CN102485897A - Method for changing petal colors by using cotton gene GbF3H - Google Patents
Method for changing petal colors by using cotton gene GbF3H Download PDFInfo
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Abstract
The invention belongs to the technical field of plant genetic engineering. The invention specifically relates to a separated and cloned GbF3H gene. As a result of function verification, the gene is a functional gene which can change the colors of cotton petals. The nucleotide sequence of the gene is represented by SEQ ID No.1 in the sequence list. A coded amino acid sequence is represented by SEQ ID No.2, wherein 368 amino acids are coded. The gene GbF3H is related to plant flavonoid metabolism and anthocyanidin synthesis. When the expression of the gene is inhibited by using RNAi, the anthocyanidin content of cotton petals is substantially reduced, such that the petal color turns lighter. When the gene is over expressed, the anthocyanidin content of cotton petals is increased. With the cloned gene provided by the invention, the colors of plant petals can be changed through conversion. The gene can be used in flower color modification. Also, molecular markers related to flower colors are possible to be developed.
Description
Technical field
The invention belongs to the cotton gene field of engineering technology.Be specifically related to a kind of separation, clone's GbF3H gene, this gene is proved and can changes the cotton petal color.Described gene GbF3H is synthetic relevant with plant flavonoids metabolism and cyanidin(e).Suppress the anthocyanidin content that this expression of gene can significantly reduce cotton petal through RNAi, make pattern thin out; And this gene of overexpression can improve the anthocyanidin content of cotton petal.Utilize gene of the present invention to thirst for changing the color of plant petals, the color of improvement flowers can be developed the molecule marker relevant with pattern simultaneously.
Background technology
The pattern of plant is one of key character of plant, in the entomophila cross-pollinatd plant, plays the effect of inducing pollination insect, and colorful pattern has also increased the sight of plant simultaneously, and pattern still is important selective marker and objective trait in many plant researches.Colourful pattern is that nature is evolved and the artificial result who selects.People have had history for a long time to the research of pattern; Cyanidin(e) be most plants petal color the major decision factor (Grotewold etc.; The Genetics and Biochemistry of Floral Pigments, Annu.Rev.Plant Biol.2006.57:761-80), and cyanidin(e) is a kind of flavonoid class material; Flavonoid is one of secondary substance main in the plant materials (D ' Auria etc.; Thesecondary metabolism of Arabidopsis thaliana:growing like a weed, Curr.Opin.Plant Biol.2005.8,308-316.).Along with biology and CHEMICAL DEVELOPMENT, to the kind of flavonoid, chemical property with and the biological metabolism path very big progress has all been arranged.The biosynthesizing of the cyanidin(e) (Lepiniec etc. that must be perfectly clear after deliberation in some model plants; Genetics and Biochemistry of SeedFlavonoids; Annu.Rev.Plant Biol.2006.57:405-30), many relevant genes are also by clone and checking.F3H (flavanone3-hydroxylase; Flavanone 3-hydroxylase) gene is the key gene (Britsch etc. at the cyanidin(e) biosynthesizing upper reaches; Molecular cloning; Sequence analysis, and in vitro expression of flavanone 3 beta-hydroxylase from Petunia hybrida.J BiolChem.1992.267:5380-5387), its catalysis is by the process of flavanone to flavanonol.Research in other plant shows the disappearance of F3H gene or the phenotype (Martin etc. that inhibition can directly have influence on the plant pattern; Control ofanthocyanin biosynthesis in flowers ofAntirrhinum majus.1991.Plant J.1,37-49.).
