CN103269578A - Ibor-Ins gene mutant from ipomoea batatas and uses thereof - Google Patents
Ibor-Ins gene mutant from ipomoea batatas and uses thereof Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
Abstract
An IbOr-Ins mutant gene having specific nucleotide sequences inserted into IbOrange gene is provided to enhance carotenoid content and to develop a transgenic plant with salt stress tolerance. An IbOr-Ins mutant gene is prepared by inserting a specific nucleotide sequence into Ipomoea batatas-derived IbOrange gene and has a base of sequence number 2. A method for producing a transgenic plant with enhanced carotenoid content comprises a transforming a recombinant vector containing IbOr-Ins mutant gene into plant cells and overexpressing IbOr-Ins mutant gene. A composition for enhancing carotenoid content of a plant contains the IbOr-Ins mutant gene.
Description
Technical field
The present invention relates to IbOr-Ins gene mutation body that is derived from sweet potato and uses thereof, relate to the IbOr-Ins gene mutation body that in the IbOrange gene that is derived from sweet potato (Ipomoea batatas), inserts specific base sequence artificially in more detail; The recombinant vector that contains described gene mutation body; Transform the host cell of described recombinant vector; Described recombinant vector is transformed in the plant cell, thereby makes the preparation method of the conversion plant corpus that carotenoid content or salt stress patience obtains increasing; The conversion plant corpus and the seed thereof that are obtained increasing by carotenoid content or the salt stress patience of described method preparation; Utilization is derived from the preparation method of the conversion plant corpus that the salt stress patience of the IbOrange gene of sweet potato obtains increasing; Conversion plant corpus and seed thereof that the salt stress patience that is prepared by described method obtains increasing.
Background technology
Sweet potato (Ipomoea batatas L.Lam) not only can be cultivated in barrenr soil, and output per hectare is about 30 tons, is a kind of representative root crop that is used as grain and feed stripped.Coloured sweet potato such as purple, yellow contains multiple polyphenoils, particularly be the carotin that yellow yellow sweet potato contains 14.7～20mg/100g, and purple sweep potato contains the anthocyan about 2.28g/100g, it can promote the aging of cell, and can remove the active oxygen as the various adult diseases causes of disease, have excellent antioxidant effect in vivo.In addition, after generation nineteen ninety, sweet potato has been attempted working in coordination with the conversion of cultivating by Agrobacterium (Agrobacterium), take place by top and lateral bud meristem inducing embryo generation cultured cell and somatic embryo, make plant corpus break up again conversion system developed (people such as Lim. " molecular breeding " 19,227-239,2007, (Lim et al.Mol Breeding19,227-239,2007)).But also there is not the relevant report to the sweet potato of little molecule polyphenoils such as a large amount of production carotenoid, anthocyan, polyphenol.Think that thus develop a kind ofly when having compound stress tolerance, the crop that can also produce little molecule polyphenoils in a large number can be used for solving grain, the energy and the environmental problem that the 21 century mankind face.
The World Health Organization (WHO) has announced that there are 100,000,000 children in the whole world owing to the A that is deficient in vitamin suffers hardships, and annual thus have the children more than 500,000 blind.Carotin is as the precursor of vitamin A, and it has the physiological active functions of nutrition fortifier and food adjuvant, we can say that therefore learning research about the metabolic engineering of accumulating of carotin in the food is to be worth necessary research object for improving alimentology.By these effort, learning direction such as applications to the molecular biology of carotenoid metabolic pathway related gene and metabolic engineering the nutritive value aspect of raising crop is being studied.
Known class carotin is as the material with high anti-oxidation activity, not only play physiological active functions, and also play an important role for the defense mechanism of the oxidative stress of plant self, there is report to point out that the biosynthesis of carotenoid is subjected to the influence of the abscisic acid (ABA) of one of plant hormone, therefore need study these polyphenoils and environment-stress recently.A lot of regions make the loss in productivity of crop reach more than 70% because of the high salt of soil in the world, or becoming can't farming barren land.Therefore, the exploitation of salt resistance kind is present urgent problem.
Disclose a kind of method that increases carotenoid content in Korean granted patent 10-0813284 number, it is that the phytoene biosynthetic enzyme genes that will be derived from oranges and tangerines is transformed in the plant corpus, thereby increases carotenoid content; Disclose a kind of method that increases salt stress patience among the Korea S publication 2010-0100097, its be will be derived from sweet potato the MuS1 genetic transformation in plant corpus, thereby increase salt stress patience.
Summary of the invention
The technical problem that solves
The present invention finishes according to above-mentioned requirements, the IbOr-Ins gene mutation body that the inventor will be derived from the IbOrange gene of sweet potato and insert specific base sequence artificially in said gene is transformed in the plant corpus, and by confirming that carotenoid content and salt stress patience obtain increasing in described plant corpus, thereby finished the present invention.
