CN106916775A - Transgenic algae strain of high-yield nano polyphosphoric acids body and preparation method thereof - Google Patents

Abstract

The invention provides a transgenic algae strain with high yield of nano-polyphosphate body and its preparation method. The transgenic algae strain is a transgene obtained by introducing a recombinant pSyn_1 plasmid containing a homologous ppk gene on the basis of Synechococcus 7002 and identifying it through screening. microalgae. The ability of the transgenic algae strain to produce polyphosphate is 90-150% higher than that of the wild strain, and the method of inducing and accumulating polyphosphate is to use 0.5-5 μM nickel ions for induction. The algae strain has strong operability, good genetic stability, and the foreign gene can be continuously expressed for more than one year. The polyphosphoric acid body produced by the algae strain is at the nanometer level, and the size is in the range of 100 nm, and a good nanometer shape can be maintained during the extraction process.

Description

Transgenic algae strain with high yield of nano polyphosphate body and preparation method thereof

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a transgenic algae strain with high yield of nanometer polyphosphoric acid body.

Background technique

Mineral nutritional deficiencies, especially calcium, iron, and zinc deficiencies, are one of the most serious public health problems in the world today. Higher than vitamin nutritional deficiencies. Negative health effects of mineral malnutrition are established early in life and can lead to stunted growth, poor cognition, lethargy, difficulty concentrating and greater susceptibility to major infectious diseases, reducing an individual's educational attainment. ability, physical capacity, and life expectancy. Mineral nutrition intervention is particularly important for people to get rid of the "poverty trap" of mineral nutrition deficiency and improve people's health.

Biomineralized nanostructures have unique application advantages in the practice of mineral nutrition intervention. Polyphosphate is a biomineralized structure mainly composed of long-chain polyphosphates present in many biological cells. Many biological cells (such as prokaryotic and eukaryotic microorganisms, protozoa, mammalian platelet cells and osteoblasts, etc.) synthesize long chains (ranging from tens to hundreds of residues) of polyphosphate (PolyP), and With this as the main component, it constitutes a nanometer or submicron-scale inclusion body - Polyphosphate bodies (PPB), also known as Volutingranules or Acidocalcisomes. In recent years, many studies have proved that polyphosphate bodies are related to various biological functions. Different from the short-chain PolyP commonly used in the food industry, long-chain PolyP from biological sources is difficult to be hydrolyzed by alkaline phosphatase in the intestinal tract of mammals, so it can reach the intestinal tract through the mouth and endocytosis mediated by caveolin It is absorbed by intestinal epithelial cells and plays a prebiotic role in protecting intestinal tissues from oxidative damage, inflammation, fibrosis and carcinogenesis. Polyphosphate can provide cells with raw materials for DNA synthesis and store or provide mineral elements needed by cells, and has the potential to be developed as a natural nano-mineral nutritional supplement or enhancer. Although the functional properties of polyphosphate bodies have been gradually recognized, there is no case of using microorganisms for large-scale fermentation production. Choosing a suitable "cell factory" for polyphosphate body production is of great significance for its development and application.

Synechococcus 7002 is a model microalgae in the sea, and it is also the fastest dividing cyanobacteria found so far. It has a natural exogenous DNA transformation system, and its whole genome has also been deciphered. It is currently recognized as an ideal "Engineering microalgae" construction platform. Multiple strains of transgenic Synechococcus sp. 7002 were successively constructed, which have the potential to produce various active substances by large-scale fermentation.

Although the method of transgenic microalgae is relatively mature, it still faces many problems and challenges such as low success rate, difficulty in inducing expression, low expression efficiency, and low quality of expression products. At the same time, the expression of some foreign genes will affect the growth of microalgae. Dynamics come with some downsides. Therefore, in the process of transgenic microalgae, it is very important to select the appropriate algae strain, plasmid and target gene. An algal strain with good genetic stability and high expression efficiency is of great significance for improving the comprehensive utilization value of microalgae.

