CN116200387A

CN116200387A - Arsenic-induced response promoter, expression vector and application thereof

Info

Publication number: CN116200387A
Application number: CN202310135575.3A
Authority: CN
Inventors: 曹越; 赵逸涵; 蔡煊; 孙丹; 冯华原; 汤叶涛; 仇荣亮
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2023-02-17
Filing date: 2023-02-17
Publication date: 2023-06-02

Abstract

The invention discloses an arsenic induction response promoter, and an expression vector and application thereof. The promoter comprises a nucleotide sequence which is selected from any one of the following groups and has a promoter function: (1) the nucleotide sequence shown in SEQ ID NO. 1; (2) A nucleotide sequence capable of hybridizing to the nucleotide sequence of (1) under stringent conditions; (3) A nucleotide sequence having at least 90% identity to the nucleotide sequence set forth in (1) or (2). The promoter provided by the invention has strong arsenic response, can specifically respond to pentavalent arsenic stress so as to improve the expression quantity of a target gene, is inhibited and started by trivalent arsenic, and has wide application prospect in the aspect of gene regulation.

Description

Arsenic-induced response promoter, expression vector and application thereof

Technical Field

The invention relates to the field of genetic engineering and biotechnology, in particular to an arsenic induction response promoter, and an expression vector and application thereof.

Background

Arsenic is a metalloid element widely existing in nature, and arsenic and its soluble compounds all have strong toxicity. Arsenic contamination is a great hazard to many life forms such as plants and animals. Related researches show that arsenic accumulation in soil reaches a certain degree, can directly influence the growth and development of plants, reduce agricultural yield, and possibly enter edible tissues of vegetables and fruits and grains of crops such as rice, wheat and the like, and when a certain amount of arsenic is ingested by a human body, various skin cancers, kidney injuries, cardiovascular diseases and the like are easily caused.

In the related art, the heavy metal soil pollution restoration method mainly comprises engineering treatment measures and physical and chemical restoration methods, but has relatively high cost, large disturbance to the environment and possibility of secondary pollution to the soil. The plant restoration technology has the advantages of low cost, simple operation, high ecological benefit, small environmental disturbance, no secondary pollution and the like, shows the advantages and development prospect in the aspect of soil restoration, and is a common effective approach for restoring arsenic-polluted soil by utilizing arsenic super-enriched plants. The arsenic super-enriched plant refers to a plant with the aboveground part of the plant absorbing more than 10 times of arsenic than that of a common plant and does not influence normal vital activities, and most of the arsenic super-enriched plants found at present belong to the pteris genus plant. Part of genes of a representative plant ciliate desert-grass of an arsenic super-enrichment plant are reported to be induced by arsenic and are related to important arsenic metabolic processes such as ciliate desert-grass arsenic transportation, detoxification and accumulation, wherein an arsenic (trivalent arsenic) antiport protein PvACR3 gene plays an important role in the arsenic super-enrichment process. Among the super-enriched plants and model plants, some key genes which are induced to express by arsenic are found, the expression enhancement of arsenic-related genes such as PvACR3 on arsenic response is limited, and the selectivity of promoters of the arsenic-related genes such as PvACR3 on trivalent and pentavalent arsenic responses is low.

Thus, there is still a need for a promoter that has high expression of arsenic response and is capable of specifically responding to pentavalent arsenic stress.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a DNA molecule which can specifically respond to the stress of pentavalent arsenic and can be used for driving the expression of a target gene.

The invention also provides a primer group for amplifying the DNA molecules.

The invention also provides a biological material.

The invention also provides application of the DNA molecule in preparing a promoter.

The invention also provides application of the DNA molecule or the biological material in culturing transgenic plants.

The invention also provides application of the DNA molecule or the biological material in driving expression of a target gene in plants.

The invention also provides a preparation method of the transgenic plant,

in a first aspect of the present invention there is provided a DNA molecule comprising a nucleotide sequence selected from any one of the following groups and having promoter function:

(1) A nucleotide sequence shown as SEQ ID NO. 1;

(2) A nucleotide sequence capable of hybridizing to the nucleotide sequence of (1) under stringent conditions;

(3) A nucleotide sequence having at least 90% identity to the nucleotide sequence set forth in (1) or (2).

