CN104152474A - Tobacco lycopene beta-cyclase gene and its application - Google Patents
Tobacco lycopene beta-cyclase gene and its application Download PDFInfo
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Abstract
The invention discloses a tobacco lycopene beta-cyclase gene. The base sequence of the tobacco lycopene beta-cyclase gene is represented by SEQIDNO:1. Researches of the transcription level expression mode of an Ntbeta-LCY gene in different organs of tobacco in different stages find that the gene is mainly expressed in young leaves; OE and RNAi methods are used to research the influences of the Ntbeta-LCY gene on the growth condition and the pigment content of tobacco plants under the change of the expression level and analyze the important functions of the Ntbeta-LCY gene in the tobacco. The gene is obtained, and then is clearly and pertinently researched, and then the functions of the gene are analyzed by adopting a stable transgenic technology.
Description
Technical field
The invention belongs to plant genetic engineering field, particularly, the present invention relates to the synthetic key gene of Tobacco Carotenoid, relate to the vital role of β-lycopene cyclase gene (Nt β-LCY) in carotenogenesis and growth and development of plants.
Background technology
Carotenoid is to be synthesized and presented yellow, an orange red and red class terpene substances by Isoprenoid pathway in organism.Carotenoid is being born the critical function of photoabsorption in photosynthesis of plant, mainly be present in the chloroplast(id) of plant leaf and the chromoplastid of many flowers and fruit, there is the ability that absorbs and transmit electronics, and play an important role aspect removing the free radical such as chlorophyll triplet state and singlet and superoxide anion producing in photosynthesis.On the other hand, carotenoid can circulate by xenthophylls under light rejection condition, and with the dissipate excess energy of Photosystem I I (PSII) of the mode of non-photochemical radiation, protection chlorophyll is avoided the destruction of oxidative damage.In addition, carotenoid is also the synthetic precursor of Abscisic Acid (ABA), ABA involved in plant growth cycle is grown widely links and degeneration-resistant process.So carotenoid has important effect in plant growth and development process.
Cyclization of lycopene is an important branching-point of Carotenoid biosynthetic pathway in plant materials.In plant materials, there are two kinds of cyclases, one is lycopene beta cyclase (β-LCY), under its catalysis, Lyeopene is converted into β-carotene, and then generate the xenthophylls such as zeaxanthin, zeaxanthin diepoxide, Neoxanthine, another kind is Lyeopene ε-cyclase (ε-LCY), and generates alpha-carotene under β-LCY acting in conjunction, and then generates lutein etc.The activity of β-LCY and two enzymes of ε-LCY has determined the ratio of carotenoid material and the total amounts of carotenoid such as two branch product β-carotenes, zeaxanthin diepoxide, zeaxanthin and lutein to a certain extent.
Tobacco is important cash crop, aspect development local economy, increase state revenue and expenditure accumulation, is having outstanding role.In tobacco leaf, between carotenoid content and quality of tobacco, there is positive correlation, tobacco leaf exterior quality is directly related with carotenoid component content on the one hand, carotenoid is the important prerequisite material of aroma components on the other hand, and the perfume quantity to tobacco and fragrance matter have direct effect.Research shows that carotenoid is the terpene compound that affects tobacco aroma quality-critical, and its degraded product accounts for the 8%-12% of tobacco leaf general volatile aroma component.The degraded of carotenoid and thermal cracking products can generate nearly hundred kinds of volatile compounds, as Megastigmatrienone, dorinone etc., these fragrance matter threshold values are relatively low, and pungency is less, large to tabacco fragrance contribution rate, be the main component that forms flue-cured tobacco exquisiteness, elegance and novel aroma.Therefore, in tobacco, find the gene playing a crucial role in carotenogenesis process and explore its application and will have great importance.
Summary of the invention
The object of the invention is the mode by homologous clone, obtain Nt β-LCY coding sequence from the large gold dollar vivo clone of cultivation tobacco safflower, and carried out expression pattern analysis.Utilize stable transgenic technology overexpression (overexpression, OE) and RNA interfere (RNA interference, RNAi) method studied Nt β-LCY gene impact of the situation of growing on tobacco plant and impact of carotenoid content under expression amount changing conditions.The present invention is intended to explore the function of Nt β-LCY gene in tobacco, whether is directly controlling the content of carotenoid, and this result of study can provide theoretical foundation and genetic resources for utilizing genetic engineering technique to cultivate improved seeds.
