CN103255120A

CN103255120A - Monoterpene/sesquiterpene synthetase CsLIS/NES gene for affecting tea aroma quality and application of gene in aroma induction of plants

Info

Publication number: CN103255120A
Application number: CN201310070031XA
Authority: CN
Inventors: 魏书; 贺志荣; 刘国峰; 阎轶峰
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2013-03-06
Filing date: 2013-03-06
Publication date: 2013-08-21
Anticipated expiration: 2033-03-06
Also published as: CN103255120B

Abstract

The invention discloses monoterpene/sesquiterpene synthetase CsLIS/NES for affecting tea aroma quality. The sequence of the monoterpene/sesquiterpene synthetase CsLIS/NES is shown as the sequence table. The invention also discloses an application of monoterpene/sesquiterpene synthetase CsLIS/NES in aroma induction of tobacco. The beneficial effects of the invention lie in the fact that CsLIS/NES is the key enzyme in the synthesis and accumulation of linalool of fresh tea leaves, has a significant impact on odor type and aroma quality of tea, has a good expression effect in tobacco and possesses the function of aroma induction.

Description

Influence the monoterpene/sesquiterpene synthetic enzyme CsLIS/NES gene of tea aroma quality and in plant, urge fragrant purposes

Technical field

The present invention relates to a kind of purposes that influences the monoterpene/sesquiterpene synthetic enzyme CsLIS/NES gene of tea aroma quality and in plant, urge perfume.

Background technology

Fragrance is one of tealeaves important quality.Aroma substance kind in the tealeaves is a lot.Wherein the volatility terpenoid is the important aroma component of all kinds of tea because having special flowers and fruits fragrance, and is particularly important to the formation of black tea, the peculiar flavouring essence quality of oolong tea.Phantol fragrance is pleasant, and two kinds of optically active enantiomorphs (R)-and (S)-Linalool has lavender perfume (or spice) (Lavender) and lemon respectively, is with sweet in the perfume (or spice).Secondly, phantol to experience threshold value low, fragrance is active high.In addition, phantol can also further transform and generate multiple aroma substance, and as linalool oxide (Hotrienol, 3,7-dimethyl-1,5,7-sarohornene-3-alcohol) and aldehyde alcohol derivative thereof, these materials are considered to oolong tea characteristic perfume material.Again secondly,, the multiple phantol epoxide of furan type and pyranose form isomer has the multiple fragrance of a flower, fruital and the banksia rose, and the flavouring essence quality of tea is also had remarkably influenced.In the bright leaf of tea in spring, the content of phantol and four kinds of oxide compounds (I, II, III and IV) thereof accounts for 17% of the bright leaf essential oil total amount of tea, is the important component of tea aroma.The nerolidol of sesquiterpene has sweet clear soft and graceful flores aurantii breath, has the fragrance of picture rose, the lily of the valley and apple flower, and fragrance is lasting, and the tea aroma quality is also had material impact.In addition, terpene volatile matter such as phantol also are the bio signal molecules between plant and between plant and other biological, can attract insect pollination, repellent herbivore insect etc.Yet these terpene alcohols materials often are stored in the bright leaf of tea with the glucosides attitude, the bright leaf of tea is bitten insect and is stung, be subjected to physical abuse etc. in harvesting and the course of processing, can cause the cell locellus to destroy, make the terpene fragrance aglycon of terpene glycoside compounds and the endogenous glycoside hydrolase in the bright leaf of tea meet and hydrolysis release aroma substance.In process for making tea, oolong tea and black tea do blue or green and fermentation procedure can make a large amount of hydrolysis release of glucosides aglycon.

