CN108795941B

CN108795941B - An inducible promoter and its application

Info

Publication number: CN108795941B
Application number: CN201810705759.8A
Authority: CN
Inventors: 赵春梅; 隋炯明; 李恩广; 王晶珊; 乔利仙; 郑春花; 孔祥远
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2018-07-02
Filing date: 2018-07-02
Publication date: 2021-04-23
Anticipated expiration: 2038-07-02
Also published as: CN108795941A

Abstract

The invention discloses an inducible promoter and an application thereof, belonging to the technical field of genetic engineering. The promoter sequence of the present invention is shown in SEQ ID NO. 1, and the present invention also provides a recombinant vector, transformant or expression cassette containing the promoter. The promoter of the present invention constructs a plant expression vector to transform plants, which can drive exogenous genes to be induced and expressed by salt stress in transgenic plants, and is suitable for constructing plant expression vectors for crop stress resistance improvement and crop high yield and quality improvement.

Description

Inducible promoter and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to an inducible promoter and application thereof.

Background

With the continuous deterioration of the environment, the stress of salt and alkali and other adversities has become a worldwide problem, and the cultivation of new varieties of crops with various stress resistance has become one of the main targets of the research of broad breeders.

At present, a large number of excellent new genes are isolated and cloned, but promoters widely used in plant genetic engineering, such as CaMV 35S promoter from cauliflower mosaic virus, and promoters from plants such as Act1 and Ubi1, are all constitutive promoters. The constitutive promoter driven gene has different expression in different tissues and organs of plant, and this raises some problems in application. In most cases, it is undesirable to express the foreign gene throughout the entire plant and throughout the entire growth period of the transgenic plant, because on the one hand, the metabolic burden on the plant is increased, and on the other hand, some foreign proteins are not necessary or even toxic to the plant and are not beneficial to the normal growth of the plant.

Successful application of plant genetic engineering techniques requires not only extensive regulation of promoters at different levels, but also promoters adapted to different plant backgrounds, different organs, tissues, transgene types to avoid adverse effects during the transgene process.

The inducible promoter can enable the exogenous gene to respond to certain signals and is only expressed under the stimulation of special signals, so that a research hotspot in the field of plant biotechnology is to discover the plant-derived inducible promoter.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to: the first is to provide an inducible promoter, the second is to provide a recombinant vector, an expression cassette and a transformant of the promoter, and the third is to provide the application of the inducible promoter, the recombinant vector, the expression cassette and the transformant.

In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:

a promoter, a nucleic acid sequence selected from any of the following groups and having promoter function:

1) the nucleic acid sequence shown in SEQ ID NO.1 or at least one section of 103 bp-1914 bp, 536 bp-1914 bp, 1250 bp-1914 bp, 1405 bp-1914 bp and 1736 bp-1914 bp of the nucleic acid sequence shown in SEQ ID NO. 1;

2) a nucleic acid sequence complementary to the nucleic acid sequence of 1);

3) a nucleic acid sequence which hybridizes with the nucleic acid sequence defined in 1) or 2) under conditions of high stringency;

4) a nucleic acid sequence obtained by modifying the nucleic acid sequence shown in 1) or 2) above by substitution, deletion or addition of one or more bases;

5) a nucleic acid sequence having more than 90% homology with the nucleic acid sequence defined in 1) or 2).

Recombinant vectors, transformants or expression cassettes containing the above promoters.

On the basis of the scheme, the recombinant vector is a recombinant plasmid inserted with the promoter at a polyclonal site.

On the basis of the scheme, the recombinant vector is a recombinant plasmid obtained by inserting the promoter sequence shown in the description into a position between HindIII and Nco I enzyme cutting recognition sites.

On the basis of the scheme, the vector is pMD18-T or pCAM 1301.

On the basis of the scheme, the expression cassette consists of a nucleic acid sequence with a promoter function, a target gene to be started and expressed and a transcription termination sequence; the nucleic acid sequence with the promoter function is connected with the target gene, and the target gene is connected with the transcription termination sequence; the nucleic acid sequence with the promoter function is the same as or different from the target gene sequence in source.

On the basis of the scheme, the transformant is a recombinant bacterium or a transgenic cell line transformed into a recombinant vector.

On the basis of the scheme, the promoter is an inducible promoter.

The promoter is applied to starting or promoting the expression of a target gene under defense, stress or stress conditions.

And a primer pair for amplifying the full length of the promoter and any fragment thereof.

