CN116769778B - Tomato cell factory fruit specific promoter NP24pro and application thereof - Google Patents

Abstract

The present invention discloses a tomato cell factory fruit-specific promoter NP24pro and its application. The present invention belongs to the field of biotechnology, and specifically relates to a tomato cell factory fruit-specific promoter NP24pro and its application. The specific promoter of the present invention is a DNA molecule of the following a), b) or c): a) a DNA molecule of a nucleotide sequence of SEQ ID No.1; b) a DNA fragment having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the nucleotide sequence defined in a), and derived from tomato and having promoter function; c) a DNA fragment hybridizing with the nucleotide sequence defined in a) or b) under strict conditions and having promoter function. The promoter is used to drive the expression of genes involved in tomato fruit ripening, such as genes involved in fruit color regulation, cell wall softening, and sweetness increase. The present invention can also be used for tomato germplasm innovation and genetic improvement.

Description

Tomato cell factory fruit specific promoter NP24pro and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a tomato cell factory fruit specific promoter NP24pro and application thereof.

Background

Tomato fruits have rich metabolites, are easy to carry out genetic transformation, and are excellent chassis of synthetic biology. Tomatoes can be genetically engineered into cell factories for the production of oral vaccines and pharmaceutical metabolites. In order to promote efficient production of the target product in tomato fruits and reduce the adverse effects of plant growth, a fruit-specific promoter may be used to drive high level expression of the target gene in tomato fruits. The tomato fruit specific promoters reported in the prior literature include E8, E4,2A11, PG and the like, but the number is limited, and the specificity is not strong enough. Efficient anabolic substances in tomato fruits often require simultaneous transfer of a series of genes of a certain metabolic pathway, which, if identical promoters are used, leads to homologous sequence mediated gene silencing. Thus a range of different fruit-specific promoters is required.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a fruit specific promoter.

The promoter provided by the invention, named NP24 gene promoter, is derived from tomato (Solanum lycopersicum), is a DNA molecule of the following a), b) or c):

a) A DNA molecule with a nucleotide sequence of SEQ ID No. 1;

b) A DNA fragment which has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with the nucleotide sequence defined in a) and which is derived from tomato and has a promoter function;

c) A DNA fragment which hybridizes under stringent conditions to the nucleotide sequence defined under a) or b) and has a promoter function.

The above 75% or more identity may be 80%, 85%, 90%, 95% or more identity.

The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65℃and then washing the membrane once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

Wherein SEQ ID No.1 consists of 1445 nucleotides.

Expression cassettes or recombinant vectors, recombinant microorganisms or transgenic cell lines containing the NP24 gene promoter are also within the scope of the invention.

The expression cassette containing the NP24 gene promoter refers to DNA capable of expressing a target gene in a host cell, and the DNA may include not only the NP24 gene promoter for promoting the target gene but also a terminator for terminating transcription of the target gene. Further, the expression cassette may also include an enhancer sequence. Such transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g., odell et al (I985) Nature 313:810; rosenberg et al (1987) Gene,56:125; guerineau et al (1991) mol. Gen. Genet. 262:141; proudfoot (1991) Cell,64:671; sanfacon et al Genes Dev.,5:141; mogen et al (1990) PLANT CELL,2:1261; munroe et al (1990) Gene,91:151; ballad et al (1989) Nucleic Acids Res.17:7891 Joshi et al (1987) Nucleic Acid Res., 15:9627).

Wherein, the recombinant vector can be a recombinant expression vector or a recombinant cloning vector.

The recombinant vector containing the NP24 gene promoter can be specifically obtained by inserting the NP24 gene promoter between two BpiI cleavage sites of pL 2-1. The recombinant microorganism may be bacteria, yeasts, algae and fungi in particular. Wherein the bacteria may be derived from Escherichia (Escherichia), erwinia (Erwinia), agrobacterium (Agrobacterium), flavobacterium (Flavobacterium), alcaligenes (Alcaligenes), pseudomonas (Pseudomonas), bacillus (Bacillus), etc. The transgenic cell line does not include plant propagation material.

Primer pairs that amplify the full length of the NP24 gene promoter or any fragment thereof are also within the scope of the present invention.

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

The invention also provides application of the DNA molecule in promoting target gene expression in plants.

In the application, the initiation of the expression of the target gene is to initiate the specific expression of the target gene in tomato fruits.

