulatory protein gene fusion homology arm PCR reaction system

[0056]

[0057] Table 6 Ferritin-like regulatory protein gene fusion homology arm PCR reaction program

[0058]

[0059] Test results such as Figure 1 As shown in B, the fused HR was amplified by PCR and its size was 1600 bp.

[0060] 3. Plasmid extraction: Extract pCpf1b plasmid with reference to Zhuangmeng Large Plasmid Extraction Kit. Detect the extracted plasmid by gel electrophoresis.

[0061] Test results such as Figure 1 As shown in C, nucleic acid electrophoresis shows that the pCpf1b plasmid was successfully extracted.

[0062] 4. Enzyme digestion: The pCpf1b obtained in step 3 was digested with BamH I at 30°C for 30 min. The digestion system was as shown in Table 7. The digestion effect was detected by gel electrophoresis. The pCpf1b after digestion was recovered by hand using the method of extracting the genome of Nostoc fusca cells with reference to the CTAB method.

[0063] Table 7 Vector pCpf1b and fusion HR restriction enzyme system

[0064]

[0065]

[0066] Test results such as Figure 1As shown in D, in nucleic acid electrophoresis, the pCpf1b after restriction digestion moves slower than the original plasmid band.

[0067] 5. Ligation: The above-mentioned recovered fragments (i.e., the fused HR recovered in step 2 and the pCpf1b recovered by enzyme digestion in step 4) were connected using the In-fusion method (Shanghai Biotech Seamless Cloning Kit). The connection system is shown in Table 8. The connection temperature is 50°C, the time is 50 min, and the ice bath is 2 min to obtain the In-fusion connection system.

[0068] Table 8 In-fushion connection system

[0069]

[0070] 6. Transformation: Use heat shock transformation to transform the ligated fragments into DH5α competent cells. Add 100 μL of DH5α competent cells to the In-fusion ligation system in step 5, place on ice for 10 min, then place in a 42°C water bath for 60 s, ice bath for 2 min, and finally add 800 μL of liquid LB, and culture at 37°C shaker (120 rpm) for 1 h to obtain the transformed bacterial solution.

[0071] 6. Colony PCR identification: Take 200 μL of the transformed bacterial solution in step 5 and spread it on the LB solid plate. Screen the resistance to spectinomycin (Spe) at a concentration of 175 μg / mL and culture at 37°C for 12 to 16 hours. Pick the white colonies for colony PCR identification, extract the plasmid (pCpf1b-HR) of the positive colonies and send it to the company for sequencing identification. The test results are as follows: Figure 1 As shown in middle E, the target band appeared after colony PCR identification, with a size of 1600 bp.

[0072] 2. Cloning the target sequence into pCpf1b-HR

[0073] 1. Target sequence fusion: Take 5 μL of the upstream and downstream primers of TargetS for fusion respectively. The TargetS sequence is shown in SEQ ID NO.5 in Table 1. The upstream and downstream primer sequences are shown in SEQ ID NO.6 and SEQ ID NO.7 in Table 1 respectively. The fusion procedure is shown in Table 9:

[0074] Table 9 Fusion procedures

[0075]

[0076] 2. Enzyme digestion: pCpf1b-HR obtained in step 1 was digested with Aar I. The pCpf1b-HR digestion system is shown in Table 10. The temperature is 37° C. and the time is 2 h. Gel electrophoresis is used to detect whether the digestion is normal. The plasmid after digestion is hand-picked and recovered. The method refers to the CTAB method for extracting the genome of Nostoc cells.

[0077] Table 10pCpf1b-HR restriction enzyme system

[0078]

[0079] Test results such as Figure 1 As shown in F, in nucleic acid electrophoresis, the pCpf1-HR after restriction digestion moved slower than the original plasmid band.

[0080] 3. Ligation: Use T4 ligation technology to connect the TargetS fused in step 1 and the pCpf1b-sp-HR recovered by enzyme digestion in step 2. The T4 ligation system is shown in Table 11. The ligation is carried out at 25°C for 1.5 h.

[0081] Table 11T4 connection system

[0082]

[0083]

[0084] 4. Transformation: Use heat shock transformation method to transform the ligation system into DH5α competent cells. Add 80 μL of DH5α competent cells to the T4 ligation system obtained in step 3, place on ice for 10 minutes, incubate in 42°C water bath for 60 seconds, incubate on ice for 2 minutes, and finally add 800 μL of liquid LB, and culture at 37°C shaker (120 rpm) for 1 hour.