In the plant, the synthetic precursor of monoterpene and sesquiterpene is respectively from geranyl tetra-sodium (GPP) and the cytoplasmic farnesene base tetra-sodium (FPP) of plastid.Methyl erythritol phosphoric acid (MEP) approach and mevalonic acid (MVA) approach are two relatively independent pathways metabolisms in the terpene metabolism.Generally believe, in plastid, by the synthetic GPP of MEP approach; In tenuigenin, then generate FPP by the MVA approach.But some reports show, have a spot of FPP in plastid, and a spot of GPP is also arranged in tenuigenin.Some terpene synthetic enzyme has the dual-use function of monoterpene/sesquiterpene synthetic enzyme, as the FaNES1 of strawberry, can be that substrate produces phantol with GPP in fruit, can be again that substrate produces nerolidol with FPP.Yet most of difunctional phantols/nerolidol synthetic enzyme is a kind of function of main enforcement in plant, AmNES/LIS1 and AmNES/LIS2 difference catalysis in spending as Common Snapdragon generate nerolidol and phantol, and this mainly is subject to the Subcellular Localization of these terpene synthetic enzyme.These enzymes discharge the fragrance of flower in plant and the generation of volatile oil, and local flavor, the quality of fruit is being formed with important effect.In the blade of plant, the expression of volatility terpene synthase gene obviously is subjected to as the inducing of environment stresses such as arid, physical abuse, and then produces volatility terpene semiochemicals, improves plant opposing adverse circumstance ability.Yet these terpene synthetic enzyme seldom are mentioned in the function that improves blade essential oil output and improve aspect the taste of fruit.

In fact, in the economic product and essence and flavoring agent Chemicals of all kinds of plants, be the aroma substance of representative with volatility terpene substances such as phantol and glucosides thereof, flavouring essence quality to product has great effect, extensive and great industrial value is arranged, the phantol that for example has peculiar fragrance occupies main status in the perfume formulation of many odor types, can be used for allocating various essence such as lily, cloves, flores aurantii.China is that phantol consumes big country, and annual requirement surpasses the phantol of 400t, but mainly by import.Early stage phantol mainly extract from essential oil, but be unable to catch up with the needs that develop far away, therefore the method for synthetic has caused people's attention, yet because the phantol of synthetic and change oxidation products and can not form abundant optically active isomer as biosynthesizing, thereby its fragrance and biological value are far away not as good as natural phantol.

Summary of the invention

Key problem in technology of the present invention is to find and prove the difunctional synthase gene CsLIS/NES of the monoterpene/sesquiterpene that influences the tea aroma quality and enzyme product thereof first.

1, monoterpene/sesquiterpene synthetic enzyme CsLIS/NES gene order is:

>CsLIS/NES?mRNA,2050bp

CCAAGAACAAACGTACTTTCCATAAAACACAAAACAAAAAAACACTTCCTAAACATTGTCCTCATCATGCAAATCTTCCACTGTGC

ATCTCCTCCTTCTCATCTTCCCATTGCTCCAAACAACATCCCACAAATCAACAAGACTAGTCTCATTGCATCATCTAGTACACTTA

AAACCCACAAATGGAGCATTGGTGATGATCTCACTTTAGTTTCCAACCCCTCAATACAGAAAGACTATCTAACTGGATATCGCAGC

TTAACCGATGACTTTTGTGTAAAGCATGAACAAAAATTGAAGGAAGCTAAGAGGATGTTGAGGAAAGTAGGAGAAAACCCATTAGA

AGGTTTGGTCATGATCGATAACCTCCAACGCCTAGGCATCGATTACCATTTCCAAGAAGAGATTGAAGCACTTCTACAAAGCCAAT

ATACGAAATCCAACGCTACTACGCTTGGTTATGACCTTTATGAAGTTTCTACTCGCTTTCGTCTACTAAGGCAAGAAGGGTACAAT

GTGCCCGCGGATGTGTTTAACAACTTTAAGGCTAAGAACGGAAAGTTCAAACCAGAACTAAATGCAGATATGAGAGGGTTGATGAG

TTTGTATGAAGCTTCACAGCTAAGCATAGAAGGAGAAGACATTCTTGATCAAGCCGCAGACTTTAGTACCCAAGTCCTTAACGGTT

TGATGCCACATCTTTCTCATCATCAAGCTCGAGTTGTCAGCAACACACTGGGAAATCCCCATCACAAGAGCCTAGCAAGGTTCATG

GCAAGAGATTTCCTTAGTGATTACACGAACCCAAGTGAATGGGAAAATGTCTTGCAAGAACTGGCGAAAATGGATTTTAACATGGT

TCAATTCACACACCAAAAAGAAATACTTCAAGTATCAAAGTGGTGGAAAGACACGGGTTTGGCGAGTGAACTGAAGTTCGCTAGAG

ACCAACCTCTAAAATGGTACATGTGGCCTATGGCAGCCCTCACAGATCCAAGGTTCTCAGAGCAAAGAGTTGAGCTCACAAAACCC

ATCTCTTTCATCTACTTAATAGATGATATTTTTGATGTTTATGGGACACTTGAAGAACTCACTCTCTTCACAGAAGTTGTCAATAG

ATGGGAACTTGGTGCTGTGGAGCAACTACCAGAGTACATGAAGATTTGTTTCAAGGCTCTTGATGACATTACAAACCATATTGCTT

ACAAGGTCTACAGAGAACATGGATGGAACTCCATAGATTCTCTTAGAAGAACGTGGGCAAGTTTATGCAATGCTTTCTTAGTAGAA

GCAAAGTGGTTTGACTCAGGGCACTTGCCAAAACCAGAAGAGTACTTGAAGAATGGTGTGATTAGTTCAGGGGTACATGTGGTATT

AGCACACATGTTCTTTCTATTAGGTCACAACATAACCAAGCAAAATGTCAATCTTGTGAATGACAATCCAGGCATTGTGACTTCTA

CAGCTACAATTCTTCGTCTTTTTGATGATTTGGGAAGTGCCAAGGATGAGAATCAAGATGGCAAAGATGGGTCCTATGTACAGTGC

TACATGAAGGAGAACAATTGCTCCTCAGTTGACACTGCAAGAAAGCAAGTCATTCACATGATATCACAAGCATGGAAGAGCCTCAA

CAAGGAATGCCTATCTCCAAATCCATTCTCACCAGTTTTCACAAAGGGTTCTCTTAATATTGCAAGGATGGTTCCTTTGATGTATA

GTTATGATGACAAACAAAACCTCCCAGTCCTTGAGGAGTACATGAAGTCTATGTTCTATGATAAATTACCTTAGGTGAGTGAGCAT

TGTTAGATGCTCTGCAACTTCTTCACTGTTGATGCTACAAAGCTAGCACCTGTAATGCTCATAAACTAGCTGTTTGGTGAGGAATA

AGTGTATAATCCAACAAGATGCAAGAAAAATGTAGTAGTTATGTTACAAGGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCT

CCCTTTTAACCCCCCTTATTTTAAACCCTGCCTATGGGTTTCGTATTTTGGAGACCTCACAAAAAAAGACTA

2, monoterpene/sesquiterpene synthetic enzyme CsLIS/NES protein sequence is:

>CsLIS/NES,575amino?acids

MQIFHCASPPSHLPIAPNNIPQINKTSLIASSSTLKTHKWSIGDDLTLVSNPSIQKDYLTGYRSLTDDFCVKHEQKLKEAKRMLRK

VGENPLEGLVMIDNLQRLGIDYHFQEEIEALLQSQYTKSNATTLGYDLYEVSTRFRLLRQEGYNVPADVFNNFKAKNGKFKPELNA

DMRGLMSLYEASQLSIEGEDILDQAADFSTQVLNGLMPHLSHHQARVVSNTLGNPHHKSLARFMARDFLSDYTNPSEWENVLQELA

KMDFNMVQFTHQKEILQVSKWWKDTGLASELKFARDQPLKWYMWPMAALTDPRFSEQRVELTKPISFIYLIDDIFDVYGTLEELTL

FTEVVNRWELGAVEQLPEYMKICFKALDDITNHIAYKVYREHGWNSIDSLRRTWASLCNAFLVEAKWFDSGHLPKPEEYLKNGVIS

SGVHVVLAHMFFLLGHNITKQNVNLVNDNPGIVTSTATILRLFDDLGSAKDENQDGKDGSYVQCYMKENNCSSVDTARKQVIHMIS

QAWKSLNKECLSPNPFSPVFTKGSLNIARMVPLMYSYDDKQNLPVLEEYMKSMFYDKLP*

The bioinformatic analysis of 3, CsLIS/NES:

By the RACE technology, we transcribe the full length sequence that the group sequence successfully clones monoterpene/sesquiterpene synthetic enzyme CsLIS/NES gene by known 303bp.This full length gene 2050bp base comprises 5 ' end 66bp UTR and 3 ' end 256bp UTR and 1728bp ORF frame, and this genes encoding contains 576 amino acid whose protein.The size of predictive coding protein is 66KDa, iso-electric point 5.93.

BlastX analyzes (with NCBI＜http://www.ncbi.nlm.nih.gov〉database serve as the analysis comparison other) and shows that this enzyme has the catalysis phantol probably and nerolidol is biosynthetic difunctional, so called after CsLIS/NES.As shown in Figure 2, analyze by evolutionary tree, CsLIS/NES belongs to TPS-g group family, the phantol synthetic enzyme of its protein sequence and this family's Kiwifruit and the consistence of nerolidol synthetic enzyme be respectively 71% and 70%(as shown in Figure 1).The principal character of TPS-g family is to lack ' RRX8W ' structure at the N end, and this is their major cause from it is generally acknowledged that monoterpene TPS-b that synthetic enzyme is in and TPS-d separate.CsLIS/NES is the same with most of terpene synthetic enzyme, has and metal ions M g ²⁺Binding site ' DDXXD ', and this enzyme also has the site ' EDXXD ' relevant with the activity of diterpene (Fig. 2).

4, CsLIS/NES is positioned at chloroplast(id)

Use TargetP1.1 at sequence of threads＜http://www.cbs.dtu.dk/services/TargetP/〉predict the Subcellular Localization of this enzyme, find that this enzyme may be positioned chloroplast(id) (p=0.839).Use this gene and reporter gene green fluorescent Protein G FP integration technology, make up plant expression vector, transform Nicotiana gossei (Nicotiana Benthamiana), and observe with laser confocal microscope, confirm that this gene product is positioned at (Fig. 3) in the chloroplast(id).

The prokaryotic expression of 5, CsLIS/NES and enzyme functional verification thereof:

CsLIS/NES contains 1728bp ORF frame sequence, after NcoI and Sal I enzyme are cut, be connected to prokaryotic expression carrier pET32a after identical restriction enzyme site enzyme is cut go up (Fig. 4, A).The carrier pET32a_CsLIS/NES that builds is transformed into BL21(DE3) intestinal bacteria in, induce down at 0.25uM isopropyl-β-D-thiogalactopyranoside(IPTG), obtain inclusion body protein (Fig. 4, B5).Under ultrasonic wave and N,O-Diacetylmuramidase acting in conjunction, extract inclusion body protein, use STET buffer(10mM Tris again, pH8.0,150mM NaCl, 1mM EDTA, 1%Triton-X100) wash-out inclusion body, until detect with SDS-PAGE almost only contain target protein (Fig. 4, B7).Then, with containing the PBS(pH7.0 of 0.3%sarkosyl (N-lauroylsarcosine)) damping fluid dissolve 4h(on ice in the middle of jog for several times), ultrafiltration is centrifugal desalts, the centrifugal 20min of 4000rpm.Measure the concentration of albumen, and place 4 ° of C to preserve.