The technical scheme of the invention has the advantages that:

the technical scheme of the invention obtains a novel inducible promoter which can drive the salt stress induced expression of the exogenous gene in the transgenic plant and is suitable for constructing a plant expression vector for improving the stress resistance of crops and improving the high yield and quality of the crops.

The invention obtains a salt stress inducible promoter which effectively plays a role in dicotyledonous plants such as tobacco, and realizes that the promoter drives the salt stress inducible expression of exogenous genes in the tobacco.

By fusing the inducible promoter disclosed by the invention with a GUS reporter gene and tracking the expression condition of GUS enzyme in a tobacco plant under the condition of salt induction treatment or not, the salt induction expression mode of the promoter in the tobacco plant can be determined.

Drawings

FIG. 1 is an electrophoretogram in which M1 and M2 in a and b are markers, lane 1 in a is an electrophoretogram of a cloned AhRabG3f-P promoter, and lane 2 in b is an electrophoretogram of enzyme digestion verification of a constructed pMD18-T-AhRabG3f-P vector;

FIG. 2, prediction of functional elements in the AhRabG3f-P promoter;

FIG. 3 is a schematic diagram of the AhRabG3f-P promoter deletion fragment;

FIG. 4, restriction enzyme digestion verification of a series length promoter plant expression vector; lane M: marker DL 2000;

lanes

1 and 2 are the pCAMBIA1301 and pCAM-RabG3f full-length promoters, respectively; lane 3-7 is the restriction enzyme digestion verification of the 5' serial deletion promoter expression vectors of P1-P5;

FIG. 5, GUS staining analysis of transgenic tobacco leaves; 1-6 respectively represent pCAM-RabG3f-P, pCAM-RabG3f-P1, pCAM-RabG3f-P2, pCAM-RabG3f-P3, pCAM-RabG3f-P4 and pCAM-RabG3f-P5 transgenic tobacco leaves;

FIG. 6 fluorescent quantitation of GUS enzyme activity in transgenic tobacco leaves under NaCl treatment (in two side-by-side columns, the left column is 0h and the right column is 12 h).

Detailed Description

Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.

The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

Example 1: cloning and sequence analysis of peanut small GTP binding protein gene promoter AhRabG3f-P

Designing a promoter primer according to the reported AhRabG3f gene (GenBank: ES752601) sequence in GenBank, and amplifying a promoter region on the promoter primer; and the restriction sites Hind III (shown in lower case) and Nco I (shown in lower case) and the protecting bases (shown in underline) (the same applies below) were added to both ends of the primer, which was constructed into pMD18-T vector.

An upstream primer: 3 f-P:ATAaagcttTATATCTCATCCGACTT(SEQ ID NO.2)

a downstream primer: 3 f-R:ATAccatggCCGATCGGGAAAAAAAAGTGTAACC(SEQ ID NO.3)

the primer sequences used were synthesized by Shanghai Biotechnology Ltd.

The genome DNA of the peanut is used as a template, and the primer is used for PCR amplification to prepare the AhRabG3f gene promoter fragment. The PCR reaction system is as follows:

PCR reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30 seconds, annealing at 58 ℃ for 50 seconds, and extension at 72 ℃ for 2.5 minutes for 35 cycles; final extension at 72 ℃ for 10 min; keeping the temperature at 4 ℃.

The amplification products obtained were electrophoretically detected on a 1% agarose gel, and the results showed that: there was a specific band at about 1900bp, the size of which corresponded to the expected theoretical value (FIG. 1). Recovering and purifying the PCR product by using a DNA agarose gel recovery kit; connecting the purified product with a pMD18-T vector to obtain a recombinant plasmid named as pMD18-T-AhRabG3f-P, transforming escherichia coli DH5 alpha competent cells, carrying out Amp and blue-white screening, selecting a recombinant plasmid, carrying out PCR and Hind III/Nco I double enzyme digestion identification on the recombinant plasmid to obtain a positive cloned bacterial liquid (shown in the attached figure 1), and sending the positive cloned bacterial liquid to TaKaRa company for sequencing; the sequence of the obtained promoter is shown in SEQ ID NO. 1:

SEQ ID NO.1

the sequencing result is searched and aligned in peanutbase, and the obtained sequence is confirmed to be an upstream regulatory sequence (AhRabG3f-P) of the AhRabG3f gene, and the length is 1930 bp. Sequence analysis of the nucleotide sequence of AhRabG3f-P (SEQ ID NO:1 in the sequence Listing) was performed using PLACE (Plant cis-acting alignment DNA-elements, PLACE) software, and the results showed that ABA response element (ABRE), drought stress response element (MBS), defense and stress response element (TC-rich repeat), and heat stress response element (HSE) and the like were present upstream of AhRabG3f-P promoter (FIG. 2).