[0085] 5. Blue-white screening: Take 200 μL of the transformed bacterial solution in step 5 and spread it on a LB solid plate (containing 175 μg / mL Spe, 40 μL 50 mg / mL IPTG, and 16 μL 20 mg / mL X-gal), and culture at 37°C for 12-16 hours. Figure 1 As shown in G, white colonies were picked for colony PCR identification. Figure 1 As shown in H, the plasmid extracted from the positive colony was sent to the company for sequencing identification, and the results showed that the knockout vector was pCpf1b-TargetS-HR.

[0086] Example 2 Transformation of Nostoc pubescens

[0087] 1. The correctly sequenced recombinant plasmid (pCpf1b-TargetS-HR) was transformed into HB101 competent cells by heat shock transformation method, and positive clones were identified by colony PCR. Positive single clones were picked and inoculated into liquid LB (containing 175μg / mL Spe, 25μg / mL Cm and 25μg / mL Amp) and cultured in a shaking incubator at 37℃ until OD 600 The value reached 0.6 to 0.8. Centrifuge the HB101 bacterial solution at 4000 rpm for 5 min, remove the supernatant, rinse three times with liquid LB without antibiotics to remove the antibiotics in the culture medium, and finally add 0.5 mL of liquid LB to resuspend the bacteria.

[0088] 2. Take 100 mL of logarithmic phase Nostoc cells, centrifuge at 8000 rpm for 10 min, remove the supernatant, rinse with 0.1 M NaCl solution 2 to 3 times, and finally add 5 mL of BG11 liquid culture medium without antibiotics to resuspend the Nostoc cells.

[0089] 3. Mix 0.5 mL of the bacterial solution from step 1 and 5 mL of the Nostoc cells from step 2 and incubate in a light incubator at 25°C with weak light (15-20 μmol photons m -2 s -1 ) was cultured for 1 hour to obtain a bacterial-algal mixed solution.

[0090] 4. Take the sterilized 0.22 μm nitrocellulose film and spread it on a BG11 solid plate without antibiotics, and blow dry the surface moisture in a clean bench.

[0091] 5. Take the bacteria-algae mixed solution obtained in step 3 and spread it evenly on the plate in step 4, and blow dry it in a clean bench.

[0092] 6. Place the plate from step 5 in a light incubator for 24 h at 25 °C and a light intensity of 40 μmolphotons m -2 s -1 .

[0093] 7. In a clean bench, transfer the nitrocellulose membrane containing algae solution in step 6 to a BG11 solid plate (containing 30 μg / mL Spe).

[0094] 8. Place the plate from step 7 in a light incubator at a temperature of 25°C and a light intensity of 40 μmolphotons m -2 s -1 , transformants will appear in about 3 to 4 weeks.

[0095] Example 3 Identification of genetically engineered Nostoc fusca strains

[0096] 1. The transformants obtained in Example 2 were streaked and cultured on a BG11 solid plate (containing 30 μg / mL Spe) at a temperature of 25°C and a light intensity of 40 μmol photons m -2 s -1 , transfer 2 to 3 generations, each generation takes about 2 weeks.

[0097] 2. The transformants from step 1 were selected and inoculated into BG11 liquid medium without antibiotics. The culture temperature was 25°C and the light intensity was 40 μmol photons m -2 s -1 , transfer 2 to 3 times, each time taking about 2 weeks, in order to discard the exogenous plasmid.

[0098] 3. Collect the algae liquid from step 2, extract the algae cell genome using the CTAB method, and perform PCR identification. The PCR identification procedure is shown in Table 12. The correct identification result of the genetically engineered algae strain of Nostoc fusca is Δcsrnf1( Figure 2 ) for subsequent experiments.

[0099] Table 12 PCR identification procedure

[0100]

[0101] Example 4 Growth phenotype analysis of genetically engineered Nostoc fusca strains at normal temperature 25°C

[0102] Will be in the logarithmic phase (OD 750 =0.4-0.5) of Nostoc cells (WT and Δcsrnf1) were dispensed into 250 mL Erlenmeyer flasks and filled to 100 mL with BG11. The initial OD 750 Controlled at around 0.1. At a light intensity of 40 μmol photos m -2 s -1 , and cultured statically at normal temperature of 30℃ for 16 days, and then samples were taken to determine the chlorophyll a content. The chlorophyll a content was determined as follows:

[0103] Take 5 mL of algae solution, centrifuge at 6000 rpm for 5 min, discard the supernatant, add 5 mL of 95% ethanol solution and mix well by pipetting, place at 4 ° C in the dark for more than 12 hours, then centrifuge at 6000 rpm for 5 min, take the supernatant and measure the absorbance at 664.1 nm and 648.6 nm. The chlorophyll a (Chl a) content is calculated as follows:

[0104] Chlorophyll a (mg / L) = 13.36A 664.1 -5.19A 648.6

[0105] The experimental results are as follows Figure 3As shown, within 16 days of treatment at normal temperature of 25℃, the chlorophyll a content of Nostoc fusca WT and Δcsrnf1 was basically the same, indicating that the knockout of the Ferritin-like regulatory protein gene did not affect the growth of algal cells under normal temperature conditions.

[0106] Example 5 Growth phenotype analysis of genetically engineered Nostoc fusca strains treated at low temperature of 15°C

[0107] Will be in the logarithmic phase (OD 750 =0.4-0.5) of Nostoc cells (WT and Δcsrnf1) were dispensed into 250 mL Erlenmeyer flasks and made up to 100 mL with BG11. The initial OD 750 Controlled at around 0.1. At a light intensity of 40 μmol photos m -2 s -1 , and cultured statically at low temperature of 15℃ for 16 days, and then samples were taken to determine the chlorophyll a content. The chlorophyll a content was determined as follows:

[0108] Take 5 mL of algae solution, centrifuge at 6000 rpm for 5 min, discard the supernatant, add 5 mL of 95% ethanol solution and mix well by pipetting, place at 4 ° C in the dark for more than 12 hours, then centrifuge at 6000 rpm for 5 min, take the supernatant and measure the absorbance at 664.1 nm and 648.6 nm. The chlorophyll a (Chl a) content is calculated as follows:

[0109] Chlorophyll a (mg / L) = 13.36A 664.1 -5.19A 648.6

[0110] The experimental results are as follows Figure 4 As shown, within 16 days of low temperature treatment at 15℃, the chlorophyll a content of Nostoc fusca WT and Δcsrnf1 was basically the same, indicating that knockout of the Ferritin-like regulatory protein gene did not affect the growth of algal cells.

[0111] Example 6 Growth phenotype analysis of genetically engineered Nostoc fusca strains treated at low temperature of 10°C

[0112] Will be in the logarithmic phase (OD 750 =0.4-0.5) of Nostoc cells (WT and Δcsrnf1) were dispensed into 250 mL Erlenmeyer flasks and made up to 100 mL with BG11. The initial OD 750 Controlled at around 0.1. At a light intensity of 40 μmol photos m -2 s -1 , and cultured statically at low temperature of 10℃ for 16 days, and then samples were taken to determine the chlorophyll a content. The chlorophyll a content was determined as follows:

[0113] Take 5 mL of algae solution, centrifuge at 6000 rpm for 5 min, discard the supernatant, add 5 mL of 95% ethanol solution and mix well by pipetting, place at 4 ° C in the dark for more than 12 hours, then centrifuge at 6000 rpm for 5 min, take the supernatant and measure the absorbance at 664.1 nm and 648.6 nm. The chlorophyll a (Chl a) content is calculated as follows:

[0114] Chlorophyll a (mg / L) = 13.36A 664.1 -5.19A 648.6

[0115] The experimental results are as follows Figure 5 As shown, within 16 days of low temperature treatment at 10℃, the chlorophyll a content of Δcsrnf1 was significantly lower than that of WT, indicating that the knockout of the Ferritin-like regulatory protein gene would affect the growth of algal cells in a low temperature environment to a certain extent.

[0116] Example 7 Analysis of the content of pseudocladactylin in genetically engineered Nostoc fusca strains at normal temperature 25°C and at different low temperature treatments

[0117] Will be in the logarithmic phase (OD 750 =0.4-0.5) of algal cells (WT and Δcsrnf1) were divided into 250 mL Erlenmeyer flasks and filled to 100 mL with BG11 (containing nitrogen). The initial OD 750 Controlled at around 0.1. At a light intensity of 40 μmol photos m -2 s -1 , and then cultured statically for 16 days at low temperatures of 15°C and 10°C, and samples were taken to determine the content of pseudocladonia solani, while the control temperature was 25°C. The steps for extracting pseudocladonia solani are as follows:

[0118] Take 5 mL of algae liquid, freeze-dry it and weigh it. Then, moisten the dried hairy Nostoc algae and add 2 mL of 100% acetone. Extract it in the dark overnight. Then filter it with a 0.22 μm organic filter membrane. The absorbance of the filtrate is measured at 384 nm, 490 nm and 663 nm. Corrected absorbance calculation method: A 384 =1.04A 384 -0.79A 663 -0.27A 490 The calculation formula of pseudoclase content is as follows:

[0119] A = KbC, where: A is the corrected absorbance; K (molar absorptivity) is 112.6 L / (gcm); b: cuvette thickness (cm); C: substance concentration (mg / mL).

