CN114672489B - Tobacco gland Mao Qidong seed pNtGGPPS2a and application thereof - Google Patents

Abstract

The application discloses a tobacco gland Mao Qidong seed pNtGGPPS2a and application thereof. The promoter has a nucleotide sequence shown as SEQ ID NO.1 or a sequence which has more than 90 percent of homology with a DNA sequence defined by SEQ ID NO.1 and has the same promoter activity. The promoter drives the target gene to be specifically expressed in the glandular hair tissue of the tobacco leaf, but not in the non-glandular hair tissue of the leaf, so that the influence on the growth and development of plants caused by the fact that a large amount of secondary metabolites are generated in the leaf is avoided. The application provides a new regulatory sequence for the efficient production of secondary metabolites by utilizing tobacco glandular hairs or the breeding of high-aroma tobacco varieties.

Description

Tobacco gland Mao Qidong seed pNtGGPPS2a and application thereof

Technical Field

The application relates to the technical field of plant genetic engineering, in particular to tobacco gland Mao Qidong seed pNtGGPPS2a and application thereof.

Background

Glandular wool is a special structure formed by differentiation of plant epidermal cells. The glandular hair synthesizes, stores and secretes a series of secondary metabolites (such as alkaloid, nicotine and terpene etc.) to protect plants from herbivores and insects, and furthermore, the glandular hair is also called a "small factory" for metabolite synthesis, and some special metabolites have become pesticides, food additives, fragrances or pharmaceutical components etc. with important commercial value, such as artemisinin in Artemisia annua (artemia annua), menthol in mint (menthol) and metabolites such as cannabinoid in Cannabis (Cannabis sativa) are mainly synthesized and stored in glandular hair.

Tobacco (Nicotiana tabacum l.) is an important commercial crop and the tobacco glandular hair secretion is mainly a mixture of cembrane diterpene, sugar ester, alkane, volatile aldehyde, ketone, acid and the like, and accounts for about 0.5-10% of the fresh tobacco leaf weight, wherein most of the components have an important contribution to the tobacco leaf aroma. Studies show that two key enzyme genes CBTS and CYP71D16 in the cembrane diterpene synthesis pathway are specifically expressed in glandular wool.

A promoter is a DNA sequence located upstream of the 5' end of a gene, which is recognized and bound by RNA polymerase and regulates the expression of the gene of interest at a specific time or space.

CaMV35S from cauliflower mosaic virus is a constitutive expression promoter commonly used in plant genetic engineering at present, and can drive the expression of a target gene in any development stage and tissue part of a plant, but if applied to the production of secondary metabolites, the constitutive expression tends to accumulate the target product in the whole plant body, so that abnormal plant growth occurs.

Disclosure of Invention

In the prior art, caMV35S from cauliflower mosaic virus is often selected as a constitutive expression promoter in plant genetic engineering, and when the CaMV35S promoter is used for producing secondary metabolites in plants, as described above, expression often causes accumulation of target products in the whole plants, so that abnormal plant growth occurs, and thus the improvement of tobacco gland Mao Fenbi to produce components with important effects on tobacco aroma is not facilitated.

In view of the above, the glandular hair tissue specific promoter can drive the target gene to specifically express in glandular hair, and effectively avoid the damage to the growth and development of plants when exogenous genes are expressed (or endogenous genes are over expressed). Thus, the present application contemplates providing a tobacco gland Mao Qidong promoter that drives the specific expression of a gene of interest in tobacco gland bristles.

In a first aspect, the embodiment of the application discloses a tobacco gland Mao Qidong seed pntggpss 2a, wherein the promoter drives target genes to be specifically expressed in the gland hair tissue of tobacco leaves, and the nucleotide sequence of the promoter is one of (I) or (II):

(I) As shown in SEQ ID NO. 1;

(II) a sequence which has more than 90% homology with the DNA sequence defined in SEQ ID NO.1 and has the same promoter activity.

In a second aspect, embodiments of the present application provide a primer pair for amplifying the promoter of the first aspect, wherein the primer pair has the sequence shown in SEQ ID NO.2 and SEQ ID NO. 3.