Method configuration enzyme reaction buffer (50mM Bis-Trispropane, pH7.5,10mM MgCl2,5mM DTT) alive with reference to Green adds 100 μ g albumen and 20 μ M substrates (GPP or FPP) then, 30 ° of C reaction 1h.The method collecting reaction product of bleeding by head space is again by the GC-MS analytical results.Interpretation of result shows: in the reaction with the GPP substrate, can detect a large amount of phantols and a spot of firpene and ocimene produce (Fig. 5, A).Yet in the reaction that contains substrate FPP, can only detect a large amount of nerolidols and produce, not have other sesquiterpene to produce.By isomers experimental results show that generation for (E)-nerolidol (Fig. 5 C, D).Catalytic constant Kcat (catalytic constant, s are used in experiment ^-1) study enzyme to substrate maximum activity, the result shows that the Kcat constant value of the GPP of CsLIS/NES and FPP reaction is respectively 0.3103 ± 0.0173 and 0.0691 ± 0.002(SE, n=3).

Expression and the catalysis checking thereof of 6, CsLIS/NES gene in tobacco:

As shown in Figure 6, the cDNA full length sequence of CsLIS/NES is connected on the plant expression vector pCamb2300 by restriction enzyme site Xba I and Sac I, the pCamb2300_CsLIS/NES1 carrier that builds is passed through Agrobacterium GV3101 transformation of tobacco respectively with contrast empty carrier pCamb2300, under the screening of kantlex, obtain regeneration plant, and verify through PCR, obtain transgene tobacco and the contrast tobacco (Fig. 6, B).Use real-time fluorescence quantitative PCR to detect the expression of CsLIS/NES at transgene tobacco in addition, find that transgene tobacco has very high expression level, have 3 strains to reach level more than 1000 times, the highest T8 is up to more than 2500 times, also have several strains can reach hundreds of times (Fig. 6, C).And tobacco T2 and T9 that PCR does not identify out, the expression level is almost consistent with contrast.Method by solid-phase microextraction SPME (Solid phase microextraction)-GC-MS is collected transgenic plant head space volatile matter, and is contrast with the standard substance of purifying, the release conditions of qualitative detection volatility terpene substances.The result shows that the blade of transfer-gen plant can discharge a large amount of phantol (linalool), and (Fig. 6, D), (Fig. 6, E) tobacco all do not detect the release of phantol and contrast.In addition, can also discharge a spot of firpene at rotaring gene plant blade.Transgene tobacco with the contrast tobacco compare, phenotype seem short and small (Fig. 6, G).

7, description of drawings

Fig. 1 is the sequence alignment of monoterpene of the present invention/sesquiterpene synthetic enzyme CsLIS/NES and other species terpene synthetic enzyme;

Fig. 2 is the analysis of monoterpene of the present invention/sesquiterpene synthetic enzyme CsLIS/NES evolutionary tree;

Fig. 3 is that monoterpene of the present invention/sesquiterpene synthetic enzyme CsLIS/NES and reporter gene GFP merge localization and expression in the experimental result of chloroplast(id).A, the fluorescent signal that fluorescence channel detects; B, the white light passage detects the subcellular structure of blade; C, the common detected fluorescent signal that is positioned on the chloroplast(id) of fluorescence and white light passage.

Fig. 4 is the monoterpene of the present invention/expression of sesquiterpene synthetic enzyme CsLIS/NES gene in intestinal bacteria.A, pET32a_CsLIS/NES vector construction synoptic diagram; B, the polyacrylamide gel electrophoresis result of prokaryotic expression CsLIS/NES.M wherein, the protein molecular mark; Before the blank plasmid IPTG of 1, pET32a induces; After the blank plasmid IPTG of 2, pET32a induces; Before 3, pET32a_CsLIS/NES induces; After 4, pET32a_CsLIS/NES induces; 5, pET32a_CsLIS/NES induces the back centrifuged supernatant; 6, recombinant protein.

