CN102851290A - ODC gene promoter for nicotine biosynthesis and application thereof - Google Patents

Abstract

The invention relates to an ODC gene promoter for nicotine biosynthesis and an application thereof. The ODC gene promoter for nicotine biosynthesis is extracted from an N. sylvestris genome sequence, and has a nucleotide sequence as shown in SEQ ID NO.1. The promoter region of the nicotine ODC gene can generate transcription with a basic level, and the regulation region can response to various ambient conditions and thus regulate the expression level of the nicotine synthesis genes correspondingly (up-regulation of down-regulation as required), which has important practical significance on the production of nicotine extraction raw materials with high quality.

Description

Nicotine Biosynthesis ODC Gene Promoter and Its Application

technical field

The present invention relates to the technical field of biological genetic engineering, in particular to a nicotine biosynthesis ODC gene promoter and its application.

Background technique

Nicotin, also known as nicotine, has a chemical name of 1-methyl-2-(2-pyridyl)pyrrolidine and a molecular formula of C ₁₀ H ₄ N ₂ . It is an alkaloid naturally present in tobacco. unique to tobacco. Nicotine can produce a certain physiological stimulating effect on people, and is the main factor that makes tobacco have commercial value. As the biological activity of nicotine has been paid more and more attention by people, its application has become more and more extensive. Their development has gradually penetrated into many fields such as food, health care, medicine and daily chemical industry, and the application prospect is very broad. . For example, the production of tobacco substitutes requires a large amount of nicotine: the current treatment methods for tobacco dependence include drug treatment, psychotherapy, acupuncture and traditional Chinese medicine treatment, etc. The treatment programs and drugs approved by the US FDA mainly include: nicotine replacement therapy such as nicotine Patches, mouth gels, nasal sprays, and inhalants, etc., with nAChR inhibitor medications such as bupropion and injectables such as varenicline tartrate, as well as mecala, nortriptyline, clonidine, and anxiolytics The production of these nicotine substitutes requires the extraction of a large amount of nicotine as a raw material; in addition, nicotine is also widely used in the development of environmentally friendly pesticides and the production of special disease drugs: nicotine series pesticides are plant insecticides, because of their Steam fumigation, stomach poisoning, contact killing function and rapid degradation without residue, etc. It is widely used as an insecticide for crops such as grain, oil, vegetables, fruits, pastures, etc. It is an ideal high-efficiency insecticide and biological pesticide for the production of green food. It is also widely used in the pharmaceutical industry and is a special raw material for the development of drugs for the treatment of cardiovascular, skin diseases, snake venom and other diseases. The nicotine citrate produced by it can also be used in the variety improver components of high-grade cigarettes, etc., and with the rapid development of the tobacco industry, fine chemicals, pharmaceuticals, organic synthesis, national defense, and pesticides. The market demand for nicotine is increasing day by day, especially for high-purity nicotine.

The nicotine molecule contains a pyrrolidine ring and a pyridine ring, and the pyridine ring is synthesized by the NAD pathway, and the pyrrolidine ring is formed from basic amino acids through the intermediate product putrescine. Proteins involved in nicotine biosynthesis include ornithine decarboxylase (ornithine decarboxylase, ODC), quinolinate synthase (quinolinate synthase, QS), aspartate oxidase (aspartate oxidase, AO), quinolinate phosphoribose quinolinate phospho-ribosyltransferase (QPT), putrescine N-methytransferase (PMT) (Katoh et al., 2005; Cane et al., 2005), diamine oxidase (diamine oxidase, DAO), the genes encoding these proteins can be found in N. sylvestris . Among them, putrescine N-methyltransferase is encoded by three genes (PMT1, PMT2, PMT3) (Shoji et al., 2000). If the synthesis of nicotine is to be regulated by the genes encoding these enzymes, the corresponding promoters should be found first.

Contents of the invention

The technical problem to be solved by the present invention is to provide an ODC gene promoter and its chimeric gene that can regulate nicotine biosynthesis.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:

A nicotine biosynthesis ODC gene promoter, its nucleotide sequence is:

(1) the nucleotide sequence shown in SEQ ID NO.1; or

(2) The nucleotide sequence shown in SEQ ID NO.1 is modified by conventional technical means such as repetition, deletion, substitution or translocation, and has the same function as the nucleotide sequence.

The pair of primers for amplifying the above-mentioned ODC gene promoter has the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3.

A chimeric gene, which comprises the above-mentioned nicotine biosynthesis ODC gene promoter and a sequence encoding a target gene operably linked thereto.

A plant transformation vector, which contains the above-mentioned chimeric gene.

A transgenic plant cell comprising the above-mentioned chimeric gene.