In a third aspect, embodiments of the present application provide a method for preparing the promoter according to the first aspect, which comprises the following steps:

extracting tobacco genome DNA;

cloning a promoter pNtGGPPS2a from the genome DNA;

the promoter pntggps 2a was subjected to cis-acting element analysis;

constructing a pCAMBIA1391Z-pNtGGPPS2 a:GUS plant expression vector;

culturing aseptic tobacco seedlings, and genetically transforming GUS vectors into the aseptic tobacco seedlings to obtain transgenic tobacco;

GUS staining is carried out on the transgenic tobacco;

qRT-PCR detects the relative expression of the NtGGPPS2a gene in tobacco glandular wool.

Further, the genetic transformation process is to transform the recombinant plasmid into tobacco through agrobacterium mediation.

In a fourth aspect, embodiments of the present application provide an application of the promoter in tobacco genetic engineering breeding, specifically:

the promoter drives the target gene to express at the glandular wool part of the tobacco, and the secondary metabolite is efficiently produced to directionally cultivate the new variety of the high-aroma tobacco.

In a fifth aspect, embodiments of the present application disclose a nucleic acid molecule comprising the sequence of the promoter of the first aspect.

In a sixth aspect, embodiments of the present application disclose chimeric genes comprising the nucleic acid molecules of the fifth aspect, which are chimeric linked to a heterologous nucleic acid molecule.

In embodiments of the present application, the heterologous nucleic acid molecule encodes a protein or peptide.

In a seventh aspect, embodiments of the present application disclose vectors comprising the nucleic acid molecules of the fifth aspect.

In an eighth aspect, embodiments of the present application disclose a host cell comprising the vector of the seventh aspect.

Compared with the prior art, the application has at least one of the following beneficial effects:

the application provides a new method for molecular breeding of tobacco. The method comprises the steps of connecting the promoter pNtGGPPS2a with a target gene and transferring the target gene into tobacco, driving the expression of the target gene in glandular hair tissues through the promoter, improving the efficiency of producing secondary metabolites, and having important values for breeding new flue-cured tobacco varieties and improving the aroma quality of the tobacco.

Drawings

FIG. 1 is a PCR amplification electrophoresis chart of the promoter pNtGGPPS2a provided in the examples of the present application.

FIG. 2 is an analysis of cis-acting elements of promoter pNtGGPPS2a provided in the examples of the present application.

FIG. 3 is a construction chart of pCAMBIA1391Z-pNtGGPPS2a:: GUS vector provided in the examples of the present application.

FIG. 4 is a GUS staining chart of GUS transgenic tobacco leaves of pCAMBIA1391Z-pNtGGPPS2a provided in the examples of the present application.

FIG. 5 shows the relative expression levels of the NtGGPPS2a gene in different tissues of tobacco provided in the examples of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.

Various changes to the exact description of the present application may be readily made by those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the present application is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the present application. Indeed, various modifications of the embodiments which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.

In this application, the term "gene" refers to a nucleic acid fragment expressing a particular protein or functional RNA molecule, which may comprise a regulatory sequence preceding (5 'non-coding region) and a regulatory sequence following (3' non-coding region) the coding sequence.

In this application, a "native gene" refers to a naturally occurring gene having its own regulatory sequences. "chimeric gene" refers to any gene that is not a native gene, comprising regulatory sequences and coding sequences that are not naturally present together. Thus, a chimeric gene may comprise regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that found in nature.

"element" in this application is meant to include (upstream promoter elements) CAAT and GC boxes, which are typically located around-70 bp, as well as upstream elements further from the transcription initiation point.

In this application, a "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. Promoters may be derived entirely from a native gene, or consist of different elements derived from different naturally occurring promoters, or even comprise synthetic DNA fragments. It will be appreciated by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that cause expression of genes in most cell types in most cases are commonly referred to as "constitutive promoters".

In this application, the term "expression" refers to the transcription and stable accumulation of coding RNA (mRNA) or functional RNA derived from a gene, and may also refer to translation of mRNA into a polypeptide or protein.

In this application, the term "messenger RNA (mRNA)" refers to RNA that is intronless and can be translated into protein by a cell.

