阅读说明：本技术 一种紫球藻叶绿体表达系统及其应用 (Porphyridium chloroplast expression system and application thereof ) 是由 崔玉琳 甄张赫 王康 任家利 秦松 于 2019-12-04 设计创作，主要内容包括：本发明涉及基因工程技术领域,具体涉及一种紫球藻叶绿体同源重组空载体及其应用。载体包括紫球藻叶绿体基因组上SEQ ID NO:1所示碱基序列的上游同源臂和SEQ ID NO:2所示碱基序列的下游同源臂,SEQ ID NO:3、SEQ ID NO:4所示碱基序列的启动子,SEQ ID NO:5、SEQ ID NO:6所示碱基序列的终止子,启动子和终止子之间插入与至少一个外源基因构成多顺反子结构的SEQ ID NO:7所示的碱基序列。采用本发明的紫球藻叶绿体同源重组空载体可实现多个外源基因在叶绿体中稳定表达。(The invention relates to the technical field of genetic engineering, in particular to a porphyridium chloroplast homologous recombination empty vector and application thereof. The vector comprises an upstream homologous arm of a base sequence shown by SEQ ID NO. 1 and a downstream homologous arm of a base sequence shown by SEQ ID NO. 2 on a porphyridium chloroplast genome, promoters of base sequences shown by SEQ ID NO. 3 and SEQ ID NO. 4, terminators of base sequences shown by SEQ ID NO. 5 and SEQ ID NO.6, and a base sequence shown by SEQ ID NO. 7 which forms a polycistronic structure with at least one exogenous gene is inserted between the promoter and the terminators. The porphyridium chloroplast homologous recombination empty vector can realize stable expression of a plurality of exogenous genes in chloroplast.)

1. A porphyridium chloroplast expression system, which is characterized in that: the expression system comprises an upstream homologous arm and a downstream homologous arm, and a promoter and a terminator are arranged between the homologous arms, and a base sequence shown as SEQ ID NO. 7 which forms a polycistronic structure with at least one exogenous gene is inserted between the promoter and the terminator; wherein, the upstream homologous arm contains a base sequence shown in SEQ ID NO. 1, and the downstream homologous arm contains a base sequence shown in SEQ ID NO. 2.

2. The porphyridium chloroplast expression system of claim 1, wherein: a selection marker gene is inserted between the homology arms.

3. The porphyridium chloroplast expression system of claim 1, wherein: at least one promoter and a terminator are inserted between the upstream homology arm and the downstream homology arm.

4. The porphyridium chloroplast expression system of any one of claims 1 to 3, wherein: the expression system comprises an upstream homology arm, at least one promoter, a selective marker gene, a base sequence shown in SEQ ID NO. 7 forming a polycistronic structure with at least one exogenous gene, a terminator and a downstream homology arm in sequence.

5. The porphyridium chloroplast expression system of claim 4, wherein: the promoter is used for regulating and controlling exogenous genes; or the promoter is a promoter for regulating and controlling the exogenous gene and a promoter for regulating and controlling the selective marker gene; wherein the promoter is a base sequence shown by SEQ ID NO. 3 and/or a base sequence shown by SEQ ID NO. 4.

6. The porphyridium chloroplast expression system of claim 4, wherein: the terminator is a terminator for regulating and controlling an exogenous gene; or, the terminator is a terminator for regulating the exogenous gene and a terminator for regulating the selective marker gene; wherein the terminator is a base sequence shown by SEQ ID NO. 5 and/or SEQ ID NO. 6.

7. The porphyridium chloroplast expression system of claim 1, wherein: the upstream homology arm is a base sequence shown by a sequence shown in SEQ ID NO. 1; or, the 3 'end of the sequence shown in SEQ ID NO. 1 begins, and extends to the 5' end to a continuous fragment of not less than 500 bp;

the downstream homology arm is a base sequence shown by a sequence shown by SEQ ID NO. 2; or, the sequence shown in SEQ ID NO. 2 starts from the 5 'end and extends to the 3' end to a continuous fragment of not less than 500 bp.

8. The use of the porphyridium chloroplast expression system of claim 1 in the chloroplast transformation of porphyridium.

9. Use according to claim 8, characterized in that: and introducing exogenous genes into the constructed expression system, introducing porphyridium cells, and culturing and screening to obtain transgenic porphyridium.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a porphyridium chloroplast expression system and application thereof.

Background

Porphyridium is a relatively primitive and the only single-cell red algae found so far, is widely distributed, and is found in seawater, fresh water and moist soil, the porphyridium cells are generally spherical and have a diameter of about 5-24 μm, the cells contain a large and asteroid vector containing abundant phycobiliproteins, wherein the phycoerythrin content is the most, so the cells are red or dark purple, the porphyridium cells also generally contain a layer of mucilaginous sheath, namely porphyridium polysaccharides, the porphyridium polysaccharides among the cells are adhered to each other to generate irregular accumulation, when in moist soil and walls, the porphyridium is in a light brown or red sheet, when water is lost, the cells are in a crust, the porphyridium cells are in a binary fission growth mode, belong to seawater, saline water, moist soil and brine, and have the capability of growing in freshwater, namely 354.5 percent of vegetative growth, and can reach the normal vegetative growth resistance.