Cotton is a kind of important cash crop, also is the wider farm crop of biotechnology applications scope simultaneously.Insect Resistant Cotton through the transformation to cotton gene obtains has been brought a lot of hope and life to traditional agricultural.Biotic component of cotton and genome are all very complicated, also are the comparison difficulties to cotton gene cloning and functional verification.The research of existing several functional genes at present all is to grow relevant (Luo etc. with fiber; GhDET2; Asteroid 5 α-reductase, plays an important role in cotton fiber cell initiation and elongation.2007.PlantJ.51:419-430).And direct and cotton formalness, particularly the clone of pattern genes involved does not also appear in the newspapers.The flavonoid class genes involved all has expression period at cotton fiber development; Research before applicant the present invention proposes just shows that sea island cotton GbF3H gene efficiently expresses period such as (Tu etc. at cotton fiber development; Genes expression analyses of sea-island cotton (Gossypium barbadense L.) during fiberdevelopment; 2007.Plant Cell Rep.26:1309-1320); And in the gene library that upland cotton fiber is grown; Also very high (the Accumulation of genome-specific transcripts such as Yang of F3H expression of gene abundance; Transcription factors and phytohormonalregulators during early stages of fiber cell development in allotetraploid cotton.Plant is J.2006.47:761-75.), F3H also is color cotton pigment synthetic key gene (Cotton flavonoid structural genes related to the pigmentation in brownfibers.Biochemical and Biophysical Research Communications.2007.358:73-78 such as Xiao).The applicant obtains a F3H gene order from the library that fiber is grown before the present invention proposes; Show that through the transgenic checking F3H gene is synthetic relevant with the pattern of cotton, the pattern synthetic research of cotton and the molecular markers development of transgene cotton are all had very important significance.
Summary of the invention
The objective of the invention is dna fragmentation (said in the present invention " dna fragmentation " and " nucleotide sequence " and " dna molecular " synonym that from sea island cotton one of separating clone comprises this functional protein homologous gene complete coding region section; Down together); Utilize this gene can change pattern, this gene can also be used as genetically modified morphological markers.The F3H gene similarity of the protein sequence of this genes encoding being carried out analysis revealed it and Arabidopis thaliana and other plants is very high, therefore is named as the GbF3H gene.
The present invention relates to separate and use a kind of dna fragmentation of the GbF3H of comprising gene, this fragment is given plant cyanidin(e) synthetic ability.Wherein, described GbF3H gene is one of following nucleotide sequences:
1) dna sequence dna shown in the 82-1188 position among the sequence table SEQ NO:1; Or
2) the protein DNA sequence that coding and 1) encoded protein matter is identical.
Can adopt the GbF3H gene of having cloned to make probe, screening obtains gene of the present invention or homologous gene from cDNA and genomic library.Equally, also can adopt PCR (polymerase chain reaction) technology, from genome, mRNA and cDNA amplification obtain GbF3H gene of the present invention and any interested section of DNA or with its homologous section of DNA.Adopt above technology, can separate the sequence that obtains comprising the GbF3H gene, this sequence is connected the back transforms plant with any carrier that can guide foreign gene in plant, to express, can obtain the higher transfer-gen plant of petal anthocyanidin content; And a part of sequence of this sequence is transformed plant as the RNA interference vector that can in plant materials, produce that target makes up, can obtain the plant that anthocyanidin content reduces.Gene of the present invention can add any strong promoter, specific promoter or inducible promoter in being building up to plant expression vector the time before its transcription initiation Nucleotide.Gene of the present invention also can use enhanser in being building up to plant expression vector the time, and these enhanser zones can be ATG initiator codon and neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the translation of whole sequence.Thereby in the gene of the present invention any one section can be as target design based on the plant RNA i carrier of hair fastener ring structure or transform plant based on the plant interference vector of miRNA and suppress this expression of gene and change the content of cyanidin(e) and change pattern.The present invention can be building up in the expression vector of plant through above dual mode, can add that before its transcription initiation Nucleotide the petal specific promoter is used as genetically modified morphological markers.
Carrying GbF3H expression carrier of the present invention or contain its segmental RNAi carrier can be through using Ti-plasmids, plant viral vector, directly DNA conversion; Microinjection, conventional biotechnological means such as electroporation imports vegetable cell (Weissbach, 1998; Method for PlantMolecular Biology VIII; Academy Press, New York, pp.411-463; Geiserson and Corey, 1998, Plant MolecularBiology (2nd Edition).
Can use to comprise that GbF3H expression carrier host transformed of the present invention is to comprise the cotton various plants, cultivate flavonoid and the high plant variety of anthocyanidin content; Can use to comprise that GbF3H gene fragment of the present invention transforms the host cotton as the RNAi carrier of target, can be used for cultivating the cotton variety of different patterns, or as genetically modified morphological markers.Or as application of flowers color improvement gene or the like.