A transgenic plant tissue comprising the above-mentioned chimeric gene.

The application of the above nicotine biosynthesis ODC gene promoter or the above chimeric gene in regulating nicotine biosynthesis.

The present invention has positive and beneficial effects:

1. Found and extracted the nicotine biosynthesis ODC gene promoter;

2. The promoter region of the nicotine ODC gene produces a basic level of transcription, and the regulatory region can respond to different environmental conditions and make corresponding adjustments to the expression level of nicotine synthesis genes (up-regulation or down-regulation as needed), and then to Tobacco planting produces tobacco leaves with moderate nicotine content and high-quality nicotine extraction raw materials have important practical significance.

Description of drawings

Figure 1 is the electrophoresis diagram of the PCR amplified nicotine gene promoter, wherein the left image uses N.tabacum genomic DNA as a template, and the right image uses N.sylvestris genomic DNA as a template;

Figure 2 is a frame diagram of the conserved transcriptional binding sites contained in ODC, QPT, AO, QS and other gene promoters; a frame diagram constructed by analyzing a single transcriptional binding site using Genomatix's FrameWorker software; the transcriptional binding site The organization (including the specificity and relative order of the DNA strands) is conserved; the distance between the transcription binding sites in different gene promoters varies very little; different colors represent the binding sites of different transcription factors; the line above the single line represents transcription The binding site is located on the sense strand, and the coincidence below the single line represents that the transcription binding site is located on the antisense strand;

Figure 3 is a schematic diagram of the transcription factors found in the promoters of ODC, QPT, AO, QS and other genes; the diagram was analyzed by the Fast software of Genomatix; distance between transcription factor binding sites.

Detailed ways

The present invention is further described below in conjunction with specific examples. The test methods or analytical methods involved in the following examples are conventional methods unless otherwise specified, and all reagents used are commercially available unless otherwise specified.

Example 1 Isolation and identification of nicotine synthesis gene

The promoter sequences of nicotine synthesis genes ODC, DAO, AO, QS, QPT, PMT1, PMT2 and PMT3 were all extracted from the N. sylvestris genome sequence, and the positions of these genes in the genome are shown in Table 1 below. The sequence of the ODC promoter of the nicotine synthesis gene is shown in SEQ ID NO.1.

Table 1 The position of each promoter in N. sylvestris genome

According to the promoter sequences of the nicotine synthesis genes ODC, QS, AO, QPT, DAO, PMT1, PMT2 and PMT3 extracted from the N. sylvestris genome, design primers (as shown in Table 2) to amplify the promoter from the N. tabacum genome The subsequence was cloned into the modified pET-43.1a vector and sequenced, wherein the sequence of >NT-ODC was shown in SEQ ID NO.4. The above sequence is very conservative compared with the promoter sequence amplified from N. sylvestris tobacco, and the regulatory elements on the promoter are consistent, thus proving that the regulatory mechanism between them is the same.

Table 2 Primers used for PCR amplification of ODC, AO, QS, QPT, PMT1, PMT2 and PMT3 promoters in N. tabacum K326 and N. sylvestris genomes

ODC-F TACTTCCAATCCATGagtgaaattacaagtacaag ODC-R TATCCACCTTTACTGtcaAAGGAAGAAAAGAGAGAGGTAA QPT-F TACTTCCAATCCATGactcctagttgttgttata QPT-R TATCCACCTTTACTGtcaTTATAAGTGAGAGTTAA QS-F1 TACTTCCAATCCATGttctacaaaaggtccatttc QS-R1 TATCCACCTTTACTGtcaTTTTTCGGGTGAGGACGAAA AO-F TACTTCCAATCCATGaagttactgtgttctagatt AO-R TATCCACCTTTACTGtcaATTACCTTAAAGTAGCAA PMT1-F TACTTCCAATCCATGattttttattaaatactatc PMT1-R TATCCACCTTTACTGtcaGATATGACTTCCATTTTC PMT2-F TACTTCCAATCCATGctcatacggattttagacag PMT2-R TATCCACCTTTACTGtcaGTAGAGCCATTTGTGTT PMT3-F TACTTCCAATCCATGaaatactatctggtgacaag PMT3-R TATCCACCTTTACTGtcaAATGGCACCATTCTTGAA

PCR reaction system: PCR reaction solution contains 10 pmol primers, 50 ng genomic DNA, 0.2 mM dNTP, 10% DMSO and Hot Start DNA polymerase KOD (Novagen), PCR reaction conditions are 95oC, pre-denaturation 5 min, 95oC, denaturation 1 min, annealing at 50oC for 1 min, extension at 68oC for 1 min, 35 cycles, and finally extension at 68oC for 10 min. Using QIAquick PCR purification kit (Qiagen), the kit was used to purify the PCR product, and the purified PCR product was cloned into the modified pET-43.1a (pLEICS-01) using the In-Fusion HD EcoDry Cloning Kit (Clontech) kit, and then Sequencing analysis.