Technical scheme of the present invention is: Tobacco Tomato red pigment β cyclase gene, its base sequence is as shown in SEQ ID NO:1.
Tobacco Tomato red pigment β cyclase gene, its RNAi sequence is as shown in SEQ ID NO:2.
The application of described Tobacco Tomato red pigment β cyclase gene, described Tobacco Tomato red pigment β cyclase gene improves Tobacco Carotenoid content.
Improve a method for Tobacco Carotenoid content, express with RNA interference technique and studied this gene function by mistake, obtain the Transformation of tobacco plant that carotenoid content improves.
The method of described raising Tobacco Carotenoid content, using the coding nucleic acid fragment of described Tobacco Tomato red pigment β cyclase gene as functional sequence, this sequence is inserted under the expression cassette that contains 35S promoter, this expression cassette belongs to a part for plant double source expression vector, builds the over-express vector of Tobacco Tomato red pigment β cyclase gene.
First, studied the transcriptional level expression pattern of Nt β-LCY gene in tobacco different times Different Organs, found that this gene mainly expresses in spire; Utilize two kinds of methods of OE and RNAi studied Nt β-LCY gene under expression amount changing conditions on the grow impact of situation and pigment content of tobacco plant, analyzed the critical function of Nt β-LCY gene in tobacco.First be first to obtain gene, can clearly and targetedly conduct a research to this gene; Next is to adopt stable transgenic technology analyzing gene function.
Brief description of the drawings
Fig. 1: each organoid (root-R, stem-S, leaf-L (5 leaf position-5L of different times, (stage1-stage4 is respectively also seedling stage, Topping Stage, full-bloom stage and ripening stage) in the common tobacco of quantitative PCR analysis, 10 leaf position-10L, 15 leaf position-15L), flower-F) in the transcriptional expression pattern of Nt β-LCY gene;
Fig. 2: Nt β-LCY gene expression pattern under quantitative PCR analysis recovery, CON is gene relative expression quantity under normal condition; LL is gene relative expression quantity under low temperature and poor light;
Fig. 3: Nt β-LCY transgenosis phenotype, CON is not transgenosis adjoining tree of tobacco; OE is that Nt β-LCY crosses expression plant; RNAi is Nt β-LCY-RNAi transfer-gen plant;
Fig. 4: Nt β-LCY-RNAi transgene silencing effect, NT is transgenosis adjoining tree not; Nt β-LCY-RNAi is transfer-gen plant;
Fig. 5: pigment content in Nt β-LCY-RNAi transgenic line, NT is transgenosis adjoining tree not; Nt β-LCY-RNAi is transfer-gen plant;
Fig. 6: Nt β-LCY-OE transfer-gen plant screening;
Fig. 7: Nt β-LCY-OE transfer-gen plant β-LCY genetic expression component analysis, NT is transgenosis adjoining tree not; Nt β-LCY-OE is transfer-gen plant;
Fig. 8: pigment content in Nt β-LCY-OE transgenic line, NT is transgenosis adjoining tree not; Nt β-LCY-OE is transfer-gen plant.
Embodiment
Tobacco Tomato red pigment β cyclase gene, its base sequence is as shown in SEQ ID NO:1.
Tobacco Tomato red pigment β cyclase gene, its RNAi sequence is as shown in SEQ ID NO:2.
The application of described Tobacco Tomato red pigment β cyclase gene, described Tobacco Tomato red pigment β cyclase gene improves Tobacco Carotenoid content.
Improve a method for Tobacco Carotenoid content, express with RNA interference technique and studied this gene function by mistake, obtain the Transformation of tobacco plant that carotenoid content improves.
The method of described raising Tobacco Carotenoid content, using the coding nucleic acid fragment of described Tobacco Tomato red pigment β cyclase gene as functional sequence, this sequence is inserted under the expression cassette that contains 35S promoter, this expression cassette belongs to a part for plant double source expression vector, builds the over-express vector of Tobacco Tomato red pigment β cyclase gene.
