CN106916775B - Transgenic algae strain for high yield of nano polyphosphate and preparation method thereof - Google Patents

Abstract

The invention provides a transgenic algal strain with high yield of nano-polyphosphate and a preparation method thereof. The transgenic algal strain is obtained by introducing a recombinant pSyn_1 plasmid containing a homologous ppk gene on the basis of Synechococcus 7002 and screening and identifying it. Microalgae. The ability of the transgenic algal strain to produce polyphosphates is increased by 90-150% compared with the wild strains, and the method for inducing and accumulating polyphosphates is to use 0.5-5 μM nickel ions for induction. The algal strain has strong operability, good genetic stability, and continuous expression of foreign genes for more than 1 year. The polyphosphate produced by this algal strain is nano-scale, with a scale in the range of 100 nm, and a good nano-morphology can be maintained during the extraction process.

Description

Transgenic algal strain with high yield of nano-polyphosphate and preparation method thereof

technical field

The invention belongs to the technical field of genetic engineering, in particular to a transgenic algal strain with high yield of nanometer polyphosphates.

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 deficiencies. The negative effects of mineral malnutrition on human health are established early in life and can lead to delayed growth, poor cognition, lethargy, difficulty concentrating, and increased susceptibility to major infectious diseases, thereby reducing individuals’ ability to receive education. capacity, physical labor capacity, and life expectancy. Mineral nutrition intervention is of great significance for people to get rid of the "poverty trap" of mineral nutrition deficiency and to improve people's health.

Biomineralized nanostructures have unique application advantages in the practice of mineral nutrition intervention. Polyphosphates are biomineralized structures mainly composed of long-chain polyphosphates that exist in many biological cells. Many biological cells (such as prokaryotic and eukaryotic microorganisms, protozoa, mammalian platelet cells and osteoblasts, etc.) will synthesize long-chain (tens to hundreds of residues) polyphosphate (PolyP), and This is the main component to form a nano- or sub-micron-scale inclusion body - polyphosphate bodies (PPB), also known as heterochromatic particles (Volutingranules) or calcium acid bodies (Acidocalcisomes). In recent years, many studies have proved that polyphosphates are related to various biological functions. Different from the short-chain PolyP commonly used in the food industry, the long-chain PolyP of biological origin is difficult to be hydrolyzed by alkaline phosphatase in the mammalian intestine, so it can reach the intestine through the mouth, and enterocytosis mediated by caveolin. Absorbed by intestinal epithelial cells and plays a prebiotic role in protecting intestinal tissue from oxidative damage, inflammation, fibrosis and carcinogenesis. Polyphosphates can provide cells with raw materials for synthesizing DNA and store or provide mineral elements needed by cells, and have the potential to be developed into a natural nano-mineral nutritional supplement or fortifier. Although the functional properties of polyphosphates are gradually recognized, there is no case of large-scale fermentation production using microorganisms. Selecting a suitable "cell factory" for the production of polyphosphates is of great significance for its development and application.

Synechococcus 7002 is a type of offshore microalgae, and it is also the fastest dividing cyanobacteria found so far. It has a natural exogenous DNA transformation system, and its entire genome has also been deciphered. "Engineered microalgae" construction platform. Multiple transgenic Synechococcus 7002 strains have been constructed successively, which have the potential to produce a variety of 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 exogenous 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 suitable algal strains, plasmids and target genes. An algal strain with good genetic stability and high expression efficiency is of great significance to improve the comprehensive utilization value of microalgae.