In the present application, the term "transformation" refers to the transfer of a nucleic acid fragment into a host organism, resulting in a genetically stable inheritance. The transformed nucleic acid may be in the form of a plasmid that remains in the host cell, or some of the transformed nucleic acid may be integrated into the host cell genome. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

In the present application, the terms "plasmid" and "vector" refer to an extrachromosomal element that normally carries a gene that is not part of the central metabolism of the cell, and is often in the form of a circular double stranded DNA molecule. Such elements may be autonomously replicating sequences, genomic integrating sequences, phage or nucleotide sequences (linear or circular) of single-or double-stranded DNA or RNA derived from any source, wherein multiple nucleotide sequences have been joined or recombined into a unique construct capable of introducing a promoter fragment of a selected gene product and the DNA sequence into a cell along with the corresponding 3' -terminal untranslated sequence.

In the present application, the term "chimeric ligation" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one nucleic acid sequence is affected by the other. For example, a promoter is chimeric to a coding sequence when it is capable of affecting the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence may be chimeric linked to the regulatory sequence in either sense or antisense orientation. Correspondingly, a "chimeric gene" refers to nucleic acid sequences that are "chimeric linked" together, for example, by "chimeric linking" a promoter to a coding sequence that it can affect.

In this application, the term "heterologous" refers to a site that does not naturally occur at the site of interest. For example, a "heterologous gene" refers to a gene that does not naturally occur in a host organism and that is introduced into the host organism by gene transfer. For example, a heterologous nucleic acid molecule present in a chimeric gene is a nucleic acid molecule that does not naturally occur with other fragments in the chimeric gene, such as a nucleic acid molecule having coding regions and promoter fragments that do not naturally associate with each other.

In this application, a "nucleic acid molecule" is a polymer of RNA or DNA that is single-stranded or double-stranded, optionally comprising synthetic, non-natural or altered nucleotide bases. The nucleic acid molecule in the form of a DNA polymer may be composed of one or more segments of cDNA, genomic DNA, or synthetic DNA.

For a better understanding of the present application, and not to limit the scope of the present application, all numbers expressing quantities, percentages, and other values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

In the examples described below, the temperature is not particularly limited, but is a normal temperature condition. The normal temperature is natural room temperature in four seasons, and is generally controlled at 10-30 ℃, preferably 15-25 ℃ without additional cooling or heating treatment.

The application discloses a tobacco gland Mao Qidong and pNtGGPPS2a and uses thereof, the plant genomic DNA extraction kit related to the present application is purchased from Axygen corporation of America; high-fidelity enzyme 2X TransStart KD Plus PCR SuperMix and seamless cloning kit-Basic Seamless Cloning and Assembly Kit, plasmid extraction kit EasyPure Plasmid MiniPrep Kit, glue recovery kit EasyPure Quick Gel Extraction Kit purchased from beijing all gold biotechnology limited; tobacco genetic transformation and screening related media were purchased from Beijing Cool Lei Bo technology Co., ltd; GUS staining fluid was purchased from Beijing Bai Rui Biotech Co., ltd; agrobacterium GV3101, E.coli DH 5. Alpha. And plasmid pCAMBIA1391Z are commercially available; RNA extraction kit EasyPure Plant RNA Kit, cDNA reverse transcription kit easy script One-Step gDNA Removal and cDNA Synthesis SuperMix, purchased from Beijing full gold biotechnology Co., ltd; qRT-PCR kit TB Green Premix DimerEraser was purchased from Takara-Bao Ri doctor Material technology (Beijing); the apparatus used for qRT-PCR experiments was the model ViiA7, available from America Applied Biosystems. Primers were synthesized by Shanghai JieRui bioengineering Co., ltd, and sequencing was performed by Biotechnology (Shanghai) Co., ltd.

The technical solutions of the present application will be clearly and completely described below in connection with the embodiments of the present application.

Cloning of the promoter pNtGGPPS2a and construction of pCAMBIA1391Z-pNtGGPPS2a: GUS vector

Genomic DNA of cultivar "K326" was extracted using plant genomic DNA extraction kit (Axygen, AP-MN-MS-GDNA-50) according to the method described herein. Selecting a nucleotide sequence of about 1.7kb upstream of the ATG 5' end of the NtGGPPS2a gene initiation codon as a candidate promoter region, and designing a specific primer for PCR amplification, wherein the primer sequence is as follows:

pNtGGPPS2a-F1 is shown as SEQ ID NO. 2;

pNtGGPPS2a-R1 is shown in SEQ ID NO. 3.