1, the homologous clone of Nt β-LCY gene
Search all est sequences of Nt β-LCY gene by NCBI-Blastn, at ATG and TGA place over-designed primer.Carry out pcr amplification taking common tobacco cDNA and genomic dna as template respectively, reclaim object fragment, T-A connects, and transforms intestinal bacteria (DH5 α) competence, 37 DEG C of cultivations, and bacterium colony PCR qualification obtains positive colony.Positive plasmid extracts, and finally carries out plasmid enzyme restriction qualification and order-checking and obtains common tobacco Nt β-LCY coding region total length and full length gene sequence.Obtain Nt β-LCY coding region (CDS) full length sequence 1503bp, 500 amino acid of encoding altogether, Nt β-LCY full length gene 1503bp, so there is not intron in Nt β-LCY gene in tobacco.
Concrete steps are as described below:
The primer is 5 '-ATGGATACATTGTTGAAAACCCCAAAT-3 ' and 5 '-TTCTGTATCCTGTAACAAATTGTTGATC-3 '.As the cDNA of template and DNA all from the cultivation prosperous long-term spire sheet of the large gold dollar of tobacco safflower (RNA and DNA extraction are carried out according to the RNA of QIAGEN company or DNA extraction test kit specification sheets).According to Reverse transcription system test kit specification sheets, synthetic cDNA the first chain taking Oligo (dT) 18 (TaKaRa company) as primer.PCR reaction conditions 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.Reaction system 50 μ L:cDNA or DNA profiling 2 μ L, the each 2.5 μ L of upstream and downstream primer (10 μ M), dNTPs 4 μ L (10 μ M), 10 × Buffer, 5 μ L, HiFi enzyme (Beijing Quanshijin Biotechnology Co., Ltd) 0.5 μ L, sterilized water 33.5 μ L.PCR product carries out agarose gel electrophoresis, with gel extraction kit (GelExtraction KIT, purchased from QIAGENE) purification of target Nt β-LCY DNA fragmentation from sepharose (method reference reagent box specification sheets), with the BioPhotometer of Eppendorf company measure after concentration for enzyme cut, connect or deposit-20 DEG C for subsequent use.Nt β-LCYDNA fragment of purifying and pMD19-T carrier (TaKaRa company, ammonia benzyl mycin resistance) connection (Solution I, TaKaRa company, according to the operation of test kit specification sheets), heat shock transforms (42 DEG C of 90s) and enters bacillus coli DH 5 alpha (TaKaRa company).From the clone's transformant obtaining, with bacterium colony PCR (primer is above-mentioned amplimer) qualification positive colony, extract positive colony plasmid (method reference reagent box specification sheets carries out for the little plasmid kit of carrying, QIAGENE).
2, Nt β-LCY expression pattern is analyzed
Gather each organoid (root, stem, the 5 leaf positions in each period (also seedling stage, Topping Stage, full-bloom stage and ripening stage) of cultivation tobacco, 10 leaf positions, 15 leaf positions, flower) sample, and extract their total RNA, reverse transcription becomes cDNA (method is as described in 1).Quantitative PCR (BIO-RAD, USA) is analyzed the expression pattern under Nt β-LCY spatial and temporal expression pattern and recovery condition.As shown in Figure 1, Nt β-LCY is higher at full-bloom stage expression amount, and multilist reaches in spire (expression level: 10 leaf position > 15 leaf position > 5 leaf position > flower >=stem > roots).Under recovery, Nt β-LCY expression amount has significant change, and illustrates that this gene is to be subject to recovery condition expression profile.Nt β-LCY expression amount is the lower trend that rises in the time of 6h, and after 12h, expression amount improves gradually, reaches maximum (seeing Fig. 2) at 24h.The result of study of transcriptional level expression pattern shows, the physiological status of this gene and plant is closely related, the tender tissue expression amount comparatively prosperous and powerful at cigarette strain photosynthetic physiology is higher, and can be induced by light rejection condition, participating in cigarette strain and resist the process that light suppresses, should there is close relationship with Photosynthetic physiological process in its biological function.
Expression pattern is analyzed tobacco organ samples used all in the sampling of land for growing field crops, Yunnan, and quantitative primer used is Nt β-LCY-Q-F/R, 5 '-GATGACAATACAACTAAAGATCTTGATAG-3 and 5 '-CATAAGCTACTTGATATCCAGGAT-3 '.Adopt 26s RNA as internal reference.Pcr amplification reaction condition: 95 DEG C of 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, the each 1 μ L of upstream and downstream primer (10 μ mol/L), mends ddH
2o to 20 μ L.Low temperature and poor light treatment condition: 100 μ mol m
– 2s
– 1, 4 DEG C.
