CN109762829A - Petunia gene PhLcyB and its application - Google Patents

Abstract

The present invention provides petunia gene PhLcyB and its application.Present invention successful clone PhLcyB full length gene sequence for the first time, and there is by VIGS technical identification PhLcyB gene the biological function of carotenogenesis in regulation plant (such as petunia) blade.Experiment shows that large area albinism occurs in the plant leaf of PhLcyB gene silencing, and petal does not have significant change.Whitening leaf carries out carotenoid content in piece biochemistry detection discovery PhLcyB gene silencing rear blade and is substantially reduced, it is known that the adjusting of Carotenoid Metabolism plays an important role in PhLcyB gene pairs petunia blade.PhLcyB gene of the invention can be used for the cultivation of new variety of plant, be conducive to the content for improving carotenoid in plant leaf blade.

Description

Petunia gene PhLcyB and its application

Technical field

The present invention relates to biotechnologys and plant genetics and breeding field, specifically, be related to petunia gene PhLcyB and It is applied.

Background technique

Petunia (Petunia Hybrida) is perennial herb, and the biennial cultivation of Chang Zuoyi, Hua Dansheng is funnel-shaped, Hose-in-hose is spherical, is Solanaceae, petunia juss plant.South America Argentina is originated in, in widespread all over the world.By Europe The continuous breeding improvement of the horticulturist of the states such as beauty, Japan, the cultivar of present petunia hundreds of.

In nineteen ninety-five, petunia CHS gene is transferred to plant by Shao Li etc. by way of mediated by agriculture bacillus, and petunia goes out The phenomenon that existing pattern albefaction, and influence the normal fertility of plant.Researcher has found that the growth cycle of petunia is short since then It is suitable for research Flower color heredity and posttranscriptional gene silencing with conditions such as the maturations of regenerating system.From 1995~2018 years, change was accounted for Bravely, many researchers such as Yang Weiyuan, Richard use petunia as the test material of verifying gene function.Such as 2016, Cellulose synthase subunit (Cellulose synthase catalytic is verified with petunia in Yang Wei garden etc. Subunits, CESAs) gene function, it has been investigated that, the plant after being infected occur in phenotype plant become short, stem, flower Stalk and filigree equal diameter are thicker, and some phenomenons of deformity occur in column cap and seed etc..It is raw in plant to verify PhCESA3 gene There is critical role in terms of long development and resistance.

Virus induced gene silencing (Virus-induced gene silencing, VIGS) refers to and carries plant mesh Gene cDNA virus, after infecting plant can induce endogenous gene mRNA occur selective degradation, make the gene send out Gene silencing after raw transcriptional level, corresponding phenotype morph, so as to pass through the change on plant phenotype or physical signs Change the function of reflecting the gene.Compared with the method for conventional genetic conversion, VIGS technology is that one kind removes Large-scale Screening mutation from Body, can the present age complete target gene verifying, have it is easy to operate, be suitable for plant gene function analyze, become function base Because of group an one of technological means for research field most attraction.VIGS technology is widely used in the growth and development, disease-resistant of plant In the functional study of the related genes such as worm, metabolic regulation.VIGS technology is also applied to different types of plant, and such as tobacco is intended Southern mustard, petunia, tomato, soybean, wheat, strawberry etc..

Summary of the invention

The object of the present invention is to provide petunia gene PhLcyB and its applications.

In order to achieve the object of the present invention, the present invention uses petunia for experimental material, and successful clone PhLcyB gene is complete Long sequence, and pass through the VIGS technical identification function of PhLcyB gene.

In a first aspect, the present invention provides petunia gene PhLcyB, which is characterized in that its are as follows:

I) nucleotide sequence shown in SEQ ID NO:1；

Ii) nucleotide sequence shown in SEQ ID NO:1 be substituted, lack and/or increase one or more nucleotide and Express the nucleotide sequence of identical function protein；

Iii) hybridize under strict conditions with sequence shown in SEQ ID NO:1 and express the nucleotide of identical function protein Sequence, the stringent condition are in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C Lower hybridization, and film is washed with the solution；Or

Iv) and i), ii) or nucleotide sequence iii) there is 90% or more homology and express identical function protein Nucleotide sequence.