Nanoscale active substances have very obvious advantages in function. Synechococcus can accumulate polyphosphates, and the particle size is nanoscale, which is easier to be absorbed and utilized by cells. The accumulation of polyphosphates is regulated by the ppk gene. The overexpression of the ppk gene in Synechococcus can be induced by introducing an exogenous plasmid of the recombinant ppk gene to achieve the purpose of excessive accumulation of nanopolyphosphates . The selection of suitable homologous genes and plasmids containing promoters that are easily activated by corresponding algal strains can greatly improve the success rate of exogenous gene expression during the transgenic process. Compared with other microorganisms, the use of Synechococcus for the expression of related exogenous genes has many incomparable advantages: (1) Synechococcus 7002 is a natural competent cell, which can absorb exogenous plasmids under natural conditions, which is convenient for exogenous gene expression. Gene introduction; (2) Microalgae, as photoautotrophs, have fast splitting speed and high production efficiency, and can use seawater for large-scale production under light, without causing problems such as encroachment of cultivated land and energy waste, and the abundance of seawater. Mineral elements can enrich the elemental composition of polyphosphates and improve their functional properties; (3) The nutritional value of microalgae itself is very high. When transgenic microalgae produce relevant active substances, the obtained algae and fermentation process produce The secondary metabolites can be used for high-value utilization of functional health food and drug development; (4) The expression of exogenous genes is stable and the genetic stability is good.

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

SUMMARY OF THE INVENTION

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

Another aspect of the present invention relates to the application of the above-mentioned transgenic algal strain for fermentation to produce nanometer polyphosphates; the nanometer polyphosphates can be applied to the development of products that improve intestinal immunity.

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

A transgenic algal strain with high-yield nano-polyphosphates, the algal strain is a transgenic Synechococcus 7002 algal strain obtained by introducing a recombinant pSyn_1 plasmid containing a ppk homologous gene on the basis of the Synechococcus 7002 algal strain.

The preparation method of the transgenic algal strain of the above-mentioned high-yield nano-polyphosphate, comprising the following steps:

1) Extraction, cloning and modification of the target gene: Extract the genome of Synechococcus 7002, design specific primers according to the sequence of the Synechococcus 7002 ppk gene, and add corresponding enzyme cleavage sites and ribosome binding sites at both ends of the primers. The target gene was obtained by PCR and purified for later use;

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

3) The above two plasmids obtained by extraction were digested with the same two restriction enzymes respectively, and the digested products were recovered and purified by gel cutting. The purified expression plasmid and ppk gene were at a molar concentration of 1:5-1. : 10 ratio, utilize DNA ligase to connect, obtain recombinant expression plasmid, carry out cloning and reproduction by Escherichia coli, extract and purify for subsequent use;

4) adding the recombinant expression plasmid purified in step 3) into Synechococcus 7002 for natural transformation, and then using a solid plate containing spectinomycin hydrochloride to screen the transformants;

5) Extract the plasmid from the screened algal strain, use ppk gene-specific primers to amplify, and identify the sequence of the amplified product to obtain the Synechococcus 7002 algal strain with the recombinant ppk gene.

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

In the above step 3), DNA ligase was used for ligation at 16°C for 1-3 h.

The above step 4) is specifically, adding the plasmid after the recombination in step 3) to the Synechococcus 7002 in a good production state and OD ₇₅₀ =1-2 washed with a fresh culture solution concentrated by 10-15 times, and performing natural transformation, After 24 h of transformation, the transformants were screened on solid plates containing antibiotics.

The application of the above-mentioned transgenic algal strain with high-yield nano-polyphosphate in high-yield nano-polyphosphate; wherein, 0.5-5 μM nickel ions are added to the culture medium of the transgenic algal strain to induce expression; and Synechococcus 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-polyphosphates, the production method is green and environmentally friendly, and the research and utilization value of by-products (algal residue, secondary metabolites, etc.) is high. 2. Using the model microalga Synechococcus 7002 for transgenic operation, the operation is simple, the success rate is high, and the genetic stability is good. 3. The imported gene is the homologous ppk gene of Synechococcus 7002. The exogenous plasmid pSyn_1 contains the promoter from cyanobacteria, which is easier to translate and express, and the polyphosphates stimulated by the homologous gene expression have no quality problems. 4. Nickel ions are used to induce exogenous plasmid promoters to promote the excessive accumulation of polyphosphates in transgenic microalgae, and the method is simple and convenient; 5. The transgenic algal strains have the ability to produce high polyphosphates, and the accumulated polyphosphates are Nanoscale, good stability, smaller particle size, easier to be absorbed and utilized.