In the application, the target gene is specifically expressed in the tomato fruits in the color breaking period and/or the red ripeness period of the tomato fruits.

The application of the NP24 gene promoter, the recombinant vector or the expression cassette, the recombinant microorganism or the transgenic cell line in the cultivation of transgenic plants also belongs to the protection scope of the invention.

The term transgenic plant is understood to encompass not only the first generation transgenic plant obtained by transforming the plant of interest with the gene of interest, but also its progeny. For transgenic plants, the gene may be propagated in that species, and may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, whole plants and cells.

In the application, the transgenic plant can specifically express the target gene in the fruit color breaking period and the red ripeness period.

In the above application, the plant may be a monocot or dicot.

Further, the dicotyledonous plant may be a plant of the family Solanaceae.

More specifically, the solanaceous plant may be tomato (Solanum lycopersicum l.).

The invention discovers 1 gene expressed at high level in tomato fruits in red ripe stage through early stage screening, and the gene codes for osmotic regulatory protein, so that the plant can resist pathogenic bacteria. A multi-gene over-expression vector is constructed by utilizing the promoter of the gene, and tomatoes are transformed by an agrobacterium-mediated method, so that the promoter can promote the high-level expression of exogenous genes in tomato fruits. The invention expands the selection range of the promoter special for tomato fruits, is favorable for synthesizing more complex metabolites or proteins in a tomato cell factory, and promotes the application of synthetic biology in tomatoes.

The invention can be effectively applied to the research of synthetic biology taking tomato fruits as the chassis, and enables the target genes to be efficiently and stably expressed in the fruits in the red ripe stage of the tomatoes so as to realize the purpose of producing beneficial substances in the fruits, such as metabolites, recombinant proteins and the like.

The invention can be used for researching molecular mechanism or gene function of tomato fruit maturation. For example, the promoter is used for driving the expression of genes involved in tomato fruit maturation, such as genes involved in fruit color regulation, cell wall softening and sweetness increase, and the effect of target genes in tomato fruit maturation is studied. The invention can also be used for innovation and genetic improvement of tomato germplasm.

Drawings

FIG. 1 shows NP24 promoter activity and the specificity for construction of a multigenic overexpression vector. Wherein (a) a dual fluorescent system identifies a promoter reporter gene structure schematic diagram of a promoter activity experiment; (b) NP24 promoter amplified nucleic acid gel electrophoresis band schematic; (c) The exogenous gene CDS driven by the NP24 promoter is expressed at the gene expression level of each tissue of tomato; (d) fruit transient transformation experiments to detect NP24 promoter activity; (e) The activity test of the double-fluorescence system promoter shows that the NP24 promoter has higher activity.

FIG. 2 shows the results of positive plant identification by PCR electrophoresis.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The pGreenII-LUC plasmid in the examples described below was offered by Liu Hongtao laboratories and has been described in ：Li Y,Shi Y,Li M,Fu D,Wu S,Li J,Gong Z,Liu H,Yang S.The CRY2-COP1-HY5-BBX7/8 module regulates blue light-dependent cold acclimation in Arabidopsis.Plant Cell.2021Nov 4;33(11):3555-3573.doi:10.1093/plcell/koab215.PMID:34427646;PMCID:PMC8566302.. public to obtain this biomaterial from the applicant, which was used only for repeated experiments of the invention and was not used for other purposes.

The vectors pL1F-3, pL1F-4, pL1F-2 and pL2-1 in the examples described below have been described in ：Weber E,Engler C,Gruetzner R,Werner S,Marillonnet S.A modular cloning system for standardized assembly of multigene constructs.PLoS One.2011 Feb 18;6(2):e16765.doi:10.1371/journal.pone.0016765.PMID:21364738;PMCID:PMC3041749. for the public to obtain the biological material from the applicant, which was used only for the experiments of the present invention and was not used for other purposes.

Agrobacterium GV3101 (containing pSoup.sup.19 plasmid) in the examples described below was purchased from Bio-only company.

Agrobacterium tumefaciens AGL1 in the examples described below was purchased from Bio-only company.

The following examples of M82 tomatoes have been described in ：Huang X,Xiao N,Zou Y,Xie Y,Tang L,Zhang Y,Yu Y,Li Y,Xu C.Heterotypic transcriptional condensates formed by prion-like paralogous proteins canalize flowering transition in tomato.Genome Biol.2022Mar 14;23(1):78.doi:10.1186/s13059-022-02646-6.PMID:35287709;PMCID:PMC8919559. publicly available from the applicant for this biomaterial, which was used only in repeated experiments of the invention and not as other uses.