Therefore gene of the present invention is synthetic relevant with flavonoid, can the flavonoid metabolism that gene of the present invention is applied to change cotton be come developing new product variety and the new mark of exploitation.
Below in conjunction with accompanying drawing and embodiment the present invention is further specified.
Description of drawings
What sequence table SEQ ID NO:1 showed is the nucleotide sequence that includes the GbF3H gene of separating clone of the present invention.
Sequence table SEQ ID NO:2 is the aminoacid sequence of GbF3H genes encoding, its 368 amino acid of encoding.
Fig. 1: adopt ClustalW software (public use software) to GbF3H gene sequencing result.Show that 3 sequence homologies are very high in GbF3H and the upland cotton.Gh1:GI 74273629 Sequence source Gossypium hirsutum upland cotton (white cotton); Gh2:GI121755803 Sequence source Gossypium hirsutum upland cotton (brown cotton T586); Gh3:GI 289470637 Sequence sourceGossypium hirsutum upland cotton (green cotton); Gb:GI 119657097 Sequence source Gossypium barbadense sea island cotton; Ipb:GI 4512593 Sequence source Ipomoea batatas (sweet potato) sweet potatoes; Eg:GI 50788697 Sequence sourceEustoma grandiflorum Lisianthus; Nt:GI 164454783 Sequence source Nicotiana tabacum (common tobacco) tobaccos; Ac:GI 219944305 Sequence source Actinidia chinensis Kiwifruits; Cit:GI 4126401 Sequence source Citrus sinensis sweet oranges; Ph:GI 2465434 Sequence source Petunia x hybrida petunias; D1:GI 134039064 Sequence sourceDimocarpus longan longans
Fig. 2: detect the expression level of GbF3H gene in the cotton different tissues with Real-time PCR.Gene all has a certain amount of expression in selected tissue, higher relatively at the expression amount of petal, in bloom back 5 days ovule and fiber, higher expression is arranged also, along with the growth expression amount decline of fiber.Organize successively and be: 1. piece; 2. cotyledon; 3. true leaf; 4. stem; 5. petal; 6.-3DPA ovule; 7.0DPA ovule; 8.5DPA ovule; 9.5DPA fiber; 10.10DPA fiber; 11.15DPA fiber.Respectively organizing GbF3H gene relative expression quantity is that GhUB7 is an internal control gene with reference to (promptly being set at 1) with the root tissue.
Fig. 3: the structure synoptic diagram of overexpression carrier p35s-GbF3H of the present invention.The GbF3H full-length gene is cut the situation after the CaMV35S promotor is inserted in the link reaction through enzyme.
Fig. 4: the structure synoptic diagram of RNAi carrier pHellsgate4-F3Hi of the present invention.GbF3H portion gene fragment (261-707bp) is inserted the pHellsgate4 carrier through recombining reaction.
Fig. 5: transgenic T2 analyzes for cotton; The plant petal color of RNAi is obviously thin out; And overexpression strain system compares not significant the variation with contrast; The cyanidin(e) quantitative analysis show the anthocyanidin content of contrast be RNAi strain system 3-4 doubly, and the anthocyanidin content comparison of overexpression strain system is according to wanting high, but difference is not remarkable.Among the figure: a) the transgenic copy number is analyzed; B) phenotype and the anthocyanidin content analysis of correspondence.GN is the transgenic negative control; Ri1, Ri2, Ri3,3 T2 strains that are respectively RNAi are; Ox1, Ox2, Ox3, the strain that is respectively 3 overexpressions is.
Fig. 6: GN, the gene expression analysis of GbF3H among Ri2, Ri3 and Ox1, the Ox2.Among the figure: GN is the transgenic negative control; Ri2, Ri3 are the T2 strain system of RNAi; Ox1, Ox2 are the strain system of overexpression.
Fig. 7: RNAi plant and color cotton T586 filial generation are analyzed, and transgenic positive plant anthocyanidin content obviously reduces, and pattern shoals.Among the figure: T is the cotton T586 of coloured silk; Ri is RNAi strain system; TF1, TF2, TF3 are T586 and Ri filial generation.