Second aspect, the present invention provide the biomaterial for containing the gene PhLcyB, and the biomaterial includes but not It is limited to recombinant DNA, expression cassette, transposons, plasmid vector, phage vector, viral vectors, engineering bacteria or non-reproducible plant Part.

The third aspect, the present invention provides the gene PhLcyB or the biomaterial containing the gene PhLcyB is regulating and controlling Application in plant leaf blade in carotenogenesis, wherein the plant includes but is not limited to petunia.

Specifically, the regulation refers to the content for improving carotenoid in plant leaf blade.

In the present invention, carotenoid includes carrotene and lutein.

Fourth aspect, the present invention provides the gene PhLcyB or prepared by the biomaterial containing the gene PhLcyB Application in genetically modified plants.

5th aspect, the present invention provide the gene PhLcyB or the biomaterial containing the gene PhLcyB in plant Application in breeding.

The purpose of the breeding include regulate and control plant leaf blade in carotenoid synthesis, wherein the plant include but It is not limited to petunia.

6th aspect, the present invention provide institute it is a kind of raising plant leaf blade in carotenoid content method, the method Include:

1) making plant includes the gene PhLcyB；Or

2) plant is made to be overexpressed the gene PhLcyB.

Wherein, the plant includes but is not limited to petunia.

Further, the method includes but be not limited to transgenosis, hybridization, backcrossing, selfing or vegetative propagation.

The method for being overexpressed gene PhLcyB can be selected from it is following 1)~4) or optional combination:

1) by importing the plasmid with the gene PhLcyB into plant；

2) pass through the copy number of the gene PhLcyB on increase plant chromosome；

3) by the way that strong promoter is operably connected with the gene PhLcyB；

4) by importing enhancer.

In the present invention, the expression vector for carrying the target gene can be by using Ti-plasmids, plant viral vector, straight Connect in the standard biologics technical methods such as DNA conversion, microinjection, electroporation importing plant cell (Weissbach, 1998, Method For Plant Molecular Biology VIII, Academy Press, New York, the 411-463 pages；Geiserson And Corey, 1998, Plant Molecular Biology, 2^nd Edition)。

By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that

Present invention successful clone PhLcyB full length gene sequence for the first time, and pass through VIGS technical identification PhLcyB gene The biological function of carotenogenesis in regulation plant (such as petunia) blade having.Experiment shows PhLcyB gene There is large area albinism in the plant leaf of silencing, and petal does not have significant change.Whitening leaf carries out the discovery of piece biochemistry detection Carotenoid content is substantially reduced in PhLcyB gene silencing rear blade, it is known that class Hu in PhLcyB gene pairs petunia blade The adjusting of radish element metabolism plays an important role.PhLcyB gene of the invention can be used for the cultivation of new variety of plant, be conducive to mention The content of carotenoid in high plant leaf blade.

Detailed description of the invention

Fig. 1 is the agarose electrophoresis testing result of petunia gene PhLcyB amplified production in present pre-ferred embodiments.

Fig. 2 is the structural schematic diagram of recombinant vector pTRV2-PhLcyB and pTRV2-PhCHS in present pre-ferred embodiments.

Fig. 3 is the recombinant vector verification result that PhCHS, PhLcyB genetic fragment are carried in present pre-ferred embodiments.

Fig. 4 is that the phenotype for the petunia plant infected in present pre-ferred embodiments by VIGS technology compares.

Fig. 5 is carotenoid total content, carrotene and lutein in petunia blade in present pre-ferred embodiments Relative amount/mgg^-1·FW。

Fig. 6 is TRV-PhLcyB gene semi-quantitative analysis knot in petunia transgenic plant in present pre-ferred embodiments Fruit.

Fig. 7 is TRV-PhLcyB gene by fluorescence quantitative expression point in petunia transgenic plant in present pre-ferred embodiments Analyse result.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW, Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.

The clone of embodiment petunia gene PhLcyB and functional analysis

1 materials and methods

1.1 vegetable material

The present invention is with petunia ' rising sun ' for material.Sowing plantation petunia ' rising sun ' in plant growth room, wait grow to 4 Experimental material when piece young leaves, as VIGS functional verification.