Cloning the target fragment from genomic DNA by using high-fidelity enzyme 2× TransStart KD Plus PCR SuperMix, and the PCR reaction system is shown in Table 1:

TABLE 1

The reaction conditions are shown in table 2:

TABLE 2

After the PCR amplification is finished, the target PCR product is recovered by using a gel recovery kit, the electrophoresis chart of the PCR product is shown in FIG. 1, and the size of the product is about 1.7kb.

The recovered product was constructed into pCAMBIA1391Z plant expression vector (digested with HindIII and BamH I) using a seamless cloning kit according to the procedure described, the recombinant product was transformed into E.coli DH 5. Alpha. Competent cells, the bacterial solution was spread on solid LB (containing 100mg/L kanamycin) plates, and cultured overnight at 37 ℃. And (3) selecting positive clone for sequencing, and after sequence comparison, naming the promoter sequence with correct sequence as pNtGGPPS2a and 1656bp in length, wherein the sequence information is shown as SEQ ID NO. 1. The constructed vector is pCAMBIA1391Z-pNtGGPPS2a:: GUS, and the structural schematic diagram is shown in figure 2.

Analysis of promoter pNtGGPPS2a cis-acting element

The obtained promoter sequence pNtGGPPS2a is subjected to cis-acting element prediction (shown in figure 3) by adopting an online website plant CARE (http:// bioinformation information. Psb. Ugent. Be/webtools/plant/html /), and the prediction finds that the pNtGGPPS2a has photoresponsive elements ACE, box4, G-Box and TCT-motif besides promoter core sequences such as TATA Box and CAAT Box; anaerobic induction response element ARE; an auxin response element AuxRR-core; drought responsive element DRE1; an ethylene response element ERE; a low temperature responsive element LTR; a transcription factor binding element STRE involved in fatty acid metabolism; MYC transcription factor binding site TCTCTTA; MYB transcription factor binding site TAACTG; a WRKY transcription factor binding site TTGACC; and other functional elements such as AAGAA-motif.

The above analysis shows that the NtGGPPS2a gene may be under complex regulation.

Culturing tobacco aseptic seedlings

Sterilizing tobacco seed surface with 75% ethanol in ultra clean bench for 30s, and using 10% (V/V) H ₂ O ₂ Soaking for 10min, washing with sterile water for 5 times, absorbing water on sterile paper, sowing seeds on MS solid culture medium, and performing genetic transformation on the obtained sterile seedlings after 30 d.

pCAMBIA1391Z pNtGGPPS2a:: GUS genetically transformed tobacco

(1) GUS was transformed into GV3101 Agrobacterium competent cells with the plasmid pCAMBIA1391Z-pNtGGPPS2a constructed as described above.

(2) Cutting the leaf of aseptic seedling obtained by the above culture to 0.8cm with a surgical knife in an ultra clean bench ² Leaf discs of left and right sizes were inoculated into co-culture medium for 2d of preculture.

(3) The tobacco leaf discs were genetically transformed with Agrobacterium containing the plasmid pCAMBIA1391Z-pNtGGPPS2a:: GUS according to the following procedure:

A. taking the agrobacterium liquid in the optimal logarithmic growth phase, centrifugally collecting thalli, diluting with sterile water, placing the leaf disc in the bacteria liquid for infection for 10min, taking out the leaf disc, sucking the bacteria liquid on the surface of the leaf disc by using sterilized filter paper, transferring to a co-culture medium, and culturing in dark at 25 ℃ for 2d.

B. She Panqing is washed 3 times with sterile water containing 160mg/L Tintin, the water is absorbed, transferred to a screening culture medium containing corresponding antibiotics, and the screening culture is carried out under the illumination condition of 25 ℃ and 16h/d, and the culture medium is replaced every 15-20 d.

C. Cutting off the resistant buds growing to more than 1cm along the basal part, transferring to a 1/2MS culture medium containing corresponding antibiotics, hardening seedlings after rooting, and transplanting.

The culture medium used in the experimental process is specifically:

co-culture medium: MS culture medium +1.5 mg/L6-BA

Screening the culture medium: MS culture +1.5mg/L6-BA +10mg/L Hyg +160mg/L Timantin

Rooting medium: 1/2MS culture medium+0.1 mg/L NAA+10mg/L Hyg+160mg/L Timentin

After the culture medium is prepared, the pH value is adjusted to 5.8-5.9, and the culture medium is sterilized at the high temperature of 121 ℃ for 20min for standby.