Example 2 Function of nicotine synthesis gene

Eight gene regulation mechanisms involved in nicotine synthesis were analyzed: the nicotine molecule contains a pyrrolidine ring and a pyridine ring, while the pyrrolidine ring is synthesized by the NAD pathway, and the pyrrolidine ring is formed from basic amino acids through the intermediate product putrescine. Pyridine rings are synthesized by catalysts such as AO, QS, and QPT. Among them, aspartate oxidase (AO) catalyzes the formation of pyridine-2,3-dicarboxylic acid from glycerol and aspartic acid. Quinolinate synthase (QS) catalyzes the formation of quinolinic acid from pyridine-2,3-dicarboxylic acid. Quinolinate phosphoribosyltransferase (quinolinate phospho-ribosyltransferase, QPT) catalyzes the production of niacin from quinolinic acid through the pyridine nucleotide cycle. The main route of biosynthesis of the N-methylpyrrolidine ring in nicotine is sequentially catalyzed by ornithine decarboxylase (ODC) to form putrescine, followed by putrescine N-methyltransferase (putrescine N-methytransferase (PMT) catalyzes the generation of N-methyl putrescine, followed by diamine oxidase (diamine oxidase, DAO) catalyzes the generation of N-methyl-pyrrolidinium salt. The final synthesis of nicotine is completed by the decarboxylation of niacin and the combination of pyrrole ring. Among them, QPT is the main regulatory enzyme of niacin required by the pyridine part in the process of nicotine biosynthesis, and is an important control point of nicotine biosynthesis. PMT is the most restrictive enzyme in the pyrrolidine ring synthesis pathway. PMT and QPT are the maximally rate-limiting enzymes in the biosynthesis of 2 intermediates of nicotine, and other enzymes in this pathway have regulatory roles in some cases. The expression levels of these enzymes are regulated by their promoter regulatory elements by binding different transcription factors.

Two transcriptional binding sites (TFBS) G-box and GGC-motif in N. tabacum mediate the response of PMT1a to methyl jasmonate. The G-box and GGC-motif are located 95bp and 55bp upstream of the tss site, respectively (Xu and Timko, 2004). A FastM model (Klingenhoff et al., 1999) was defined using the information of these two loci and their distance and relative orientation to represent this mode of regulation (Fig. 3).

The inventors extracted the promoter sequence of the nicotine synthesis gene, and analyzed and compared the occurrence types of TFBS using the FastM model. The promoter sequences of PMT1, PMT2, and PMT3 fit this model. The distance and relative orientation of the two elements is consistent with conservation in N. tabacum . The GCC-motif in N. sylvestris is 54-60 bp away from the hypothetical transcription start site, which can also be compared with that in N. tabacum . The promoter sequences of other nicotine gene synthesis genes do not contain the TFBS motif.

The jasmonic acid response is mediated by the MYC2 transcription factor (Chini et al., 2007). Therefore, FrameWorker was used to analyze the MYC2 binding sites contained in the TFBS of the eight promoter sequences (D?hr et al. 2005, Cohen et al., 2006). For the promoter sequences of AO, QS and QPT, six different TFBSs could be identified, suggesting that these genes contain a common regulatory pattern (Fig. 2). The similarity of each pair of promoters is <50%, except that the organization of TFBS (including distance, relative orientation) is highly conserved. Therefore, we detected that the framework was not due to the high conservation of nucleotide sequence, but was conserved at the transcription factor level.

The conserved motifs contain binding sites for the transcript families MYCL, DOFF, GTBX, L1BX, OCSE, and GBOX. In addition to MYCL and GBOX, transcription factors of the OCSE family are also involved in defense responses and plant hormone signaling (salicylic acid) transduction (Chen et al., 1996). The interaction between transcription factors has also been found in other genes before, such as GBOX-DOFF (Norre et al., 2002), OCSE-DOFF (Chen et al., 1996), GTBX-MYCL (Simpson et al. , 2003).

Example 3 Expression of genes related to nicotine synthesis

Nicotine is synthesized in the roots of tobacco and then transported to the leaves and other aerial parts (Cane et al., Shoji et al.). The gene expression results showed that all nicotine synthesis genes were present in the roots of N. sylvestris , but not or lowly expressed in stems, leaves or flowers (Table 3).