At the same time, polyphosphates can enrich the abundant mineral elements in seawater and may have better functional properties. Obviously, the present invention obtains a transgenic algal strain with high yield of nano-polyphosphates with high research and utilization value, and obtains a method for preparing the algal strain and inducing expression of exogenous genes, which has a very broad market prospect.

Description of drawings

Figure 1 is the electrophoresis diagram of recombinant plasmid construction; wherein, A: lane a: ppk and pEASY-Blunt zero recombinant clone plasmid; lane b: cloned plasmid double digestion; swimming lane c: pSyn_1 expression plasmid; d: pSyn_1 plasmid double digestion; Lane M: 1 Kb DNA Ladder; B: Lane a: ppk and pSyn_1 recombinant plasmids; Lane b: plasmid single digestion; Lane c: plasmid double digestion; Lane M: 1 Kb DNA Ladder.

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

Figure 3 is a graph showing the particle size and electron microscope analysis of the nano-polyphosphates.

Figure 4 shows the effect of nickel ion concentration on the yield of nanopolyphosphates.

Detailed ways

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 examples can usually be carried out under normal conditions.

Example 1:

1) Obtaining the target gene

Specific primer design:

ppk pre-primer: CCCAAGCTTGAAGGAGATAAAAAGTATATGTCCTCTGCG

Primer after ppk : CGGGGTACCCTAATCCAAGCTGTCCTGGAG

The front and rear primers added Hind III and Kpn I restriction sites respectively, and the front primer added the RBS sequence of GAAGGAG sequence.

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

The target gene was purified and cloned by conventional methods.

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

3) Construction and screening of recombinant transgenic algal strains: 100 ng of purified recombinant plasmids were added to 100 μL of Synechococcus 7002 algal solution ( OD ₇₅₀ =1) washed with 15-fold concentrated fresh medium. 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 by screening were identified to obtain transgenic algal strains with recombinant ppk gene.

4) The transgenic algal strains were cultured in 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 nanopolyphosphates was detected after culturing for 1 week. The content of nanopolyphosphates in the induced algal strain was 90% higher than that in the wild strain.

Example 2:

1) Obtaining the target gene

Specific primer design:

ppk pre-primer: CCCAAGCTTGAAGGAGATAAAAAGTATATGTCCTCTGCG

Primer after ppk : CGGGGTACCCTAATCCAAGCTGTCCTGGAG

The front and rear primers added Hind III and Kpn I restriction sites respectively, and the front primer added the RBS sequence of GAAGGAG sequence.

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

The target gene was purified and cloned by conventional methods.

2) Construction of recombinant pSyn_1 plasmid: pSyn_1 plasmid and target gene were double digested by 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, and T4 DNA ligase was ligated at 16 °C for 1 h , the ligated plasmid was cloned by conventional methods, the correctness of the plasmid was identified by enzyme digestion, and it was purified for later use.

3) Construction and screening of recombinant transgenic algal strains: 100 ng of purified recombinant plasmids were added to 100 μL of Synechococcus 7002 algal solution ( OD ₇₅₀ = 2) washed with 10-fold concentrated fresh medium. 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 by screening were identified to obtain transgenic algal strains with recombinant ppk gene.

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 nanopolyphosphates was detected after culturing for 1 week. The content of nanopolyphosphates in the induced algal strain was 150% higher than that in 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 the 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 b in lane d is the cut plasmid. There is only one band in lane d because the cut part of the other fragment is very short and cannot be observed by electrophoresis. As shown in Figure 1.B, the recombinant pSyn_1 plasmid containing ppk gene was digested with a single enzyme to obtain a fragment of about 6000 bp, which was about 2000 bp for the target gene and about 4000 bp for the pSyn_1 plasmid. After double digestion, the expected molecular weight was obtained respectively. The size of the plasmid and the target gene, and the results of enzyme digestion identification proved that the plasmid was successfully constructed. Combined with the results of gene sequencing, the recombinant plasmid was successfully constructed.

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

As shown in Figure 3, Figure 3.A shows the particle size analysis results of nano-polyphosphates obtained by boiling, purification and other methods. more particle size. Electron microscope observation of the extracted nano-polyphosphates, Figure 3.B, it can be seen that the nano-polyphosphates maintain a good nano-shape after extraction.

