CN102154314A - Photoinduced cotton anthocyanidin synthesis regulatory gene GhMYBAP (Gossypium hirsutum Anthocyanin Promoting MYB) and application thereof - Google Patents
Photoinduced cotton anthocyanidin synthesis regulatory gene GhMYBAP (Gossypium hirsutum Anthocyanin Promoting MYB) and application thereof Download PDFInfo
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Abstract
The invention discloses a photoinduced anthocyanidin synthesis regulatory gene GhMYBAP (Gossypium hirsutum Anthocyanin Promoting MYB) cloned from cotton. Synthesis and accumulation of anthocyanidin in transgenic cotton and other plants can be promoted by expressing the gene in the plants. By applying the gene and a plant expression vector thereof, plant strains (varieties) with high anthocyanidin content can be bred, and nutritive values and appearance quality of plant products can be improved.
Description
Technical field
The invention belongs to plant genetic engineering field, be specifically related to the synthetic and cumulative gene of a kind of promotion cotton and other plant flowers pigments.The invention still further relates to the polypeptide of this genes encoding, contain this expression carrier, and the application of this gene in promoting the plant flowers pigment to synthesize and accumulating.
Background technology
Cyanidin(e) is the non-photosynthetic pigments of the important plant of a class.It extensively exists in natural plant, is the main pigment to leaf, flower and fruit color.Cyanidin(e) mainly accumulates in the vacuole of vegetable cell, and tool is water-soluble, under the effect of pH or other factors, can make plant present variation from pink to purple.Cyanidin(e) is a kind of important antioxidant simultaneously, is widely used in industries such as health care, food.Just because of this, color is not only most important in the gardening ornamental plant, also is important quality trait in crops such as grain, fiber, vegetables, fruit.Utilize genetic engineering technique, it is synthetic to activate cyanidin(e) in plant, obtains colored plant prod, and wide application prospect is arranged.
The synthetic branch road that belongs to phenylpropyl alcohol alkane approach of the cyanidin(e) of higher plant is a complex biological route of synthesis of being made up of the multistep biochemical reaction from phenylalanine to the final product anthocyanin.Per step reaction is by the enzyme catalysis of different structure genes encoding.The genetics evidence shows, the synthetic R that mainly is subjected to of plant flowers pigment
2R
3-MYB, WD40 and the regulation and control of bHLH three class transcription factors.These transcription factor interactions form complex body and are attached on the structure gene promotor, activate the expression of one or more structure genes in the cyanidin(e) biosynthetic pathway, thereby promote cyanidin(e) in the intravital synthetic and accumulation of plant, make plant organ or tissue colour developing.With respect to bHLH and WD40 albumen, R
2R
3-MYB albumen specificity is stronger, generally regulates and control cyanidin(e) synthetic R
2R
3-MYB albumen only participates in cyanidin(e) synthetic regulation and control, and bHLH and WD40 then may participate in the regulation and control of other physiological processs (as proanthocyanidin synthetic and epidermal hair decision etc.) simultaneously.Therefore, R
2R
3-MYB albumen and encoding gene thereof have the center regulating and controlling effect in the plant flowers pigment is synthetic.
In the early-stage Study, we find that intense light irradiation can inducing cotton blade and the synthetic cyanidin(e) (Fig. 1) of stem.The present invention utilizes the method for homologous clone to clone a R from cotton leaf
2R
3-MYB protein gene (GhMYBAP, Gossypium hirsutum anthocyanin promoting MYB).Expression analysis shows that the GhMYBAP expression of gene is induced by high light, may synthesize relevant (Fig. 1) with photoinduced cyanidin(e) in the cauline leaf.Transgenic research shows that the up-regulated expression of this gene can promote tobacco and the intravital cyanidin(e) of cotton is synthetic and accumulation, makes corresponding organ and tissue show red (Fig. 4,5 and 6).The clone of GhMYBAP gene and functional verification are that the cyanidin(e) genetic engineering regulation of cotton and other plant is laid a good foundation, utilize cotton fiber specific promoter to control might be in the cotton fiber special synthetic and accumulation cyanidin(e) of this expression of gene, thereby create genetically modified colored cotton.
Summary of the invention
An object of the present invention is to provide the synthetic and cumulative gene of a kind of promotion plant flowers pigment.
Another object of the present invention provides said gene encoded protein matter.
A further object of the present invention provides the expression vector that contains said gene.
Further purpose of the present invention provides the application of said gene in agricultural, forestry crop breeding, is used to obtain the vegetable material of tool high-content cyanidin(e), the nutritive value and the exterior quality of improvement plant prod.
