CN117126851A

CN117126851A - Pepper light mottle virus infection inducible promoter and application thereof

Info

Publication number: CN117126851A
Application number: CN202210557610.6A
Authority: CN
Inventors: 鲁宇文; 陈益; 罗朝鹏; 彭杰军; 郑红英; 陈剑平; 燕飞
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2022-05-19
Filing date: 2022-05-19
Publication date: 2023-11-28

Abstract

The application discloses an inducible promoter for infection of pepper light mottle virus, which is a newly discovered promoter for infection induction expression of pepper light mottle virus, and the sequence of the inducible promoter is shown as SEQ ID NO.1. And combining the inducible promoter with a target gene to construct an expression vector, transferring the expression vector into a host cell, and activating the inducible promoter under the condition of infection of pepper light mottle virus to express the target gene at the downstream. The inducible promoter has potential application value in genetic engineering disease-resistant breeding, in particular to the aspect of improving the resistance of plants to pepper light mottle virus.

Description

Pepper light mottle virus infection inducible promoter and application thereof

Technical Field

The application belongs to the technical field of genetic engineering, and particularly relates to a plant virus infection inducible promoter, in particular to a pepper light mottle virus infection inducible promoter and application thereof.

Background

A promoter is a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription. Promoters in plants are one of the important molecular elements of gene expression, consisting of a number of cis-regulatory elements, controlled by a variety of molecules and physiological stimuli. Promoters mainly include constitutive promoters, inducible promoters, and tissue-specific promoters, depending on the mode of action and function. Among them, inducible promoters (Inducible promoter) are capable of greatly increasing the level of transcription of genes downstream of the promoter upon stimulation by certain specific physical, chemical signals or pathogens.

The inducible promoter and an exogenous gene (such as a resistance gene) are combined to form an expression vector and transferred into a plant, so that the exogenous gene can be expressed under the condition that the plant is induced or stimulated, and the expression level of the exogenous gene in transgenic rice is improved under the condition that the rice stem borer is a pest inducible promoter as disclosed in Chinese patent 201811137886.9; the low-temperature inducible promoter disclosed in Chinese patent 201510085874.6 can improve the expression of exogenous gene GFP in tobacco under low-temperature stress, and the like.

The pepper light mottle virus (Pepper mild mottle virus, PMMoV) belongs to a member of the genus Tobamovirus, the virion is in the shape of a straight rod, is a positive single strand RNA virus, the genome consists of 6357 nucleotides, and has 4 open reading frames (Open reading frames, ORFs) in total, encodes 4 proteins, encoding viral replicase protein p126 (70-3421 nt), viral replicase protein p183 (70-4906 nt), motor protein MP (4909-5680 nt) and Coat protein CP (Coat protein,5685-6156 nt), respectively. PMMoV is one of the important viruses that endanger peppers, plants show mottled or yellow-green alternate flowers She Zhengzhuang after being infected, fruits show malformations after being infected, mottled, causing the peppers to fall leaves, flowers and fruits, and seriously threatening the production of peppers worldwide.

At present, the research of the pepper light mottle virus inducible promoter is less, and the pepper light mottle virus inducible promoter has important significance for resisting pepper light mottle virus infection, improving the resistance or yield of crops such as peppers and the like.

Disclosure of Invention

The application aims to provide a pepper light mottle virus infection inducible promoter and application thereof.

The application discloses a pepper light mottle virus infection inducible promoter, which is named PMMoV-Rpro promoter, and the sequence of the promoter is shown in SEQ ID NO.1. The promoter is obtained by the inventors by the following method: extracting the Nicotiana benthamiana DNA by adopting an SDS-DNA extraction method; PCR amplification is carried out by taking DNA as a template and adopting an upstream primer F1 (SEQ ID NO. 2) and a downstream primer R1 (SEQ ID NO. 3); the nucleic acid sequence was determined by TA cloning.

