CN104152474B - Tobacco Tomato red pigment β cyclase gene and its application - Google Patents
Tobacco Tomato red pigment β cyclase gene and its application Download PDFInfo
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Abstract
The invention discloses a kind of Tobacco Tomato red pigmentsβCyclase gene, base sequence is as shown in SEQ ID NO:1.The present invention has studiedNtβ‑LCYTranscriptional level expression pattern of the gene in tobacco different times Different Organs, it is found that the gene is mainly expressed in spire；It is had studied using two methods of OE and RNAiNtβ‑LCYInfluence of the gene under expression quantity situation of change to tobacco plant growth and development situation and pigment content, analyzesNtβ‑LCYCritical function of the gene in tobacco.It is first to obtain gene first, can defines and targetedly conduct a research to the gene；Gene function is followed by analyzed using stable transgenic technology.
Description
Technical field
The invention belongs to plant genetic engineering fields, in particular it relates to which Tobacco Carotenoid synthesizes crucial base
Cause is related to important work of the β-lycopene cyclase gene (Nt β-LCY) in carotenogenesis and plant growth and development
With.
Background technique
Carotenoid is that yellow, orange red and red one is presented by Isoprenoid pathway synthesis in organism
Class terpene substances.Carotenoid carries the critical function of light absorption in photosynthesis of plant, is primarily present in leaves of plants
In the chromoplast of the chloroplaset of piece and many flowers and fruit, there is the ability for absorbing and transmitting electronics, and removing photosynthesis
It plays an important role in terms of the free radicals such as the chlorophyll triplet state and singlet of middle generation and superoxide anion.On the other hand,
Carotenoid can be by Zeaxanthin cycle under the conditions of Xanthophyll cycle, and dissipated Photosystem I I in a manner of non-photochemical radiation
(PSII) excess energy protects chlorophyll from the destruction of oxidative damage.In addition, carotenoid is also that plant hormone falls off
The precursor of sour (ABA) synthesis, ABA widely participate in the links and degeneration-resistant process that plant growing cycle is developed.So class
Carrotene plays an important role in plant growth and development process.
Cyclization of lycopene is an important branching-point of Carotenoid biosynthetic pathway in plant.In plant
There are two kinds of cyclases, and one is lycopene beta cyclase (β-LCY), and under its catalysis, lycopene is converted into β-Hu Luo
Bu Su, and then generating lutein, the another kinds such as zeaxanthin, violaxanthin, neoxathin is lycopene ε-cyclase (ε-LCY),
With generate alpha-carotene under β-LCY collective effect, and then generate lutern etc..The activity of two enzymes of β-LCY and ε-LCY is one
Determine to determine the carotenoid substances such as two branch product beta carotenes, violaxanthin, zeaxanthin and lutern in degree
Ratio and carotenoid total amount.
Tobacco is important industrial crops, has outstanding role in terms of developing local economy, increasing state revenue and expenditure accumulation.Cigarette
There are positive correlation, one side tobacco leaf exterior quality and carotenoid between carotenoid content and quality of tobacco in leaf
Component content is directly related, and another aspect carotenoid is the important prerequisite substance of aroma components, to the fragrance of tobacco
Amount and aroma quality have direct effect.Research shows that carotenoid is the terpene compound for influencing tobacco aroma quality-critical,
Its catabolite accounts for the 8%-12% of tobacco leaf general volatile fragrance component.The degradation of carotenoid and thermal cracking products produce
Nearly hundred kinds of volatile compounds, such as Megastigmatrienone, dorinone, these fragrance matter threshold values are relatively low, and irritation is smaller,
It is big to tabacco fragrance contribution rate, it is to form flue-cured tobacco exquisiteness, graceful and novel aroma main component.Therefore, it is found in tobacco
The gene that plays a crucial role during carotenogenesis simultaneously explores its application and will have great importance.
