阅读说明：本技术 一种用于嗜热微生物遗传操作的诱导型毒素-抗毒素元件 (Inducible toxin-antitoxin element for genetic manipulation of thermophilic microorganisms ) 是由 徐俊 宋庆浩 于 2020-06-23 设计创作，主要内容包括：本发明公开了一种用于嗜热微生物遗传操作的诱导型毒素-抗毒素元件；所述元件携带有诱导型启动子、热稳定的毒素基因和抗毒素基因；所述制备方法包括构建正向选择标记和诱导型毒素-抗毒素反向选择标记；该原件可在嗜热微生物中进行遗传操作。本发明提供的热稳定的毒素抗毒素元件可在嗜热微生物中进行遗传操作时作为高效的选择性标记,该元件可应用于嗜热微生物的分子遗传学研究。(The invention discloses an inducible toxin-antitoxin element for genetic manipulation of thermophilic microorganisms; the elements carry inducible promoters, heat-stable toxin genes and antitoxin genes; the preparation method comprises the steps of constructing a forward selection marker and an inducible toxin-antitoxin reverse selection marker; the elements can be genetically manipulated in thermophilic microorganisms. The heat-stable toxin antitoxin element provided by the invention can be used as a high-efficiency selective marker during genetic manipulation in thermophilic microorganisms, and the element can be applied to molecular genetic research of thermophilic microorganisms.)

1. A heat-stable toxin-antitoxin selective selection marker HHP-TAC, wherein the selective selection marker HHP-TAC comprises a high hydrostatic pressure inducible promoter from hyperthermophilic archaea, a heat-stable toxin gene and an antitoxin gene, a constitutive promoter, and an HMG-CoA reductase gene.

2. The selectable marker HHP-TAC for use according to claim 1, wherein the constitutive promoter comprises the constitutive promoter P_hmtBAnd constitutive promoter P_gdh。

3. The selective selection marker HHP-TAC according to claim 2, wherein the constitutive promoter P_hmtBAnd constitutive promoter P_gdhDerived from plasmid pTS 535.

4. The selective selection marker HHP-TAC according to claim 1, wherein the inducible promoter is the high hydrostatic pressure inducible promoter P from the hyperthermophilic archaea Pyrococcus yayanosii_hhp(ii) a Genes encoding heat stable toxins and antitoxins are derived from the PF0776 gene and PF0775 gene of hyperthermophilic archaea p.

5. The selective screening marker HHP-TAC according to claim 1, wherein the base sequence of the selective screening marker HHP-TAC is shown in SEQ ID No. 1.

6. A process for the preparation of the selective selection marker HHP-TAC of claim 1, comprising the following steps:

and (2) using a fusion PCR amplification technology to sequentially fuse a high hydrostatic pressure inducible promoter with a heat-stable toxin gene, a constitutive promoter with an antitoxin gene and an HMG-CoA reductase gene to obtain the selective screening marker HHP-TAC.

7. The method for producing the selective selection marker HHP-TAC of claim 6, wherein the high hydrostatic pressure inducible promoter is the high hydrostatic pressure inducible promoter P derived from Pyrococcus yayanosii, a hyperthermophilic archaea_hhp。

8. The method for producing the selectable marker HHP-TAC according to claim 6, wherein the thermostable toxin gene is PF0776 gene from P.furiosus of hyperthermophilic archaea; the antitoxin gene is PF0775 gene from hyperthermophilic archaea P.furiosus.

9. Use of the selective selection marker HHP-TAC according to claim 1, in thermophilic microorganisms, wherein said selective selection marker HHP-TAC is directly amenable to molecular genetic manipulation in the prototrophy of thermophilic microorganisms.

10. Use according to claim 9, characterized in that the high hydrostatic pressure inducible promoter in the selective selection marker HHP-TAC is used to control the expression of the protein.

Technical Field

The invention relates to a method for constructing a genetic screening marker by utilizing a heat-stable toxin-antitoxin element and application thereof, in particular to a method for inducing genetic manipulation of the toxin-antitoxin element in a hyperthermophilic microorganism by virtue of high hydrostatic pressure and application thereof.