It should be emphasized that simple transformations or modifications of the DNA sequence of the PMMoV-Rpro promoter are made, for example: deletion, addition or substitution of a base at a certain site; or the modification of a base at a certain position, such as phosphorylation, methylation, amination, sulfhydrylation and the like, does not influence the function of the PMMoV-Rpro promoter, or the PMMoV-Rpro promoter after simple transformation or modification can still regulate or start the expression of a downstream gene under the infection of the pepper light mottle virus. These simple transformations or modifications do not require the inventive effort and thus the PMMoV-Rpro promoter after these simple transformations or modifications is still within the scope of the present application. In addition, a nucleotide sequence which has at least 60%, 80% or 90% or more than 95% homology with the DNA sequence of the PMMoV-Rpro promoter and has the function of the infection-inducing promoter of the pepper light mottle virus belongs to the protection scope of the application.

Further, the application provides an expression cassette, an expression vector and a recombinant host cell, which are specifically as follows:

(1) an expression cassette comprising a PMMoV-Rpro promoter.

(2) An expression vector comprising a PMMoV-Rpro promoter. In some embodiments, the expression vector comprises a gene of interest, and the PMMoV-Rpro promoter is upstream of the gene of interest, the PMMoV-Rpro promoter being capable of expressing or increasing the amount of expression of the gene of interest when PMMoV infection is activated.

(3) An expression vector comprising the PMMoV-Rpro promoter is introduced into a host cell to obtain a recombinant host cell comprising the expression vector, e.g., a plasmid comprising the PMMoV-Rpro promoter is introduced into an Agrobacterium cell by electrotransformation.

Further, the application provides the use of the PMMoV-Rpro promoter.

In some ways, the PMMoV-Rpro promoter can mark the event of PMMoV infection, for example, GFP (Green fluorescent protein ) is spliced downstream of the PMMoV-Rpro promoter and introduced into the plant as in one embodiment of the application, and when PMMoV is infected, PMMoV infection can be indicated by GFP expression.

In some embodiments, the PMMoV-Rpro promoter is capable of regulating or promoting expression of a gene of interest.

Preferably, the gene of interest is a PMMoV-resistant gene, such as an L-series allele. For example, an expression vector comprising an L-series allele and a PMMoV-Rpro promoter is constructed and the L-series allele is located downstream of the PMMoV-Rpro promoter, and the expression vector is introduced into a plant cell, and when the plant cell is infected by PMMoV, the PMMoV-Rpro promoter can increase the expression of the L-series allele, thereby increasing the resistance of the plant to PMMoV. The plants herein are preferably plants susceptible to PMMoV infection, such as peppers, tomatoes, pumpkins, cucumbers, zucchini, melons etc. [ mid-week of poplar etc. ], review and hope of the study of pepper light mottle virus, cucurbits, 2021, 34 (9): 1-6

The application has the advantages that: the application provides a PMMoV inducible promoter, namely a PMMoV-Rpro promoter, which is an inducible promoter newly discovered in Nicotiana benthamiana and related to PMMoV infection, and the inducible promoter improves the expression of a downstream gene under the condition of PMMoV stimulation. The PMMoV inducible promoter has important significance in vegetable breeding, especially for plants which are easy to be infected by PMMoV, such as capsicum, tomato and the like.

Drawings

Fig. 1: schematic construction of recombinant plasmid pCV-PMMoV-Rpro promoter-GFP.

Fig. 2: the results of the pCV-GFP BamHI+HindIII double cleavage were verified (2K PlusII DNA ladder as a Marker for DNA molecular weight).

Fig. 3: pGEM-PMMoV-Rpro BamHI/Hind III cleavage confirmed the results.

Fig. 4: the results were verified by pCV-PMMoV-Rpro master-GFP agarose gel electrophoresis.

Fig. 5: the PCR amplification of PMMoV-Rpro master-GFP colonies verifies the results (2K PlusII DNA ladder is the molecular weight Marker of DNA).

Fig. 6: the PMMoV-Rpro promoter function verifies the results.

Detailed Description

The process according to the application is described below by way of specific embodiments. The technical means used in the present application are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the application, which is defined solely by the claims. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagent companies.

First, the inventors of the present application extracted Nicotiana benthamiana DNA by SDS-DNA extraction, amplified PCR with the DNA as a template and the upstream primer F1 (SEQ ID NO. 2) and the downstream primer R1 (SEQ ID NO. 3), and determined the nucleic acid sequence by TA cloning to obtain PMMoV-Rpro promoter.