According to an aspect of the present invention, the present invention adopts genetic engineering technique, isolate the synthetic and synthetic regulatory gene GhMYBAP of cumulative cotton cyanidin(e) of a kind of promotion plant flowers pigment, derive from cotton (Gossypium hirsutum), it has the dna sequence dna shown in SEQ ID NO.1.
In addition, have 80% above homology, and coding identical function protein DNA sequence also belongs to scope of the present invention with the dna sequence dna shown in the SEQ ID NO.1.
According to a further aspect in the invention, a kind of by said gene GhMYBAP encoded protein matter, it is the aminoacid sequence shown in SEQ ID NO.2.
In addition, with the aminoacid sequence of SEQ ID NO.2 through one or several amino acid whose replacement, disappearance or interpolation and have that aminoacid sequence with SEQ ID NO.2 is identical bioactively also to belong to scope of the present invention by SEQ ID NO.2 deutero-protein.
Dna sequence dna shown in the SEQ ID NO.1 is by 835 based compositions, the protein sequence SEQ ID NO.2 that is made up of 251 amino-acid residues in the code sequence tabulation.
According to a further aspect in the invention, the present invention also provides the recombinant vectors that comprises said gene, and the host cell that comprises above-mentioned recombinant vectors.Described recombinant vectors is the plant expression vector that contains above-mentioned one or more genes and composing type or organizing specific type promotor at least.In one embodiment, the cDNA with SEQ IDNO.1 gene is structured in CaMV35S promotor downstream, the plant expression vector that obtains plant expression vector (its structure as shown in Figure 3) or obtain with organizing specific type promotor replacement CaMV35S promotor.Further, the transformant that obtains by described plant expression vector transfecting host also belongs to scope of the present invention.Plant expression vector of the present invention is utilized methods such as electric shocking method and freeze-thaw method transform the agrobacterium strains that Agrobacterium promptly can obtain containing this expression vector.
The present invention also provides the application in the synthetic and accumulation of described new gene GhMYBAP cyanidin(e) in promoting plant, is used to improve the nutritive value and the exterior quality of plant prod typically.By analysis to transgene tobacco and transgene cotton, find that overexpression GhMYBAP gene can promote the synthetic and accumulation of cyanidin(e), cyanidin(e) content significantly improves, and corresponding histoorgan presents redness (Fig. 4,5 and 6).Therefore may to activate cyanidin(e) in the histoorgan of transgenic plant synthetic for this expression of gene, thereby obtain the vegetable material of tool high-content cyanidin(e), improves the nutritive value and the exterior quality of plant prod.This is significant for cultivating good crop varieties in agricultural, the forestry.
Description of drawings
Fig. 1: the cotton cyanidin(e) synthesizes the photoinduction characteristic with GhMYBAP genetic expression
A, B and C are respectively the variation that colour-change, cyanidin(e) (Anthocyanin) content and the GhMYBAP genetic expression of (L-S) and (L-W) blade that is untreated are handled in the sunlight direct projection.D, E and F are respectively sunlight direct projection (S-S) and cover the variation of colour-change, cyanidin(e) content and the GhMYBAP genetic expression of (S-W) stem section.Detect cyanidin(e) content (Q=(A with colorimetry
530-0.25 * A
657) * M
-1, wherein M is the fresh weight that takes by weighing).Detect relative expression's level (REL, relative expression level) of GhMYBAP gene with quantitative RT-PCR method.
Fig. 2: the structure schema of the cotton GhMYBAP expression vector under the constitutive promoter CaMV35S regulation and control
NPTII: neomycin phosphotransferase gene; GUS: β-gluconic acid glycoside enzyme gene; CaMV 35S: the plant composition promotor that derives from cauliflower mosaic virus; The NosTer:Nos terminator.The skeleton carrier that is used to make up plant expression vector is pBI121, has CaMV 35S promoter regulation and control NPTII gene down, is convenient in the process of plant genetic conversion transformant be carried out the screening of kantlex (Kan) resistance.
Fig. 3: the structural representation of the cotton GhMYBAP expression vector under the constitutive promoter CaMV35S regulation and control
Fig. 4: overexpression GhMYBAP gene promotes that the cyanidin(e) of tobacco is synthetic
A, the blade of wild-type (WT) and GhMYBAP overexpression (AP-5) tobacco; B, the cyanidin(e) content (Q=(A of 4 GhMYBAP overexpression tobacco plants (AP-1 ,-3 ,-5 and-7) and wild-type blade
530-0.25 * A
657) * M
-1, wherein M is the fresh weight that takes by weighing); C, relative expression's level of GhMYBAP gene (REL, relative expression level) in 4 GhMYBAP overexpression tobacco plants (AP-1 ,-3 ,-5 and-7) and the wild-type blade.