Summary of the invention
The purpose of the present invention is obtain Nt β-from cultivation tobacco Hongda tobacco vivo clone by way of homologous clone
LCY coding sequence, and carried out expression pattern analysis.Utilize stable transgenic technology overexpression
(overexpression, OE) and RNA interference (RNA interference, RNAi) technique study Nt β-LCY gene is in table
The influence of influence and carotenoid content in the case of up to amount variation to the growth and development situation of tobacco plant.The present invention is directed to
Explore function of the Nt β-LCY gene in tobacco, if directly control the content of carotenoid, which can be
It is cultivated improved seeds using technique for gene engineering and theoretical foundation and genetic resources is provided.
The technical scheme is that Tobacco Tomato red pigment β cyclase gene, base sequence such as SEQ ID NO:1 institute
Show.
Tobacco Tomato red pigment β cyclase gene, RNAi sequence is as shown in SEQ ID NO:2.
The application of the Tobacco Tomato red pigment β cyclase gene, the Tobacco Tomato red pigment β cyclase gene improve cigarette
Careless carotenoid content.
A method of Tobacco Carotenoid content being improved, by being overexpressed and the RNA interference technical research gene
Function obtains the Transformation of tobacco plant that carotenoid content improves.
The method of the raising Tobacco Carotenoid content, with the volume of the Tobacco Tomato red pigment β cyclase gene
The sequence is inserted under the expression cassette containing 35S promoter, which belongs to plant by code nucleic acid fragment as functional sequence
A part of double source expression vector constructs the over-express vector of Tobacco Tomato red pigment β cyclase gene.
Firstly, having studied transcriptional level expression pattern of the Nt β-LCY gene in tobacco different times Different Organs, find
The gene is mainly expressed in spire；Nt β-LCY gene is had studied in expression quantity situation of change using two methods of OE and RNAi
Under influence to tobacco plant growth and development situation and pigment content, analyze critical function of the Nt β-LCY gene in tobacco.
It is first to obtain gene first, can defines and targetedly conduct a research to the gene；Followed by use stable transgenosis
Technology analyzes gene function.
Detailed description of the invention
Fig. 1: different times in quantitative PCR analysis Nicotiana tabacum, (stage1-stage4 be respectively also seedling stage, Topping Stage,
Full-bloom stage and maturity period) each organoid (root-R, stem-S, leaf-L (5 leaf position -5L, 10 leaf position -10L, 15 leaf position -15L), flower -
F) in Nt β-LCY gene transcriptional expression mode；
Fig. 2: Nt β-LCY gene expression pattern under quantitative PCR analysis recovery, CON be it is normal under the conditions of gene
Relative expression quantity；LL is gene relative expression quantity under low temperature and poor light；
Fig. 3: Nt β-LCY transgenic phenotypes, CON are the non-non-transgenic control lines of tobacco；OE is that Nt β-LCY is overexpressed plant
Strain；RNAi is Nt β-LCY-RNAi transgenic plant；
Fig. 4: Nt β-LCY-RNAi transgene silencing effect, NT are non-non-transgenic control lines；Nt β-LCY-RNAi is to turn
Gene plant；
Pigment content in Fig. 5: Nt β-LCY-RNAi transgenic line, NT are non-non-transgenic control lines；Ntβ-LCY-
RNAi is transgenic plant；
The screening of Fig. 6: Nt β-LCY-OE transgenic plant；
The analysis of Fig. 7: Nt β-LCY-OE transgenic plant β-LCY gene expression amount, NT are non-non-transgenic control lines；Ntβ-
LCY-OE is transgenic plant；
Pigment content in Fig. 8: Nt β-LCY-OE transgenic line, NT are non-non-transgenic control lines；Nt β-LCY-OE is
Transgenic plant.
Specific embodiment
Tobacco Tomato red pigment β cyclase gene, base sequence is as shown in SEQ ID NO:1.
Tobacco Tomato red pigment β cyclase gene, RNAi sequence is as shown in SEQ ID NO:2.
The application of the Tobacco Tomato red pigment β cyclase gene, the Tobacco Tomato red pigment β cyclase gene improve cigarette
Careless carotenoid content.