Nanoscale active substances have very obvious advantages in function. Synechococcus can accumulate polyphosphate, and the particle size is nanoscale, which is easier to be absorbed and utilized by cells. The accumulation of polyphosphate bodies is regulated by the ppk gene, and the overexpression of the ppk gene in Synechococcus can be induced to achieve the purpose of excessive accumulation of nano-polyphosphate bodies by introducing the recombinant ppk gene exogenous plasmid. Selecting appropriate homologous genes and plasmids containing promoters that can be easily activated by the corresponding algae strains during the transgenic process will greatly increase the success rate of exogenous gene expression. Compared with other microorganisms, using Synechococcus to express related exogenous genes has many incomparable advantages: (1) Synechococcus 7002 is a natural competent cell, which can absorb exogenous plasmids in a natural state, which is convenient for exogenous gene expression. Gene introduction; (2) As a photoautotrophic organism, microalgae have fast division speed and high production efficiency, and can use seawater for large-scale production under light without causing problems such as encroaching on cultivated land and wasting energy. Mineral elements can enrich the elemental composition of polyphosphate and improve its functional characteristics; (3) The nutritional value of microalgae itself is very high. While the transgenic microalgae produces related active substances, the obtained algae and the fermentation process produce The secondary metabolites can be used in functional health food and drug development for high-value utilization; (4) The expression of exogenous genes is stable and the genetic stability is good.

Obtaining high-yielding nano-polyphosphate algae strains through transgenic methods is of great significance for improving the research and utilization value of microalgae and the wide application of nano-polyphosphate.

Contents of the invention

The purpose of the present invention is to provide a transgenic algae strain with high yield of nano polyphosphate and its preparation method. In the present invention, on the basis of the Synechococcus 7002 strain, a recombinant pSyn_1 plasmid containing a ppk homologous gene is introduced, and the purpose of accumulating nanometer polyphosphoric bodies is achieved by inducing the expression of the target gene. The algae strain can rapidly accumulate a large amount of nanometer polyphosphate bodies, has good genetic stability and strong operability.

Another aspect of the present invention relates to the application of the above-mentioned transgenic algae strain for fermenting and producing nano-polyphosphate; the nano-polyphosphate can be applied to the development of functional products for improving intestinal immunity.

In order to realize the object of the invention, the concrete technical scheme that the present invention takes is:

A transgenic algae strain with high yield of nanometer polyphosphoric body, the algae strain is a transgenic Synechococcus algae 7002 algae strain obtained by introducing a recombinant pSyn_1 plasmid containing a ppk homologous gene on the basis of the Synechococcus algae 7002 algae strain.

The preparation method of the above-mentioned transgenic algae strain with high yield of nano-polyphosphate comprises the following steps:

1) Extraction, cloning and modification of the target gene: extract the genome of Synechococcus sp. 7002 , design specific primers according to the sequence of the Synechococcus sp. The target gene was obtained by PCR and purified for later use;

2) The target gene is recombined into the cloning plasmid, the cloning plasmid and the expression plasmid are propagated by Escherichia coli, and the two plasmids are extracted and purified respectively;

3) Digest the above two kinds of plasmids obtained by extraction with two identical restriction enzymes respectively, and the digested products are recovered and purified by gel cutting, and the purified expression plasmid and ppk gene are purified according to the molar concentration of 1:5-1 :10 ratio, using DNA ligase to connect to obtain recombinant expression plasmid, carry out cloning and propagation by Escherichia coli, extract and purify for subsequent use;

4) Add the recombinant expression plasmid purified in step 3) to Synechococcus sp. 7002 for natural transformation, and then use a solid plate containing spectinomycin hydrochloride to screen the transformed strain;

5) Plasmid extraction was performed on the screened algal strains, amplified with ppk gene-specific primers, and sequence identification was performed on the obtained amplified products to obtain Synechococcus 7002 strains with recombinant ppk genes.

The expression plasmid in the above step 2) is preferably a pSyn_1 expression plasmid.

In the above step 3), use DNA ligase to ligate at 16°C for 1-3 h.