Fig. 8: be the nucleotide sequence of the present invention's GbF3H gene of cloning, underscore partly is the coding region (82-1188bp) of this gene.
Embodiment
Following examples have defined the present invention, and have described the present invention and include the dna fragmentation of GbF3H gene complete coding section and the method for checking GbF3H gene function at separating clone.According to following description and these embodiment; Those skilled in the art can confirm essential characteristic of the present invention; And under the situation that does not depart from spirit and scope of the invention, can make various changes and modification to the present invention, so that it is suitable for different purposes and conditions.
Embodiment 1:GbF3H gene isolation clone and at the expression characterization of cotton different tissues
From the cotton fiber development library of setting up, island, isolate one in the previous work of the applicant's crop genetic improvement National Key Laboratory and (see Genes expression analyses of sea-island cotton (Gossypium barbadense L.) during fiber development.Plant Cell Rep.2007 such as Tu in detail with other plant F3H height homologous EST EST; 26:1309-1320) (see Fig. 1 a) with the evolutionary analysis of other species F3H gene; Bioinformatic analysis shows; This EST comprises a complete ORF; With its called after GbF3H; Its accession number in the NCBI gene pool is DQ912945, and this gene efficiently expresses at the fiber developmental stage, further 3 sequence homologies very high (seeing Fig. 1 b) in analysis revealed GbF3H and the upland cotton.(it is international cotton Sea Island Cotton standard system from sea island cotton 3-79; Can ask for from the germplasm resource bank of Cotton Inst., Chinese Agricultural Academy) extract the total RNA of 0DPA ovule (process for extracting be according to An improved simpleprotocol for isolation of high quality RNA from Gossypium spp.suitable for cDNA library construction.ActaAgronomica Sinica.2005 such as Zhu; 31.1657-1659.); Utilize ThermoScript II SuperscriptIII (available from Invitrogen company; The U.S.) with the synthetic cDNA of its reverse transcription; Reaction conditions is: 65 ℃ of 5min, 50 ℃ of 60min, 70 ℃ of 10min.Amplify the full-length cDNA (1433bp) of GbF3H gene with primers F 3HF (5 ' AGGTTTAAGTTCAGAGTCATAAG3 ') and F3HR (5 ' TAAGGATTGAAGAATAGTTACAG 3 ').The PCR reaction conditions is: 94 ℃ of preparatory sex change 3min; 94 ℃ of 30sec, 56 ℃ of 30sec, 72 ℃ of 2min, 32 circulations; 72 ℃ are extended 10min.The PCR product that amplification is obtained is connected into pGEM-T carrier (available from Promega company, the U.S.), and screening positive clone and order-checking obtain required full length gene cDNA.
With sea island cotton 3-79 is material, extracts the RNA of following 11 different tissues, detects GbF3H expression of gene level with Rea-time PCR.Organize respectively for 11 that choose and be: 1. piece; 2. cotyledon; 3. true leaf; 4. stem; 5. petal; 6.-3DPA ovule; 7.0DPA ovule; 8.5DPA ovule; 9.5DPA fiber; 10.10DPA fiber; 11.15DPA fiber.The cotton method for extracting total RNA is according to (An improved simple protocol for isolation of high quality RNA from Gossypium spp.suitable for cDNA libraryconstruction.ActaAgronomica Sinica.2005 such as above-mentioned Zhu; 31.1657-1659.) document delivered; Handled with DNaseI (available from Promega company) back after RNA extracted and accomplishes, and the RNA integrity is through 1.2% (w/v) agarose gel (EtBr) electrophoresis detection (5V/cm).Being determined on the Beckman DU800 spectrophotometer of nucleic acid concentration carried out.RNA 260/280 ratio is between 1.9 to 2.1, and 260/230 ratio is used for next step analysis greater than 2.0 RNA.CDNA synthetic is to be masterplate with the total RNA of 3 μ g, with 1 μ l, 500 μ g/ml oligo-dT (15) primers (available from Promega company, the U.S.), and 1 μ l10mM dNTP, DEPC-water mixes, and TV is 12 μ l; 65 ℃ of sex change 5min quenchings on ice then; Add 8 μ l again and contain 4 μ l RT buffer; 2 μ l 0.1M dithiothreitol; 40units of