The MicroTom tomatoes in the examples described below have been described in ：Kai W,Wang J,Liang B,Fu Y,Zheng Y,Zhang W,Li Q,Leng P.PYL9 is involved in the regulation of ABA signaling during tomato fruit ripening.J Exp Bot.2019Nov 18;70(21):6305-6319.doi:10.1093/jxb/erz396.PMID:31504753;PMCID:PMC6859720. and the biological material is publicly available from the applicant and is used only for repeated experiments of the invention and is not available for other uses.

The quantitative experiments in the following examples were performed in triplicate unless otherwise indicated.

EXAMPLE 1 cloning of the NP24 Gene promoter and construction of the transgenic vector

1. Cloning of the NP24 Gene promoter

The DNA of tomato M82 leaf is extracted by CTAB extraction method, and the DNA of tomato M82 genome is used as template, KOD plus high-fidelity enzyme is used to amplify promoter, and specific primer is designed to amplify NP24 gene promoter in 5' non-coding region. The NP24 gene number was Solyc g080640. The primer sequences were as follows:

NP24-F:5'-gtcgacggtatcgataagcttGGTGTGTTTGCTACGAAGG-3' (lower case letters indicate sequences required for homologous recombination with pGreenII 0800-LUC)

NP24-R:5'-gctctagaactagtggatccGTTTGTTGAAATGTATATAT-3' (lower case letters indicate sequences required for homologous recombination with pGreenII 0800-LUC)

The NP24 gene promoter PCR reaction system is as follows:

TABLE 1 NP24 Gene promoter PCR reaction System

The PCR reaction procedure was carried out for 2min at 94℃for denaturation, 15s at 94℃for denaturation, 30s at 55℃for annealing, and 1min at 68℃for amplification for 34 cycles; extending at 68℃for 5min. The amplified NP24 gene promoter fragment was subjected to gel electrophoresis. mu.L of the PCR product was aspirated, 1. Mu.L of loading buffer was added to the reaction tube, and electrophoresis was performed on 1% (5. Mu.L of gel red,1g of agarose/100 mL of 0.5 XTAE buffer) agarose gel. After electrophoresis, photographing in a gel imaging system, determining that the size of a band is consistent with that of a target fragment (see (b) in fig. 1), and carrying out PCR product recovery by using a Axygen PCR clean up kit (product number: AP-PCR-250G) to obtain an NP24 gene promoter DNA fragment, wherein the nucleotide sequence is shown as a sequence 1 in a sequence table, and the nucleotide sequence shown as the sequence 1 is named as NP24pro and consists of 1445 nucleotides.

Taking 20 mu L of PCR product, adding 100 mu L of buffer A and 100 mu L of isopropanol, mixing, passing the mixture through an adsorption column, adding 700 mu L of wash buffer II, washing twice, centrifuging for 1 min at 12000rmp, adding 20 mu L elute buffer of eluting DNA, and measuring the fragment concentration to obtain the NP24 gene promoter fragment.

2. PGreenII0800-LUC-NP24pro construction

And carrying out enzyme digestion on pGreenII0800-LUC plasmid to obtain a vector after enzyme digestion. The vector cleavage system is shown in Table 2. The enzyme was digested at 37℃for 2 hours, followed by enzyme inactivation at 80℃for 20 minutes.

TABLE 2 vector cleavage System

Using VazymeUltra One Step Cloning Kit homologous recombination kit (company: northenzan, cat# C115-01) was subjected to vector ligation. The configuration system is shown in table 3 below. Reacting for 15min at 50 ℃ to obtain the homologous recombination product.

TABLE 3 homologous recombination ligation reaction System

Taking 5 mu L of homologous recombination products, adding 80 mu L of DH5 alpha competent cells, carrying out ice bath for 30min, then carrying out heat shock for 90s at 42 ℃, placing on ice for 5min, adding 400 mu L of antibiotic-free liquid LB culture medium, culturing at 37 ℃ and 220rpm for 1h, centrifuging competent cells, coating on an LB solid culture medium resistant plate containing 50mg/L kanamycin, placing the plate in a 37 ℃ incubator for culturing for 16h, picking clones for colony PCR detection, selecting colonies with correct fragment size, adding 4mL of liquid LB culture medium containing Canada for shake culture at 37 ℃ for 16h, and extracting plasmids. The recombinant vector which showed the DNA molecule containing SEQ ID No.1 was designated pGreenII-0800-LUC-NP 24pro.