Background

The genetic manipulation of hyperthermophilic microorganisms has been studied for over 20 years, but the number of genetic tools available has been very limited due to the limitations of high temperature conditions. For example, Thermococcus kodakaraensis KOD1 was the first strain to develop a gene knockout system using the pryF gene as a selection marker and a uracil-deficient strain as the starting strain in combination with a synthetic medium. Several amino acid defect markers were subsequently developed: like tryptophan-deficient strains, histidine-deficient strains, these markers can only be used in synthetic media. Until the development of an over-expression system for simvastatin/HMG-CoA reductase, it was indeed achieved to operate in a nutrient-rich medium. However, no scientist has developed a genetic system for traceless knockout directly in prototrophic strains of hyperthermophilic archaea so far. The original strains of the existing hyperthermophile genetic operation are amino acid/nucleoside defective strains, and compared with prototrophic strains, the defective strains have certain interference on metabolic network and adaptability research, thereby increasing the research difficulty. Furthermore, genetic manipulation in synthetic media takes longer due to the slower growth of the strain. There is therefore a great need to develop a genetic system that can be genetically manipulated in prototrophic strains.

Recently, the Toxin Antitoxin (TA) system has achieved genetic manipulation in mesophilic bacteria. In particular, type II TA systems, since toxins and antitoxins are both proteins and interact directly, toxin proteins inhibit bacterial growth by transcription or translation, antitoxins neutralize this toxicity, and the TA system is widely distributed among bacteria and archaea, and it is a viable option to find an available TA system for genetic manipulation, particularly in hyperthermophiles that can tolerate higher temperatures.

Disclosure of Invention

The invention aims to provide a selective element which can be directly applied to genetic manipulation of prototrophic hyperthermophilic microorganisms, in particular to a high hydrostatic pressure induced toxin-antitoxin element for genetic manipulation of hyperthermophilic microorganisms; the invention also provides a method for preparing the selective element and application thereof.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the invention relates to a heat-stable toxin-antitoxin selective selection marker HHP-TAC comprising a high hydrostatic pressure inducible promoter from hyperthermophilic archaea, a heat-stable toxin gene and antitoxin gene, a constitutive promoter, and an HMG-CoA reductase gene.

Preferably, the constitutive promoter comprises a constitutive promoter P_hmtBAnd constitutive promoter P_gdh。

Further preferably, said constitutive promoter P_hmtBAnd P_gdhThe region was derived from plasmid pTS 535.

Preferably, the inducible promoter is a high hydrostatic pressure inducible promoter P from the hyperthermophilic archaea Pyrococcus yayanosii_hhp(ii) a Genes encoding heat stable toxins and antitoxins are derived from the PF0776 gene and PF0775 gene of hyperthermophilic archaea p.

Preferably, the base sequence of the selective screening marker HHP-TAC is shown as SEQ ID NO. 1.

In a second aspect, the invention relates to a process for the preparation of the selective screening marker HHP-TAC, comprising the following steps:

and (2) using a fusion PCR amplification technology to sequentially fuse a high hydrostatic pressure inducible promoter with a heat-stable toxin gene, a constitutive promoter with an antitoxin gene and an HMG-CoA reductase gene to obtain the selective screening marker HHP-TAC.

Preferably, the high hydrostatic pressure inducible promoter is a high hydrostatic pressure inducible promoter P derived from the hyperthermophilic archaea Pyrococcusyayanosii_hhp. The stress-inducible promoter P_hhpThe length of the element sequence is 488bp, and the base sequence is shown as SEQ ID NO. 13.

Preferably, the heat-stable toxin gene is PF0776 gene from hyperthermophilic archaea p.furiosus; the antitoxin gene is PF0775 gene from hyperthermophilic archaea P.furiosus. The lengths of the sequences of the toxin gene PF0776 and the antitoxin gene PF0775 are 327bp and 324bp respectively; the base sequence of the heat-stable toxin gene is shown as SEQID NO. 14; the base sequence of the antitoxin gene is shown in SEQ ID NO. 15. The inhibition of the growth of the strain can be realized by controlling the expression of the toxin gene.

It is further preferred that the first and second liquid crystal compositions,sequentially amplifying high hydrostatic pressure inducible promoter P by using fusion PCR amplification technology_hhpFusion with heat-stable toxin gene PF0776, constitutive promoter P_hmtBFusion with antitoxin gene PF0775, and constitutive promoter P_gdhFused with HMG-CoA reductase gene to obtain the selective screening marker HHP-TAC.

Preferably, the HMG-CoA reductase gene is derived from the hyperthermophilic archaea P. Overexpression of the gene can confer simvastatin resistance to the hyperthermophilic archaea. The base sequence is shown in SEQ ID NO. 16.