Ribonuclease Inhibitor (Promega); Mixed solution with 200units of SuperscriptIII RT (available from Invitrogen company, the U.S.); 50 ℃ of temperature are bathed synthetic first chain of 1h; Reaction finishes back 75 ℃ of processing 15min makes SuperscriptIII RT inactivation.-20 ℃ of preservations were for use after every part of cDNA was diluted to 300 μ l.With above-mentioned reverse transcription synthetic cDNA is template, with primers F 3Hin-f (5 ' TGGTTTCATCGTCTCCAGCC3 ') and F3Hin-r (5 ' CATGGCCTCTGACAACACCTC3 ') the GbF3H gene is carried out special pcr amplification (the long 204bp of amplified production).(the GenBank number of landing: DQ116441) gene is done specific amplified (the long 198bp of amplified production), to carry out relative quantitative assay as internal reference to cotton GhUbiquitin7 to use primer UB7-f (5 ' GAAGGCATTCCACCTGACCAAC3 ') and UB7-R (5 ' CTTGACCTTCTTCTTCTTGTGCTTG3 ') simultaneously.The quantitative PCR appearance is ABI7500, and quantitative PCR reagent is available from Bio-Rad company.PCR reaction system (20 μ l) comprising: the cDNA (equaling the initial total RNA of 10ng) after the 1 μ l dilution, 10 μ l, 2 * PCR Master Mix, the primer of 200nM.Reaction conditions is: 95 ℃ of 30sec; 95 ℃ of 5sec, 60 ℃ of 35sec, 40 circulations.Carry out the fluoroscopic examination real-time quantitative analysis in the reaction process.The result shows: cloned genes GbF3H of the present invention all has a certain amount of expression in selected tissue; But the expression amount at petal is higher relatively; And in bloom back 5 days ovule and fiber, higher expression is arranged also, along with the growth expression amount decline (see figure 2) of fiber.
Embodiment 2: the structure of overexpression carrier and RNAi and conversion
In order to verify the function of GbF3H gene in cotton, the applicant has made up overexpression and RNAi carrier converting cotton.
According to obtaining GbF3H cDNA design overexpression primer among the embodiment 1; Add restriction enzyme site and the protection base of BamHI and SalI respectively at the primer two ends; Respectively with this primer called after primers F 3Hoef and F3Hoer; CDNA with the GbF3H gene is that template is carried out pcr amplification again, and the method that the PCR product that obtains is cut link through enzyme is connected into overexpression vector p35s-GbF3H, and (intermediate carrier is pCAMBIA2300, and Australian CAMBIA gives in the laboratory; CaMV35S promotor on this carrier is cut on the MCS that connection oppositely is incorporated into pCAMBIA2300 through enzyme, promptly obtain overexpression vector p35s-GbF3H of the present invention).The overexpression carrier p35s-GbF3H that makes up sees (Fig. 2).According to the comparison result of GbF3H gene and upland cotton F3H at homologous region designated rna i primer; Add attB site sequence and the protection base that is used to recombinate respectively at the two ends of primer; Respectively with this primer called after primers F 3Hrif and primers F 3Hrir; Be that template is carried out pcr amplification to obtain the GbF3H gene cDNA among the embodiment 1 again, the product that obtains imports RNAi carrier pHellsgate4 through the BP recombining reaction, and (recombinase is available from Invitrogen company, the U.S.; The RNAi carrier is seen Construct design for efficient such as Wesley; Effective and high-throughput genesilencing in plants.Plant J.2001.27; 581-590.), the RNAi carrier pHellsgate4-F3Hi of structure sees (Fig. 4).