The promoter pNtGGPPS2a drives GUS gene to specifically express in glandular hair cells

And taking T0 generation transgenic positive plant leaves for GUS staining. The specific operation is as follows:

adding 90% (V/V) pre-cooled acetone into tobacco leaf, fixing for 20min, and adding ddH ₂ O washing acetone, adding GUS dye solution and allowing the GUS dye solution to pass through the leaves, and placing the leaves in a 37 ℃ incubator for overnight dyeing. After staining, leaves were decolorized with 50%, 75% and 100% ethanol, respectively, to colorless the solution to exclude color interference, and observed with a split microscope and photographed.

GUS staining results are shown in FIG. 4, in which the GUS gene was expressed in secretory glandular hairs of leaf blades (grey dots in the figure), but not in other non-glandular hair tissues. The promoter pNtGGPPS2a can drive the specific expression of the target gene in the tobacco gland hair tissue.

Analysis of tissue expression Properties of NtGGPPS2a Gene

In order to further determine the specific expression condition of the NtGGPPS2a gene in leaf glandular hairs, selecting root, stem, leaf, flower, glandular hairs and glandular hair removing leaf tissues of tobacco for cultivation "K326"; the glandular wool was collected as follows: the upper leaf of tobacco in bud period 10d is taken out, the glandular hair tissue is gently scraped from the surface of the leaf by adopting a liquid nitrogen freezing brush method, and the leaf scraped with glandular hair is the gland Mao Shepian removed.

Extracting total RNA of each tissue and reversely transcribing the total RNA into cDNA, adopting beta-action as an internal reference gene, and adopting real-time fluorescence quantitative PCR (Quantitative Real-time PCR, qRT-PCR) to detect the relative expression quantity of the NtGGPPS2a gene in different tissues of tobacco.

The qRT-PCR amplification primer sequences were as follows:

NtGGPPS2a-Q-F1 is shown in SEQ ID NO. 4;

NtGGPPS2a-Q-R1 is shown in SEQ ID NO. 5;

Actin-Q-F1 is shown as SEQ ID NO. 6;

Actin-Q-R1 is shown as SEQ ID NO. 7.

The qRT-PCR reaction system is shown in Table 3:

TABLE 3 Table 3

Reagent(s)	Usage amount
		TB Green Premix DimerEraser (premix)	10μl
Rox Reference Dye II (reference dye)	0.4μl
		Primer 1	6μM
Primer 2	6μM
		cDNA template	2μl
ddH 2 O	Mix the solution to 20. Mu.l

The reaction conditions are shown in Table 4:

TABLE 4 Table 4

The PCR reaction products were analyzed by melting curve, and the results are shown in FIG. 5, from which the following conclusions can be seen:

the NtGGPPS2a gene has small expression in leaves and flowers, and the stem is the next;

the NtGGPPS2a gene is hardly expressed in roots;

the NtGGPPS2a gene is expressed in a large amount in glandular wool, and the expression amount in glandular wool removing leaves is extremely low;

the above results indicate that the NtGGPPS2a gene is specifically expressed in the glandular hair tissue of tobacco leaf.

In conclusion, the tobacco gland Mao Qidong promoter pNtGGPPS2a and application thereof have the nucleotide sequence shown in SEQ ID NO.1 or a sequence which has more than 90% of homology with the DNA sequence defined by SEQ ID NO.1 and has the same promoter activity; the promoter drives the target gene to specifically express in the tobacco leaf gland hair tissue. The promoter pNtGGPPS2a is connected with a target gene and then transferred into tobacco, and the promoter drives the expression of the target gene in glandular hair tissue, so that the efficiency of producing secondary metabolites is improved, and the promoter has very important values for breeding new flue-cured tobacco varieties and improving the aroma quality of tobacco.

The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.

Sequence listing

<110> institute of tobacco science in Guizhou province

<120> tobacco gland Mao Qidong seed pNtGGPPS2a and application thereof

<160> 7

<170> SIPOSequenceListing 1.0

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<211> 1656

<212> DNA

<213> tobacco (Nicotiana tabacum L.)