1.1.1 petunia Total RNAs extraction and reverse transcription

It is extracted with the EASYspin Plus plant RNA rapidly extracting kit of Beijing Ai Delai Biotechnology Co., Ltd The total serum IgE of plant sample, petunia total serum IgE carry out agarose gel electrophoresis, the results showed that total serum IgE is more complete, can be used for subsequent Experiment.With the TransScript First-Strand cDNA Synthesis SuperMix kit of Beijing Quan Shi King Company Reverse transcription is carried out to RNA and obtains cDNA template.

The clone of 1.2 petunia PhLcyB genes and measurement

According to the Unigene information of LCYB gene in petunia ' rising sun ' transcript profile sequencing result, carried out in GenBank It compares, finds the initial position of LCYB gene.In conjunction with Unigene sequence design specific forward primer Primer PHLCYB- F3 and downstream primer Primer PHLCYB-R3.PCR program: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 45sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 1min, 32 circulations；72 DEG C of extension 10min；4 DEG C of heat preservation.PCR product is detected through agarose gel electrophoresis Afterwards, gel extraction target fragment carries out purpose clone and sequencing.

1.3 vector construction

1.3.1PhLcyB with the clone of PhCHS genetic fragment

The result being sequenced according to the petunia transcript profile library constructed and the PhLcyB gene order cloned, The primer of design amplification PhLcyB and PhCHS gene (PhCHS gene GenBank:X14599.1) segment, when design primer, introduce EcoR I, BamH I restriction enzyme site sequence, primer are shown in Table 1.Pcr amplification product detects through Ago-Gel and recycles purpose item Band, for testing in next step after sequence verification.

1.3.2pTRV2-PhLcyB with the building and verifying of pTRV2-PhCHS recombinant vector

Using Takara QuickCut^TMBamH I and EcoR I to the amplified production of PhLcyB and PhCHS genetic fragment and PTRV2 plasmid carries out double digestion, by double enzyme digestion product respectively after Ago-Gel detects and recycles purpose band, uses Takara T4DNA ligase carries out staying overnight connection, by connection product Transformed E .coli DH5 α, chooses positive colony bacterium solution and carries out Sequence verification, and recombinant plasmid pTRV2-PhLcyB and pTRV2-PhCHS are extracted, carry out PCR amplification verifying.

1.4 infect method

Plasmid pTRV2, recombinant plasmid pTRV2-PhLcyB and pTRV2-PhCHS are converted into Agrobacterium by electroporated method GV3101 is containing the Agrobacterium GV3101 bacterium solution respectively containing pTRV1, pTRV2, pTRV2-PhLcyB and pTRV2-PhCHS 36-48h, picking are cultivated on the LB solid medium for having 50mg/L kanamycins, 50mg/L gentamicin and 50mg/L rifampin Respective single colonie for 24 hours in the LB liquid medium culture that 5ml contains identical type and concentration is transferred to that is mould containing 50mg/L card Element, 50mg/L gentamicin, 200 μM of AS, 10mM MES 200mlLB culture medium in overnight incubation.It is about to bacterium solution OD600 When 0.8,4000rpm be centrifuged 10min, with contain 200 μM of AS, 10mM MES, 10mM MgCl₂Outstanding bacterium solution collect bacterium respectively Liquid.Adjusting each bacterium solution OD600 is 1.0, by pTRV1 bacterium solution and pTRV2 bacterium solution by volume 1:1 mix, pTRV1 bacterium solution with 1:1 is mixed by volume for pTRV2-PhLcyB 1:1 mixing by volume and pTRV2-PhCHS bacterium solution, is protected from light and is stored at room temperature activation It is infected after 4h.The petunia plant for being infected for 4 leaf phases using injection method is rinsed 2 times after infecting with sterile deionized water.Dark treatment It after for 24 hours, is placed in phytotron and cultivates, carry out subsequent experimental use after petunia adult.