Fig. 5: overexpression GhMYBAP gene specific promotes the expression of the structure gene of tobacco cyanidin(e) approach
The relative expression's level (REL, relative expressionlevel) that has compared the cyanidin(e) pathway structure gene in GhMYBAP overexpression (AP-5) and wild-type (WT) tobacco leaf with quantitative RT-PCR method.Discovery belongs to the looking into youngster's ketone synthetic enzyme (CHS), look into youngster's ketone isomerase (CHI) of the synthetic branch road of cyanidin(e), flavonoid 3-hydroxylase (F3H), flavanonol reductase enzyme (DFR) and the significantly rise in the overexpression plant of cyanidin(e) synthetic enzyme (ANS) gene; The total phenylalanine ammonia of phenylpropyl alcohol alkane approach is separated enzyme (PAL) and the expression amount of coumaric acid CoA ligase (4CL) gene in the overexpression plant is lower than wild-type and belong to.
Fig. 6: overexpression GhMYBAP gene promotes that the cyanidin(e) of cotton is synthetic
A, the cotton healing tissue of non-transgenic (WT) and GhMYBAP overexpression (GAP-5); B, relative expression's level (REL, relative expression level) of GhMYBAP gene in 3 GhMYBAP overexpression healing cell systems (GAP-2 ,-3 and-5) and the non-transgenic cotton callus; C, the cyanidin(e) content (Q=(A of 3 GhMYBAP overexpression healing cell systems (GAP-2 ,-3 and-5) and non-transgenic cotton callus
530-0.25 * A
657) * M
-1, wherein M is the callus fresh weight that takes by weighing).
Embodiment
Below in conjunction with accompanying drawing the present invention is further described in detail, but following explanation does not limit the present invention, any to distortion of the present invention and change, only otherwise break away from spirit of the present invention, all should belong to the defined scope of claims of the present invention.
It is common commercially available that reagent chemicals in the example of the present invention is not done being of specifying, and the material method is not done the equal reference " molecular cloning experiment guide " (Sambrook and Russell, 2001) that specifies.
In following example of the present invention, used cotton experiment material is cotton No. 14 (the Gossypiumhirsutum cv Jimian 14) in Ji, from from cotton genetic breeding institute of Agricultural University Of Hebei.Used tobacco experiment material is Nicotiana tabacum cv Samsun, for Southwestern University's biotechnology center is preserved.
The clone of [embodiment one] GhMYBAP gene order
1, the CTAB method is extracted cotton RNA
Choose about 0.5g fresh cotton floral material, in liquid nitrogen, wear into fine powder rapidly, the 10mL centrifuge tube of packing into adds RNA extracting solution [2%CTAB (W/V), the 2%PVP (W/V) of 65 ℃ of preheatings of 4mL, 100mmol/L Tris-HCl (pH8.0), 0.5g/L Spermidine, 2.0mol/L NaCl, 2% mercaptoethanol (V/V, add before using)], put upside down mixing.65 ℃ of water-bath 3～10min, during mixing 2～3 times.The equal-volume chloroform: primary isoamyl alcohol (24: 1) extracting 2 times (12,000r/min, room temperature, 5min).Get supernatant, add 1/4 volume 10mol/L LiCl solution, place 2h for 4 ℃.12,000r/min, 4 ℃ of centrifugal 10min abandon supernatant, with 400 μ L SSTE[1mol/L NaCl, 0.5%SDS (W/V), 10mmol/L Tris-HClpH8.0,1.0mmol/L EDTA] dissolution precipitation.With the saturated phenol of acid (pH 4.5): chloroform: primary isoamyl alcohol (25: 24: 1) and chloroform: each extracting of primary isoamyl alcohol (24: 1) 1 time (12,000r/min, room temperature, 5min).The dehydrated alcohol that adds 2 times of volumes is more than-70 ℃ of refrigerator precipitation 30min.12,000r/min, 4 ℃ of centrifugal 10min abandon supernatant.Precipitation with 70% alcohol rinsing once, and is air-dry.Add the DEPC treating water dissolving of 200 μ L.Detect the quality of RNA with non-sex change agarose gel electrophoresis.
