阅读说明：本技术 一种普通小球藻叶绿体同源重组空载体及其应用 (Chlorella vulgaris chloroplast homologous recombination empty vector and application thereof ) 是由 崔玉琳 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,具体涉及一种普通小球藻叶绿体同源重组空载体及其应用。重组空载体包括上下游同源臂,以及同源臂间设至少一个启动子和终止子,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；其中,上游同源臂含SEQ ID NO:1所示碱基序列,下游同源臂含SEQ ID NO:2所示碱基序列。采用本发明的普通小球藻叶绿体稳定表达系统,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 chlorella vulgaris chloroplast homologous recombination empty vector and application thereof. The recombinant empty vector comprises upstream and downstream homologous arms, and at least one promoter and a terminator are arranged between the homologous arms, and a base sequence shown in 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. The chlorella vulgaris chloroplast stable expression system can realize stable expression of a plurality of exogenous genes in chloroplasts.)

1. A chlorella vulgaris chloroplast homologous recombination empty vector is characterized in that: the recombinant empty vector comprises upstream and downstream homologous arms, and at least one promoter and a terminator are arranged between the homologous arms, and a base sequence shown in 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 chlorella vulgaris chloroplast homologous recombination empty vector of claim 1, wherein: a selection marker gene is inserted between the homology arms.

3. The chlorella vulgaris chloroplast homologous recombination empty vector of claim 1 or 2, wherein: the recombinant empty vector sequentially 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.

4. The chlorella vulgaris chloroplast homologous recombination empty vector of claim 3, 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.

5. The chlorella vulgaris chloroplast homologous recombination empty vector of claim 3, wherein: the terminator is a terminator for regulating and controlling an exogenous gene; or, the terminator is a promoter for regulating and controlling the exogenous gene and a terminator for regulating and controlling the selective marker gene; wherein the terminator is a base sequence shown by SEQ ID NO. 5 and/or a base sequence shown by SEQ ID NO. 6.

6. The chlorella vulgaris chloroplast homologous recombination empty vector of claim 1, wherein: 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.

7. The use of the chlorella vulgaris chloroplast homologous recombination empty vector of claim 1 in chlorella vulgaris chloroplast transformation.

8. Use according to claim 7, characterized in that: and introducing an exogenous gene into the constructed homologous recombination empty vector, then introducing into chlorella vulgaris cells, and culturing and screening to obtain transgenic chlorella vulgaris.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a chlorella vulgaris chloroplast homologous recombination empty vector and application thereof.

Background

Chlorella is a single-cell microalgae belonging to Chlorophyta (Chlorphyta), Chlorophyceae (Chlorophyceae), Chlorococcales (Chlorococcales), Chlorella (Chlorella aceae) and Chlorella (Chlorella). There are currently known about 10 kinds of Chlorella in the world, and common Chlorella vulgaris (Chlorella vulgaris), Chlorella vulgaris (Chlorella zofinensis), Chlorella ellipsoidea (Chlorella ellipsoidea), and Chlorella protothecoides (Chlorella protothecoides) are known.

The chlorella is a unicellular green alga, which is spherical or elliptical in shape, has a diameter of 3-12 mu m, has single swimming cells and is also aggregated into a group. The chlorella is widely distributed in the nature, has high growth and reproduction speed, is the only organism which can be increased by 4 times in 20 hours in the animals and plants on the earth, and can obtain higher cell density and biomass; the chlorella is easy to culture, and can utilize light energy and CO₂The organic carbon source can be used for heterotrophic growth while photoautotrophy is carried out, and the organic carbon source is a good choice for the bioreactor. Chlorella varies greatly in size and appearance color from time to time. Under the observation of a microscope (1000 x oil microscope), chlorella individuals in the logarithmic growth phase are small and green, and the pigments in vivo are mainly green pigments for photosynthesis of chlorophyll and the like; after the stable period, the cell individual grows, if the cell is stressed by the outside, the color of the chlorella becomes yellow or orange, and the main pigments in the cell are carotenoids such as zeaxanthin, lutein and astaxanthin, etc., so that the cell is protected against the outside stress. Chlorella is propagated as asexual reproduction, which forms similar spores through protoplast division, each similar spore is released after the cell wall of a parent body is broken. The sporophyte grows freely to form vegetative cells.