The DNA molecule according to the embodiment of the invention has at least the following beneficial effects: the DNA molecule of the invention is derived from non-super-enriched plants, is a promoter of PeACR3, can specifically respond to the stress of pentavalent arsenic, and can be used for driving the high expression of target genes. In addition, the DNA molecule can be activated by trivalent arsenic inhibition.

Compared with the promoter of the conventional arsenite inverse transport protein gene PvACR3, the DNA molecule provided by the invention has stronger response to arsenic as a promoter, has better specificity and is more beneficial to regulating and controlling the expression of a target gene.

In some embodiments of the invention, the DNA molecule is derived from a non-arsenic hyper-enriched plant.

In a second aspect of the invention, there is provided a primer set for amplifying said DNA molecule.

In some embodiments of the invention, the primer set comprises an upstream primer set forth in nucleotide sequence SEQ ID NO.5 and a downstream primer set forth in nucleotide sequence SEQ ID NO. 6.

In a third aspect of the present invention, there is provided a biomaterial, which is any one of the following 1) to 8):

1) The DNA molecule described above;

2) An expression cassette comprising 1) said DNA molecule;

3) A recombinant vector comprising 1) said DNA molecule;

4) A recombinant vector comprising 2) said expression cassette;

5) A recombinant microorganism or cell line comprising 1) said DNA molecule;

6) A recombinant microorganism or cell line comprising 2) said expression cassette;

7) A recombinant microorganism or cell line comprising 3) said recombinant vector;

8) A recombinant microorganism or cell line comprising the recombinant vector of 4).

In a fourth aspect of the invention there is provided the use of a DNA molecule as described above as and/or in the preparation of a promoter.

In some embodiments of the invention, the promoter is a pentavalent arsenic-induced response promoter.

In a fifth aspect of the invention there is provided the use of a DNA molecule or biological material as described above in the cultivation of a transgenic plant.

In a sixth aspect of the invention there is provided the use of a DNA molecule or biological material as described above for driving expression of a gene of interest in a plant.

In some embodiments of the invention, the gene of interest is the PeACR3 gene.

In a seventh aspect of the present invention, there is provided a method of preparing a transgenic plant, the method comprising the steps of: the above biological material is introduced into plants.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The invention is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a graph showing the statistics of the expression of the response of the pentavalent arsenic to the PeACR3 gene according to the embodiment of the invention;

FIG. 2 shows the result of agarose gel electrophoresis of four pairs of primer PCR products used in cloning the PeACR3pro promoter according to the example of the present invention.

FIG. 3 is a schematic diagram showing the structure of recombinant plasmid 1300GN-Unigene0002384 according to an embodiment of the present invention.

FIG. 4 shows the result of agarose gel electrophoresis of transgenic Arabidopsis lines screened by hygromycin according to the example of the present invention.

FIG. 5 is a graph showing GUS staining results of transgenic plants of Arabidopsis according to an embodiment of the present invention;

FIG. 6 is a statistical chart of downstream gene expression of the PeACR3Pro promoter induced and started by different concentrations of pentavalent arsenic and trivalent arsenic according to the embodiment of the invention.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.

In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1 acquisition of the PeACR3Pro promoter sequence

In the present invention, early studies on ACR3 homologous gene (No. Unigene0002384, hereinafter referred to as the PeACR3 gene) of the root system of the non-hyperaccumulated plant Pteridium silver vein, pteridium silver vein (Pteris ensiformis), showed that the PeACR3 gene was strongly induced by pentavalent arsenic and up-regulated about 50 times (specifically, as shown in A of FIG. 1).

Further using SYBR Green PCR Master Mix kit (Vazyme Biotech, nanjin, china) for real-time fluorescent quantitative PCR (qRT-PCR) experiment verification, the result adopted 2 ^-ΔΔCT Statistics of the method show that the expression of the PeACR3 gene in the root system is induced by pentavalent arsenic, and the expression is up-regulated by about 14 times (shown as B in figure 1).