2, the pcr amplification of the synthetic and GhMYBAP gene cDNA sequence of cDNA
The relevant R of cyanidin(e) regulation and control according to plants such as Arabidopis thaliana, petunia, Common Snapdragons
2R
3The proteic homologous sequence design of-MYB degenerated primer MYBAP-D (5 '-AACGATGTTAARAAYTAYTGGAA-3 ', R=A or G, Y=C or T, SEQ ID NO.3).With 3 '-the cDNA 3 '-end sequence of the method amplification cotton GhMYBAP gene of RACE.According to 3 '-two reverse nested primer MYBAP-R1 (5 '-GTCACTTACTATCAA TGAC-3 ' of end sequence design, SEQ ID NO.4) and MYBAP-R2 (5 '-CTGGCTATGGGTTGAACAC-3 ', SEQ ID NO.5), obtain the cDNA 5 '-end sequence of GhMYBAP gene with the amplification of Y-RACE method.Use at last according to ATG upstream sequence design forward primer MYBAP-F (5 '-CACCAAGCTAG CAAGCTAAC-3 ', SEQ IDNO.6), increasing with downstream primer obtains the complete encoding sequence of GhMYBAP gene.
Concrete grammar is as follows:
(1) one chain cDNA is synthetic and 3 '-the RACE amplification
Extracted total RNA the blade of sunlight direct projection processing (seeing embodiment two) from the back side.Getting the total RNA of about 2 μ g blades is template, with degenerated primer MYBAP-D and 3 '-RACE test kit (TaKaRa) provide 3 '-the site primer amplification obtains the cDNA 3 '-end sequence of cotton GhMYBAP gene.Operation steps by 3 '-RACE test kit (TaKaRa) specification sheets carries out.
(2) Y-RACE amplification
Get the total RNA of 20 μ g blades cDNA synthetic agent box (TaKaRa) synthetic double chain cDNA, and with the terminal flush endization of cDNA.Concrete operations are all undertaken by cDNA test kit (TaKaRa) specification sheets.
Get 100 μ mol/L the joint long-chain (5 '-CGGTAGGATCCCGCAGAACGACGGCCAG-3 ', SEQ ID NO.7) and the joint short chain (5 '-pCTGGCCGTCCAAGACGC-3 ', SEQ ID NO.8) each 2 μ L, add 2 μ L, 10 * annealing buffer (1mol/L NaCl, 100mmol/LTris-HCl, 10mmol/L EDTA) and 16 μ L distilled water, 65 ℃ of water-bath 10min, slowly cool to room temperature, promptly obtain joint.
Get joint and double-stranded cDNA sets up following linked system:
10 * T4DNA connects damping fluid 1 μ L
Double-stranded cDNA 2 μ L
T4DNA ligase enzyme 1 μ L
The linked system of supplying volume to 10 μ L with distilled water connects 12h, 70 ℃ of deactivation 10min for 16 ℃.
The amplification the first step is a linear amplification, contains 1 μ L in the 25 μ L systems and connects product, 1 * PCR Buffer, 200 μ mol/L dNTPs, 1.5mmol/L MgCl
2, 200nmol/L special primer MYBAP-R1,1U TaqDNA polysaccharase (when 94 ℃ of pre-sex change, adding).The temperature cycle parameter is 94 ℃ of pre-sex change 5min; 94 ℃, 30s, 56 ℃, 30s, 72 ℃, 2min30s, 40 circulations.Get 1 μ L linear amplification product and carry out the second step index amplification, remove primer and be outside joint primer (5 '-CGGTAGGATCCCGCAGAAC-3 ', SEQ ID NO.9) and the special primer MYBAP-R2, other composition colinearitys increase in the reaction system.After 25～35 circulations of reaction amplification, 72 ℃ are extended 10min.
(3) amplification of full-length cDNA
Contain 1 μ L, one chain cDNA in the 25 μ L systems, 1 * PCR Buffer, 200 μ mol/L dNTPs, 1.5mmol/L MgCl
2, each 200nmol/L primer MYBAP-F and MYBAP-R1,1U Taq archaeal dna polymerase (when 94 ℃ of pre-sex change, adding).The temperature cycle parameter is 94 ℃ of pre-sex change 5min; 94 ℃, 30s, 56 ℃, 30s, 72 ℃, 1min30s, 35 circulations; Last 72 ℃ are extended 10min.