Example 2: promoter function verification

2.1 construction of recombinant expression vectors

The vector plasmid pCAMBIA1301 (a strain purchased from Bio Inc. or CAMBIA) was extracted from E.coli, and the pCAMBIA1301 plasmid was double-digested with HindIII/NcoI to recover a large vector fragment (containing the GUS reporter gene sequence). The plasmid pMD18-T-AhRabG3f-P prepared in example 1 was digested simultaneously with Hind III/Nco I, and the AhRabG3f-P promoter fragment was recovered by agarose gel electrophoresis. The AhRabG3f-P promoter fragment recovered above was ligated with the pCAMBIA1301 digested large vector fragment overnight at 16 ℃ under ligase catalysis to complete the construction of the AhRabG3 f-P: GUS fusion gene on the pCAMBIA1301 vector, and the recombinant plasmid was named pCAM-RabG3 f-P.

A connection system:

the ligation product was transformed into E.coli DH 5. alpha. competent cells in the same manner as in example 1. White colonies on a transformation plate (Kan resistance) are selected, plasmids are extracted according to a conventional method, and PCR and HindIII/NocI double enzyme digestion identification are carried out on recombinant plasmids. And (3) transforming the identified positive clone plasmid into agrobacterium Gv3101 by a freeze-thaw method to obtain the engineering agrobacterium for plant transformation.

2.2 genetic transformation of plants

Transforming tobacco leaf (size 0.5 multiplied by 0.5) by using agrobacterium tumefaciens bacterial liquid leaf disc method containing pCAM1301-RabG3f-P plasmid, co-culturing for 2 days, then transferring to a differentiation medium (containing 50mg/L hygromycin) for resistance screening, subculturing the resistant callus once every two weeks until resistant plantlets appear, transferring to a rooting medium (containing 50mg/L hygromycin) for rooting, hardening seedlings and transplanting until T is harvested₁And (5) seed generation. The obtained resistant plants are subjected to positive detection by a PCR method, the primer is an internal primer of the gus gene, and the size of the product is 220 bp.

2.3 RabG3f-P drives GUS expression of transgenic plants

GUS histochemical staining is carried out on transgenic tobacco T0 generation leaves. Treating plant leaves by using X-Gluc reaction liquid, adding a sample material into the X-Gluc reaction liquid to react for 24 hours at 37 ℃, sucking out the reaction liquid, decoloring for 2-3 times by using 75% ethanol until a negative material is white, and observing by naked eyes or performing photomicrography, wherein the result shows that: the promoter of the invention can drive GUS protein to express in leaf tissue, and although the expression is weaker, the sequence shown by SEQ ID NO.1 has promoter activity. (FIG. 5).

Example 3 truncated promoter Activity assay

Construction of 3.1 series deletion expression vectors

According to the analysis and prediction result of software on AhRabG3f-P promoter functional elements, pCAM1301-RabG3f-P plasmid is used as a template, deletion primers are designed to delete cis-acting elements in sequence, PCR amplification is carried out to obtain 5 promoter 5' end gradient deletion fragments, Hind III and Noc I are used for double enzyme digestion, the deletion fragments are constructed on a vector of pCAMBIA1301 after enzyme digestion to obtain a series of deletion expression vectors which are named as pCAM-RabG3f-P respectively₁、pCAM-RabG3f-P₂、pCAM-RabG3f-P₃、pCAM-RabG3f-P₄And pCAM-RabG3f-P₅The recombinant vector was subjected to HindIII/NocI double restriction assay (FIG. 4). The specific operation process can be referred to example 2, and the deletion primer sequences are as follows:

TABLE 1 deletion primer sequences

The primer sequences used were synthesized by Shanghai Biotechnology Ltd.

3.2 Agrobacterium-mediated genetic transformation

The concrete operation process can be referred to example 2

3.3GUS Activity assay

3.3.1 histochemical staining for GUS

The specific operation process can refer to example 2.

As shown in FIG. 5, for the transgenic tobacco transformed by the 6 recombinant expression vectors (pCAM-RabG3f-P, pCAM-RabG3f-P1, pCAM-RabG3f-P2, pCAM-RabG3f-P3, pCAM-RabG3f-P4 and pCAM-RabG3f-P5), the young leaves were stained by GUS, but the leaves of the plants transformed by the promoters with different deletion lengths were stained in different depths, wherein the GUS staining of the tobacco leaves transformed by the deletion promoter P1 and the tobacco leaves transformed by the P3 was deeper, which indicated that the promoter ability was stronger, and even the shortest promoter (length of 186bp) with the deletion of P5 also had the driving ability. In contrast, the young leaves of tobacco transformed by the full-length promoter AhRabG-3f-P stain the lightest and the promoter capacity is relatively weak.