3, gene silencing research Nt β-LCY gene function of RNAi mediation in tobacco
For the function of more definite understanding Nt β-LCY gene in tobacco, we utilize stable transgenic technology to adopt RNAi method to carry out functional study in body to this gene.Build Nt β-LCY gene RNAi carrier and transform Agrobacterium GV3101, utilize agriculture bacillus mediated genetic transfoumation to transform the common tobacco of SR1, adopt that resistant gene of card to carry out positive plant screening, obtain transforming successful tobacco plant.There is obvious photobleaching or etiolation in transgenic tobacco plant blade, plant is seriously downgraded (Fig. 3), the carrying out of growth, and blade is bleached all the more, part plant dying gradually.And the cigarette strain sample surviving is cultivated, until then inoculation carries out Nt β-LCY gene transcript expression analysis and metabolite analysis with T2 for cigarette strain tobacco leaf.The demonstration of quantitative PCR result, in the strain of RNAi transgenosis cigarette, Nt β-LCY gene expression amount significantly reduces (Fig. 4), and silence efficiency is up to 90%.Utilize HPLC to analyze carotenoids content, find β-carotene, zeaxanthin diepoxide, Neoxanthine and lutein (Lutein) carotenoid and chlorophyll a and b content obviously decline (Fig. 5).Transgenic RNAi result shows, the generation of the dicyclo xenthophylls of Nt β-LCY gene pairs cigarette strain has material impact, and dicyclo xenthophylls plays vital effect to vine growth and development, it is the important component part that organism is indispensable, it is synthetic that the coloring matters such as Carotenoid in Plants in major control, and then affect the photosynthesis of plant.
Concrete grammar is as described below:
Adopt Gateway recombinant technology, utilize the RNAi carrier of pHellsgate2 (spectinomycin resistance) vector construction transformation of tobacco.Primer is 5 '-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACGAGGAAGCTAAATCTATGC-3 ' (with attB1 joint) and 5 '-GGGGACCACTTTGTACAAGAAAGCTGGGTATACGTAATCCAGGACGAGCAAC-3 ' (with attB2 joint).PCR reaction is increased taking Nt β-LCY-T in 1 as template, and product glue reclaims purifying.Then carry out BP reconstruction 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, BP clonase II enzyme mix 2 μ L, supplement TE Buffer (pH 8.0) 5.1 μ L to 10 μ L systems, 25 DEG C of reaction 3h, add 1 μ L K albumen, 37 DEG C of temperature are bathed 10min, then get 1 μ L reaction solution heat shock and transform bacillus coli DH 5 alpha, through bacterium colony PCR qualification positive colony, and upgrading grain carries out enzyme and cuts checking, confirm correct after, obtain pHellsgate2-Nt β-LCY-RNAi plasmid.Then proceed in Agrobacterium competence GV3101, after the appropriate conversion bacterial plaque PCR of picking qualification is errorless, shake bacterium transformation of tobacco plant.
Plasmid transforms Agrobacterium: get pHellsgate2-Nt β-LCY-RNAi plasmid 1 μ L, add in the competent EP pipe of GV3101 Agrobacterium containing 0.1mL, place 30min on ice; The Agrobacterium competence that contains this plasmid is put into liquid nitrogen flash freezer 1min, then 37 DEG C of incubation 5min; Add 1mL LB liquid nutrient medium, 3h is cultivated in 28 DEG C of concussions; The centrifugal 1min of 5,000rpm, supernatant discarded, adds 200 μ L LB liquid nutrient mediums, resuspended precipitation; Get the resuspended liquid of 200 μ L (being converted product) and be applied to equably on the LB flat board containing 25mg/mL Rifampin (Rif, Sigma) and 50mg/mL spectinomycin (Spe, Sigma), cultivate 2-3 days for 28 DEG C; After the appropriate conversion bacterial plaque PCR of picking qualification is errorless, shakes bacterium and transform the common cigarette seedling of SR1.