Primer sequence is following:
F3Hoef：5′CTCGGATCCAGGTTTAAGTTCAGAGTCATAAG?3′；
F3Hoer：5′CTGGTCGACTAAGGATTGAAGAATAGTTACAG?3′；
F3Hrif：5′GGGGACAAGTTTGTACAAAAAAGCAGGCTGCTGTTGAGGCTTGTGAGGAT3′；
F3Hrir：5′GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGGTTGAGGGCATTTAG?3′。
The carrier that makes up is transformed agrobacterium strains LBA4404 (Octopine Ti-plasmid deletion mutants of Agrobacteriumtumefaciens with emphasis on the right side of the T-region; Plasmid; 1982; 7:15-29), through agriculture bacillus mediated method for transformation converting cotton, the transformation receptor material that is adopted is YZ-1 again; This material is the material with very high embryo's generating ability that this laboratory is found through a large amount of screenings; The method for transformation of agriculture bacillus mediated converting cotton and program are with reference to efficient transformation system (Identification of a novel elite genotype in vitro culture and genetic transformation of cotton.BiologiaPlantarum, 2006, the 50:519-524 of foundation such as Jin; An efficient grafting system for transgenic plant recovery in cotton (Gossypiumhirsutum L.) .Plant Cell, Tissue and Organ Culture, 85:181-185,2006; Factors affecting stable transformation andplant regeneration during transforming embryogenic callus of Upland cotton (Gossypium hirsutum L.) viaAgrobacterium tumefaciens; Plant Cell; Tissue and Organ Culture, 81:229-237,2005); And done corresponding adjustment and modification, concrete grammar and flow process are following:
1, the cultivation of aseptic seedling
Just cotton strain YZ-1 is (referring to the document of Jin etc. (Identification of a novel elite genotype in vitroculture and genetic transformation ofcotton.Biologia Plantarum; 2006; 50:519-524) cotton seed hulls removes; With 0.1/100 mercuric chloride sterilization 10min, aseptic water washing three times is inoculated on the aseptic seedling substratum that (prescription as follows: 1/2MS macroelement+glucose 15g/L+ vegetable jelly phytagel is (available from sigma company; The U.S.) 2.5g/L), 28 ℃ of dark cultivations 3-6 days.
2. activation of Agrobacterium and preservation
2.1 prepare:
Agrobacterium LBA4404, preparation contains the MGL liquid nutrient medium (prescription: tryptone 5g/L, sodium-chlor 5g/L, MgSO of kantlex 50mg/L
4.7H
2O 0.1g/L, KH
2PO
40.25g/L, N.F,USP MANNITOL 5g/L, glycocoll 1.0g/L transfers pH to 7.0), aseptic triangular flask, LB (solid, liquid) substratum (containing kantlex 50mg/L), sterile glycerol, aseptic rifle head, aseptic 1.5mL centrifuge tube.
2.2 operation:
2.2.1 activation suspends:
Take out in the Ultralow Temperature Freezer preserve bacterial strain the glycerine pipe melting on ice, rule 26.5 ℃ of dark 36-48h that cultivate on the LB plate; Treat to grow in the ware single clearly bacterium colony, picking list bacterium colony is secretly cultivated 36-48h for 26.5 ℃ in other LB plate line; Treating to grow in the ware enough bacterium colonies finishes to cultivate; Scrape the media surface bacterium colony in the MGL substratum in the triangular flask, 27 ℃, 200rpm shake 2h, and the OD value promptly can be used for infecting between 0.5-1.5.
2.2.2 the preservation of bacterial strain:
Picking list bacterium colony is connected to 150rpm the LB liquid nutrient medium in petridish, and 26 ℃ are shaken 48h, is 1: 1 adding 1.5mL centrifuge tube mixing by bacterium liquid and glycerine volume ratio ,-70 ℃ of preservations.
3. contaminate, cultivate altogether:
3.1 prepare:
The dark YZ-1 seedling of cultivating about 5 days young tender stalwartnesses, activated Agrobacterium, sterile petri dish and aseptic filter paper etc.
3.2 operation:
Under the aseptic condition YZ-1 seedling hypocotyl is cut into the long segment of 0.5-1cm with sharp blade; Be transferred in the activated Agrobacterium bacterium liquid, stir, leave standstill 5-10min; Outwell bacterium liquid; Filter paper blots remaining bacterium liquid, blows 5min and makes the surface dry a little, divides to intersperse among (prescription: MS inorganic salt+B in the common culture medium that is lined with filter paper
5Organism+2,4-D 0.1mg/L+KT0.1mg/L+ glucose 30g/L+phytagel2.5g/L, pH7.0), 20 ℃ of dark 38-42h that cultivate.