PGreenII0800-LUC-NP24pro is a recombinant vector obtained by replacing the fragment between 5'-gtcgacggtatcgataagctt-3' and 5'-gctctagaactagtggatcc-3' of pGreenII0800-LUC with the DNA molecule of nucleotide sequence SEQ ID NO.1 (NP 24pro promoter), keeping the other nucleotides of pGreenII0800-LUC unchanged (the structural schematic diagram of the promoter reporter gene is shown in FIG. 1 (a)). pGreenII0800-LUC-NP24pro contains a promoter NP24pro, which promoter NP24pro drives transcription of the downstream luciferase gene (luciferase).

The tobacco mosaic virus 35S promoter is used as a positive control to construct a recombinant vector pGreenII0800-LUC-35S. Recombinant vector pGreenII0800-LUC-35S is obtained by replacing the fragment between 5'-gtcgacggtatcgataagctt-3' and 5'-gctctagaactagtggatcc-3' of pGreenII0800-LUC with a DNA molecule (containing tobacco mosaic virus 35S promoter and Arabidopsis ADH gene 5' -UTR sequence) with the nucleotide sequence of SEQ ID NO.2, keeping the other nucleotides of pGreenII0800-LUC unchanged.

Vector transient transformation was achieved by fruit injection experiments using recombinant vector pGreenII0800-LUC-35S as positive control, empty vector pGreenII0800-LUC as negative control, and Agrobacterium GV3101 (containing pSoup plasmid) transformed with pGreenII0800-LUC, pGreenII0800-LUC-35S and pGreenII0800-LUC-NP24 pro.

Fruit injection experimental procedure:

Three vectors, pGreenII0800-LUC-NP24pro, pGreenII0800-LUC-35S and pGreenII0800-LUC, were transformed into Agrobacterium GV3101, and single clones were picked up on LB resistant medium containing 50mg/L kanamycin and 50mg/L rifampicin, and 5mL of YEB Agrobacterium medium containing 50mg/L kanamycin and 50mg/L rifampicin were added for overnight culture until the bacterial solution was shaken to orange red (OD _600nm value was 2-3), after which 100. Mu.L of bacterial solution was aspirated into 5mL of induction medium (the induction medium was formulated as (1L) 0.5% bovine extract, 0.1% yeast extract, 0.5% peptone, 0.5% sucrose, 2mM MgSO ₄, 20. Mu.M acetosyringone, 10mM MES, pH5.6, 100. Mu.M kanamycin, 100. Mu.M rifampicin, the balance was water), and cultured at 28℃for 16-18 hours, so that the bacterial solution was shaken to OD _600nm value was about 2. After reaching the corresponding OD _600nm value, the culture was centrifuged (5000 g,10 min), the supernatant removed and the cells were suspended in an osmotic medium (osmotic medium was prepared (1L) 10mM MgCl ₂, 10mM mes,200 μm acetosyringone, balance water, pH 5.6) to give a bacterial solution OD _600nm =1.0. Incubate at room temperature for 2 hours at 20rpm under light-protected conditions. GV3101 bacterial solutions containing pGreenII-LUC-NP 24pro, pGreenII-0800-LUC-35S and pGreenII-0800-LUC vectors were obtained, respectively.

The tomato fruits in the M82 red ripe stage were taken and injected with a 1mL syringe (tip 0.5X16 mm needle). The needle head is inserted into the tomato fruit by 3-4mm deep through the top of the flower column, so as to avoid the injection of bacterial liquid into the cavity of the tomato fruit. The specific processing packets are as follows:

NP24 promoter group: 1mL of GV3101 bacterial liquid containing pGreenII-0800-LUC-NP 24pro vector is injected into each tomato fruit;

35S promoter group: 1mL of GV3101 bacterial liquid containing pGreenII-0800-LUC-35S carrier is injected into each tomato fruit;

negative control group: each tomato fruit was injected with 1mL of GV3101 broth containing pGreenII-0800-LUC vector.

The bacterial liquid needs to be slowly pushed, so that the cracking of fruits is reduced. The experiment was repeated three times with 3 red ripe stage fruits of tomato injected each time.