The chlorella has important commercial application value and is widely applied to the fields of food, medicine, energy, aquaculture and the like. Chlorella contains abundant protein with content of 50% -60%, can be used as an important source of single cell protein, and also contains various essential amino acids, dietary fiber, vitamins, etc. The chlorella also has the advantages of low fat, low sugar, low calorie and the like, is a high-quality green nutrient source food, and has great economic value in the industries of food, health-care food and the like. The chlorella can also be used as feed for animals such as fish and shrimp, and can be widely applied to aquaculture industry.

The chlorella is found to accumulate astaxanthin, and may become a high-quality natural astaxanthin algae source following haematococcus pluvialis. Chlorella contains various bioactive substances such as Chlorella growth factor COF, polysaccharide and glycoprotein, and has significant effects in resisting tumor, enhancing immunity, resisting virus infection, etc. It has also been reported that some chlorella cells have high contents of fats and fat-soluble compounds and can be used for preparing bio-oil. As early as 1993, the National Renewable Energy Laboratory (NREL) in the united states established and developed programs to genetically engineer microalgae to increase hydrocarbon production.

The genetic engineering research of chlorella is also earlier, and is second only to chlamydomonas reinhardtii in microalgae. At present, the genetic transformation methods suitable for chlorella include an electric shock transformation method, a gene gun transformation method, an agrobacterium transfection method and the like, and have higher efficiency. The neomycin phosphotransferase gene (NPT II) is used as a screening marker gene, and a stable transformation system of the chlorella karyon nucleus is established by an electric shock transformation method and is widely applied. However, the chlorella cell nuclear transformation method has some problems, such as: the nuclear genome has complex structure and function and is difficult to realize directional recombination and site-directed mutagenesis; the expression efficiency of the exogenous gene is low, and the expression is unstable.

Chloroplasts are organelles of chlorella which carry relatively independent genetic material, and there is and only one large cupped chloroplast in chlorella. In 1988, Boynton et al established a chloroplast transformation system of Chlamydomonas reinhardtii by the biolistic method, leading to the recognition that chloroplasts can be used as a new transformation expression receptor in genetic engineering. Compared with the nuclear transformation system, chloroplast transformation has the following advantages: prokaryotic expression system, chloroplast genome site-specific transformation, no gene silencing, high exogenous gene expression efficiency and small variation. Therefore, the development of a chloroplast genetic transformation system of chlorella has important value for basic research and application development of chlorella.

At present, research aiming at a chlorella chloroplast transformation system has been advanced to a certain extent. One of them is to introduce the exogenous gene into the nuclear genome with the nuclear transformation vector, and add the chloroplast-localized peptide leader to the N section of the exogenous gene, and introduce the exogenous gene into the chloroplast to make it function, which is an indirect chloroplast expression. In the other method, the experience of chlamydomonas reinhardtii chloroplast transformation is used for reference, a gene (such as chlL) which does not influence the photosynthetic growth of cells is taken as an insertion site, and a promoter, a terminator and the like of the chlamydomonas reinhardtii are taken as regulatory elements to establish a chloroplast genetic transformation vector of the chlorella. However, the vector is based on mutating a functional gene, photosynthesis is not affected, but dark reaction is affected, and the chlorella mutant strain obtained after transformation cannot be applied to large-scale culture; meanwhile, in the mutant strain, the exogenous gene is regulated and controlled by a regulatory element derived from chlamydomonas reinhardtii, and the expression efficiency is low. Although the chloroplast genome has been sequenced and constructed with chloroplast vectors of some algal strains, due to the inter-species variability and the specificity of chlorella vulgaris itself, the difficulty of the chloroplast expression vector is the selection of endogenous elements and their upstream and downstream nodes, where the homology arms and insertion sites, the upstream node of the promoter are critical and not obvious.

Disclosure of Invention

The invention aims to provide a chlorella vulgaris chloroplast homologous recombination empty vector and application thereof.

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

a chlorella vulgaris chloroplast homologous recombination empty vector comprises an upstream homologous arm and a downstream homologous arm, at least one 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.