Preferably, the production method further comprises a step of verifying the resulting selective element.

Preferably, the validation specifically comprises inserting the selective element into a traceless knockout plasmid pUS776TA1369 for plasmid amplification in E.coli DH5 alpha and sequencing validation, and then transferring the selected element into the archaea thermophila P.yayanosii A1 for validation of successful knockout experiments.

In a third aspect, the invention provides the use of said selective selection marker HHP-TAC in thermophilic microorganisms, which allows direct molecular genetic manipulation in the prototrophy of thermophilic microorganisms.

Preferably, the high hydrostatic pressure inducible promoter in the selective selection marker HHP-TAC is used to control expression of the protein.

The high-pressure induction type toxin-antitoxin element for the genetic manipulation of the hyperthermophilic microorganisms constructed by the invention can be used as a genetic manipulation tool for directly knocking out genes in prototrophic hyperthermophilic microorganisms and greatly expands the selection range of negative selection markers. Meanwhile, the element carries a simvastatin resistance marker gene available at high temperature, so that resistance screening of the simvastatin resistance marker gene at high temperature is possible. Enriches the variety of the genetic tools of the hyperthermophilic microorganisms and is beneficial to the functional genomics research of the strains.

Compared with the prior art, the invention has the following beneficial effects:

(1) the selective element HHP-TAC can efficiently perform forward selection and high-pressure induction direction selection of simvastatin resistance in a hyperthermophilic archaea strain P.yayanosii A1, and can be applied to gene function identification research as gene genetic operation of the hyperthermophilic archaea.

(2) The pressure-inducible promoter of the invention is a high hydrostatic pressure-inducible promoter P from hyperthermophilic archaea Pyrococcus yayanosii_hhp(ii) a The stress-inducible promoter P_hhpThe length of the element sequence is 488 bp. The stress-inducible promoter is induced by physical (stress), is easy to operate and has low application cost.

(3) The selective element HHP-TAC of the invention has many effects: high-pressure induced expression can be used for expressing toxic proteins, can also be used for controlling expression of other proteins, and can be used for overexpression purification of proteins and the like; toxin-antitoxin elements are widely distributed in hyperthermophilic microorganisms, and the genetic marker does not have the problem of high-temperature inactivation. Furthermore, the presence of multiple toxin antitoxin elements in the same microorganism allows for the development of different selectable markers.

(4) The toxin-antitoxin element based on pressure induction can be directly used for genetic manipulation in prototrophic strains, and the interference of auxotrophic genes is eliminated.

Drawings

Other features, objects and advantages of the invention will become apparent upon reading the following detailed description of alternative embodiments with reference to the accompanying drawings in which:

FIG. 1 is an explanatory diagram of selective element HHP-TAC construction;

FIG. 2 is a schematic diagram of a pUS776TA1369 traceless knockout plasmid;

FIG. 3 is a traceless knockout strain Δ 1369^-A genome level PCR verification result graph; wherein, the picture A is PCR fragment analysis between upstream and downstream regions of a target gene PYCH _1369 gene, and M is 1kb DNA ladder; 1, A1; 2, Δ 1369^-(ii) a FIG. B is an internal PCR fragment analysis of the target gene PYCH _1369 gene, M is a 1kb DNA ladder; 1, A1; 2, Δ 1369^-(ii) a FIG. C is a Lugol iodine solution method residual starch content analysis, 1, TRM containing 2 ‰ (M/V) soluble starch; 2, culturing the strain A1 in a TRM medium containing 2 per mill (M/V) of soluble starch; 3, traceless knockout strain Δ 1369^-Culturing in TRM medium containing 2 ‰ (M/V) soluble starch; 4, TRM.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

The strains involved in the invention are all known strains, and the disclosure conditions are as follows:

the hyperthermophilic archaea p.yayanosii has been described in the literature: pyrococcus yayanosii sp. nov, anobiligate bipolar hyperthermophilic archaea isolated from a deep-sea hydrothermal vent, 2011;

fusiosus sp.thermophilus is already in the literature: pyrococcus furiosus sp. nov. expression and gene of marine microbiological archaebacteria growing opportunity at 100 ℃, 1986;

the hyperthermophilic archaea p.yayanosii a1 has been disclosed in the literature: the P.yayanosii A1 strain has been published in Genetic tools for the piezoelectric hyperthermophilic archaon pyrococcus yuyanosii 2015;