[0120] The experimental results are as follows Figure 6As shown in the figure, within 16 days of normal temperature treatment at 25℃, Nostoc (WT and Δcsrnf1) did not produce pseudocladina, while low temperature treatment could induce the synthesis of pseudocladina. After the csrnf1 gene was deleted, the content of pseudocladina could be further increased. After low temperature treatment at 15℃, Δcsrnf1 first achieved a breakthrough from zero to a certain amount of pseudocladina. After low temperature treatment at 10℃, the content of pseudocladina was further increased, and its content was 2.38 times that of WT.

[0121] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. The use of knocking out the Ferritin-like regulatory protein gene of Nostoc pubescens in increasing the production of pseudocladactylin, characterized in that: The nucleotide sequence of the Ferritin-like regulatory protein gene is shown in SEQ ID NO.1, and the amino acid sequence of the Ferritin-like regulatory protein is shown in SEQ ID NO.

2.

2. A carrier for increasing the yield of pseudoclase, characterized in that: The vector contains a base sequence that causes the knockout of the Ferritin-like regulatory protein gene in the genome of Nostoc tricholoma; Among them, the nucleotide sequence of the Ferritin-like regulatory protein gene is shown in SEQ ID NO.1, and the amino acid sequence of the Ferritin-like regulatory protein is shown in SEQ ID NO.

2.

3. A carrier for increasing the production of pseudomycotina according to claim 2, characterized in that: The vector uses the pCpf1b vector as a template, and recombines the target sequence of the Ferritin-like regulatory protein gene and the homologous sequences at both ends thereof into the pCpf1b vector.

4. A genetically engineered strain of Nostoc for increasing the production of pseudocladonia, characterized in that: The Ferritin-like regulatory protein gene on the genome of the genetically engineered Nostoc tricholoma strain has been knocked out; the nucleotide sequence of the Ferritin-like regulatory protein gene is shown in SEQ ID NO.1, and the amino acid sequence of the Ferritin-like regulatory protein is shown in SEQ ID NO.

2.

5. The method for constructing a genetically engineered strain of Nostoc for improving the production of pseudocladonia solani according to claim 4, characterized in that: The vector for increasing the production of pseudocladonia solani according to claim 2 or 3 is introduced into Nostoc solani cells in the logarithmic phase for homologous recombination, and a genetically engineered Nostoc solani strain producing pseudocladonia solani is obtained through resistance screening and verification.

6. The method for constructing a genetically engineered strain of Nostoc for improving the production of pseudocladonia according to claim 5, characterized in that: The recombinant vector for increasing the yield of pseudocladonia solani according to claim 2 or 3 is introduced into Nostoc solani cells in the logarithmic phase by conjugation transfer, and a genetically engineered Nostoc solani strain producing pseudocladonia solani is obtained by resistance screening and verification.

7. Use of the recombinant vector for increasing the production of pseudocladinapine according to claim 2 or 3 or the genetically engineered strain of Nostoc fusca according to claim 4 in increasing the production of pseudocladinapine.

8. A method for improving the cold stress-induced synthesis of pseudocladactyl by Nostoc fusca by knocking out the Ferritin-like protein gene, characterized in that: The genetically engineered Nostoc strain for increasing the production of pseudocladonia solani according to claim 4 or the genetically engineered Nostoc strain obtained by the construction method according to claim 5 or claim 6 is inoculated into a BG11 liquid culture medium, and synthesizes pseudocladonia solani under induction at 10° C. to 20° C.; Among them, the nucleotide sequence of the Ferritin-like regulatory protein gene is shown in SEQ ID NO.1, and the amino acid sequence of the Ferritin-like regulatory protein is shown in SEQ ID NO.

2.

9. The method of claim 8, wherein the method comprises: The light intensity during induction was 40 μmolphotos m -2 s -1 .

10. The method of claim 8, wherein the method comprises: The induction time is 8 to 16 days.

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