Fig. 5 is monoterpene of the present invention/sesquiterpene synthetic enzyme CsLIS/NES enzyme functional verification; A:CsLIS/NES catalysis geranyl tetra-sodium (GPP) product GC-MS result wherein, B: phantol standard specimen GC-MS result, C:CsLIS/NES catalysis method Thessaloniki tetra-sodium (FPP) product GC-MS result, a:3-allyl group-6-methoxyphenol, b: butyl phenyl ester, D:(Z, E) nerolidol isomers standard specimen GC-MS result.

Fig. 6 is that CsLIS/NES is in tobacco expressed expression, A: plant expression plasmid makes up B:PCR checking transfer-gen plant, C:CsLIS/NES is at the expression of transgene tobacco, D transgene tobacco aroma substance discharges E: contrast (changeing unloaded) tobacco aroma material discharges F: phantol standard specimen G: the phenotype (left side: transgene tobacco of transgene tobacco and wild-type plant; Right: wild-type tobacco).

Fig. 7 is the application synoptic diagram of CsLIS/NES.

8, embodiment

The present invention is described in further detail with embodiment with reference to the accompanying drawings below.

Case study on implementation 1:

Use PCR primer 5 ' AAA TCTAGACCAAGAACAAACGTACTTTCC (underscore is Xba I restriction enzyme site) and 5 ' AAA CCCGGGThe CTAAGGTAATTTATCATAGAACATAGAC(underscore is Sac I restriction enzyme site), cDNA full length sequence by PCR method clone CsLIS/NES gene, after Xba/Sac I enzyme is cut, be connected to after same group of enzyme enzyme cut to plant expression vector pCamb2300, obtain expression vector.After containing the CsLIS/NES gene by double digestion (Xbal I/Sac I) and PCR (above-mentioned same primers as) checking expression vector, the pCamb2300_CsLIS/NES1 carrier that builds is transformed Agrobacterium GV3101 respectively with contrast empty carrier pCamb2300, Agrobacterium after the conversion receives the LB solid medium of mycin and screens containing anti-card, and the resistance bacterium colony is again through the above-mentioned same primers as of PCR() the having or not of checking CsLIS/NES gene.Contain the Agrobacterium of goal gene again through the cultivation of liquid LB, make bacterium liquid OD ₆₀₀Value reaches 0.8-1.2.Meanwhile, under aseptic condition, cutting is into about 1cm with the young leaflet tablet of aseptic tobacco tissue cultured seedling ²Leaf disk, and submergence 1 minute in the bacterium liquid of aforementioned ready Agrobacterium.After leaf disk after the submergence of bacterium liquid blots bacterium liquid with aseptic filter paper, place and cultivated altogether on the 1/2MS solid medium two days.Afterwards, leaf disk goes in the resistance screening substratum of the hormone that contains 6-benzyladenine and indolebutyric acid and kantlex and Pyocianil and cultivates, to obtain transfer-gen plant.Regeneration plant with anti-kantlex characteristic is seeded on the new resistance screening substratum one by one, carries out succeeding transfer culture until taking root.Plant after taking root is extracted the DNA of plant before transplanting the open air, PCR checks to confirm that plant contains goal gene.Plant after the affirmation is collected head space gas, carries out GC-MS and detects.Confirm that it discharges aroma substances such as phantol.

The concrete application of CsLIS/NES is with reference to Fig. 7.

Above-described embodiment only is explanation technical conceive of the present invention and characteristics, and its purpose is to allow the personage that is familiar with this art can understand content of the present invention and is also implemented, and can not limit protection scope of the present invention with this.All equivalences that spirit is done according to the present invention change or modify, and all should be encompassed in protection scope of the present invention.

Claims

1. monoterpene/sesquiterpene synthetic enzyme CsLIS/NES that influences the tea aroma quality, the sequence of its gene and protein is shown in sequence table.

2. monoterpene as claimed in claim 1/sesquiterpene synthetic enzyme CsLIS/NES urges application in the perfume (or spice) plant.

3. monoterpene as claimed in claim 2/sesquiterpene synthetic enzyme CsLIS/NES urges application in the perfume (or spice) tobacco.