A method of Tobacco Carotenoid content being improved, by being overexpressed and the RNA interference technical research gene
Function obtains the Transformation of tobacco plant that carotenoid content improves.
The method of the raising Tobacco Carotenoid content, with the volume of the Tobacco Tomato red pigment β cyclase gene
The sequence is inserted under the expression cassette containing 35S promoter, which belongs to plant by code nucleic acid fragment as functional sequence
A part of double source expression vector constructs the over-express vector of Tobacco Tomato red pigment β cyclase gene.
1, the homologous clone of Nt β-LCY gene
All est sequences of Nt β-LCY gene are searched by NCBI-Blastn, over-designed draws at ATG and TGA
Object.PCR amplification is carried out using Nicotiana tabacum cDNA and genomic DNA as template respectively, recycles target fragment, T-A connection, conversion is greatly
Enterobacteria (DH5 α) competence, 37 DEG C of cultures, bacterium colony PCR identify to obtain positive colony.Positive plasmid extracts, and finally carries out plasmid
Digestion identification and sequencing obtain Nicotiana tabacum Nt β-LCY code area overall length and full length gene sequence.Obtain the code area Nt β-LCY
(CDS) full length sequence 1503bp encodes 500 amino acid, Nt β-LCY full length gene 1503bp, so Nt β-LCY gene exists altogether
Introne is not present in tobacco.
It is described that specific step is as follows:
The primer is 5 '-ATGGATACATTGTTGAAAACCCCAAAT-3 ' and 5 '-
TTCTGTATCCTGTAACAAATTGTTGATC-3'.It is prosperous that cDNA and DNA as template both are from cultivation tobacco Hongda tobacco
Long-term spire piece (RNA and DNA are extracted and carried out according to QIAGEN company RNA or DNA extraction kit specification).According to
Reverse transcription system kit specification is primer synthesis with Oligo (dT) 18 (TaKaRa company)
The first chain of cDNA.PCR reaction condition is 94 DEG C, 3min；94 DEG C, 30s, 58 DEG C, 30s, 72 DEG C, 1.5min, 35cycles；72
DEG C, 10min；4 DEG C, pause.50 μ L:cDNA of reaction system or DNA profiling 2 μ L, each 2.5 μ L of upstream and downstream primer (10 μM),
4 μ L of dNTPs (10 μM), 10 × Buffer 5 μ L, HiFi enzyme (Beijing Quanshijin Biotechnology Co., Ltd) 0.5 μ L, sterile water
33.5μL.PCR product carries out agarose gel electrophoresis, and with gel extraction kit, (GelExtraction KIT, is purchased from
QIAGENE) purification of target Nt β-LCY DNA fragmentation (method is referring to kit specification) from Ago-Gel, is used
Eppendorf company BioPhotometer measurement concentration after for digestion, connect or deposit -20 DEG C it is spare.The Nt β-of purifying
(Solution I, TaKaRa company, presses for LCYDNA segment and pMD19-T carrier (TaKaRa company, ammonia benzyl chloramphenicol resistance) connection
Operated according to kit specification), heat-shock transformed (42 DEG C of 90s) enters bacillus coli DH 5 alpha (TaKaRa company).From obtained clone
With bacterium colony PCR (primer is above-mentioned amplimer) identification positive colony in transformant, positive colony plasmid (small upgrading grain examination is extracted
Agent box, QIAGENE, method are carried out referring to kit specification).