Three specific primers SP1, SP2, SP3 were designed based on the CDS sequence known for the above-mentioned PeACR3 gene using a chromosome walking kit (TAKARA, japan).

The specific primer sequences are shown in Table 1:

TABLE 1 primer nucleotide sequences

Primer(s)Name of the name	Primer sequence (5 '-3')
		SP1 Primer	CCACCGAGCTTTTCATATTGCACCTT(SEQ ID NO.2)
SP2 Primer	AGCTACTTGGAATGCCTTCTTCAC(SEQ ID NO.3)
		SP3 Primer	TCGAGCAGCGAAAGCTGTTTGAAA(SEQ ID NO.4)

After 1st PCR reaction is carried out and genome DNA is accurately quantified by OD measurement, a proper amount of genome DNA is taken as a template, AP Primer1/2/3/4 (random Primer with lower annealing temperature provided by a kit) is taken as an upstream Primer, and SP1 Primer is taken as a downstream Primer.

The 1st PCR reaction system is shown in Table 2:

table 2:1st PCR reaction system

Reagent(s)	Volume (mu L)
		Templite (genomic DNA)	2
dNTP Mixture(2.5mM each)	3.2
		10×LA PCR BufferⅡ(Mg ²⁺ plus)	2
TaKaRa LA Taq(5U/μL)	0.2
		AP1/2/3/4Primer(100pmol/μL)	0.4
SP1 Primer	0.4
		ddH ₂ O	41.8

The 1st PCR amplification procedure was: (1) pre-denatured at 94℃for 1min and at 98℃for 1min. (2) Denaturation at 94℃for 1min, annealing at 50℃for 1min, and extension at 72℃for 2min. (2) 5 reaction cycles were carried out. (3) Denaturation at 94℃for 30sec, annealing at 25℃for 3min and extension at 72℃for 2min. (4) Denaturation at 94℃for 30sec, annealing at 60℃for 1min, and extension at 72℃for 2min. Denaturation at 94℃for 30sec, annealing at 60℃for 1min, and extension at 72℃for 2min. Denaturation at 94℃for 30sec, annealing at 44℃for 1min, and extension at 72℃for 2min. (4) 15 reaction cycles were carried out. Extending at 72℃for 10min.

The 2nd PCR reaction was performed by taking 1. Mu.L of the 1st PCR reaction solution as a template for the 2nd PCR reaction, and AP Primer1/2/3/4 as an upstream Primer and SP2 Primer as a downstream Primer.

The 2nd PCR reaction system is shown in Table 3:

table 3:2nd PCR reaction system

Reagent(s)	Volume (mu L)
		Templite (1 st PCR reaction solution)	1
dNTP Mixture(2.5mM each)	3.2
		10×LA PCR BufferⅡ(Mg ²⁺ plus)	2
TaKaRa LA Taq(5U/μL)	0.2
		AP1/2/3/4Primer(100pmol/μL)	0.4
SP2 Primer	0.4
		ddH ₂ O	42.8

The 2nd PCR amplification procedure was: (1) denaturation at 94℃for 30sec, annealing at 60℃for 1min, and extension at 72℃for 2min. Denaturation at 94℃for 30sec, annealing at 60℃for 1min, and extension at 72℃for 2min. Denaturation at 94℃for 30sec, annealing at 44℃for 1min, and extension at 72℃for 2min. (1) 15 reaction cycles were carried out. (2) Annealing at 72 ℃ for 10min.

3rd PCR reaction was performed, 1. Mu.L of 2nd PCR reaction solution was used as a template for the 3rd PCR reaction, AP Primer1/2/3/4 was used as an upstream Primer, and SP3 Primer was used as a downstream Primer.