The above step 4) is specifically, adding the recombined plasmid in step 3) to Synechococcus sp. 7002 in a good production state, OD ₇₅₀ =1-2, washed with 10-15 times concentrated fresh culture medium, and performing natural transformation, Transformation was enough for 24 h, and then the transformed strains were screened on a solid plate containing antibiotics.

The application of the above-mentioned transgenic strains of high-yielding nano-polyphosphate bodies on high-yield nano-polyphosphate bodies; wherein, 0.5-5 μM nickel ions are added to the culture medium of the transgenic strains to induce expression; and Synechococcus sp. When the 7002 transgenic algal strain was cultured, 10 μg/L spectinomycin hydrochloride was added to the medium.

The advantages of the present invention are:

1. The present invention skillfully utilizes microalgae to produce nano-polyphosphate, the production method is green and environmentally friendly, and the by-products (algal residues, secondary metabolites, etc.) are of high research and utilization value. 2. Using the model microalgae Synechococcus sp. 7002 for transgenic operation, the operation is simple, the success rate is high, and the genetic stability is good. 3. The introduced gene is the homologous ppk gene of Synechococcus sp. 7002. The exogenous plasmid pSyn_1 contains a cyanobacteria-derived promoter, which is easier to translate and express. There is no quality problem in the polyphosphate body stimulated by the expression of the homologous gene. 4. Nickel ions are used to induce the start of the exogenous plasmid promoter to promote the excessive accumulation of polyphosphate in transgenic microalgae. The method is simple and convenient; Nanoscale, good stability, smaller particle size, easier to be absorbed and utilized.

At the same time, polyphosphate bodies can enrich mineral elements abundant in seawater, and may have better functional properties. Apparently, the present invention has obtained the transgenic algae strain with high yield of nano-polyphosphate which is of high value for research and utilization, and obtained the method for preparation of algae strain and induced expression of exogenous gene, which has a very broad market prospect.

Description of drawings

Figure 1 is the electropherogram of recombinant plasmid construction; among them, A: lane a: ppk and pEASY-Blunt zero recombinant cloning plasmid; lane b: double digestion of cloning plasmid; lane c: pSyn_1 expression plasmid; d: double digestion of pSyn_1 plasmid; Lane M: 1 Kb DNA Ladder; B: Lane a: ppk and pSyn_1 recombinant plasmid; Lane b: single digestion of plasmid; lane c: double digestion of plasmid; lane M: 1 Kb DNA Ladder.

Fig. 2 is an electron microscope picture of Synechococcus sp. 7002 cells; wherein A: wild type; B: transgenic type.

Fig. 3 is the particle size of nano polyphosphate body and electron microscope analysis diagram.

Figure 4 is the effect of nickel ion concentration on the yield of nano polyphosphate.

detailed description

The method of the present invention will be further described below in conjunction with examples, and the experimental methods that do not indicate specific conditions in the embodiments can usually be carried out under conventional conditions.

Example 1:

1) Acquisition of the target gene

Specific primer design:

ppk pre-primer: CCCAAGCTTGAAGGAGATAAAAAGTATATGTCCTCTGCG

ppk post primer: CGGGGTACCCTAATCCAAGCTGTCCTGGAG

Hind III and Kpn I restriction sites were added to the front and back primers respectively, and the RBS sequence of the GAAGGAG sequence was added to the front primer.

PCR conditions: 50 μL reaction system, 94°C for 5 min, (94°C for 30 s, 60°C for 40 s, 1 min and 72°C) for 30 cycles; 72°C for 10 min.

The target gene was purified and cloned by conventional methods for later use.

2) Construction of recombinant pSyn_1 plasmid: pSyn_1 plasmid and target gene were double digested with Hind III and Kpn I, and the digested product was purified and then ligated. 100 ng of pSyn_1 plasmid and 500 ng of ppk gene were ligated with T4 DNA ligase at 16 °C for 3 h, The ligated plasmid was cloned by conventional methods, digested with restriction enzymes to identify the correctness of the plasmid, and purified for later use.