2, Nt β-LCY expression pattern analysis
Each period (also seedling stage, Topping Stage, full-bloom stage and maturity period) of acquisition cultivation tobacco each organoid (root, stem,
5 leaf positions, 10 leaf positions, 15 leaf positions, flower) sample, and their total serum IgE is extracted, reverse transcription becomes cDNA (method is as described in 1).It is fixed
Measure the expression pattern under the conditions of PCR (BIO-RAD, USA) analysis Nt β-LCY spatial and temporal expression profile and recovery.It is such as attached
Shown in Fig. 1, Nt β-LCY is higher in full-bloom stage expression quantity, and multilist reaches the (expression: the 15 leaf position 10 leaf position > in spire
5 leaf position > flower >=stem > root of >).Under recovery, Nt β-LCY expression quantity has significant change, illustrate the gene be by
Recovery condition expression profile.Nt β-LCY expression quantity is in lower liter of trend in 6h, and the later expression quantity of 12h is gradually
It improves, is reaching maximum (see Fig. 2) for 24 hours.The result of study of transcriptional level expression pattern shows the physiology shape of the gene and plant
State is closely related, higher in the more prosperous and powerful tender tissue expression quantity of cigarette strain photosynthetic physiology, and can be induced by Xanthophyll cycle condition,
The process that cigarette strain resists Xanthophyll cycle is participated in, biological function should there are close relationships with Photosynthetic physiological process.
Tobacco organ samples used in expression pattern analysis sample in Yunnan crop field, and quantitative primer used is Nt β-LCY-
Q-F/R, 5 '-GATGACAATACAACTAAAGATCTTGATAG-3 and 5 '-CATAAGCTACTTGATATCCAGGAT-3 '.Using
26s RNA is as internal reference.Pcr amplification reaction condition: 95 DEG C of initial denaturation 3min；95 DEG C of 20s, 60 DEG C of 20s, 40 circulations.Reaction
System: 1 μ L cDNA, 10 μ L SYBR Green, each 1 μ L of upstream and downstream primer (10 μm of ol/L) mends ddH2O to 20 μ L.Low temperature is weak
Light processing condition: 100 μm of ol m–2s–1, 4 DEG C.
3, the gene silencing that RNAi is mediated in tobacco studies Nt β-LCY gene function
For function of the understanding Nt β-LCY gene in tobacco more precisely, we are adopted using stable transgenic technology
In vivo functionality research is carried out to the gene with RNAi method.It constructs Nt β-LCY gene RNAi carrier and converts Agrobacterium GV3101, benefit
With Agrobacterium-mediated genetic transformation technical transform SR1 Nicotiana tabacum, positive plant screening is carried out using that resistant gene of card, is obtained
To the successful tobacco plant of conversion.There is apparent photobleaching in transgenic tobacco plant blade or aetiolation, plant are seriously short
Change (Fig. 3), the progress of growth, blade is more bleached, and plant part is gradually died.And the cigarette strain sample to survive is carried out
Culture, until inoculation, then carries out the analysis of Nt β-LCY gene transcript expression and metabolite analysis for cigarette strain tobacco leaf with T2.It is fixed
PCR is measured the results show that Nt β-LCY gene expression amount significantly reduces (Fig. 4) in RNAi transgenosis cigarette strain, silence efficiency is up to
90%.Carotenoids content is analyzed using HPLC, discovery beta carotene, violaxanthin, neoxathin and lutern
(Lutein) carotenoid and chlorophyll a and b content are decreased obviously (Fig. 5).Transgenic RNAi the result shows that, Nt β-LCY base
Because the generation to the bicyclic lutein of cigarette strain has a major impact, and bicyclic lutein vine growth and development is played it is vital
Effect, is the indispensable important component of organism, mainly controls the synthesis of the coloring matters such as Carotenoid in Plants, into
And affect the photosynthesis of plant.
It is described that the specific method is as follows:
Using Gateway recombinant technique, pHellsgate2 (Spectinomycin resistance) vector construction transformation of tobacco is utilized
RNAi carrier.Primer is 5 '-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACGAGGAAGCTAAATCTATGC-3 ' (band
Have attB1 connector) and 5 '-GGGGACCACTTTGTACAAGAAAGCTGGGTATACGTAATCCAGGACGAGCAAC-3 ' (have
AttB2 connector).PCR reaction is expanded using Nt β-LCY-T in 1 as template, product glue recovery purifying.Then BP recombination is carried out
Experiment.System is as follows: attB-PCR product (100ng/ μ L) 2.5 μ L, pHellsgate 2 vector (400ng/ μ L) 0.4 μ L,
2 μ L of BP clonase II enzyme mix, supplement TE Buffer (pH 8.0) 5.1 μ L to 10 μ L systems, 25 DEG C of reaction 3h,
1 μ L K albumen is added, then 37 DEG C of warm bath 10min take the 1 heat-shock transformed bacillus coli DH 5 alpha of μ L reaction solution, identify through bacterium colony PCR
Positive colony, and upgrading grain carries out digestion verification and obtains pHellsgate2-Nt β-LCY-RNAi plasmid after confirmation is correct.So
After be transferred in Agrobacterium competence GV3101, shake bacterium transformation of tobacco plant after picking suitable conversion bacterial plaque PCR identification is errorless.