3, amplified fragments reclaims, and connects, and intestinal bacteria transform
(1) electrophoresis and recovery
Amplified production is carried out electrophoretic separation in 1.2% (W/V) sepharose.The purpose band is downcut from gel, reclaim test kit (Bioflux) with DNA and reclaim purifying purpose fragment, recycling step carries out according to the test kit specification sheets.It is quantitative to reclaim fragment electrophoresis on sepharose.
(2) clone and order-checking
The fragment that reclaims is quantitative through agarose gel electrophoresis.Press the test kit specification sheets, fragment transforms with the intestinal bacteria that are connected, connect product of cloning vector by reclaiming, the cultivation and the plasmid enzyme restriction of positive bacterium colony are verified, will reclaim fragment cloning to pMD19-T (TaKaRa) carrier.
The fragment and pMD19-T (TaKaRa) carrier that reclaim are set up following linked system:
10 * T4DNA connects damping fluid 1 μ L
External source connects product D NA fragment 1 μ L
T4DNA ligase enzyme 1 μ L
Supply the linked system of volume to 10 μ L with distilled water
Carrier DNA and external source fragment dna fragmentation mol ratio are 1: 3, and 16 ℃ connect 12h.To connect product transformed into escherichia coli DH5a afterwards.Choose white colony and cultivate, the upgrading granzyme is cut checking.Correct clone sends extra large handsome company and carries out sequencing.The final cDNA sequence (SEQID NO.1) that obtains cotton GhMYBAP gene.
[embodiment two] cotton cyanidin(e) synthesizes the photoinduction Characteristics Detection with GhMYBAP genetic expression
Select unfolded blade just on the cotton plants of normal growth, the leaf back that makes half sheet blade from centre turnover and is fixed with paper clip up, is handled 3 days by sunlight direct projection vacuum side of blade at fine day.Take off blade and carry out cyanidin(e) assay and gene expression analysis with direct projection processing and untreated half sheet leaf respectively.
In the cotton field of field planting, choose top by the relatively cyanidin(e) content and the analysis of GhMYBAP expression of gene of branch of sunlight point-blank branch and bottom concealment.
Adopt the cyanidin(e) content of colorimetric method for determining blade.Get the 0.2g blade, be cut into small pieces and pack in the 1.5mL centrifuge tube, add 1mL acidifying methyl alcohol (1% hydrochloric acid, 80% methyl alcohol), room temperature is extracted 18h to the material redfree.Centrifugal 12000g under the normal temperature, 3min.Get 0.2mL, measure the light absorption value (A under 530nm and the 657nm
530And A
657).Total cyanidin(e) Q=(A
530-0.25 * A
657) * M
-1, wherein M is the fresh weight of the plant tissue that takes by weighing.
Extract total RNA of cotton stem section and blade with CTAB method (seeing embodiment one).Strand cDNA with these total RNA reverse transcriptions is that template is carried out quantitative PCR analysis.Analyze the expression of goal gene in the transfer-gen plant with quantitative RT-PCR method.The GhMYBAP gene increases with primer MYBAP-F (SEQ IDNO.4) and MYBAP-R1 (SEQ ID NO.6), do interior mark with cotton Histone gene, primer be His-up (5 '-GAAGCTGCAGAGGCATACC-3 ', SEQ ID NO.10) and His-down (5 '-CTACCACTACCATCATGGC-3 ', SEQ ID NO.11).Reverse transcription and quantitative PCR analysis all adopt the SYB Green RT-PCR assay kit of TaKaRa company to finish on iCycle quantitative PCR instrument.Respectively go on foot concrete operations by test kit and instrument specification sheets.With the relative expression level of the GhMYBAP gene that records with the ratio value representation goal gene GhMYBAP of the initial copy number of Histone.
Detected result shows that the color of handling the blade of (L-S) through the sunlight direct projection presents redness, the content height of cyanidin(e), relative expression's level height of GhMYBAP gene; And the color of the blade of unprocessed (L-W) is green, and the content of cyanidin(e) is low, relative expression's level of GhMYBAP gene low (Fig. 1, A-C); , the color of handling the stem section of (S-S) through the sunlight direct projection takes on a red color the content height of cyanidin(e), relative expression's level height of GhMYBAP gene fully; And the color that covers the stem section of (S-W) is green fully, and the content of cyanidin(e) is very low, relative expression's level of GhMYBAP gene very low (Fig. 1, D-F).