Agrobacterium conversion tobacco: the errorless Agrobacterium of qualification is inoculated in to (containing 25mg/mL Rifampin and 50mg/mL spectinomycin) in 5mL LB liquid nutrient medium, 28 DEG C of concussion overnight incubation, be transferred in 50mL LB liquid nutrient medium (containing 25mg/mL Rifampin and 50mg/mL spectinomycin) with the ratio of 1:100 next day, and 28 DEG C of concussions are cultured to OD
600it is 0.8 left and right; Centrifugal collection thalline, with 50mL conversion substratum MS
0(pH 5.9, Murashige & Shoog Medium, purchased from Duchefa Biochemia) this Agrobacterium that suspends, adopt infusion method transformation of tobacco blade, the blade that is about to be cut into sheet is immersed in 2min in the Agrobacterium substratum that contains destination carrier.Then take out blade and be attached to not containing 28 DEG C of dark cultivations 2 days on antibiotic MS solid medium, after 2 days, move on on the MS division culture medium that contains kantlex and cephamycin (purchased from Sigma) and wait for differentiation, within 7-10 days, change a subculture.After differentiation, transfer on root media until after sending out roots, transplanting, in soil, is cultivated plant to solid by normal method, results mature seed.
The normal culture condition of tobacco: culture condition is (28 ± 1) DEG C, illumination 16h, dark 8h, the relative humidity of (60 ± 2) %.
4, overexpression Nt β-LCY gene in tobacco, can increase carotenoid content
Carotenoid is the material impact that grown to plant not only, and the fragrance quality of tobacco is had to important contribution, in order more fully to understand the function of Nt β-LCY gene in tobacco, explore how to increase carotenoid content in tobacco, utilize stable transgenic technology to adopt the method for overexpression to carry out further research to the function of Nt β-LCY.First build Nt β-LCY gene OE carrier and transform Agrobacterium GV3101, utilize agriculture bacillus mediated genetic transfoumation to transform the common tobacco of SR1, adopt and carry out positive plant screening (Fig. 6) across target gene and label protein Flag gene primer, and β-LCY expression amount is carried out to quantitative PCR analysis, find that transgenosis cigarette strain β-LCY expression amount significantly raises (Fig. 7), obtains transforming successful tobacco plant.T2 is carried out to metabolic analysis for Nt β-LCY gene overexpression transgenosis cigarette strain tobacco sample, find that β-carotene, zeaxanthin diepoxide, Neoxanthine and lutein carotenoid and chlorophyll a and b content obviously increase (Fig. 8), tobacco plant growth conditions good (Fig. 3).Transgenic RNAi and OE result show, Nt β-LCY gene pairs cigarette strain carotenogenesis and grow most important, be the key gene on carotenogenesis path, its expression level is directly connected to the upgrowth situation of tobacco, and then affects output and the quality of tobacco tobacco leaf.Good basis has been laid in the research work that the functional study of carrying out Nt β-LCY gene in tobacco is other plant, this result of study can improve quality of tobacco from source simultaneously, provides theoretical foundation and genetic resources for tobacco utilizes genetic engineering technique to cultivate improved seeds.
Nt β-LCY-OE vector construction concrete grammar is as described below:
Building Nt β-LCY-OE the primer is Nt β-LCY-sp1300-SpeI-F:5 '-GCACTAGTATGGATACATTGTTGAAAACCCCAAATAAG-3 ' (with SpeI restriction enzyme site) and Nt β-LCY-sp1300-KpnI-R:5 '-TAGGTACCTTCTGTATCCTGTAACAAATTGTTGATCAT-3 ' (with KpnI restriction enzyme site).PCR reaction is increased taking Nt β-LCY-T in 1 as template, product glue reclaims purifying, use SpeI and KpnI enzyme to cut PCR and reclaim fragment and spCAMBIA1300 carrier (by pCAMBIA1300, carrier transformation, after polyclone restriction enzyme site, add Flag label protein), reclaim object fragment and connect, transform bacteria (DH5 α, TaKaRa company), bacterium colony PCR identifies positive colony, upgrading grain obtains object carrier Nt β-LCY-OE, and enzyme is cut and verified and check order and confirm that sequence correctly there is no frameshit situation.Then proceed in Agrobacterium competence GV3101, after the bacterium colony PCR qualification positive, shake bacterium, according to step transformation of tobacco plant in 3.Across target gene and label protein Flag gene primer sequence be: Nt β-LCY-OE-JC-F5 '-CGGTATGGATATTCTTCTGAAGCTTG-3 and Flag-R5 '-CTTATCGTCATCGTCCTTGTAATCG-3 '.