Next, the inventors constructed a plasmid vector containing PMMoV-Rpro promoter and downstream GFP gene, electrotransformed the plasmid vector into agrobacterium, injected agrobacterium into nicotiana benthamiana plants, and injected PMMoV, found that: after PMMoV infects Benshi smoke, GFP gene expression on leaves of plants containing PMMoV-Rpro promoter and GFP gene is obviously increased. This result demonstrates that the PMMoV-Rpro promoter can increase expression of its downstream genes in the event of PMMoV infection.

Example 1 obtaining the PMMoV-Rpro promoter

Using NCBI database (http:// www.ncbi.nlm.nih.gov /) to obtain a sequence of approximately 2700bp upstream of PMMoV-induced expression genes, specific primers were designed, namely upstream primer F1 (SEQ ID NO. 2) and downstream primer R1 (SEQ ID NO. 3). PCR amplification was performed using Nicotiana benthamiana DNA as a template. Connecting the DNA fragment obtained by PCR amplification to a T carrier, and sequencing to obtain a nucleotide sequence shown in SEQ NO:1, namely PMMoV-Rpro promoter sequence.

The specific experimental steps comprise:

1. taking a sample of the Nicotiana benthamiana, and extracting the Nicotiana benthamiana DNA by an SDS-DNA extraction method

(1) An appropriate amount of sample was placed in a 2ml centrifuge tube, quickly frozen with liquid nitrogen, and then immediately ground, 200 μl of DNA extraction buffer (DNA extraction buffer) with ph=7.5 was added, the DNA extraction system was shown in table 1, mixed with vigorous shaking, and left at room temperature for 5min. Centrifuge at 4 ℃,12,000rpm for 5min.

(2) 300. Mu.l of the supernatant was taken in a 1.5ml doffer tube (Ependorf tube), and 300. Mu.l of isopropyl alcohol was added thereto, followed by vigorous mixing for 15s and standing at room temperature for 2 to 3 minutes. Centrifuge at 4 ℃,12,000rpm for 5min.

(3) Removing supernatant, air-drying at room temperature, adding 30 μl dd H ₂ O is dissolved.

TABLE 1DNA extraction System (50 ml)

Type(s)	Parameters (parameters)
		Total volume of	50ml
1M Tris HCl	10ml
		NaCl	0.75g
EDTA	0.365g
		10％SDS	2.5ml
dd H ₂ O	Constant volume to 50ml
		pH value of	7.5

2. PCR amplification and DNA sequencing

And (3) taking the DNA extracted in the step (1) as a template for PCR amplification. The amplification system is shown in Table 2 below, and specifically comprises: 10 XKOD NEO buffer 5. Mu.l, 2.5mM dNTPs 5. Mu.l, 10. Mu.M each of the upstream primer F1 and the downstream primer R1 2. Mu.l, KOD FX NEO polymerase (5U/. Mu.l) 1. Mu.l (purchased from Dain Takara Co., ltd.), DNA template 1. Mu.l, and sterile water was made up to 50. Mu.l. The reaction conditions are as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72℃for 10min.

The size of the target PCR product was 2700bp, and the PCR product was purified by Qiagen gel purification kit (Qiagen, germany) and TA cloning of the PCR fragment was performed by using Promega pGEM-T EASY Vector (Promega, USA). The conditions were as follows: 2X Promega ligase buffer. Mu.l, pGEM-T EASY Vector 1. Mu.l, PCR recovery fragment 2. Mu.l, promega T4-ligase 1. Mu.l (Promega Co., USA), sterile water 1. Mu.l, and after mixing, reaction at 4℃for 16h. After the reaction, 10. Mu.l of the reaction solution was added to a centrifuge tube containing 100. Mu.l of competent cells of E.coli, and after being gently mixed and placed on ice for 30min, the mixture was heat-shocked at 42℃for 90s, immediately placed on ice for 2-3min, and 890. Mu.l of LB liquid medium was added to the centrifuge tube. After shaking the centrifuge tube on a shaking table at 37℃for 1 hour, 300. Mu.l of the culture solution was spread on a solid LB medium containing 100mmol/L kanamycin, and cultured at 37℃for 14-16 hours. Then 6 clones positive in PCR detection are selected and sent to a Kanga company for sequencing, the sequences detected by the 6 clones are identical, the sequence SEQ ID NO.1 (namely PMMoV-Rpro promoter) is finally obtained, and a T vector pGEM-PMMoV-Rpro containing the PMMoV-Rpro promoter is obtained.