The structure and the tobacco genetic transformation of [embodiment three] overexpression carrier
1, the structure of overexpression carrier
The pMD19-GhMYBAP carrier makes up when clone GhMYBAP gene, and the GhMYBAP fragment on it checks order.For overexpression GhMYBAP gene in transgenic plant, need GhMYBAP that the gene forward is inserted in the plant expression vector, and start expression with suitable promotor.We have designed vector construction route as shown in Figure 2 according to direction of insertion and the multiple clone site on the plant expression vector (pBI121) and the direction of CaMV 35S promoter of restriction enzyme site on the pMD19-GhMYBAP carrier and GhMYBAP for this reason.According to this plant expression vector construction route, made up the plant expression vector pBI121-GhMYBAP (Fig. 3) of overexpression GhMYBAP gene.
2, tobacco genetic transformation
With electrization the plant expression carrier plasmid that makes up is imported Agrobacterium LBA4404 and tobacco genetic transformation.
With reference to Bio-RAD MicroPulser instruction manual book, above-mentioned carrier is swashed conversion method by electricity import Agrobacterium LBA4404.
Above-mentioned plant expression vector imports tobacco by the method for agriculture bacillus mediated leaf disk infection.Conversion sees Table 1 with substratum and prescription.Concrete grammar is as follows:
Tobacco seed with the sterilization of 1% clorox after, on the MSB minimum medium, sprout, culture condition is 25 ℃, photoperiod of 16hr illumination/8hr dark.The aseptic seedling of robust growth promptly can be used as the conversion explant after about one month.
Adopt agriculture bacillus mediated leaf disc transformation method.The agrobacterium strains that contains plant expression vector is inoculated in liquid YEB, and 28 ℃ of 200rpm shaking table overnight incubation are drawn 1～2mL activation once more in 20～25mL liquid YEB again from the bacterium liquid that has shaken.Treating that bacterium liquid shakes to OD600 is about at 0.8 o'clock, takes out and is diluted to OD with YEB
600Value is 0.05～0.2 standby.
Table 1: Agrobacterium tumefaciens mediated tobacco genetic transformation substratum
MS：Murashige&Skoog，1962；B5：Gamborg，1986
With the healthy and strong blade of aseptic seedling, be cut into the big leaflet dish of about 0.5cm * 0.5cm, put into OD
600Value is in 0.05～0.2 the Agrobacterium bacterium liquid, and after 5min was infected in vibration gently, the bacterium liquid that inclines at once inserted the surface with explant and is covered with on the common substratum of one deck filter paper, 24 ℃ of dark 3d that cultivate.
After cultivation was finished altogether, explant inserted in the screening culture medium and carries out differentiation culture, and per 2～3w subculture once.After regeneration bud occurring, its cutting-out changed in the root media take root.
When root growth to the 2～3cm of resistance seedling is long, clean the agar of root, water planting hardening 2～3d is transplanted to nutrition pot, grows under natural condition.
GhMYBAP expression of gene in [embodiment four] transgene tobacco, cyanidin(e) is synthetic and the detection of related gene expression
Transgene tobacco generally shows as the red plant of different depths.Adopt the cyanidin(e) content (seeing embodiment two) of colorimetric method for determining blade.
Extract total RNA of transgene tobacco and wild-type tobacco blade with CTAB method (seeing embodiment one).Strand cDNA with these total RNA reverse transcriptions is that template is carried out quantitative PCR analysis.Analyze the expression of goal gene in the transfer-gen plant with quantitative RT-PCR method.The GhMYBAP gene increases with primer MYBAP-F (SEQ ID NO.4) and MYBAP-R1 (SEQ ID NO.6), do interior mark with the Actin gene, primer be Actin-up (5 '-GATGGTGTCAGCCACACTGTC-3 ', SEQ ID NO.12) and Actin-down (5 '-ATGCTGCTAGGAGCCAGTGC-3 ', SEQ ID NO.13).Reverse transcription and quantitative PCR analysis all adopt the SYB Green RT-PCR assay kit of TaKaRa company to finish on iCycle quantitative PCR instrument.Respectively go on foot concrete operations by test kit and instrument specification sheets.With the relative expression level of the GhMYBAP gene that records with the ratio value representation