Table 3 Expression of nicotine synthesis genes

Gene root leaf flowers stem QPT 1.18142 0.34834 0.28289 0.24329 QS 1.08342 0.21988 0.11682 0.11265 AO 0.00200 0.00000 0.00000 0.00000 ODC 0.09851 0.01137 0.03978 0.02068 PMT1 1.12893 0.00007 0.00366 0.00064 PMT2 0.00102 0.00000 0.00000 0.00000 PMT3 0.06579 0.00000 0.00092 0.00000 DAOs 0.28119 0.00000 0.01128 0.00092

Although, the present invention has been described in detail with general descriptions and specific embodiments above, but on the basis of the present invention, some modifications or improvements can be made to it (such as modifications to the gene sequence of the present invention, etc.), which are important to this invention. obvious to those skilled in the art. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

sequence listing

SEQUENCE LISTING

<110> Henan Agricultural University

<120> Nicotine biosynthesis ODC gene promoter and its application

<130> /

<160> 4

<170> PatentIn version 3.2

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<212> DNA

<213> N. sylvestris

<400> 1

agtgaaatta caagtacaag attacaactt taattatgta atttacaaat ataatacata 60

tcctatttaa actaaactaa gcaatttaat tttaaaagtc acaagttaaa ataaaaacac 120

acattaatta atatgattaa tgatagacat ttatttttaa aaatactccg gtgcaaacac 180

aatatacgag taattatatg tggattgact tccactgatt ttttcttgat cctctaatat 240

gtcgtagaaa aaatcatgca ccgttggata atatagattt aattatcatt taccaacaa 300

aaagtacatc acttcatcag atttacataa atgacttatt gtaattaaat tcaataccgt 360

tgaccaccta ccccttcaac agctatttct ctaaaaaaaa aaaaaaagaa gaaaatacta 420

cgtagattac acaatattat cagtagtagt atcacttttc gtccctctat ataatgataa 480

acattttttg aggtttcccc gtctcaaagg gaacaagaga aacattcata ttattgaatc 540

cctagtttct tttctttccc tttgattcct tcctctcatt tacctctctc ttttcttcct 600

t 601

<210> 2

<211> 35

<212> DNA

<213> Synthetic

<400> 2

tacttccaat ccatgagtga aattacaagt acaag 35

<210> 3

<211> 40

<212> DNA

<213> Synthetic

<400> 3

tatccacctt tactgtcaaa ggaagaaaag agagaggtaa 40

<210> 4

<211> 597

<212> DNA

<213> N. tabacum

<400> 4

agtgaaatta caagtacaag attacaactt taattatgta atttacaaat ataatacata 60

tcctatttaa actaaactaa gcaatttaat tttaaaagtc acaagttaaa ataaaaacac 120

acattaatta atatgattaa tgatagacat ttatttttaa aaatactccg gtgcaaacac 180

aatatacgag taattatatg tggattgact tccactgatt ttttcttgat cctctaatat 240

gtcgtagaaa aaatcatgca ccgttggata atatagattt aattatcatt taccaacaa 300

aaagtacatc acttcatcag atttacataa atgacttatt gtaattaaat tcaataccat 360

tgaccaccta ccccttcaac agctatttct ctaaaaaaaa aaagaagaaa atactacgta 420

gattacacaa tattatcagt agtagtatca cttttcgtcc ctctatataa tgataaacat 480

tttttgaggt ttccccgtct caaagggaac aagagaaaca ttcatattat tgaatcccta 540

gtttcttttc tttccctttg attccttcct ctcatttacc tctctctttt cttcctt 597

the

Claims (7)

1. nicotine biosynthesizing ODC gene promoter, its nucleotides sequence is classified as:

(1) nucleotide sequence shown in the SEQ ID NO.1; Or

(2) nucleotide sequence shown in the SEQ ID NO.1 is modified the nucleotide sequence with same function that forms through repeating, lack, replace or being shifted to transform.

2. the primer pair of the described ODC gene promoter of amplification claim 1 is characterized in that having the nucleotide sequence shown in SEQ ID NO.2 and the SEQ ID NO.3.

3. a mosaic gene wherein comprises the described nicotine biosynthesizing of claim 1 ODC gene promoter and sequence that be operatively connected with it, the coding goal gene.

4. a plant conversion carrier wherein comprises mosaic gene claimed in claim 3.

5. a transgenic plant cells wherein comprises mosaic gene claimed in claim 3.

6. a transgenic plant tissue wherein comprises mosaic gene claimed in claim 3.

7. the described nicotine biosynthesizing of claim 1 ODC gene promoter or the described mosaic gene of claim 3 application in the biosynthesizing of regulation and control nicotine.

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