Plasmid converts Agrobacterium: taking 1 μ L of pHellsgate2-Nt β-LCY-RNAi plasmid, the GV3101 containing 0.1mL is added
In the EP pipe of Agrobacterium competence, 30min is placed on ice；Agrobacterium competence containing the plasmid is put into liquid nitrogen flash freezer
1min, then 37 DEG C of incubation 5min；1mL LB liquid medium, 28 DEG C of shake culture 3h are added；5,000rpm centrifugation 1min, are abandoned
Supernatant is removed, 200 μ L LB liquid mediums are added, precipitating is resuspended；It takes 200 μ L re-suspension liquids (i.e. converted product) to be equably applied to contain
On the LB plate of 25mg/mL rifampin (Rif, Sigma) and 50mg/mL spectinomycin (Spe, Sigma), 28 DEG C are cultivated 2-3 days；
After the suitable conversion bacterial plaque PCR identification of picking is errorless, the bacterium conversion common tobacco seedlings of SR1 are shaken.
Agrobacterium conversion tobacco: it will identify errorless Agrobacterium inoculation in 5mL LB liquid medium (containing 25mg/mL benefit
The gentle 50mg/mL spectinomycin of good fortune), 28 DEG C of shake cultures are stayed overnight, and next day is transferred to 50mL LB liquid with the ratio of 1:100 and trains
It supports in base (rifampin containing 25mg/mL and 50mg/mL spectinomycin), 28 DEG C of shake cultures to OD600It is 0.8 or so；It is collected by centrifugation
Thallus converts culture medium MS with 50mL0(pH 5.9, Murashige&Shoog Medium are purchased from Duchefa Biochemia)
Suspend the Agrobacterium, using infusion method transformation of tobacco blade, i.e., the blade for being cut into sheet is immersed in the agriculture containing destination carrier
2min in baccilus medium.It then takes out blade and is attached to 28 DEG C dark culture 2 days, 2 days on not antibiotic MS solid medium
It moves back and waits differentiation on the MS differential medium of containing kanamycin and cephalosporin (purchased from Sigma), change within 7-10 days primary
Culture medium.It after being transferred to after differentiation on root media until sending out roots, transplants in soil, cultivates plant extremely by normal method
It is solid, harvest mature seed.
Tobacco regular culture conditions: condition of culture is (28 ± 1) DEG C, and illumination 16h, dark 8h, (60 ± 2) %'s is relatively wet
Degree.
4, overexpression Nt β-LCY gene in tobacco, can increase carotenoid content
Carotenoid not only has a major impact the growth and development of plant, and has to the fragrance quality of tobacco important
Contribution, in order to more fully understand function of the Nt β-LCY gene in tobacco, how exploration, which increases carotenoid in tobacco, contains
Amount is further studied the function of Nt β-LCY using the method for overexpression using stable transgenic technology.It is first
First building Nt β-LCY gene OE carrier converts Agrobacterium GV3101, general using Agrobacterium-mediated genetic transformation technical transform SR1
Logical tobacco carries out positive plant screening (Fig. 6) using across target gene and label protein Flag gene primer, and to β-LCY table
Quantitative PCR analysis is carried out up to amount, discovery transgenosis cigarette strain β-LCY expression quantity significantly increases (Fig. 7), obtains converting successful tobacco
Plant.To T2 for Nt β-LCY gene overexpression transgenosis cigarette strain tobacco sample carry out metabolic analysis, discovery beta carotene,
Violaxanthin, neoxathin and lutern carotenoid and chlorophyll a and b content obviously increase (Fig. 8), tobacco plant growth conditions
Well (Fig. 3).Transgenic RNAi and OE the result shows that, Nt β-LCY gene pairs cigarette strain carotenogenesis and growth and development are extremely
It closing important, is the key gene on carotenogenesis access, expression is directly related to the upgrowth situation of tobacco, into
And affect the yield and quality of tobacco tobacco leaf.The functional study for carrying out Nt β-LCY gene in tobacco is grinding for other plants
Study carefully work and laid excellent basis, while the result of study can improve quality of tobacco from source, utilizes gene for tobacco
Engineering technology, which is cultivated improved seeds, provides theoretical foundation and genetic resources.