The lipid content in the porphyridium can account for 9 ~ 16% of biomass, wherein the polyunsaturated fatty acid accounts for more than half of the total lipid, the content of eicosapentaenoic acid (EPA) and arachidonic acid (ARA) is the most, the content of the fatty acid and the composition in the cell are easy to change under the influence of the growth environment, and the metabolic products with higher added value, such as ARA and EPA, can be directionally produced by controlling the culture condition.

The porphyridium grows through four periods of a lag phase, a logarithmic growth phase, a stationary phase, a decay phase and the like, when cells enter the growth stationary phase, a large amount of algal polysaccharides can be secreted to the outside of the cells, a thick viscous sheath membrane is formed, and the rest polysaccharides are mutually soluble with a culture medium, so that the culture solution is viscous, the porphyridium cells are protected, and the difficulty in harvesting the porphyridium is increased. In addition, the colloidal state of algal polysaccharide has stability, and can be widely applied to industries such as clinic, food, cosmetics and the like. The porphyridium polysaccharide is usually sulfate polysaccharide, mainly consists of monosaccharides such as xylose, glucose and galactose, and has the function of preventing virus adsorption, so that the porphyridium polysaccharide has a special effect on treating tumors. In addition, the porphyridium polysaccharide also has the functions of preventing and treating cardiovascular diseases, reducing blood fat, treating diseases of liver, gallbladder, pancreas and the like, and is widely applied to the health product industry, so the porphyridium polysaccharide is a metabolite with wide application range and obvious functional effect.

Another important product of porphyridium is phycobiliprotein. Phycobiliprotein is a water-soluble chromoprotein with bright color, and aggregates into supramolecular complexes called phycobilisomes, which aggregate on the outer surface of the thylakoid membrane of chloroplasts. The color of phycobiliproteins is derived primarily from covalently bound subunit groups, which are open-chain tetrapyrrole chromophores with A, B, C and D rings, known as phycobilisins. Phycobiliproteins are present in prokaryotic cyanobacteria and eukaryotic red algae. In blue and red algae, four major phycobiliproteins are produced: allophycocyanin (APC, blue-green), phycocyanin (PC, blue), phycoerythrin (PE, purple), and phycoerythrin (PEC, orange). Phycoerythrin accounts for more than 70% of phycobiliprotein in most red algae, while phycocyanin is abundant in blue algae. Phycobiliprotein can be used as food pigment and food additive. The food pigment contained in pudding is mostly made of chemicals, and phycobiliprotein is used as natural pigment, can be used as pudding colorant and is widely used in food. Analytical-grade phycobiliprotein (purity is more than or equal to 4.0) is reported to have obvious biological activities of oxidation resistance, liver protection, anti-inflammation and the like. Currently, related studies report different biosynthetic pathways of phycobiliproteins, which use δ -aminolevulinic acid, heme or biliverdin as precursors.

At present, large-scale culture and application of porphyridium have been started, but great difficulties are still existed in biomass and harvesting. Research results in the aspect of metabolic mechanism of porphyridium are many, and the chloroplast genome of porphyridium is also completed, so that data support is provided for chloroplast genetic transformation. However, the lack of tools for genetic transformation, including homologous insertion sites for chloroplast transformation, transformation methods, regulatory sequences, etc., has hindered the development and application of metabolites of porphyridium.

Disclosure of Invention

The invention aims to provide a porphyridium chloroplast expression system and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a porphyridium chloroplast expression system, which comprises an upstream homologous arm and a downstream homologous arm, and a promoter and a terminator arranged between the homologous arms, wherein a base sequence shown as SEQ ID NO. 7 which forms a polycistronic structure with at least one exogenous gene is inserted between the promoter and the terminator; wherein, the upstream homologous arm contains a base sequence shown in SEQ ID NO. 1, and the downstream homologous arm contains a base sequence shown in SEQ ID NO. 2.

A selection marker gene is inserted between the homology arms.

At least one promoter and a terminator are inserted between the upstream homology arm and the downstream homology arm.

The expression system comprises an upstream homology arm, at least one promoter, a selective marker gene, a base sequence shown in SEQ ID NO. 7 forming a polycistronic structure with at least one exogenous gene, a terminator and a downstream homology arm in sequence.

The promoter is used for regulating and controlling exogenous genes; or the promoter is a promoter for regulating and controlling the exogenous gene and a promoter for regulating and controlling the selective marker gene; wherein the promoter is a base sequence shown by SEQ ID NO. 3 and/or a base sequence shown by SEQ ID NO. 4.