After 5 days of fruit injection, the treated fruit slices were taken, thin disks were cut with the injection point as the center, 4-5 layers were cut, each layer had a thickness of about 4mm, a luciferase substrate (Promega Co., ltd., cat. No. E1605) was coated on the surface of each layer, and the fruits were left in the dark for 2 minutes, and then fluorescence of the fruits was detected using a plant living molecular imaging system (manufacturer: berthold, instrument model: LB 985).

Quantitative experiment step of promoter activity: using Dual-purpose-Fluorescence intensity quantification experiments were performed with a Reporter 1000Assay Systems kit (Promega, cat. E1960) and a multi-labeled microplate detector (manufacturer: PERKIN ELMER, instrument model: envision). And (5) taking the pulp of the part with the strongest fluorescence in the living body fluorescence imager, freezing the pulp with liquid nitrogen, and grinding the pulp into powder. 100mg of pulp powder is taken, 100 mu L of protein extract 1 XPLB is added, 14000g is centrifuged for 15min at 4 ℃,20 mu L of supernatant is sucked into a 96-well ELISA plate, 50 mu L of luciferase substrate solution LAR II is added, fluorescence is detected, and the firefly luciferase activity with the value of the sample is obtained. After the measurement was completed, 50. Mu.L of Stop was added to the reaction systemReagent (Promega Co., ltd.; cat# E1960) again detects fluorescence to obtain a Renilla luciferase activity having a value of the sample. The firefly luciferase activity is divided by the Renilla luciferase activity, and the firefly luciferase activity is the activity of the corresponding promoter.

As a result, as shown in FIG. 1 (d), significant fluorescence was detected in fruits injected with Agrobacterium containing pGreenII0800-LUC-NP24pro recombinant vector (NP 24 promoter group for short), and the fluorescence intensity cps was in the range of 6631-65535, which was higher overall than in fruits injected with Agrobacterium containing pGreenII0800-LUC-35S recombinant vector (35S promoter group for short), and the fluorescence intensity cps was in the range of 5181-25904. Quantitative analysis of luciferase activity revealed that the NP24 gene promoter-driven luciferase activity was about 3 times that of the 35S promoter (e in FIG. 1, table 4), indicating that the promoter had higher activity.

TABLE 4 quantitative data on the activity of the NP24 promoter

Group of	35S promoter	NP24 promoter	Negative control
				LUC/REN ratio	1.12±0.17	2.82±0.06	0.03±0.01

Example 2, NP24 Gene promoter drives overexpression of exogenous Gene and quantitative PCR results of transgenic Material

In order to determine the specificity of the NP24 promoter, an exogenous gene overexpression vector pL2-1-NP24pro-CDS driven by the NP24 promoter was constructed, and a stably transformed plant was obtained by the Agrobacterium transformation method.

The pL2-1-NP24pro-CDS vector construction procedure was as follows:

1. Exogenous gene and promoter fragment amplification: amplifying to obtain a triterpene synthesis pathway gene cucurbitadienol synthase gene coding sequence (CDS) by taking a fructus momordicae cDNA as a template, wherein the nucleotide sequence of the coding sequence is shown as SEQ ID No.3 in a sequence table, and the coding amino acid sequence is shown as SEQ ID No. 4; the vector pGreenII-LUC-NP 24pro obtained in example 1 was used as a template for amplification to obtain the NP24 promoter sequence, and the amplified fragment was subjected to PCR product recovery.

2. First round Golden gate reaction: the NP24 gene promoter was ligated to vector pL1F-3 in a unit that driven the expression of the coding gene of cucurbitadienol synthase. The Golden gate reaction system is shown in Table 5 below.

TABLE 5 first round Golden gate reaction System

Reagent(s)	Dosage (mu L)
		pL1F-3(100ng/μL)	1
NP24pro(50ng/μL)	1
		Gene fragment(50ng/μL)	1
terminator(30ng/μL)	1
		BsaI-HF	1
T4 ligase	1
		BSA(2mg/mL)	1.5
10×T4 ligase buffer	1.5
		ddH2O	6
Total reaction System	15

The following PCR procedure was then run: the two steps are carried out for 20 cycles at 37 ℃ for 3min and 16 ℃ for 4min, 50 ℃ for 5min and 80 ℃ for 10min. Adding the reaction system into competent cells DH5 alpha, carrying out heat shock for 50s at 42 ℃, adding 400 mu L of non-antibiotic LB liquid medium, shaking and culturing at 37 ℃ for 1h at 220rpm, coating bacterial liquid on an LB solid medium resistant plate containing 50mg/L of ampicillin, placing the plate in a 37 ℃ incubator for 16 hours, picking up bacteria, sequencing, placing a clone extracted plasmid with correct sequencing at-20 ℃ for preservation, and naming the plasmid with correct sequencing as pL1F-3-NP24pro-CDS.