goal gene GhMYBAP of the initial copy number of Actin.Detected result shows that the blade of wild-type (WT) tobacco is emerald green, and cyanidin(e) content is very low, and relative expression's level of GhMYBAP gene is very low; And the blade of the tobacco strain of GhMYBAP overexpression presents redness to a certain degree, and cyanidin(e) content has raising (Fig. 4) in various degree.Choose the higher transformant of GhMYBAP gene expression dose (MYBAP-5) and compare, detect the expression level of each tobacco cyanidin(e) synthase gene in the blade with the non-transgenic plant.The gene that detects comprises: phenylalanine ammonia is separated the enzyme gene, and primer is PAL-up (5 '-ATTGAGGTCATCCGTTCTGC-3 ', SEQ ID NO.14) and PAL-down (5 '-ACCGTGTAACGCCTTGTTTC-3 ', SEQ IDNO.15); Coumaric acid CoA ligase gene, primer are 4CL-up (5 '-TCATTGACGAGGATGACGAG-3 ', SEQ ID NO.16) and 4CL-down (5 '-TGGGATGGTTGAGAAGAAGG-3 ', SEQ ID NO.17); Look into youngster's ketone synthase gene, primer is CHS-up (5 '-TTGTTCGAGCTTGTCTCTGC-3 ', SEQ ID NO.18) and CHS-down (5 '-AGCCCAGGAACATCTTTGAG-3 ', SEQ ID NO.19); Look into youngster's ketone isomerase gene, primer is CHI-up (5 '-GTCAGGCCATTGAAAAGCTC-3 ', SEQ ID NO.20) and CHI-down (5 '-CTAATCGTCAATGCCCCAAC-3 ', SEQ ID NO.21); Flavonoid 3-'-hydroxylase gene, primer are F3H-up (5 '-CAAGGCATGTGTGGATATGG-3 ', SEQ ID NO.22) and F3H-down (5 '-TGTGTCGTTTCAGTCCAAGG-3 ', SEQ ID NO.23); Flavanonol reductase gene, primer are DFR-up (5 '-AACCAACAGTCAGGGGAATG-3 ', SEQ ID NO.24) and DFR-down (5 '-TTGGACATCGACAGTTCCAG-3 ', SEQ IDNO.25); Cyanidin(e) synthase gene, primer are ANS-up (5 '-TGGCGTTGAAGCTCATACTG-3 ', SEQ ID NO.26) and ANS-down (5 '-GGAATTAGGCACACACTTTGC-3 ', SEQ ID NO.27).RNA extraction, reverse transcription and quantifying PCR method are the same.Found that and belong to the looking into youngster's ketone synthetic enzyme (CHS), look into youngster's ketone isomerase (CHI) of the synthetic branch road of cyanidin(e), flavonoid 3-hydroxylase (F3H), flavanonol reductase enzyme (DFR) and the significantly rise in the overexpression plant of cyanidin(e) synthetic enzyme (ANS) gene; Separate enzyme (PAL) and the expression amount of coumaric acid CoA ligase (4CL) gene in the overexpression plant than wild-type low (Fig. 5) and belong to the total phenylalanine ammonia of phenylpropyl alcohol alkane approach.
The cyanidin(e) content of [embodiment five] cotton genetic transformation and conversion callus
1, cotton genetic transformation substratum
The cotton genetic transformation is used always to join to support base and fill a prescription and is seen Table 2.
Table 2: cotton genetic transformation substratum
MS：Murashige&Skoog，1962；B5：Gamborg，1986
2, the acquisition of aseptic seedling
Cotton seeds shells, seed benevolence 0.1%HgCl
2(mercuric chloride) sterilization 8-12min, aseptic tap water flushing 5～6 times, aseptic tap water soaks about 30min, is inoculated in the 1/2MSB solid medium 28 ℃ of dark 3～5d that cultivate.Hypocotyl segment is used for agriculture bacillus mediated genetic transformation.
3, Agrobacterium-mediated Transformation
The agrobacterium strains that contains plant expression vector is inoculated in liquid YEB, and 28 ℃ of 200rpm shaking table overnight incubation are drawn 1～2mL activation once more in 20～25mL liquid YEB again from the bacterium liquid that has shaken.Treating that bacterium liquid shakes to OD600 is about at 1 o'clock, takes out 8000rpm, centrifugal 2min.MSB (containing 100 μ mol/L AS) the resuspended thalline of liquid with the original bacteria liquid volume.Resuspended liquid is contaminated the long hypocotyl segment 20min of 3～5mm (vibration gently frequently), blots bacterium liquid.Hypocotyl after the dip-dye is inoculated in common substratum (not place mat aseptic filter paper), and 24 ℃, the dark place is cultivated 2d altogether.
4, callus induces and subculture
The screening of transferring of hypocotyl after cultivating is altogether taken off and is taken off bacterium on the bacterium culture medium and carry out inducing of callus.About 20d subculture once, the hypocotyl section is with the callus subculture.After callus is more, callus can be scraped from hypocotyl, cultivate on the kanamycin-resistant callus tissue subculture medium, per 14～20d subculture once.