The terminator is a terminator for regulating and controlling an exogenous gene; or, the terminator is a terminator for regulating the exogenous gene and a terminator for regulating the selective marker gene; wherein the terminator is a base sequence shown by SEQ ID NO. 5 and/or SEQ ID NO. 6.

The upstream homology arm is a base sequence shown by a sequence shown by SEQ ID NO. 1; or, the 3 'end of the sequence shown in SEQ ID NO. 1 begins, and extends to the 5' end to a continuous fragment of not less than 500 bp;

the downstream homology arm is a base sequence shown by a sequence shown by SEQ ID NO. 2; or, the sequence shown in SEQ ID NO. 2 starts from the 5 'end and extends to the 3' end to a continuous fragment of not less than 500 bp.

An application of the expression system of the chloroplast of porphyridium in the transformation of the chloroplast of porphyridium.

Specifically, exogenous genes are introduced into the constructed expression system, then the cells of the porphyridium are introduced, and the transgenic porphyridium is obtained through culture and screening.

The expression system is introduced into porphyridium by gene gun transformation, the bombardment pressure of the gene gun is 650psi, and the bombardment distance is 9 cm.

The method successfully constructs a stable chloroplast expression system of the porphyridium. The invention can effectively recombine a plurality of exogenous genes into the chloroplast genome of the porphyridium and obtain the transgenic algal strain through screening. Compared with the prior art, the invention realizes key breakthrough of porphyridium gene engineering technology and has the following beneficial effects:

1. the invention provides technical parameters for transforming porphyridium chloroplast by using a particle gun method. The gene gun transformation method has high transformation efficiency, simple equipment operation and high result reproducibility.

2. The invention provides a screening marker gene for porphyridium chloroplast transformation. The background effect of the herbicide glufosinate as the screening pressure is low, and the false positive rate of the mutant strain generated by drug resistance is low, so that the experimental efficiency can be improved.

3. By utilizing the method and the parameters provided by the invention, the chloroplast transformation of the porphyridium can be stably realized, and a positive mutant strain can be obtained. On the basis of the platform, the basic research process of the porphyridium can be promoted, such as the research on the photosynthesis in the porphyridium chloroplast and the structure and the function of light-collecting complex protein, particularly phycocyanin and phycoerythrin; or high value-added products such as astaxanthin and the like are synthesized in chloroplasts, and a large amount of accumulation can be realized.

4. The invention provides two segments of porphyridium chloroplast genome sequence segments which are used for constructing chloroplast homologous recombination vectors, the two segments are directly connected on a chloroplast genome, and tRNA sequences are arranged at two ends of a cutting site. the tRNA is multicopy on the chloroplast genome, where loss of function of one or both rRNAs does not affect the function of the entire cell. Therefore, the mutant strain obtained by homologous recombination using this site has no influence on any function of the cell.

5. The present invention provides a multicistronic chloroplast ribosome binding site of porphyridium in which a plurality of foreign genes are connected in series, so that a plurality of foreign proteins can be obtained by introducing a vector into porphyridium.

6. The invention provides a high-efficiency porphyridium endogenous control sequence. Compared with the conventional universal exogenous regulatory sequence, the endogenous regulatory sequence has the functions of high efficiency, no rejection and the like, and can promote the expression of exogenous protein and improve the protein content.

7. The porphyridium mutant strain obtained by the invention can simultaneously express functional protein genes such as functional proteins of synthesis routes of fatty acid and astaxanthin, or structural proteins such as phycobiliprotein and the like, or proteins for improving biological activity such as antibacterial peptide and neuropeptide and the like, and the mutant strain has higher protein content, lipid content, high value-added products, bait performance and the like.

Drawings

FIG. 1 is a porphyridium empty vector map provided in an embodiment of the present invention.

FIG. 2 is a map of the porphyridium expression vector provided in the embodiment of the present invention.

FIG. 3 is an electrophoretogram of PCR products provided in the present invention (wherein M is molecular marker DL5000; lane Wild is Wild strain; lane Mutant is transgenic algal strain).

FIG. 4 shows an electrophoretogram of PCR products provided in an embodiment of the present invention (where M is molecular marker DL8000; lane Wild is a Wild strain; lane Mutants is a transgenic algal strain).

FIG. 5 is the Southern hybridization pattern of the transgenic porphyridium provided in the example of the present invention (wherein the lane wild is the wild strain; and the lane Mutant is the transgenic algal strain).

FIG. 6 is a Western hybridization diagram of transgenic porphyridium provided in the embodiments of the present invention (wherein, lane wild is a wild strain; lane Mutant is a positive transgenic strain).

Detailed Description

The invention is further described in the following with reference to the figures and examples