3. The second round Golden gate reaction: the foreign gene expression cassette NP24pro-CDS was ligated to vector pL2-1 to form the final vector pL2-1-NP24pro-CDS.

TABLE 6 second round Golden gate reaction System

Reagent(s)	Dosage (mu L)
		pL1F-3-NP24pro-CDS(100ng/μL)	1
pL1F-4(100ng/μL)	1
		pL1F-2(100ng/μL)	1
pL1F-1-NPTII(100ng/μL)	1
		end-linker(100ng/μL)	1
pL2-1(100ng/μL)	1
		T4 ligase	1
BpiI	1
		10×T4 ligase buffer	1.5
ddH2O	5.5
		Total reaction System	15

The following PCR procedure was then run: the two steps are carried out for 20 cycles at 37 ℃ for 3min and 16 ℃ for 4min, 50 ℃ for 5min and 80 ℃ for 10min.

In the reaction system, the pL1F-1-NPTII vector is a recombinant vector obtained by replacing the sequence between two BsaI enzyme cutting sites of the vector pL1F-1 with a DNA molecule shown by a nucleotide sequence SEQ ID NO.5 and keeping other nucleotides of the pL1F-1 unchanged.

Adding the reaction system into competent cells DH5 alpha, performing heat shock for 50s at 42 ℃, adding 400 mu L of antibiotic-free LB liquid medium, coating bacterial liquid on a kanamycin LB solid medium resistant plate with the concentration of 50mg/L at 37 ℃ for 1h, placing the plate in a 37 ℃ incubator for 16 hours, picking up bacteria, sequencing, and storing the clone extraction plasmid with correct sequence at-20 ℃. The plasmid with correct sequencing was named: pL2-1-NP24pro-CDS.

The structure of the pL2-1-NP24pro-CDS plasmid is described as follows: the segment between the two BpiI enzyme cutting sites of pL2-1 is replaced by a DNA molecule shown in a nucleotide sequence SEQ ID NO.6, and other nucleotides of pL2-1 are kept unchanged to obtain the recombinant vector. pL2-1-NP24pro-CDS contains the promoter NP24pro, and the promoter NP24pro drives transcription of the downstream cucurbitadienol synthase gene.

4. The recombinant plasmid pL2-1-NP24pro-CDS obtained in the step 3 is transformed into Agrobacterium tumefaciens AGL1 competent cells to obtain recombinant Agrobacterium tumefaciens AGL1/NP24pro-CDS. Plant genetic transformation mediated by agrobacterium AGL1/NP24pro-CDS is carried out, so that a plant with NP24 promoter for driving the stable overexpression of cucurbitadienol synthase gene is obtained.

The genetic transformation process of agrobacterium is as follows: seed of MicroTom tomato was sown in medium, after seedlings had grown for 6-8 days, cotyledons were cut to approximately square shape and precultured for 2 days, then agrobacteria AGL1/NP24pro-CDS was adjusted to an OD value in the range of 0.6-0.8, cotyledons were infested for 10min, co-cultivated for 2 days, and cotyledons were transferred to germination medium containing 75mg/L kanamycin. Transferring to rooting culture medium containing 75mg/L kanamycin for 2-4 weeks after bud length exceeds 1.5cm, and taking materials with good rooting condition for genotype identification by adopting the following identification primers: NPT-F5'-CTATTCGGCTATGACTGGGC-3', NPT-R5'-AATATCACGGGTAGCCAACG-3', positive seedlings were identified by PCR electrophoresis, and the specific results are shown in FIG. 2. As can be seen from FIG. 2, the target band was obtained around 500bp, which was determined as a transgenic positive plant, and the transgenic positive plant was transplanted to a greenhouse.