That the specific method is as follows is described for Nt β-LCY-OE vector construction:
Constructing Nt β-LCY-OE the primer is Nt β-LCY-sp1300-SpeI-F:5 '-GCACTAGTATGGATACATT
GTTGAAAACCCCAAATAAG-3 ' (having SpeI restriction enzyme site) and Nt β-LCY-sp1300-KpnI-R:5 '-TAGGTACCT
TCTGTATCCTGTAACAAATTGTTGATCAT-3 ' (has KpnI restriction enzyme site).PCR reaction is using Nt β-LCY-T in 1 as mould
Plate is expanded, product glue recovery purifying, using SpeI and KpnI digestion PCR recycle segment and spCAMBIA1300 carrier (by
Flag label protein is added in pCAMBIA1300, vector modification after polyclone enzyme enzyme site), recycle target fragment and company
It connects, converts bacterium (DH5 α, TaKaRa company), bacterium colony PCR identifies that positive colony, upgrading grain obtain purpose carrier Nt β-LCY-
OE, digestion verification and be sequenced confirmation sequence correctly have no frameshit situation.Then it is transferred in Agrobacterium competence GV3101, bacterium colony
After PCR identification is positive, bacterium is shaken, according to step transformation of tobacco plant in 3.Across target gene and label protein Flag gene primer sequence
It is classified as: Nt β-LCY-OE-JC-F5 '-CGGTATGGATATTCTTCTGAAGCTTG-3 and Flag-R5 '-
CTTATCGTCATCGTCCTTGTAATCG-3’。
Plasmid converts Agrobacterium: Nt β-LCY-OE plasmid is transferred to Agrobacterium according to step in 3, it is then that converted product is equal
It is applied on the LB plate of rifampin containing 25mg/mL (Rif, Sigma) and 50mg/mL kanamycins (kana, Sigma) evenly, 28
DEG C culture 2-3 days；After the suitable conversion bacterial plaque PCR identification of picking is errorless, the bacterium conversion common tobacco seedlings of SR1 are shaken.
Agrobacterium conversion tobacco: it will identify that the errorless Agrobacterium containing Nt β-LCY-OE plasmid converts cigarette according to method in 3
Grass cultivates rifampin containing 25mg/mL and 50mg/mL kanamycins in bacterium used medium.
Claims (1)
1. a kind of method for improving Tobacco Carotenoid content, it is characterised in that: with recovery condition evoking tobacco
The expression of lycopene beta cyclase gene obtains the tobacco plant that carotenoid content improves, the Tobacco Tomato red pigment β ring
Change the base sequence of enzyme gene as shown in SEQ ID NO:1.
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| CN106367410B (en) * | 2016-08-29 | 2019-08-20 | 中国科学院华南植物园 | A kind of genetic engineering bacterium producing compound carotenoid and its construction method and application |
| CN108913715A (en) * | 2018-07-19 | 2018-11-30 | 中国农业科学院北京畜牧兽医研究所 | A kind of construction method of plant expression plasmid carrier containing FLAG protein fusion label and its carrier |
| CN110423766B (en) * | 2019-08-29 | 2021-06-29 | 南京大学 | Lycopene beta-cyclase gene and coding protein and application thereof |
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