4. callus induces
To infect hypocotyl segment after the common cultivation is inoculated on the inducing culture (prescription as follows: MS inorganic salt+B
5Organism+2,4-D 0.1mg/L+KT0.1mg/L+ glucose 30g/L+phytagel 2.5g/L, pH 5.8).
5, the propagation of non-embryonic callus tissue
The proliferated culture medium of non-embryonic callus tissue is as follows: MS inorganic salt (saltpetre doubles, and an ammonium nitrate reduces by half)+B
5Organism+2,4-D0.05mg/L+KT0.1mg/L+ glucose 30g/L+phytagel 2.5g//L, pH 5.8.
6, differentiation of calli
Callus through subculture (a month subculture once) several times after, the callus that has changes into grain of rice shape particle, it is changed on the division culture medium over to (prescription: MS minimum medium+B
5Organism+kinetin (KT) 0.15mg/L+ indolebutyric acid (IBA) 0.5mg/L+ glucose 30g/L+phytagel 2.5g/L transfers pH to 5.8), further be divided into embryoid.
7, the subculture of embryo callus
The subculture medium of embryo callus is as follows:
MS inorganic salt (wherein saltpetre doubles, and an ammonium nitrate reduces by half)+B
5Organism+KT0.15mg/L+IBA0.5mg/L+Gln (glutamine) 1.0mg/L+ l-asparagine (Asn) 0.5mg/L+ glucose 30g/L+phytagel2.5g/L, pH 5.8.
8, become seedling rooting to cultivate
The seedling subculture that differentiates is become (prescription: 1/2MS inorganic salt+B on the seedling growth medium in the 1/2MS substratum
5Organism+glucose 15g/L+phytagel 2.5g/L, pH 5.8).
9, acclimatization and transplants
The good seedling of will taking root is opened triangular flask and is sealed film, and refining seedling 2-3 days is transplanted in the little native alms bowl then, and the slow seedling that shades is transplanted the land for growing field crops about one week.
Attach: the preparation of MS substratum mother liquor:
1. various medicines weighing respectively when preparing macroelement, abundant respectively dissolving is added to one by one in the volumetric flask and (adds CaCl at last
2Otherwise be easy to generate deposition), constant volume to a liter.
2. several kinds of denier medicines can be made into one-level mother liquor (concentrating 10000 times) earlier when trace element preparation, and redilution becomes secondary mother liquor (concentrating 100 times), are positioned over after the mother liquor preparation finishes under the room temperature more than the 10h, see if there is deposition and produce, and could use then.
3. when preparing molysite, two kinds of salt dissolve respectively with hot water, mix then, are positioned over to see if there is the deposition generation under the room temperature more than the 10h, could use then.
4. after various tethelin generally can be used NaOH or the HCl dissolving of 1mol/L, constant volume again.
5. prepare B
5To prepare with sterilized water during organism, once not join too big volume, in time use up with anti-pollution
6. should leave in after various mother liquors prepare in 4 ℃ of refrigerators, finding has post precipitation, must not use.
Macroelement (20 times) mother liquors (g/L)
KNO3 38；
NH3NO3 33；
MgSO47H2O (anhydrous MgSO4) 7.4 (3.8);
KH2PO4 3.4；
CaCl22H2O (anhydrous CaCl2) 8.8 (6.6).
Trace element (100 times) mother liquors (g/L)
CoCl2·6H2O 0.0025；
CuSO4·5H2O 0.0025；
H3BO3 0.62；
KI 0.083；
MnSO4·4H2O 2.23；
NaMO4·2H2O 0.025；
ZnSO4·7H2O 0.86。
Molysite (100 times) mother liquors (g/L)
FeSO
4·7H
2O 2.78；
Na2EDTA 3.73。
B
5Organism
VB1 (VitB1) 10mg/L;
VB5 (pyridoxine hydrochloride) 1mg/L;
VB6 (nicotinic acid) 1mg/L;
Inositol 100mg/L;
Glycocoll 2mg/L.