1.5 petunia carotenoid total contents, carrotene and lutein content measurement

Using organic solvent method measurement petunia carotenoid total content, carrotene and lutein in the present embodiment Content.It is repeated by biology of 5 samples, after grinding, accurately weighs 0.25g plant sample, be rapidly added 25ml acetone: Dehydrated alcohol is the mixed liquor of 1:1 (volume ratio), and piping and druming mixes, and 4h is kept in dark place.With acetone: dehydrated alcohol is 1:1 (volume Than) mixed liquor be blank control, using D30 nucleic acid-protein analyzer, in A_{(light absorption value)}Program under measurement wavelength be 470,474, 485, the light absorption value of 642.5,649,665nm.

Pass through following formula:

Chlorophyll a (mg/L)=9.99A₍₆₆₅₎-0.0872A_(642.5)

Chlorophyll b (mg/L)=17.7A_(642.5)-3.04A₍₆₆₅₎

Carotenoid total content (mg/L)=4.92A₍₄₇₄₎-0.0255[a]-0.225[b]

Carrotene (mg/L)=12.6A₍₄₈₅₎-6.00A₍₄₇₀₎-0.0298[a]+0.336[b]

Lutein (mg/L)=10.2A₍₄₇₀₎-11.5A₍₄₈₅₎-0.0036[a]-0.652[b]

The concentration of chlorophyll a and chlorophyll b uses [a], [b] to indicate respectively, calculates as follows:

Chlorophyll a (mgg^-1FW)=chlorophyll a (mg/L) × extracting solution total volume (L)/sample fresh weight (g)

Chlorophyll b (mgg^-1FW)=chlorophyll b (mg/L) × extracting solution total volume (L)/sample fresh weight (g)

Carotenoid total content (mgg^-1FW)=total carotinoid (mg/L) × extracting solution total volume (L)/sample Product fresh weight (g)

Carotene carotene content (mgg^-1FW)=carrotene (mg/L) × extracting solution total volume (L)/sample fresh weight (g)

Lutein content (mgg^-1FW)=lutein (mg/L) × extracting solution total volume (L)/sample fresh weight (g)

The expression status analysis of 1.6 petunia PhLcyB genes

1.6.1 the semiquantitive PCR expression analysis of petunia PhLcyB gene

Phenotypic character observation statistics is carried out to the petunia plant of adult.According to the petunia PhLcyB gene sequence expanded Column as a result, design petunia PhLcyB gene special primer, and according to TRV1 sequence design expand TRV1 required for Primer (table 1) expands to PhLcyB and TRV1 and carries out semi-quantitative expressed analysis, the use of 26S is reference gene.

PCR system (25 μ L): 1 μ L of cDNA template, 1 μ L of upstream primer (10 μM), downstream primer (10 μM) 1 μ L, 2 × Taq PCR Master Mix 12.5 μ L, ddH₂O is mended to 25 μ L.

PCR program: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 40s, anneal 55 DEG C of 30s, extends 72 DEG C of 40s, 32 circulations； Extend 72 DEG C of 10min；4 DEG C of heat preservation.Relative expression quantity is analyzed to 1% agarose gel electrophoresis method for detecting of PCR product.

1.6.2 the fluorescent quantitation expression analysis (qRT-PCR) of petunia PhLcyB gene

Drawn according to the petunia PhLcyB full length gene sequence design fluorescent quantitation expression analysis (qRT-PCR) expanded Object is analyzed using qRT-PCR, using 26S as reference gene, using 2^-ΔΔCTMethod, primer information are shown in Table 1.

1 primer information of table

2 results and analysis

The clone of 2.1 petunia PhLCYB genes and sequence are analyzed

RNA is extracted by experimental material of petunia blade, clone obtains the overall length 1662bp of PhLcyB gene.PhLcyB base The agarose electrophoresis testing result of gene-amplification product is shown in Fig. 1.