<400> 1

cttgaccatg aatttctggt ccatgatatt aaatggtgca gtagctagct tttgattttg 60

gaaaaactaa aaatattcta tatatataat ttggactgta atagaaacag tgtcattcta 120

cacatccact aacataaatt tattgaatct ttaattaccc tcgttatgaa gatactttca 180

ataaaaagta gaattaatag tgtcttagct attaatcaaa gaattgcaga ttttaggtac 240

agcggatttt agcctaacat attgaaagct tgtattgaag aaggagatcc tttgcgcaca 300

ggtaaaaact gtcgtgatgt gaccgggagg ttacgaataa ctgcgagaac aacctcttgc 360

ataaacccaa gataaaaata cttacataag actcaatatg gtcctgccat tctttgaccc 420

cgggcatatt tgaagtttag tgtgcttttc ttttttttta atttaaagcg tgcgacacat 480

cactataatt tttgttgaat aaaacactag aaaactcact aaagtccaag ttgtaattga 540

gggaataatt gataaacctt atgcaagagg aatcaaagca tcacttacct ataaaagtat 600

aagtaggtgt acaaaggaaa ccgataaatc gcaccaaccc gataatccaa gtcaaaccga 660

gaaaaaaacc cgactatggg ttggtttgag ttggtttggt attggaaaaa aagaacccga 720

tcataattgg tttggtttgg ttttaactaa agaaagtcaa actaaaacca aactaacccg 780

acattacata tatagaaatt ttagatatat ttaatatata aatatactta ttgtgaggta 840

atttataaat atctcttaaa agatttcata attttatttt ttgaggtatt atttcaaggt 900

tggacttgga acttttgaat gttctaataa gttttatagt cattaatatt agtaaattaa 960

atagtgctaa caaaagtcca aaccaaaatc agatcaatat taatgctaac aaaagacatt 1020

taattcaata ctacgaatag gaatgtattg aatatctatt ttttgttttg caataattta 1080

gataaaaatg cataatttat ttttattttt tctttagcgt ttaattatgt atttaatact 1140

cccttattag tctacttatt tcagcatgac ttagtactct tagattatgt ttatttttat 1200

tatggctttt taattagcaa tatttatgtt acataatttt attgtcttta ttgttgaata 1260

ttttaggata atgccatgac atatctcata ttttgtatta tattcttgga aaatacttta 1320

tatagttgta tcttactagg actaaagaaa tattttgagc ataatttatg tattttgttc 1380

tacgaagatt ttaccgagaa aaaaaccccg aataacccga aaacccaaga aaccccgaga 1440

aaaaccgaaa ccggaaaacc cgagttttat tggtttggtc tttagattta ataatctgac 1500

acaattagtt tggtttggta attgtaaaat ccgaaccaac ccgacctatg tacaccccta 1560

agtacaaaag tacaagtata taagaatggc cttgttcctt caccaccaac accattgaca 1620

taaaaacttt cttcagtatt ctaaaagctc atagca 1656

<210> 2

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 2

accatgatta cgccacttga ccatgaattt ctggtccat 39

<210> 3

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 3

cagtgaattc ccgggtgcta tgagctttta gaatactgaa 40

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 4

tgtggtttgt ggggcaataa t 21

<210> 5

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 5

aacagtccaa ggcatctagc atagt 25

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 6

cggaatccac gagactacat ac 22

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence

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gggaagccaa gatagagc 18

Claims (5)

1. The tobacco gland Mao Qidong seed pNtGGPPS2a is characterized in that the nucleotide sequence of the promoter is shown in SEQ ID NO. 1.

2. The primer pair for amplifying the promoter of claim 1, wherein the sequences of the primer pair are shown in SEQ ID NO.2 and SEQ ID NO. 3.

3. A method of driving the specific expression of a GUS gene in tobacco glandular hairs by using the promoter of claim 1, wherein the method comprises the steps of:

extracting tobacco genome DNA;

cloning a promoter pNtGGPPS2a from the genome DNA;

the promoter pntggps 2a was subjected to cis-acting element analysis;

constructing a pCAMBIA1391Z-pNtGGPPS2 a:GUS plant expression vector;

culturing aseptic tobacco seedlings, and genetically transforming GUS vectors into the aseptic tobacco seedlings to obtain transgenic tobacco;

GUS staining is carried out on the transgenic tobacco;

qRT-PCR detects the relative expression of the NtGGPPS2a gene in tobacco glandular wool.

4. A method according to claim 3, wherein the genetic transformation process is agrobacterium-mediated transformation of the recombinant plasmid into tobacco.

5. Use of the promoter according to claim 1 in tobacco genetic engineering breeding.