TABLE 2PCR amplification conditions

Parameters (parameters)	Volume of
		Total volume of	50.0μl
10×KOD NEO buffer	5.0μl
		2.5mM dNTPs	5.0μl
Upstream primer F1 (10. Mu.M) (SEQ ID NO. 2)	2.0μl
		Downstream primer R1 (10. Mu.M) (SEQ ID NO. 3)	2.0μl
Template	1.0μl
		KOD FX NEO Polymerase (DNA polymerase)	1.0μl
ddH ₂ O	Make up to 50.0. Mu.l

EXAMPLE 2 expression vector construction

FIG. 1 is a schematic diagram showing the construction of an expression vector, i.e., recombinant plasmid pCV-PMMoV-Rpro promoter-GFP. The vector selected pCV-GFP plasmid (stored and supplied by Ningbo university plant virus laboratory) as backbone, in which GFP was transcribed from the 35S promoter before transformation, the plasmid vector was digested with BamHI/HindIII for replacement of the 35S promoter, and the large fragment after digestion of 10.1kb pCV-GFP BamHI/HindIII was obtained by recovery and purification by gel cutting, as shown in FIG. 2.

pGEM-PMMoV-Rpro (same as in example 1) of the DNA fragment containing the PMMoV-Rpro promoter was obtained, and the PMMoV-Rpro promoter fragment was obtained by treating with BamHI/HindIII enzyme, and then subjecting to gel-cutting purification, as shown in FIG. 3.

The large fragment after cleavage of pCV-GFP BamHI/HindIII was ligated in vitro to the PMMoV-Rpro promoter fragment using a recombinase to obtain a recombinant plasmid pCV-PMMoV-Rpro promoter-GFP (FIG. 4).

EXAMPLE 3 recombinant plasmid Agrobacterium tumefaciens

1. Experimental method

(1) The electrode cup is soaked in 75% ethanol for 24 hours, then soaked in absolute ethanol for 4 hours, dried in a baking oven at 37 ℃ and placed in ice bath.

(2) GV3101 Agrobacterium competent cells were thawed on ice, 1. Mu.l of the recombinant plasmid pCV-PMMoV-Rpro master-GFP of example 2 was added to the competent cells, and after mixing, transferred to a pre-chilled electrode cup, and placed in an electroporation cuvette for pulsing at 200. OMEGA.and 1800V.

(3) Immediately taking out the electric rotating cup, adding 1ml of LB into the electric rotating cup, uniformly mixing, transferring the bacterial liquid to a 2ml sterile EP tube, 180r/min, and resuscitating at 28 ℃ for 1h.

(4) 200 μl of the bacterial liquid is coated on an LB plate containing Kan+/Rif+, and the bacterial liquid is cultured for 2 days at 28 ℃ in an inverted way, and the transformant grows a monoclonal.

(5) Single colonies were picked for colony PCR verification of band size, primers were upstream primer F1 (SEQ ID NO. 2) and downstream primer R1 (SEQ ID NO. 3), and bands were verified by agarose gel electrophoresis.

2. Experimental results

As shown in FIG. 5, the band size obtained by agarose gel electrophoresis was about 2700bp, and the result shows that PMMoV-Rpro was introduced into Agrobacterium, i.e., the recombinant plasmid pCV-PMMoV-Rpro promoter-GFP electrotransferred to Agrobacterium was successful.

Example 4 functional verification of PMMoV-Rpro promoter

1. Experimental method

(1) The recombinant plasmid pCV-PMMoV-Rpro promoter-GFP-transformed Agrobacterium single colony of example 2 was picked up and shake-cultured in 5mL LB liquid containing 100. Mu.g/mL Kan+/Rif+, at 28℃for 180r/min for 20-24h.

