阅读说明：本技术 一种调控目的基因表达和cca效应的元件 (Element for regulating and controlling target gene expression and CCA effect ) 是由 石贵阳 李由然 肖丰旭 张梁 丁重阳 徐沙 顾正华 张玉鹏 王瀚容 于 2020-06-28 设计创作，主要内容包括：本发明公开了一种调控目的基因表达和CCA效应的元件,属于生物工程技术领域。所述元件包括载体、启动子和靶基因结合序列,所述靶基因结合序列为AGCTTT-Y-AAAGCT或AAAGCT-Y-AGCTTT(Y代表长度为0-14bp的核苷酸序列)。含有AGCTTT-Y-AAAGCT的元件,响应葡萄糖存在时的CCA效应,甘油存在时的CCR效应；含AAAGCT-Y-AGCTTT的元件,响应葡萄糖存在时的CCR效应,甘油存在时的CCA效应。本发明为后续地衣芽孢杆菌表达原件的开发提供了高效的、靶向的分子手段。(The invention discloses an element for regulating and controlling target gene expression and CCA effect, belonging to the technical field of biological engineering. The element comprises a carrier, a promoter and a target gene binding sequence, wherein the target gene binding sequence is AGCTTT-Y-AAAGCT or AAAGCT-Y-AGCTTT (Y represents a nucleotide sequence with the length of 0-14 bp). An element comprising AGCTTT-Y-AAAGCT, responsive to the CCA effect in the presence of glucose, the CCR effect in the presence of glycerol; an AAAGCT-Y-AGCTTT containing element is responsive to the CCR effect in the presence of glucose and the CCA effect in the presence of glycerol. The invention provides an efficient and targeted molecular means for the subsequent development of a bacillus licheniformis expression element.)

1. An element for regulating the expression of a target gene and/or the effect of CCA, wherein the element comprises a promoter, a target gene binding sequence and a vector; the target gene binding sequence is positioned at the upstream of a transcription initiation site of a promoter, the promoter and the target gene binding sequence are positioned in a multi-cloning site region of a vector, and the target gene binding sequence is AGCTTT-Y-AAAGCT or AAAGCT-Y-AGCTTT, wherein Y represents a nucleotide sequence with the length of 0-14 bp.

2. The element of claim 1, wherein the vector is pHY-PLK 300.

3. The element of claim 1, wherein the promoter is a trehalose promoter having the nucleotide sequence shown in SEQ ID No. 5.

4. The element according to claim 1, wherein the nucleotide sequence of AGCTTT-Y-AAAGCT is represented by any one of SEQ ID No.1 and SEQ ID No. 2.

5. The element according to claim 1, wherein the nucleotide sequence of AAAGCT-Y-AGCTTT is represented by any one of SEQ ID No.3 and SEQ ID No. 4.

6. A recombinant plasmid comprising the element of any one of claims 1-5.

7. A strain expressing the recombinant plasmid of claim 6.

8. The strain according to claim 7, wherein the strain is a host Bacillus licheniformis (Bacillus licheniformis) ATCC 14580.

9. Use of the element of any one of claims 1 to 5 or the recombinant plasmid of claim 6 or the strain of claim 7 or 8 for regulating the expression of a gene of interest and/or the CCA effect.

Technical Field

The invention relates to an element for regulating and controlling target gene expression and CCA effect, belonging to the technical field of biological engineering.

Background

In 1942, Jacques Monod found that Escherichia coli preferentially utilizes glucose under a mixed carbon source of glucose and galactose, and utilizes galactose only when glucose is consumed, so that a significant secondary growth phenomenon exists. Later, similar effects were found successively between different microorganisms and different carbohydrates, indicating that this hierarchical metabolic phenomenon occurs widely in bacteria, a growth strategy for bacteria in complex environments. Although this hierarchical metabolic phenomenon exists in both gram-positive and gram-negative bacteria, the transcription factors primarily responsible for regulation are quite different. In gram-positive bacteria, the major transcription factor responsible for fractional metabolism is the CcpA protein, while in gram-negative bacteria, the major transcription factor responsible for fractional metabolism is the CRP protein.

The CcpA protein is a member of the LacI-GalR protein family and structurally comprises an N-terminal helix-turn-helix (HTH) motif and a carboxy-terminal domain that binds to an effector. As a main regulatory factor responsible for regulating carbohydrate metabolism in gram-positive bacteria, the protein plays an important role in the fractional metabolism of complex carbohydrates, and is also responsible for regulating nitrogen metabolism, sporulation and some important physiological processes such as expression of a solvent-producing agent and toxic genes.