5, transform the evaluation and the detection of callus
Overexpression carrier (pBI121-GhMYBAP) converting cotton obtains a plurality of red healing cells, but these callus all can not be converted into embryo callus subculture or Cheng Miao.Therefore we have detected GhMYBAP expression of gene and cyanidin(e) synthetic situation with these red callus.The detection of GhMYBAP expression of gene level and cyanidin(e) Determination on content are with embodiment two.
Detected result shows that non-transgenic (WT) cotton GhMYBAP expression of gene level is very low, and cyanidin(e) content is low, and it is green that callus is; And during GhMYBAP gene overexpression, cyanidin(e) content height obtains red callus, so overexpression GhMYBAP gene promotes the cyanidin(e) synthetic (Fig. 6) of cotton.
Claims (8)
1. an isolating cotton cyanidin(e) synthesizes regulatory gene GhMYBAP, and its nucleotide sequence is shown in SEQID NO.1.
2. by the protein of the described genes encoding of claim 1, its aminoacid sequence is shown in SEQ ID NO.2.
3. contain the described expression carrier of claim 1.
4. expression vector according to claim 3 is characterized in that, described expression vector contains the described gene of claim 1 and plant composing type or tissue-specific promoter at least.
5. expression vector according to claim 3, it has structure as shown in Figure 3.
6. the host cell that contains the described expression vector of claim 3.
7. the application of the described gene of claim 1 in promoting the plant flowers pigment to synthesize and accumulating.
8. the application of the described gene of claim 1 in farm crop, forest genetics.
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| CN 201110020787 CN102154314B (en) | 2011-01-18 | 2011-01-18 | Photoinduced cotton anthocyanidin synthesis regulatory gene GhMYBAP (Gossypium hirsutum Anthocyanin Promoting MYB) and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110452912A (en) * | 2019-08-26 | 2019-11-15 | 西南大学 | Cotton long fibre cance high-expression gene GhLFHE3 and its transgene cotton preparation method and application |
| CN111315764A (en) * | 2019-12-12 | 2020-06-19 | 中国农业科学院生物技术研究所 | Petal purpurin and coding gene thereof |
| CN111826386A (en) * | 2020-07-30 | 2020-10-27 | 西南大学 | Fusion gene for regulating and controlling color development of cotton fibers, expression vector and application thereof |
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| EP1240380A1 (en) * | 1999-12-22 | 2002-09-18 | Unilever N.V. | Method of treating fabrics and apparatus used therein |
| US20030106089A1 (en) * | 1995-06-07 | 2003-06-05 | Mcbride Kevin | Cotton fiber transcriptional factors |
| CN1491962A (en) * | 2002-10-25 | 2004-04-28 | 中国科学院遗传与发育生物学研究所 | Myb transcription factor of cotton and its encoding gene and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030106089A1 (en) * | 1995-06-07 | 2003-06-05 | Mcbride Kevin | Cotton fiber transcriptional factors |
| EP1240380A1 (en) * | 1999-12-22 | 2002-09-18 | Unilever N.V. | Method of treating fabrics and apparatus used therein |
| CN1491962A (en) * | 2002-10-25 | 2004-04-28 | 中国科学院遗传与发育生物学研究所 | Myb transcription factor of cotton and its encoding gene and use |
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| 《TRENDS in Plant Science》 20060630 Laura Serna1 and Cathie Martin Trichomes: different regulatory Trichomes: different regulatory networks lead to convergent structures 274-280 1-8 第11卷, 第6期 2 * |
| 《重庆市生物化学与分子生物学学术会议论文摘要汇编》 20091231 肖月华等 棉花棕色纤维色素的成分分析和合成基因表达 35 1-8 , 2 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110452912A (en) * | 2019-08-26 | 2019-11-15 | 西南大学 | Cotton long fibre cance high-expression gene GhLFHE3 and its transgene cotton preparation method and application |
| CN111315764A (en) * | 2019-12-12 | 2020-06-19 | 中国农业科学院生物技术研究所 | Petal purpurin and coding gene thereof |
| CN111315764B (en) * | 2019-12-12 | 2021-03-16 | 中国农业科学院生物技术研究所 | Petal purpurin and coding gene thereof |
| CN111826386A (en) * | 2020-07-30 | 2020-10-27 | 西南大学 | Fusion gene for regulating and controlling color development of cotton fibers, expression vector and application thereof |
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| CN102154314B (en) | 2013-04-17 |
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