3) Construction and screening of recombinant transgenic algae strains: 100 ng of the purified recombinant plasmid was added to 100 μL of Synechococcus 7002 algae liquid ( OD ₇₅₀ =1) washed with 15 times concentrated fresh medium, and statically After culturing for 24 h, 5 μL was spread on a solid plate containing 10 μg/L spectinomycin hydrochloride for screening, and the monoclonal algal strains obtained from the screening were identified to obtain transgenic algal strains with recombinant ppk genes.

4) The transgenic algal strains were cultured in a liquid medium containing 10 μg/L spectinomycin hydrochloride, and 2 μM nickel nitrate was added to induce the transgenic algal strains, and the content of nano-polyphosphate was detected after 1 week of cultivation. The content of nano polyphosphate in the induced algae strain increased by 90% compared with the wild strain.

Example 2:

1) Acquisition of the target gene

Specific primer design:

ppk pre-primer: CCCAAGCTTGAAGGAGATAAAAAGTATATGTCCTCTGCG

ppk post primer: CGGGGTACCCTAATCCAAGCTGTCCTGGAG

Hind III and Kpn I restriction sites were added to the front and back primers respectively, and the RBS sequence of the GAAGGAG sequence was added to the front primer.

PCR conditions: 50 μL reaction system, 94°C for 5 min, (94°C for 30 s, 60°C for 40 s, 1 min and 72°C) for 30 cycles; 72°C for 10 min.

The target gene was purified and cloned by conventional methods for later use.

2) Construction of recombinant pSyn_1 plasmid: pSyn_1 plasmid and target gene were double digested with Hind III and Kpn I, and the digested product was purified and then ligated. 100 ng of pSyn_1 plasmid and 1 μg of ppk gene were ligated with T4 DNA ligase at 16 °C for 1 h , the ligated plasmid was cloned by conventional methods, digested to identify the correctness of the plasmid, and purified for later use.

3) Construction and screening of recombinant transgenic algae strains: 100 ng of the purified recombinant plasmid was added to 100 μL of Synechococcus 7002 algae liquid ( OD ₇₅₀ =2) washed with 10-fold concentrated fresh medium, and statically After culturing for 24 h, 5 μL was spread on a solid plate containing 10 μg/L spectinomycin hydrochloride for screening, and the monoclonal algal strains obtained from the screening were identified to obtain transgenic algal strains with recombinant ppk genes.

4) The transgenic algal strains were cultured in liquid medium containing 10 μg/L spectinomycin hydrochloride, and 1 μM nickel nitrate was added to induce the transgenic algal strains, and the content of nano-polyphosphate was detected after 1 week of cultivation. The content of nano polyphosphate in the induced algae strain increased by 150% compared with the wild strain.

As shown in Figure 1, after the two plasmids in Figure A were double digested, they were cut into two segments containing corresponding restriction sites. The fragment of about 2000 bp in lane b is the ppk fragment of the target gene, and the fragment of about 4000 bp in lane d is the cut plasmid. There is only one band in lane d because the other cut part is very short and cannot be observed in the electrophoresis results. As shown in Figure 1.B, the recombinant pSyn_1 plasmid containing the ppk gene was digested with a single enzyme to obtain a fragment of about 6000 bp, the target gene was about 2000 bp and the pSyn_1 plasmid was about 4000 bp, and the expected molecular weight was obtained after double digestion The size of the plasmid and the target gene, and the results of enzyme digestion and identification prove that the plasmid was successfully constructed. Combined with the results of gene sequencing, it indicated that the recombinant plasmid was constructed successfully.

As shown in Figure 2, the black spots in the figure are polyphosphoric bodies, and it can be seen that the number of polyphosphoric bodies in transgenic cells (8-11) is significantly more than that in wild-type cells (2-5), and The particle size of polyphosphate is mostly below 100 nm.