The exogenous gene expression level is identified by taking the root, stem, leaf, flower, green ripe fruit, broken color fruit, red ripe fruit and seed tissue of MicroTom tomato stable genetic material, and the specific experimental steps are as follows:

Taking each tissue of the transgenic material, quickly freezing the tissue by liquid nitrogen, and grinding the tissue into powder by a mortar at a low temperature. 600-800mg of tomato fruits in the color breaking period and the red ripe period and 100-200mg of the rest tissues. Each tissue RNA was extracted using EASYspin plant RNA extraction kit (Aidlab, cat No. RN 09). 10 volumes (1 mL) of tissue lysate RLT were mixed with 1 volume (100. Mu.L) of PLANTaid to remove the polysaccharide polyphenol component, and then tissue powder was added thereto, and the mixture was thoroughly mixed and centrifuged at 13000rpm at room temperature for 8min. Taking supernatant, adding 0.5 volume of absolute ethyl alcohol, adding the mixture into an RNA adsorption column, centrifuging at 13000rpm for 2min, adding 700 mu L of deproteinized liquid into the RNA adsorption column, centrifuging at 13000rpm for 30s to remove impurities, adding 500 mu L of rinsing liquid, centrifuging at 13000rpm for 30s once, centrifuging at 13000 min to remove residual liquid in the adsorption column, finally adding 30 mu LRNA-free water, centrifuging at 12000rpm for 1min, and obtaining RNA of each tissue of tomatoes.

Subsequently, DNA was removed from the RNA using the DNA free ^TM kit (Thermo, cat# AM 1906). Taking a 20 mu LRNA sample, adding 1 mu L rDNase and 2 mu L DNase I buffer, uniformly mixing, incubating for 20-30min at 37 ℃, adding 2.3 mu L DNase inactivating agent, uniformly mixing, incubating for 2min at room temperature, centrifuging for 1.5min at 10000g, sucking the supernatant, and determining the RNA concentration, wherein the RNA concentration is higher than 100 ng/. Mu.L.

After RNA was obtained, the digested RNA was reverse transcribed using FASTKING RT KIT (WITH GDNASE) cDNA first strand synthesis kit (Tiangen, cat# KR 116) to obtain cDNA for each tissue of the transgenic material. Specific experimental procedures are referred to the specification. And (3) carrying out a fluorescent quantitative PCR experiment by taking cDNA of each tissue of the obtained transgenic material as a template, and identifying the expression level of the target gene. The fluorescent quantitative PCR experiment system is configured as follows:

the procedure for fluorescence quantitative PCR was as follows:

After the Cq value of the target gene of each tissue is obtained, the expression level of the target gene is calculated by a method of 2 ^-△△ct. The sequences of the fluorescent quantitative PCR primers involved in this experiment are shown in the following table:

Primer name	Primer sequence 5'-3'
		CDS-qF	GACAGCGGGAGTGAAATTGA
CDS-qR	CCAGTTCCCATCGCTTGTCT
		Slubi-qF	CGTGGTGGTGCTAAGAAGAG
Slubi-qR	ACGAAGCCTCTGAACCTTTC

The results are shown in FIG. 1 (c): the exogenous gene cucurbitadienol synthase gene is expressed at high level in the fruit color breaking period and the red ripening period, and is also expressed in trace amount in roots, and the gene expression level in other tissues is extremely low. The experiment shows that the NP24 gene promoter can drive the specific expression of the exogenous gene in the tomato fruit color breaking period and the red ripe period.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims (7)

1. The nucleotide sequence is the DNA molecule of SEQ ID No. 1.

2. An expression cassette or recombinant vector comprising the DNA molecule of claim 1.

3. A recombinant microorganism comprising the DNA molecule of claim 1.

4. Use of a DNA molecule according to claim 1 for promoting expression of a gene of interest in a plant, said plant being tomato, said use being for promoting specific expression of a gene of interest in tomato fruits during the color break period and/or the red ripe period of tomato fruits.

5. The use of the DNA molecule of claim 1 in tomato breeding, said use being for the specific expression of a gene of interest in tomato fruits in the color breaking stage and/or the red ripening stage of tomato fruits, resulting in a tomato variety with a strong specific expression capacity.

6. The use of the recombinant vector or the expression cassette according to claim 2 in tomato breeding, wherein the use is to start the specific expression of a target gene in tomato fruits in the color breaking period and/or the red ripening period of tomato fruits, so as to obtain a tomato variety with strong specific expression capability.

7. The use of the recombinant microorganism of claim 3 in tomato breeding, wherein the use is to start the specific expression of a target gene in tomato fruits in the color breaking period and/or the red ripe period of the tomato fruits, so as to obtain a tomato variety with strong specific expression capability.