Studies have shown that the CcpA protein performs the activation or inhibition process by binding at a specific site (cre site), and most of the effects are inhibition, whereas the prerequisite for achieving the activation or inhibition effect is binding to a specific CcpA protein recognition site. The classical cre site is a 14-bp pseudo-symmetric palindromic sequence with the sequence TGWNANCGNTNWCA (W represents base A or T, and N represents any base). The classical cre site reveals the molecular basis of the function of the CcpA protein in gram-positive bacteria, and is a commonality of CcpA protein regulation. However, an increasing number of non-common traits are found in different bacterial strains, for example, recognition site RGAAAANGTTTTCWW for the CcpA protein (R stands for a or G, N stands for any nucleotide, W stands for a or T) is found in Clostridium difficile (Clostridium difficile); recognition site TGTAAAYTTTACA for the CcpA protein was found in clostridium solventogenes (Y represents a variable length nucleotide sequence). These phenomena all indicate that the CcpA protein has commonality and uniqueness in the regulation of different strains, and the analysis of the connection between the commonality and the uniqueness is the molecular basis for disclosing the overall regulation of the CcpA protein.

Thus, taking the industrially advantageous bacterium (i.e., bacillus licheniformis) as an example, the target site binding motif of the CcpA protein in bacillus licheniformis was defined, revealing the uniqueness of the CcpA protein in regulation in bacillus licheniformis.

Disclosure of Invention

Aiming at the defects of the CcpA protein research in the prior stage, the invention provides an element for regulating and controlling the target gene expression and CCA effect, which is obtained based on the target gene binding sequence of the CcpA protein, and provides an efficient and targeted molecular means for developing an efficient expression platform in the future.

The technical scheme of the invention is as follows:

the invention provides an element for regulating and controlling the expression of a target gene and/or CCA effect, which comprises a promoter, a target gene binding sequence and a vector; the target gene binding sequence is positioned at the upstream of a transcription initiation site of a promoter, the promoter and the target gene binding sequence are positioned in a multi-cloning site region of a vector, and the target gene binding sequence is AGCTTT-Y-AAAGCT or AAAGCT-Y-AGCTTT, wherein Y represents a nucleotide sequence with the length of 0-14 bp.

Further, the vector is pHY-PLK 300.

Further, the promoter is a trehalose promoter with nucleotide shown as SEQ ID NO. 5.

Further, the nucleotide sequence of the AGCTTT-Y-AAAGCT is shown as any one of SEQ ID NO.1(AGCTTTATAAAGCT) and SEQ ID NO.2 (AGCTTTAAGGTTCAGAAAAGCT).

Further, the AAAGCT-Y-AGCTTT has a nucleotide sequence shown in any one of SEQ ID NO.3(AAAGCTATAGCTTT) and SEQ ID NO.4 (AAAGCTAAGGTTCAGAAGCTTT).

The invention also provides a recombinant plasmid containing the element and a bacterial strain expressing the plasmid.

Further, the strain is based on Bacillus licheniformis ATCC 14580.

The invention also provides the application of the element or the recombinant plasmid or the strain in regulating and controlling the expression of target genes and/or CCA effect.

The invention has the beneficial effects that:

the invention provides an element for regulating and controlling target gene expression and CCA effect, which comprises a carrier, a promoter and a target gene binding sequence, wherein the target gene binding sequence is AGCTTT-Y-AAAGCT or AAAGCT-Y-AGCTTT (Y represents a nucleotide sequence with the length of 0-14 bp). An element comprising AGCTTT-Y-AAAGCT, responsive to the CCA effect in the presence of glucose, the CCR effect in the presence of glycerol; an AAAGCT-Y-AGCTTT containing element is responsive to the CCR effect in the presence of glucose and the CCA effect in the presence of glycerol.

Drawings

FIG. 1: SDS-PAGE gel electrophoresis of the purified CcpA protein.

FIG. 2: EMSA gel migration electrophoresis chart.

FIG. 3: EMSA gel migration electrophoresis chart.

FIG. 4: and detecting the fluorescence intensity of the green fluorescent protein.

Detailed Description

The present invention will be further illustrated with reference to examples, wherein the nucleotide sequences used in the present invention can be produced by synthetic methods, and the instruments and reagents used in the purification of molecular fragments, construction of recombinant plasmids, selection of transformants, and the like are commercially available.

The primers referred to in the following examples are shown in Table 1.

TABLE 1 primers

Note: the underlined points are the mutation sites.