阅读说明：本技术 一种雨生红球藻叶绿体表达系统及其应用 (Haematococcus pluvialis chloroplast expression system and application thereof ) 是由 崔玉琳 王寅初 任庆敏 王康 任家利 焦绪栋 秦松 于 2019-12-04 设计创作，主要内容包括：本发明涉及生物基因工程技术领域,是一种构建雨生红球藻叶绿体表达系统的方法。以雨生红球藻叶绿体基因组上SEQ ID NO:1所示序列和SEQ ID NO:2所示的序列为同源臂,以SEQ ID NO:3所示的序列、SEQ ID NO:4所示的序列为启动子,以SEQ ID NO:5所示的序列、SEQ ID NO:6所示的序列为终止子,以<I>bar</I>基因为筛选标记基因构建同源重组载体,并利用基因枪法将载体导入雨生红球藻细胞,经除草剂草丁膦筛选获得转基因藻株。采用本发明的雨生红球藻叶绿体稳定表达系统,可实现外源基因在叶绿体中稳定表达。(The invention relates to the technical field of biological genetic engineering, in particular to a method for constructing a haematococcus pluvialis chloroplast expression system. Taking the sequence shown in SEQ ID NO. 1 and the sequence shown in SEQ ID NO. 2 on the haematococcus pluvialis chloroplast genome as homologous arms, taking the sequence shown in SEQ ID NO. 3 and the sequence shown in SEQ ID NO. 4 as promoters, taking the sequence shown in SEQ ID NO. 5 and the sequence shown in SEQ ID NO. 6 as terminators, and taking the sequence shown in SEQ ID NO. 6 as terminators bar The gene is used as a screening marker gene to construct a homologous recombinant vector, the vector is introduced into haematococcus pluvialis cells by using a gene gun method, and a transgenic alga strain is obtained by screening a herbicide glufosinate. The stable expression system of haematococcus pluvialis chloroplast can realize the stable expression of exogenous genes in the chloroplast.)

1. A haematococcus pluvialis chloroplast expression system, which comprises a homology arm, a promoter and a terminator, and is characterized in that: the homology arm comprises an upstream homology arm of a sequence shown in SEQ ID NO. 1 and a downstream homology arm of a sequence shown in SEQ ID NO. 2.

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

3. The haematococcus pluvialis 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 haematococcus pluvialis chloroplast expression system of claim 1, wherein: the recombinant empty vector sequentially comprises an upstream homology arm, at least one promoter, a selective marker gene, at least one terminator and a downstream homology arm.

5. The haematococcus pluvialis chloroplast expression system of claim 1, wherein: the promoter is used for regulating and controlling exogenous genes; or, a promoter regulating a foreign gene and a promoter regulating a selectable 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 haematococcus pluvialis chloroplast expression system of claim 1, wherein: the terminator is a terminator for regulating and controlling an exogenous gene; or, a terminator which regulates the foreign gene and a terminator which regulates the selectable 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.

7. The haematococcus pluvialis chloroplast expression system of claim 1, wherein: the selective marker gene is glufosinate-resistance genebarA gene.

8. The haematococcus pluvialis chloroplast expression system of claim 1, wherein: the upstream homology arm is shown by a base 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 shown by a base 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.

9. Use of the haematococcus pluvialis chloroplast expression system of claim 1 to transform haematococcus pluvialis chloroplasts.

10. Use according to claim 9, characterized in that: introducing an exogenous gene into the constructed empty vector, and then introducing the vector into haematococcus pluvialis by using a gene gun transformation method, wherein the bombardment pressure of a gene gun is 650psi, and the bombardment distance is 9 cm; the transformed haematococcus pluvialis is screened by using the herbicide glufosinate.

Technical Field

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

Background

Haematococcus pluvialis (Haematococcus pluvialis) is a unicellular green alga which lives in fresh water and belongs to the phylum Chlorophyta, class Chlorophyceae, order Volvocales, family Rhodococcus, genus Haematococcus. Although Haematococcus pluvialis is a unicellular organism, its life history is complicated. Generally, the growth process is divided into two phases, namely motile cells and immobile cells. The motile cells are oval or elliptical and vary in size from about 5 μm to 20-30 μm. There is a large space between protoplasts and the cell wall, which are connected by many branched or unbranched cytoplasmic links, and the space between them is filled with hyaline jelly-like material. Protoplasts are ovoid in shape, often with a papilla at the leading end. The motile cell has two top-born flagella with equal length, the flagella extend out of the cell wall through the forked colloid tube at the front end, and the length in vitro is approximately equal to the length of the cell body. The nucleus is located in the center of the cell and an eyepoint is located on one side near the nucleus. Chloroplasts are cupped, complex, reticulated or granular, with a typical chlorella protein nucleus. The cell wall is composed of two layers, the inner layer is mainly cellulose and the outer layer is pectin. The motile cells are mostly green. The immobile cell is circular, has no flagellum and does not move, the change range of the cell size is large, and the average diameter is 20-30 μm.

The vegetative propagation of H.pluvialis is cell division, producing 2, 4, 8, or even more daughter cells. When asexual reproduction is environmentally undesirable, chlamydospores develop. There has been controversy over the existence and type of sexual reproduction. There are three vegetative growth types of haematococcus pluvialis: (1) photoautotrophic type, that is, photoautotrophic growth is carried out by absorbing carbon dioxide with light energy; (2) heterotrophic growth, which utilizes organic carbon source such as acetate to perform heterotrophic growth under dark conditions, but the growth rate and the maximum biomass of the heterotrophic growth grow under the complete heterotrophic conditions, and the growth rate and the maximum biomass of the heterotrophic growth are lower than those of the photoautotrophic and mixotrophic growth; (3) mixed nutrition type, namely, mixed nutrition growth is carried out by simultaneously utilizing an organic carbon source and carbon dioxide under the illumination condition. The growth of haematococcus pluvialis can be obviously promoted by adding sodium acetate under the condition of illumination, and the growth rate of the haematococcus pluvialis is about the sum of photoautotrophic growth rate and heterotrophic growth rate.