A selection marker gene is inserted between the homology arms.

The recombinant empty vector sequentially 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.

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 promoter for regulating and controlling the exogenous gene and a terminator for regulating and controlling the selective marker gene; wherein the terminator is a base sequence shown by SEQ ID NO. 5 and/or a base sequence shown by 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 a chlorella vulgaris chloroplast homologous recombination empty vector in chlorella vulgaris chloroplast transformation.

Specifically, an exogenous gene is introduced into the constructed homologous recombination empty vector, then introduced into chlorella vulgaris cells, and the transgenic chlorella vulgaris is obtained through culture and screening.

The exogenous genes are functional protein genes, structural protein genes, nutritional protein genes and the like; wherein, the functional protein gene can be fatty acid synthetic protein gene, photosynthesis related protein gene, etc., and the structural protein gene can be cell membrane protein gene calmodulin gene, metal ion binding protein gene, etc.

The invention has the advantages that:

the invention utilizes the endogenous sequence of chlorella chloroplast to construct a vector which can recombine a plurality of exogenous genes to chlorella vulgaris chloroplast genome, and obtains the chlorella mutant strain expressing the exogenous genes. Compared with the prior art, the invention realizes key breakthrough of the chlorella vulgaris gene engineering technology and has the following beneficial effects:

1. the invention provides two sequences on a chlorella vulgaris chloroplast genome which are used as homologous arms for constructing chlorella vulgaris chloroplast homologous recombination vectors. The two sequences are adjacent on the chlorella chloroplast genome, the region is not assembled to express other genes, and the insertion site is located in a non-coding region between two genes at the upstream and downstream. The insertion of foreign genes into the site does not affect the expression and function of any gene on the genome, and thus does not affect the growth of algal cells.

2. The invention provides a ribosome binding site on a chlorella vulgaris chloroplast genome, which is used for connecting a plurality of exogenous genes in series on a chloroplast expression vector to form polycistrons, can realize the serial expression of a plurality of genes simultaneously, and is beneficial to obtaining a chlorella vulgaris mutant strain with a composite function.

3. The invention provides a promoter and a terminator of a high-efficiency expression gene on a chlorella vulgaris chloroplast genome, which are used for constructing a chlorella vulgaris chloroplast expression vector. The endogenous high-efficiency regulatory sequence of chlorella can improve the expression of exogenous genes and realize large-scale accumulation in chloroplasts.

4. The chlorella vulgaris is a kind of algae which can be subjected to photoautotrophy and heterotrophy, the growth is rapid, the nutritive value is high, and high added value products such as astaxanthin and the like are produced while a large amount of nutritive protein is accumulated in the algae chloroplast, so that high nutritive algae cells or a high-efficiency bioreactor can be obtained.

5. The invention can modularize the synthetic elements such as polyunsaturated fatty acid, astaxanthin, sterol, long-chain hydrocarbon and the like, express in chlorella chloroplasts respectively or simultaneously, develop the synthetic biology of the chlorella chloroplasts and promote the comprehensive development of the chlorella.

Drawings

FIG. 1 is a Chlorella vulgaris chloroplast homologous recombination empty vector map provided by the embodiment of the invention.

FIG. 2 is a Chlorella vulgaris chloroplast homologous recombination expression profile provided by an embodiment of the invention.

FIG. 3 is an electrophoresis diagram of PCR products provided by the present invention (wherein M is molecular marker DL2000; lane wild is wild strain; lane mutations is positive transgenic algal strain).

FIG. 4 is an electrophoresis diagram of PCR products provided by the present invention (wherein M is molecular marker DL5000; lane wild strain; lane Mutants is positive transgenic algal strain).

FIG. 5 is a southern hybridization pattern of transgenic Chlorella vulgaris provided in the examples of the present invention (wherein lane W is a wild strain; lane M is a positive transgenic algal strain).

FIG. 6 shows the western hybridization pattern of transgenic chlorella vulgaris provided in the examples of the present invention (wherein lane wild represents the wild strain; lane Mutant represents the positive transgenic strain).

Detailed Description

The invention is further described below with reference to the figures and examples.