The 3rd PCR reaction system is shown in Table 4:

table 4:3rd PCR reaction system

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The 3rd PCR amplification procedure was: (1) denaturation at 94℃for 30sec, annealing at 60℃for 1min, and extension at 72℃for 2min. Denaturation at 94℃for 30sec, annealing at 60℃for 1min, and extension at 72℃for 2min. Denaturation at 94℃for 30sec, annealing at 44℃for 1min, and extension at 72℃for 2min. (1) 15 reaction cycles were carried out. (2) Annealing at 72 ℃ for 10min.

mu.L of each of the 1st,2nd and 3rd PCR reaction solutions was subjected to electrophoresis using 1% agarose gel, and the result of the electrophoresis is shown in FIG. 2. The results showed that the obtained product, possibly the target fragment, was amplified with the AP3 and SP3 primers. Products from 3rd PCR using AP3 and SP3 primer amplification were selected, run on a 1% agarose gel, gel recovered using a gel recovery kit, the products were ligated with 007S vector using 007S-Topo kit (Tsingke, nanjin), DH 5. Alpha. E.coli competence (Tsingke, nanjin) was transformed, and picked up for sequencing. The sequence thus determined was aligned with a CDS sequence DNA fragment (5' end) known to PeACR3 to determine the product sequence as the sequence of the promoter region of PeACR3, with a total length of 1304bp. Primers Primer1 and Primer2 were designed based on the determined sequence of the PeACR3 promoter region, and the Primer sequences are shown in Table 5. Use of high fidelity DNA polymerase I-5 ^TM PCR was performed with 2 Xhigh-Fidelity Master Mix, and the PCR system is shown in Table 6.

Table 5: primer nucleotide sequence

Primer name	Nucleotide sequence (5 '-3')
		Primer1	CAAGAAGAACGTAGATGGGT(SEQ ID NO.5)
Primer2	CTCCCTTGACATAATACCCG(SEQ ID NO.6)

Table 6: PCR reaction system

Component (A)	Volume (mu L)
		Primer1	2
Primer2	2
		DNA polymerase I-5	25
Promoter DNA fragment	1
		ddH ₂ O	20

The PCR amplification procedure was: (1) pre-denaturation at 98℃for 2min. (2) Denaturation at 98℃for 10sec, annealing at 55℃for 15sec, elongation at 72℃for 30sec. (2) 34 reaction cycles were performed. (3) Annealing at 72 ℃ for 5min.

The amplified product was gel purified and the promoter sequence was recloned using 007BS-Topo cloning kit (Tsingke, nanjing). The resulting DNA fragment was named PeACR3Pro. The total length of the promoter is 1304bp.

The nucleotide sequence of PeACR3Pro is as follows:

TGCAACACAATCGTCAAATGCCCAATAACACAATCACGAGACAACATGCAGGAGAT

AAATTAGACGCCCATGCAGATTCTAAACTAAAACATTCTTTTTACAGCAAATAGATGACC

ACCATCACAAATATTTTTTATGTAGAAATTGGTCATGTAACCTGGTCCAAATTCAAACAC

AATGCTAAGGCTGCCGAGATGTGCGAAAAACGTTGCCAACGAATTTAGTTTTCCAAAAC

ACTGAACTCAAGATCAAAATTTAGTTTTTACAGATTAATGACCAAAATTGGCCAACAACC

CCCCATTTAAATGTGGACCATAGATCCAACACTTAAAACGGAGGAACAGCGTGTCTGAG

ACTTGTGTGGGGGCGGGAGTTCTAAACCTTCTCCAATATCCGAATCTTGCCCATTGGCTC

TTCCCCCATCGCAAGAGCGCTTCTTCCTCTCATTCCAAAATTCTTTTCCGCCCTATTAAAA

CCCTCGCTCTCTGCCAGCAGCGTGTGAGAGTGAGAGGGAGCGAGAGACGAGGCGGAC

GGGCGAGCGAGAGAGGAGGCAGCGTAGCAGTCGCAGCAGGTATGGCTAACTCCAGTGC

AGAGCGAAAGCAGCAAATGGCCCTGGACATTGCTGATGGGAACGACCCGTCAGACGCT

GAAAAAACCGCTGACGAAGGCATAAAACGTGAGGTTATCCCTCTCCCTCTCCCTCTTGC

GCCCCCGCGCGCGCGCGCGCGCACACACACACACACATTAAGATAACCTTCCTTTCTCT

GTCTCACTGACGACATGCATTCTCTCTCTCTCTCTCTCTCTCGTCGTGGCTTTGCTTCTCT

GTCGGCTCCCTGCTGTTTTCTACACGCATGCATCTCAAGAAATTGGGCGTGTGTCGTCTG

GTTTCTTGATTTTTGTCCGTTCGTCCTCCTCTTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC

TCTCTCACACACACACACACACACACACACACACACACGTACGTCATGCGGCTCATGCC

TTTGCTTGTCTGTCTGGTCCCTCTGTAGGCTACTACACCCATACAGTTTAGGAAGTTAGG

TGCTGTGTGTGCTCACATTTGAAATTGTGTAGGTGCAATCGCTGCTTTTTGTGTTTGTGT

GAGAGAGAGAGATTGTGTGACCTGCGAAATTTCATGTGTGTGCTGTGTTCTTTGTACGCT

TGTTTTCTTGTTGGGGGGGGAGGGGGGGGAGACAGAGAGAGAGAATTTATTGTAGTGA

AAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGTTCGTGTAACTTACGGAACTTCA

T(SEQ ID NO.1)。

EXAMPLE 2 construction of recombinant expression vectors

1. Cleavage 1300GN vector and amplification product

(1) Plasmid pCAMBIA1300GN (given by Nanjing agricultural university) was digested with restriction enzymes BamH I and Kpn I, respectively, and the digestion system was as shown in Table 7:

table 7: enzyme cutting system

(2) The recovered amplified product prepared in example 1 was subjected to PCR amplification with I5 high fidelity enzyme, and the product was recovered and purified with gel. The PCR system is shown in Table 8:

table 8: PCR reaction system

Component (A)	Volume (mu L)
		PeACR3Pro-F(20μM)	2
PeACR3Pro-R(20μM)	2
		DNA polymerase I-5	25
PeACR3 template DNA	1
		ddH ₂ O	20

The PCR amplification procedure was: (1) pre-denaturation at 98℃for 2min. (2) Denaturation at 98℃for 10sec, annealing at 55℃for 15sec, elongation at 72℃for 30sec.

(2) 34 reaction cycles were performed. (3) Annealing at 72 ℃ for 5min.

Wherein the primer sequences are shown in Table 9:

table 9: primer nucleotide sequence

2. Construction of recombinant vectors

The digested vector backbone and amplified product were ligated with Premix ligase (Yesen, shanghai) to give recombinant plasmid 1300GN-Unigene0002384 recombinant vector. The connection system is shown in Table 10:

table 10: connection system

Component (A)	Volume (mu L)
		Premix ligase	5
1300GN vector	2.5
		Amplified product fragment	2.5
ddH ₂ O	10

3. Transformation of competent cells with recombinant plasmid vectors

mu.L ligation product was used to transform 100. Mu.L DH 5. Alpha. Competent cells: mixing the product with competent cells, ice-bathing for 30min, heat-shocking at 42 deg.C for 90s, immediately placing on ice for 2min, adding 500 μL LB culture medium preheated to room temperature, shaking at 180rpm and 37 deg.C for 1h, centrifuging at 5000rpm for 3min, discarding 500 μL culture supernatant, mixing the rest 100 μL with a pipettor, uniformly coating on LB plate containing 50 μg/mL kana resistance, inverting, and culturing overnight in a 37 deg.C constant temperature incubator.

4. Sequencing identification

Positive single colonies were selected and sent to the Probiotechnological engineering (Shanghai) Co., ltd for sequencing.

Sequencing results show that the sequence of the promoter of the PeACR3Pro recombinant vector in the obtained 1300GN-Unigene0002384 particle vector is correct.

5. Extraction of plasmids from properly sequenced positive strains

Positive strains with correct sequencing were subjected to expansion culture, and added to 20ml LB medium containing kana resistance for overnight culture at 37℃to extract plasmids. The schematic structure of recombinant plasmid 1300GN-Unigene0002384 is shown in FIG. 3.

Example 3 acquisition and verification of transgenic plants

1. Transformation of recombinant plasmids

The recombinant plasmid 1300GN-Unigene0002384 constructed in example 2 was subjected to electric shock transformation with Agrobacterium strain GV3101, and after two days of culture on kan-resistant medium, single colonies were picked up and positive clones were identified by shaking.