As shown in Figure 3, Figure 3.A shows the particle size analysis results of nano-polyphosphoric acid bodies obtained by boiling, purification, etc. The particle size results show that most of the particle sizes are below 100 nm. There are many kinds of particle sizes. Electron microscope observation was carried out on the extracted nano-polyphosphate, as shown in Figure 3.B. It can be seen that the nano-polyphosphate maintained a good nano-morphology after extraction.

As shown in Figure 4, nickel ions can induce the expression of pSyn_1 plasmid, and the induction effects of different concentrations of nickel ions are different. The wild group is the wild-type control group, and other groups have added different concentrations of nickel ions. The content of phosphoric acid body, but the DAPI fluorescence method reflects the PolyP content on the surface of polyphosphoric acid body, so the actual yield is greater than the value reflected by the fluorescence value.

The yield of nano polyphosphates prepared by the transgenic algal strains obtained in the present invention is 90-150% higher than that of stramenopile strains, and the transgenic microalgae have better environmental adaptability, including temperature tolerance and heavy metal ion tolerance ability. The PPB accumulated by the algae strains in the present invention is in the nanometer scale, and the particle size ranges from more than ten nanometers to one hundred nanometers. Compared with polyphosphoric acid bodies from other biological sources, the particle size is smaller, and the isolated nanometer The stability of the polyphosphate body is good. The algae strain in the present invention has good genetic stability and has been stably expressed for more than one year at present.

sequence listing

<110> Ocean University of China

<120> Transgenic algae strain with high yield of nano polyphosphate and its preparation method

<130> 2017

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Claims (7)

1. A transgenic algae strain of high-yield nanometer polyphosphoric acid body, it is characterized in that, this algae strain is on the basis of Synechococcus 7002 algae strain, imports the transgenic aggregate that contains the recombinant pSyn_1 plasmid of ppk homologous gene and obtains algae 7002 strains. 2. the preparation method of the transgenic algae strain of high-yield nano polyphosphate body described in claim 1, is characterized in that, comprises the steps: 1) Extraction, cloning and modification of the target gene: extract the genome of Synechococcus sp. 7002 , design specific primers according to the sequence of the Synechococcus sp. The target gene was obtained by PCR and purified for later use; 2) The target gene is recombined into the cloning plasmid, the cloning plasmid and the expression plasmid are propagated by Escherichia coli, and the two plasmids are extracted and purified respectively; 3) Digest the above two kinds of plasmids obtained by extraction with two identical restriction enzymes respectively, and the digested products are recovered and purified by gel cutting, and the purified expression plasmid and ppk gene are purified according to the molar concentration of 1:5-1 :10 ratio, using DNA ligase to connect to obtain recombinant expression plasmid, carry out cloning and propagation by Escherichia coli, extract and purify for subsequent use; 4) Add the recombinant expression plasmid purified in step 3) to Synechococcus sp. 7002 for natural transformation, and then use a solid plate containing spectinomycin hydrochloride to screen the transformed strain; 5) Plasmid extraction was performed on the screened algal strains, amplified with ppk gene-specific primers, and sequence identification was performed on the obtained amplified products to obtain Synechococcus 7002 strains with recombinant ppk genes. 3. The preparation method according to claim 2, wherein the expression plasmid in the above step 2) is a pSyn_1 expression plasmid. 4. The preparation method according to claim 2, characterized in that, in the above step 3), DNA ligase is used to ligate at 16°C for 1-3 h. 5. The preparation method according to claim 2, characterized in that the above-mentioned step 4) is specifically, adding the recombined plasmid in step 3) to the fresh culture medium concentrated 10-15 times and washed with good production status, Synechococcus sp. 7002 with OD ₇₅₀ =1-2 was naturally transformed for 24 hours, and then the transformed strains were screened using a solid plate containing spectinomycin hydrochloride. 6. The application of the transgenic algae strain of high-yield nano-polyphosphate as claimed in claim 1 on high-yield nano-polyphosphate. 7. The application according to claim 6, characterized in that, during the high-yield nano-polyphosphate process of the transgenic algae strain, 0.5-5 μM nickel ions are added to induce expression in its culture medium; 10 µg/L spectinomycin hydrochloride was added to the base.