As shown in Figure 4, nickel ions can induce the expression of pSyn_1 plasmid. Different concentrations of nickel ions have different induction effects. The wild group is the wild-type control group, and the other groups are added with different concentrations of nickel ions. The ordinate fluorescence value can reflect the poly The content of phosphate, but the DAPI fluorescence method reflects the PolyP content on the surface of polyphosphate, so the actual yield is greater than the value of the fluorescence value reaction.

The yield of the nano-polyphosphates prepared by the transgenic algal strain obtained by the present invention is increased by 90-150% compared with the stramenopile strain, and the transgenic microalgae has better environmental adaptability, including temperature tolerance and heavy metal ion tolerance ability. The PPB accumulated by the algae strain in the present invention is of nanometer scale, and the particle size ranges from ten nanometers to one hundred nanometers. The stability of the polyphosphate is good. The algal 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 algal strain with high yield of nano-polyphosphate and its preparation method

<130> 2017

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 39

<212> DNA

<213> Artificial sequences

<400> 1

cccaagcttg aaggagataa aaagtatatg tcctctgcg 39

<210> 2

<211> 30

<212> DNA

<213> Artificial sequences

<400> 2

cggggtaccc taatccaagc tgtcctggag 30

Claims (7)

1. A transgenic algae strain for high-yield nano-poly-phosphate is characterized in that the algae strain is a transgenic synechococcus 7002 algae strain obtained by introducing a recombinant pSyn _1 plasmid containing ppk gene on the basis of a synechococcus 7002 algae strain; CCCAAGCTTGAAGGAGATAAAAAGTATATGTCCTCTGCG as a primer of the ppk gene; ppk rear primer CGGGGTACCCTAATCCAAGCTGTCCTGGAG.

2. The method for preparing transgenic algae strain capable of producing nano-polyphosphate body with high yield according to claim 1, comprising the following steps:

1) extracting, cloning and modifying a target gene: extracting genome of synechococcus 7002, designing specific primers according to the sequence of the synechococcus 7002ppk gene, adding corresponding enzyme cutting sites and ribosome binding sites at two ends of the primers, obtaining a target gene through PCR, and purifying for later use;

2) recombining the target gene to clone plasmid, utilizing colibacillus to reproduce the clone plasmid and expression plasmid, and respectively extracting and purifying the two plasmids;

3) performing enzyme digestion on the two extracted plasmids by using two same restriction enzymes respectively, performing gel cutting, recovering and purifying on a product after enzyme digestion, connecting the purified expression plasmid and the ppk gene by using DNA ligase according to the molar concentration of 1:5-1:10 to obtain a recombinant expression plasmid, performing clone propagation by using escherichia coli, and extracting and purifying for later use;

4) adding the recombinant expression plasmid purified in the step 3) into synechococcus 7002 for natural transformation, and screening the transformant by using a solid plate containing spectinomycin hydrochloride;

5) and (3) carrying out plasmid extraction on the screened algae strain, amplifying by utilizing a ppk gene specific primer, and carrying out sequence identification on the obtained amplification product to obtain the synechococcus 7002 algae strain of the recombinant ppk gene.

3. The method according to claim 2, wherein the expression plasmid in the step 2) is a pSyn _1 expression plasmid.

4. The method according to claim 2, wherein the ligation is performed at 16 ℃ for 1 to 3 hours in step 3) using DNA ligase.

5. The method according to claim 2, wherein the step 4) is a step of adding the recombinant plasmid of the step 3) to a fresh culture medium concentrated 10 to 15 times and washed to obtain a good-quality OD₇₅₀The synechococcus 7002 of 1-2 was transformed naturally for 24 hours, and the transformants were selected on a solid plate containing spectinomycin hydrochloride.

6. The use of the transgenic algal strain of claim 1 for producing nano polyphosphate body in high yield.

7. The use of claim 6, wherein the transgenic algal strain is induced to express by adding 0.5-5 μ M nickel ions into the culture medium during the high yield of nano-polyphosphates; and 10. mu.g/L spectinomycin hydrochloride was added to the medium.

Images