2. Screening of transgenic plants of Arabidopsis thaliana

(1) Agrobacterium infection

Wild type Arabidopsis thaliana is planted in substrate soil, and when the Arabidopsis thaliana starts bolting, the constructed Agrobacterium strain GV3101 is streaked and cultivated, after the Arabidopsis thaliana has green buds, the Arabidopsis thaliana is expanded and cultivated by using LB culture medium containing kana and RIF resistance, after the Agrobacterium is shaken to the optimal OD (0.8-1.0), the thallus is enriched, and the agrobacteria are transferred into an invasion dye solution (5% sucrose, 0.02% sliwet 77). The unopened (slightly dew point white) leaves of Arabidopsis thaliana were soaked in the dye liquor for 45s.

(2) Planting of Arabidopsis thaliana

Firstly, placing Arabidopsis seeds into a 1.5ml centrifuge tube for preparation and disinfection, adding 1ml of ethanol with the concentration of 75%, and shaking the centrifuge tube for 1-2min. 1ml of 5% sodium hypochlorite was added and the tube was washed upside down for about 10min and the solution was aspirated. Sterile deionized water was added, the washes were reversed, and the wash was repeated 5 times. Uniformly spot-seeding the sterilized Arabidopsis seeds on a 1/2MS culture medium, shading and standing for 3 days at 4 ℃, and transferring to a lighting incubator for 7 days. After two cotyledons of Arabidopsis seed germination were observed, they were transplanted into a pot containing sand and vermiculite. Covering with fresh-keeping film, and culturing in greenhouse. Culture conditions: the illumination period is 14 h/day, the day/night average temperature is 26 ℃/20 ℃, the relative humidity is kept to 60 percent and the average temperature is 350 mu mol m ^-2 s ^-1 Is a light intensity of the light source. And uncovering the preservative film after one week, and pouring the nutrient solution once in 3 days in the growth period. Finally, waiting for the mature seed collection of the arabidopsis thaliana, drying, and storing in a shade and dry place.

(3) Screening of positive Arabidopsis transgenic plants

The T1 generation seeds of the arabidopsis transgenic line are obtained through culture propagation, the T1 generation arabidopsis seedlings are germinated on a 1/2MS culture medium added with 30mg/L hygromycin, and positive transformation plants are screened.

(4) Identification of transgenic Arabidopsis lines by qRT-PCR

After positive seedlings are screened out, the positive seedlings are continuously cultured on a 1/2MS culture medium containing 30mg/L hygromycin until the biomass reaches a certain amount, plant tissues are collected to extract plant RNA, and transgenic Arabidopsis strains are identified through semi-quantitative PCR (taking hygromycin as a target product). The arabidopsis transgenic lines Ex-1, ex-2 and Ex-3 with good growth conditions under the resistant condition are obtained through hygromycin screening.

The primers for the semi-quantitative PCR reaction are shown in Table 11, and the PCR reaction system is shown in Table 12.

Table 11: primer nucleotide sequence

Primer name	Nucleotide sequence (5 '-3')
		Primer1	ATGCTCAACACATGAGCGAA(SEQ ID NO.9)
Primer2	CCACTATCCTTCGCAAGACC(SEQ ID NO.10)

Table 12: PCR reaction system

Component (A)	Volume (mu L)
		Primer1(10μM)	0.4
Primer2(10μM)	0.4
		2×ChamQ SYBR Color qPCR Master Mix	10
cDNA	2
		ddH ₂ O	7.2

The PCR amplification procedure was: (1) pre-denatured at 95 ℃ for 30sec. (2) Denaturation at 95℃for 10sec and annealing at 60℃for 30sec. (2) 40 reaction cycles were carried out. (3) Denaturation at 95℃for 15sec, annealing at 60sec, extension at 95℃for 15sec.

After completion of the PCR reaction, electrophoresis was performed on a 1% agarose gel, and the result of the electrophoresis was as shown in FIG. 4, and the result was consistent with the expectation.