3.3.2 fluorescent quantitation of GUS Activity

The activity of the GUS gene is measured by a fluorescence quantitative method, the GUS gene generates a product 4-MU with fluorescence by taking 4-MUG as a substrate of action, and the activity of the GUS gene is detected by detecting the fluorescence intensity of the product. GUS activity was measured in pmol 4-MU produced per minute per mg of soluble protein.

The specific method comprises the following steps: taking 6-leaf-stage transgenic tobacco seedlings to carry out NaCl (250mmol/L) stress treatment, and taking leaves to store at-70 ℃ at different treatment time periods. Taking 100mg of leaves, adding 100 mu L of extraction buffer solution, centrifuging for 10 minutes at 8000g, taking 20 mu L of supernate, adding preheated 200 mu L of reaction buffer solution (containing 1mmol/L MUG), mixing uniformly, reacting for 1 hour at 37 ℃, taking 100 mu L, adding 900 mu L of stop solution to terminate the reaction, and measuring the Ex340/Em465 value of each sample by using a fluorescence spectrophotometer. A standard curve was prepared using 4-MUP as a standard. Determination of protein content of samples Using the Bradford method, a standard curve was prepared with BSA and the enzyme activity was calculated in units of 4-MU pmol/mg/protein/min in conjunction with the protein content of each sample.

As shown in FIG. 6, when the transgenic tobacco was not stressed, GUS protein expression could be detected in the transgenic tobacco into which GUS gene was driven by each length fragment of AhRabG3f promoter, and the GUS fluorescence quantitative analysis result was consistent with the histochemical staining result (FIGS. 5 and 6). As compared with each structure, in the transgenic tobacco into which the full-length promoter AhRabG-3f-P was introduced, GUS expression activity was the lowest and the structure P was deleted₁And P₃Retains a high promoter activity, and it is presumed that a cis-element inhibiting the promoter activity exists in the sequence between 1930 to 1829, 1395 and 682 of SEQ ID NO. 1. The shortest construct P5 still retained some activity, indicating that it contained the core element of the promoter. After the salt stress induction, the GUS activity is improved in the transgenic tobacco of other length segment promoters except P5, which indicates that the promoter is induced by NaCl, wherein the GUS activity is improved by about 3.3 times in the transgenic tobacco of the full-length promoter AhRabG-3 f-P.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Sequence listing

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Claims

1. A promoter selected from the following nucleic acid sequences having promoter function:

the nucleic acid sequence shown in SEQ ID NO.1 or at least one section of the nucleic acid sequence shown in SEQ ID NO.1 from 103bp to 1914bp, 536bp to 1914bp, 1250bp to 1914bp, 1405bp to 1914bp and 1736bp to 1914 bp.

2. A recombinant vector, transformant or expression cassette comprising the promoter of claim 1.

3. The recombinant vector according to claim 2, characterized in that: the recombinant vector is a recombinant plasmid in which the promoter of claim 1 is inserted at a multiple cloning site of a cloning vector or an expression vector.

4. A recombinant vector according to claim 3, characterized in that: the vector is pMD18-T or pCAM 1301.

5. The recombinant vector according to claim 3, wherein: the recombinant vector is obtained by inserting the promoter sequence shown in claim 1 into the HindIII and NcoI restriction recognition sites of a cloning vector or an expression vector.

6. The expression cassette according to claim 2, characterized in that: the expression cassette consists of a nucleic acid sequence with a promoter function, a target gene to be started and expressed and a transcription termination sequence; the nucleic acid sequence with the promoter function is connected with the target gene, and the target gene is connected with the transcription termination sequence; the nucleic acid sequence with the promoter function is the same as or different from the target gene sequence in source.

7. The transformant according to claim 2, characterized in that: the transformant is a recombinant bacterium or a transgenic cell line transformed into a recombinant vector.

8. The promoter according to claim 1, wherein: the promoter is an inducible promoter.

9. Use of the promoter of claim 1 to promote or promote expression of a gene of interest under NaCl stress conditions.

10. The primer pair for amplifying the promoter of claim 1 is a combination of any one of SEQ ID NO.2, SEQ ID NO.4-8 and SEQ ID NO. 3.