2.2 building TRV recombinant vectors and qualification result

According to experimental design building pTRV2-PHLcyB and pTRV2-PHCHS recombinant vector schematic diagram (Fig. 2, wherein The nucleic acid sequence of the Insert Fragment PhCHS of the Insert Fragment PhLcyB and pTRV2-PHCHS of pTRV2-PHLcyB are respectively such as SEQ Shown in ID NO:2 and 3).After pTRV2-PHLcyB and pTRV2-PHCHS recombinant vector Transformed E .coli DH5 α, extract positive The bacterium solution plasmid of clone uses TRV2 carrier design primer using pTRV2-PHLcyB and pTRV2-PHCHS recombinant plasmid as template PCR amplification is verified.(Fig. 3) is detected through agarose gel electrophoresis, Fig. 3 shows that purpose band is about 722bp and 532bp, symbol Close target, it was demonstrated that the success of pTRV2-PHLcyB and pTRV2-PHCHS construction of recombinant vector.

The phenotype of the 2.3 petunia plant infected by VIGS system compares

The petunia plant for infecting for 4 leaf phases using injection method, as plant grows up gradually after infecting, character mutation is also gradually Show (Fig. 4), for TRV-PhLcyB plant in the maturity period, the albinism of large area occurs in blade, and obviously becoming does not occur in petal Change；There is not significant change in positive control TRV-PhCHS plant leaf, and large area albinism occurs in petal；It is negative right Do not occur according to TRV empty vector control plant and blank control (Mock control, i.e. wild type) plant compared to leaf and Hua Jun Apparent variation.

The measurement of 2.4 carotenoid contents

It is planted with the petunia of petunia Mock control (blank control) plant leaf, TRV empty carrier (negative control) dip dyeing The petunia plant leaf that strain blade, TRV-PhLcyB disseminate is experimental material, measures carotenoid total content, carrotene With the relative amount (Fig. 5) of lutein, show carotenoid total content, carrot in the blade of TRV-PhLcyB dip dyeing plant The plant that the relative amount of element and lutein is significantly lower than Mock adjoining tree, TRV empty carrier is disseminated, and Mock adjoining tree It is differed not with the relative amount of carotenoid total content, carrotene and lutein in the plant leaf of TRV empty carrier dip dyeing Greatly.

The quantitative expression analysis of 2.5 petunia PhLcyB genes

With TRV-PhLcyB, the blade of TRV empty vector control and the mature plant of Mock control is experimental material, extracts RNA Carry out semiquantitive PCR and qRT-PCR analysis.Fig. 6 semiquantitive PCR as the result is shown plant by TRV-PhLcyB plant, TRV empty vector control Strain detects TRV1 carrier, shows that Agrobacterium infects success.PhLcyB gene expression amount is obviously low in TRV-PhLcyB plant The PhLcyB gene expression amount in TRV empty vector control and Mock adjoining tree.Fig. 7 qRT-PCR analyzes TRV- as the result is shown PhLcyB gene expression amount is significantly lower than PhLcyB gene table in TRV empty vector control and Mock adjoining tree in PhLcyB plant Up to amount, this is consistent with semiquantitive PCR result.And PhLcyB gene expression amount phase in TRV empty vector control and Mock adjoining tree Than there is not significant change.

The successful clone of the present invention full length sequence of PhLcyB gene constructs TRV-PhLcyB and TRV-PhCHS recombination and carries Body infects petunia by injection method using VIGS technology.The function of PhLcyB gene is demonstrated, it can be with from phenotype Find out that large area albinism occurs in the plant leaf of PhLcyB gene silencing, petal does not have significant change.Whitening leaf carries out Carotenoid content is substantially reduced in piece biochemistry detection discovery PhLcyB gene silencing rear blade, it is known that PhLcyB gene pairs is short The adjusting of Carotenoid Metabolism in blade of leading a cow plays an important role.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Sequence table

<110>Beijing Agricultural College

<120>petunia gene PhLcyB and its application

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<212> DNA

<213>petunia (Petunia Hybrida)

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gttccctttg tcattatctc tattctagaa gtatttgaag cttgagagaa aagggacagc 240

ccaaaaaata taagatcggg aagaaacagc cgagatgaca agaagccatg ccaataacga 300

ggttctagat caaagaaggc atcgaaaaac cttctcgtag caggcaagtc aagcttcaga 360

aggatatcca taccaaagca aaagaattct ctttgacgtc tccgttctat gggccacaag 420

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tgaattattg cattagcaac aataggagct gcagctaggg tccttgctac catataacca 540