3. Planting of transgenic lines of Arabidopsis thaliana

Seeds of the positive transgenic Arabidopsis line Ex-1 obtained above were germinated on sterile 1/2MS medium containing 30mg/L hygromycin. After 5 days, arabidopsis seedlings of consistent growth were transferred to CK (without As V addition) and 1/2MS medium containing three treatments of 50. Mu.M As V and 50. Mu.M As III, respectively, for treatment.

4. GUS staining analysis

After 10 days of greenhouse culture, GUS staining experiments were performed on the lower part of Arabidopsis thaliana, DNA was extracted from the upper part and PCR and gel electrophoresis were performed using primers for the nucleic acid sequence.

The GUS staining result is shown in figure 5, and the result shows that the staining result is blue after the infected arabidopsis is cultivated on a pentavalent arsenic plate, which indicates that the reporter gene GUS is induced to start by pentavalent arsenic; the staining results after incubation on the CK plate and the trivalent arsenic plate are not developed, which shows that the PeACR3Pro promoter sequence can start the downstream gene expression under the induction of pentavalent arsenic and is not started under the induction of trivalent arsenic.

To further verify that the promoter sequence was induced by pentavalent arsenic to initiate downstream gene expression, arabidopsis transgenic lines Ex-1, ex-2, ex-3 were selected and treated with different concentrations of pentavalent arsenic (20. Mu.M and 100. Mu.M) and different concentrations of trivalent arsenic (20. Mu.M and 100. Mu.M), and RNA of each Arabidopsis transgenic line was extracted for reverse transcription, and GUS gene expression was detected by qRT-PCR.

As shown in FIG. 6, the results show that the GUS expression level is highest in the transgenic strain of Arabidopsis under the action of pentavalent arsenic stress, the average GUS gene expression level is up-regulated from about 0.008 to 0.014-0.031 under the concentration of 20 mu M pentavalent arsenic, and the average GUS gene expression level is up-regulated from about 0.008 to 0.020-0.034 under the concentration of 100 mu M pentavalent arsenic; the GUS expression level in the transgenic strain of Arabidopsis thaliana under the action of trivalent arsenic stress is extremely low and lower than that of the wild type, wherein the average GUS gene expression level is reduced from about 0.008 to 0.002 at the concentration of 10 mu M trivalent arsenic, and the average GUS gene expression level is reduced from about 0.008 to 0.002-0.004 at the concentration of 50 mu M trivalent arsenic, which indicates that trivalent arsenic can inhibit GUS gene expression.

The experiment shows that the PeACR3Pro promoter obtained from the PeACR3 gene has the capacity of being induced and started by pentavalent arsenic and possibly being inhibited and started by trivalent arsenic.

While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims

1. A DNA molecule comprising a nucleotide sequence selected from any one of the group consisting of:

(1) A nucleotide sequence shown as SEQ ID NO. 1;

2. A primer set for amplifying the DNA molecule of claim 1.

3. The primer set of claim 2, wherein the primer set comprises an upstream primer shown in nucleotide sequence SEQ ID NO.5 and a downstream primer shown in nucleotide sequence SEQ ID NO. 6.

4. A biomaterial, which is any one of the following 1) to 8):

1) The DNA molecule of claim 1;

2) An expression cassette comprising 1) said DNA molecule;

3) A recombinant vector comprising 1) said DNA molecule;

4) A recombinant vector comprising 2) said expression cassette;

5) A recombinant microorganism or cell line comprising 1) said DNA molecule;

5. Use of a DNA molecule according to claim 1 as and/or for the preparation of a promoter.

6. The use according to claim 5, wherein the promoter is a pentavalent arsenic-induced response promoter.

7. Use of a DNA molecule according to claim 1 or a biological material according to claim 4 for the cultivation of transgenic plants.

8. Use of a DNA molecule according to claim 1 or a biological material according to claim 4 for driving expression of a gene of interest in a plant.

9. The use according to claim 8, wherein the gene of interest is the PeACR3 gene.

10. A method for preparing a transgenic plant, said method comprising the steps of: introducing the biological material of claim 4 into a plant.