(2) Agrobacterium transformed with PMMov infectious clone plasmid was obtained by electrotransformation of Agrobacterium with recombinant plasmid and cultured in LB plate of Kan+/Rif+ at 28℃for 1d in an inverted manner. The PMMoV Agrobacterium plaque is scraped in 5ml LB liquid containing 100 mug/ml Kan+/Rif+, 28 ℃,180r/min and shake cultivated for 20-24h.

(3) Culturing the empty agrobacterium as in (1) as a control.

(4) The agrobacteria solutions cultivated in (1), (2) and (3) were collected in 2ml sterile EP tubes, 6000r/min, centrifuged for 1min and the supernatant discarded. Adding 1ml of sterile water for resuspension, centrifuging for 1min at 6000r/min, discarding the supernatant, and adding 2ml of transient transfection solution, which are respectively marked as PMMoV-Rpro-GFP agrobacterium solution, PMMoV agrobacterium solution and control bacteria.

(5) Adjusting OD 260nm of the PMMoV-Rpro-GFP agrobacterium solution to be 0.1, taking 1ml to mix with 1ml of PMMoV agrobacterium solution to obtain a PMMoV-Rpro-GFP+PMMoV mixed bacterial solution with the final concentration OD 260nm of 0.05; 1ml of the control bacteria were mixed with 1ml of the control bacteria to obtain PMMoV-Rpro-GFP control bacteria liquid with a final concentration OD 260nm of 0.05.

(6) 3 healthy Nicotiana benthamiana plants with proper size are selected, PMMoV-Rpro-GFP control bacterial liquid (left side, CK) and PMMoV-Rpro-GFP+PMMoV mixed bacterial liquid (right side, PMMoV) are respectively injected on two sides of the same leaf in the fourth and third leaf positions of the plants, the infiltration injection area is ensured to be the same as much as possible, and 3 plants are biologically repeated.

(7) The GFP gene expression in the leaves was observed by irradiation with UV light for 3-4 days of the co-injection experiment. Further, both left and right sides of the leaf were collected, and the accumulation amount of GFP protein was measured by WB (Western blot).

2. Experimental results

As shown in FIG. 6, under UV light, the left side (CK) of the leaf is weak or even non-fluorescent, and the right side (PMMoV) has obvious fluorescence, i.e., GFP gene is expressed in large quantity on the right side of the leaf, but little or no expression is observed on the left side of the leaf. WB results also showed that GFP protein was significantly higher in the left (CK) leaf than in the right (PMMoV) leaf.

The above results indicate that: in the present leaf of Nicotiana benthamiana with PMMoV and recombinant plasmid pCV-PMMoV-Rpro promoter-GFP, GFP gene was expressed in large amount, while in the present leaf of Nicotiana benthamiana with recombinant plasmid pCV-PMMoV-Rpro promoter-GFP, GFP gene was expressed in small amount or even not. Further proving that: the PMMoV-Rpro promoter is an inducible promoter of PMMoV; in the case of PMMoV induction, the PMMoV-Rpro promoter can increase the expression of genes downstream thereof.

Sequence listing

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<120> infection-induced promoter of pepper light mottle virus and application thereof

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ttaatctaga atttaagtgt atgaccaaca ttctgaattc taggggaagt tctaacacta 960

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Claims

1. The infection-induced promoter of the pepper mild mottle virus is characterized in that the sequence of the induced promoter is shown as SEQ ID NO.1.

2. The inducible promoter of claim 1, wherein said inducible promoter is derived from nicotiana benthamiana.

3. An expression cassette comprising an inducible promoter according to any one of claims 1 to 2.

4. An expression vector comprising the inducible promoter of any one of claims 1 to 2.

5. The expression vector of claim 4, wherein the expression vector comprises a gene of interest and the inducible promoter is upstream of the gene of interest.

6. A recombinant host cell comprising the expression vector of claim 4 or 5.

7. Use of an inducible promoter according to one of claims 1 to 2.

8. The use according to claim 7, wherein the inducible promoter is used for marker infection by pepper mild mottle virus.

9. The use according to claim 7, wherein the inducible promoter regulates or initiates expression of the gene of interest.

10. The use according to claim 9, wherein the gene of interest, when expressed, increases the resistance of the plant to pepper's light mottle virus.