Plasmid transforms Agrobacterium: Nt β-LCY-OE plasmid is proceeded to Agrobacterium according to step in 3, then converted product is applied to equably containing 25mg/mL Rifampin (Rif, Sigma) and on the LB flat board of 50mg/mL kantlex (kana, Sigma), cultivate 2-3 days for 28 DEG C; After the appropriate conversion bacterial plaque PCR of picking qualification is errorless, shakes bacterium and transform the common cigarette seedling of SR1.
Agrobacterium conversion tobacco: the errorless Agrobacterium that contains Nt β-LCY-OE plasmid of qualification, according to method transformation of tobacco in 3, is cultivated in bacterium used medium containing 25mg/mL Rifampin and 50mg/mL kantlex.
Claims (5)
1. Tobacco Tomato red pigment
βcyclase gene, its base sequence is as shown in SEQ ID NO:1.
2. Tobacco Tomato red pigment
βcyclase gene, its RNAi sequence is as shown in SEQ ID NO:2.
3.. Tobacco Tomato red pigment as claimed in claim 1
βthe application of cyclase gene, is characterized in that: described Tobacco Tomato red pigment
βcyclase gene improves Tobacco Carotenoid content.
4. a method that improves Tobacco Carotenoid content, is characterized in that: expressed with RNA interference technique and studied this gene function by mistake, obtain the Transformation of tobacco plant that carotenoid content improves.
5. the method for raising Tobacco Carotenoid content as claimed in claim 4, is characterized in that: with described Tobacco Tomato red pigment
βthe coding nucleic acid fragment of cyclase gene, as functional sequence, is inserted into this sequence under the expression cassette that contains 35S promoter, and this expression cassette belongs to a part for plant double source expression vector, builds Tobacco Tomato red pigment
βthe over-express vector of cyclase gene.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480124A (en) * | 2014-11-27 | 2015-04-01 | 郑州大学 | Indicator gene used in TRV-mediated gene silencing system as well as construction method and application of carrier thereof |
| CN106367410A (en) * | 2016-08-29 | 2017-02-01 | 中国科学院华南植物园 | Genetically engineered bacteria for producing compound carotenoids as well as construction method and application of genetically engineered bacteria |
| 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 |
| CN110423766A (en) * | 2019-08-29 | 2019-11-08 | 南京大学 | A kind of lycopene beta cyclase gene and its coding albumen and application |
| CN110656114A (en) * | 2019-10-18 | 2020-01-07 | 云南中烟工业有限责任公司 | Tobacco pigment synthesis related gene and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070253933A1 (en) * | 2002-05-14 | 2007-11-01 | Martek Biosciences Corporation | Carotene Synthase Gene and Uses Therefor |
| CN102286504A (en) * | 2011-07-01 | 2011-12-21 | 浙江大学 | Lycopene cyclase gene in sphingomonas sp. and use thereof |
-
2014
- 2014-08-18 CN CN201410405713.6A patent/CN104152474B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070253933A1 (en) * | 2002-05-14 | 2007-11-01 | Martek Biosciences Corporation | Carotene Synthase Gene and Uses Therefor |
| CN102286504A (en) * | 2011-07-01 | 2011-12-21 | 浙江大学 | Lycopene cyclase gene in sphingomonas sp. and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| LU,S.ET AL: "ACCESSION NUMBER:KC484706.1", 《GENBANK》 * |
| 马超 等: "构建番茄红素β/ε环化酶基因RNAi植物表达载体及其表达分析", 《核农学报》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480124A (en) * | 2014-11-27 | 2015-04-01 | 郑州大学 | Indicator gene used in TRV-mediated gene silencing system as well as construction method and application of carrier thereof |
| CN106367410A (en) * | 2016-08-29 | 2017-02-01 | 中国科学院华南植物园 | Genetically engineered bacteria for producing compound carotenoids as well as construction method and application of genetically engineered bacteria |
| 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 |
| CN110423766A (en) * | 2019-08-29 | 2019-11-08 | 南京大学 | A kind of lycopene beta cyclase gene and its coding albumen and application |
| CN110423766B (en) * | 2019-08-29 | 2021-06-29 | 南京大学 | Lycopene beta-cyclase gene and coding protein and application thereof |
| CN110656114A (en) * | 2019-10-18 | 2020-01-07 | 云南中烟工业有限责任公司 | Tobacco pigment synthesis related gene and application thereof |
| CN110656114B (en) * | 2019-10-18 | 2022-07-01 | 云南中烟工业有限责任公司 | Tobacco pigment synthesis related gene and application thereof |
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