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ggaagggggc ctcccattgg aattacacaa cgttcatcct cttcaatgct cttaattttt 660

atacccaagt gatctaaacg agccaccatt ctttcttgaa tatcgtccat acgtaaacca 720

ggacgagcta caagggaggt ttcttcaaga aatattctgt ttgatgaaaa tggcattgca 780

tataaaaaag ttggaatttt actatttctt tccttcagct ccatgttatt atcaagatgc 840

gaatctcgcc aatccatgaa aaccatctta tttgtatcaa agggatgctc ctccacttct 900

gccaaaatac cataagctac ttgataccca ggattatatg gcttgtcata ctgaacaaga 960

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gaccatgtag cgtcgaggca atccaacaaa tccatagcct caaattcatc cacccaaaca 1260

ccatagctat taggccatat caatttaggc gacgggtcaa tcgaacaaac cgacagtcca 1320

gcctcggata cctgctgagc aacacagcaa gacctgcagg tccaccacca accacagcta 1380

aatctacaac aacccctttt gaagggtcat acataggaag ctcaaaatca agattttcct 1440

ttttagtctc aggtacaagc tccaaaagtg tactactccc tgccataaca caaacccttt 1500

taccccaatt ttcacaaaat tttcttgaac caaacttctg aggcttcaca tagctaaaag 1560

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atgcacttct gcatcatttt agacttcagt tgcttcctat taacccttcc ataaggccta 240

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aggcaatcca acaaatccat agcctcaaat tcatccaccc aaacaccata gctattaggc 360

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tttattttgg atgaaatgag aaaggcttca tcaaaagaag ggcttggtac cactggtgaa 240

ggtcttgaat g 251

Claims (10)

1. petunia gene PhLcyB, which is characterized in that its are as follows:

I) nucleotide sequence shown in SEQ ID NO:1；

Ii) nucleotide sequence shown in SEQ ID NO:1 is substituted, lacks and/or increases one or more nucleotide and expression The nucleotide sequence of identical function protein；

Iii) hybridize under strict conditions with sequence shown in SEQ ID NO:1 and express the nucleotides sequence of identical function protein Column, the stringent condition are in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C Hybridization, and film is washed with the solution；Or

Iv) and i), ii) or nucleotide sequence iii) there is 90% or more homology and express the nucleosides of identical function protein Acid sequence.

2. the biomaterial containing gene PhLcyB described in claim 1, the biomaterial includes recombinant DNA, expression cassette, turns Stand, plasmid vector, phage vector, viral vectors, engineering bacteria or non-reproducible plant part.

3. the carotenoids in regulation plant leaf blade of biomaterial described in gene PhLcyB or claim 2 described in claim 1 Application in element synthesis, wherein the plant includes petunia.

4. application according to claim 3, which is characterized in that the regulation, which refers to, improves carotenoid in plant leaf blade Content.

5. application of the biomaterial in prepare transgenosis plant described in gene PhLcyB or claim 2 described in claim 1.

6. application of the biomaterial in plant breeding described in gene PhLcyB or claim 2 described in claim 1.

7. application according to claim 6, which is characterized in that the purpose of the breeding includes class Hu in regulation plant leaf blade The synthesis of radish element, wherein the plant includes petunia.

8. a kind of method for improving carotenoid content in plant leaf blade, which is characterized in that the described method includes:

1) making plant includes gene PhLcyB described in claim 1；Or

2) plant is made to be overexpressed gene PhLcyB described in claim 1；

Wherein, the plant includes petunia.

9. according to the method described in claim 8, it is characterized in that, the method includes transgenosis, hybridization, backcrossing, selfing or Vegetative propagation.

10. according to the method described in claim 8, it is characterized in that, be overexpressed gene PhLcyB method be selected from it is following 1)~ Or optional combination 4):

1) by importing the plasmid with the gene PhLcyB into plant；

2) pass through the copy number of the gene PhLcyB on increase plant chromosome；

3) by the way that strong promoter is operably connected with the gene PhLcyB；

4) by importing enhancer.