Embodiment 4:GbF3H gene-based changes for the phenotype of family because of overexpression and RNAi transgenic T2
GbF3H gene overexpression and RNAi transgenic are each got 3 T2 for family, and Southern result shows two RNAi and an overexpression family (is seen Fig. 5 a) for single copy.Phenotype is observed it is thus clear that the plant petal color of RNAi is obviously thin out; And overexpression strain system compares not significant the variation with contrast; The cyanidin(e) extraction and analysis has also shown corresponding result; To cyanidin(e) carry out quantitative analysis show the anthocyanidin content of contrast be RNAi strain system 3-4 doubly, and the anthocyanidin content comparison of overexpression strain system is according to wanting high, but difference not significantly (seeing Fig. 5 b).To the T2 family all is the family compound sample of getting, and the result shows that the content of cyanidin(e) is associated with the phenotype of petal.Choose transgenic line and extract RNA, RT-PCR shows that the present invention clone's GbF3H gene expression dose also is (operating identical with the embodiment 3 correlation tests) (see figure 6) of being correlated with the content of cyanidin(e).Experiment shows that the GbF3H gene is one and synthesizes relevant gene with cyanidin(e), through anthocyanidin content and the pattern of genetic manipulation GbF3H gene-based because of changing petal.It is carried out the more remarkable effect that RNAi suppresses, and the GbF3H gene this in petal, efficiently express, so be not very remarkable to the result of its overexpression.
DNA extraction and Southern experiment are with reference to works such as J. Sa nurse Brookers, and Huang Peitang etc. translate, molecular cloning experiment guide (third edition), Science Press, 2002 editions.The extraction of cyanidin(e) and measure (The photoregulation of anthocyaninsynthesis.IX.The photosensitivity of the response in dark and lightgrown tomato seedlings.Plant CellPhysiol.1984 such as Mancinelli, 25:93-105) reported method with reference to Mancinelli etc.To the T2 family all is the family compound sample of getting, and each strain system gets 3-5 individual plant at least.Take by weighing and bloom the same day or bloom one day after petal 0.5g in liquid nitrogen, pulverize with mortar, add the cold acidifying methyl alcohol of 5ml (1%HCl, V/V), in 4 ℃ of vibration extracting 48h.Low-temperature centrifugation is got the 1ml supernatant is cyanidin(e) with the OD value of spectrophotometer (BackmanDU800) detection 530nm relative content value.
The gene constructed RNAi carrier of embodiment 5:GbF3H can be used for the improvement of pattern
Suppress the color that GbF3H gene expression of gene can obviously change petal; (the upland cotton standard is the near isogenic line of TM-1 with the plant of GbF3H gene gene interference and color cotton T586; Have a plurality of marker traits; Ask for from Cotton Inst., Chinese Agricultural Academy germplasm resource for cotton storehouse) hybridization; In the segregating population of filial generation, detect transgenic mark NPTII gene with PCR, DNA extraction is NPT II-f (5 ' CGTAAAGCACGAGGAAGCG 3 ') and NPT II-r (5 ' GGCACAACAGACAATCGGC 3 ') with the primer that embodiment 4 and PCR use.The result shows that the phenotype of transgene cotton positive plant and petal and the content of cyanidin(e) are isolating altogether (Fig. 7), and transgene cotton positive plant anthocyanidin content obviously reduces, and pattern shoals.This shows that the RNAi carrier of GbF3H gene that the present invention clones and structure thereof can be used for the improvement of pattern.
Claims (5)
1. give the GbF3H gene of plant cyanidin(e) synthesis capability, the dna sequence dna of its nucleotide sequence shown in 82-1188 position among the sequence table SEQ ID NO:1; Or coding and 1) the protein DNA sequence that encoded protein matter is identical.
2. give the GbF3H gene of plant cyanidin(e) synthesis capability, its amino acid sequence coded shown in sequence table SEQ ID NO:2, its 368 amino acid of encoding.
3. RNAi expression vector pHellsgate4-F3Hi; It is characterized in that; The 261-707 position dna fragmentation of claim 1 or 2 described GbF3H genes be inserted among the carrier pHellsgate4 through recombining reaction form, its include 261-707 bit base among the sequence table SEQ ID NO:1 to shown in nucleotide sequence.
4. the application of the described gene of claim 1 in regulation and control plant pattern.
5. the application of claim 4 is comprising the application in regulation and control cotton pattern.
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