Haematococcus pluvialis can accumulate a large amount of carotenoids under stress or induction conditions, the content of carotenoids can be 2% -5% of its dry weight, wherein more than 80% is astaxanthin with high economic value. Haematococcus pluvialis is considered the best source of astaxanthin. Although astaxanthin production by utilizing haematococcus pluvialis has a wide development prospect, a plurality of key technologies and problems need to be broken through in application and development, such as the haematococcus pluvialis has high requirements on culture environment and is easy to pollute mixed bacteria and other miscellaneous algae; the contradiction between astaxanthin accumulation and biomass accumulation, and the like.

The method of genetic transformation to improve the characteristics of Haematococcus pluvialis is an important way to solve these problems. At present, a haematococcus pluvialis cell nucleus transformation system is constructed and completed through a gene gun method, an electric shock transformation method and an agrobacterium transfection method, but as the haematococcus pluvialis genome sequence is not completely sequenced, the structural complexity of the haematococcus pluvialis cell nucleus transformation system is far greater than that of most green algae, the cell nucleus transformation system is difficult to use, and a plurality of problems exist, such as: it is difficult to perform site-directed mutagenesis, to express a plurality of exogenous genes, and to achieve a high exogenous gene mutation rate. Under such circumstances, it is imperative to develop a Haematococcus pluvialis chloroplast transformation system.

Haematococcus pluvialis contains a cupped chloroplast, which is very large in its chloroplast genome, about 1.1G. Similar to the chloroplast genome of other haematococcus pluvialis, the gene distribution in haematococcus pluvialis chloroplasts is very scattered and contains the structure of introns, and the structure of polycistrons has not been found so far. The phenomena show that haematococcus pluvialis chloroplast has some eukaryotic properties, and whether a chloroplast transformation system with prokaryotic expression characteristics is applicable or not is unknown. At present, no research report of the chloroplast expression system of the alga exists, which seriously hinders the further research and application of the alga.

Disclosure of Invention

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

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

a haematococcus pluvialis chloroplast expression system comprises a homology arm, a promoter and a terminator, wherein the homology arm comprises an upstream homology arm of a sequence shown in SEQ ID NO. 1 and a downstream homology arm of a 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 recombinant empty vector sequentially comprises an upstream homology arm, at least one promoter, a selective marker gene, at least one terminator and a downstream homology arm.

The promoter is used for regulating and controlling exogenous genes; or, a promoter regulating a foreign gene and a promoter regulating a selectable 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, a terminator which regulates the foreign gene and a terminator which regulates the selectable 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 selective marker gene is glufosinate-resistance genebarA gene.

The upstream homology arm is shown by a base 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 shown by a base 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 haematococcus pluvialis chloroplast expression system in the transformation of haematococcus pluvialis chloroplasts.

The method specifically comprises the following steps: introducing an exogenous gene into the constructed empty vector, and then introducing the vector into haematococcus pluvialis by using a gene gun transformation method, wherein the bombardment pressure of a gene gun is 650psi, and the bombardment distance is 9 cm; the transformed haematococcus pluvialis is screened by using the herbicide glufosinate.

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

1. the invention provides technical parameters for transforming haematococcus pluvialis chloroplast by using a particle gun method.

2. The invention provides a screening marker gene for haematococcus pluvialis chloroplast transformation. The false positive rate of the haematococcus pluvialis mutant strain screened by the method is much lower than that of the conventional antibiotic screening marker.

3. The invention provides two segments of haematococcus pluvialis chloroplast genome segments, which are used for constructing a haematococcus pluvialis chloroplast transformation vector. In the haematococcus pluvialis chloroplast genome, these two fragments are directly linked and have two copies, one within each of the two inverted repeats of the chloroplast genome. The insertion of the foreign gene between the two fragments increases the probability of homologous recombination, and the insertion of the foreign gene does not affect the expression and function of other genes.

4. The invention provides a promoter and a terminator of a high-efficiency expression gene on a haematococcus pluvialis chloroplast genome, which are used for constructing a chloroplast expression vector of a foreign gene. The promoter and the terminator are necessary elements for expressing the exogenous gene, and the endogenous regulatory sequence, especially the promoter of the endogenous high-expression gene, can promote the expression of the exogenous gene.

5. The haematococcus pluvialis chloroplast stable expression system can promote and improve the growth performance of the haematococcus pluvialis, improve the growth speed, enhance the antibacterial property and have more superior large-scale culture characters, thereby improving the yield and the yield of the astaxanthin in the production angle.

6. The stable expression system of haematococcus pluvialis chloroplast can express and accumulate a large amount of nutritional proteins or animal vaccines in the haematococcus pluvialis chloroplast, so that the bait performance of the haematococcus pluvialis is improved.

Drawings

FIG. 1 is a haematococcus pluvialis empty vector map provided by an embodiment of the present invention.

FIG. 2 is a haematococcus pluvialis expression vector map provided by an 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 a Wild strain; lane Mutants is a transgenic algal strain).

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

FIG. 5 is a Southern hybridization pattern of transgenic Haematococcus pluvialis provided in an embodiment of the present invention (wherein lane Wild represents a Wild strain and lane Mutant represents a transgenic algal strain).

FIG. 6 shows Western hybridization of transgenic Haematococcus pluvialis according to the present invention (wherein Lane Wild represents a Wild strain; Lane Mutant represents a positive transgenic strain).

Detailed Description

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