Technical scheme
In order to solve above-mentioned problem, the invention provides a kind of IbOr-Ins gene mutation body that in being derived from the IbOrange gene of sweet potato, inserts specific base sequence artificially.
In addition, the invention provides a kind of recombinant vector that contains the said gene mutant.
In addition, the invention provides a kind of host cell that transforms above-mentioned recombinant vector.
In addition, the invention provides and a kind of above-mentioned recombinant vector is transformed in the plant cell, thereby make the preparation method of the conversion plant corpus that carotenoid content or salt stress patience obtains increasing.
In addition, the invention provides conversion plant corpus and the seed thereof that a kind of carotenoid content by method for preparing or salt stress patience obtain increasing.
In addition, the invention provides the preparation method of the conversion plant corpus that salt stress patience that a kind of utilization is derived from the IbOrange gene of sweet potato obtains increasing.
In addition, the invention provides a kind of conversion plant corpus and seed thereof that is obtained increasing by the salt stress patience of method for preparing.
In addition, the invention provides the composition of a kind of carotenoid content for increasing plant corpus or salt stress patience, it contains the IbOr-Ins gene mutation body that inserts specific base sequence in being derived from the IbOrange gene of sweet potato artificially.
In addition, the invention provides a kind of composition of the salt stress patience for increasing plant corpus, it contains the IbOrange gene that is derived from sweet potato.
Beneficial effect
According to the present invention, in the transformed plant cells that transforms IbOrange gene and IbOr-Ins gene mutation body, carotenoid content and salt stress patience obtain increasing, and this effect is more outstanding in the IbOr-Ins transformant.Therefore, utilize IbOrange gene of the present invention and IbOr-Ins gene mutation body, not only can improve the functional of conversion plant corpus, and can expect to develop the strong conversion plant corpus of salt stress patience.
Description of drawings
The IbOrange that Fig. 1 represents to be derived from sweet potato (kind: " Xin Huangmei ") be blank (
), the base sequence that carries out the IbOr-Ins gene of artificial mutation reaches the amino acid sequence of inferring thus.Part with box indicating is the part different with existing IbOrange, has namely inserted the part of the amino acid whose specific base sequence of preparation-KSQNPNL-, 307 amino acid of cDNA coding of whole 924bp.
Fig. 2 represents that the amino acid sequence to the amino acid sequence that is derived from sweet potato IbOrange and IbOr-Ins compares and confirms.Part with box indicating is the part with difference.
Fig. 3 represents to be derived from the plant expression vector of sweet potato IbOrange gene and IbOr-Ins gene.
Fig. 4 transforms the sweet potato cultured cell that is derived from sweet potato IbOrange gene and IbOr-Ins gene because the increase of carotenoid content shows yellow phenotypic result.
Fig. 5 is that to transform cultured cell be object, analyzes the result that carotenoid contents such as carotin are analyzed by HPLC.
Fig. 6 is derived from the sweet potato cultured cell of sweet potato IbOrange gene and IbOr-Ins gene in conversion, with the expression status of the oligogene of carotenoid biosynthesis pathway, by implementing the result that RT-PCR and electrophoresis are represented.
Fig. 7 represents with 150 and after the conversion of NaCl of the 200mM sweet potato cultured cell that is derived from sweet potato IbOrange gene and IbOr-Ins gene handles, the phenotype that cultured cell is dyeed with diaminobenzidine (DAB) (on) and the suffered oxidative stress (descending) of cultured cell.
Fig. 8 represent to transform the arabidopsis that is derived from sweet potato IbOrange gene and IbOr-Ins gene the seed phenotype (on) and with the carotene carotene content in the leaf of spectrophotometric determination (descending).WT: wild type (Col-0).
Fig. 9 is derived from the orange gene of carotenoid biosynthesis related genes, arabidopsis in the arabidopsis of sweet potato IbOrange gene and IbOr-Ins gene and IbOrange expression of gene state by implementing the result that RT-PCR and electrophoresis are represented with conversion.
Figure 10 is in the arabidopsis that transforms IbOrange gene and IbOr-Ins gene, will be as the expression status of the NCED of one of stress-inducing gene by implementing the result that RT-PCR and electrophoresis are represented.
The arabidopsis that Figure 11 represents to transform IbOrange gene and IbOr-Ins gene respectively with 0,50,100 and the medium handled of the NaCl of 150mM in germination rate.
Figure 12 is expression when handling with the NaCl of multiple concentration, in contrast the live body weight of the arabidopsis of Zu wild type and conversion IbOrange and IbOr-Ins.
Figure 13 is illustrated under the 100mM NaCl condition, the result that the phenotype of the root of the arabidopsis that transforms IbOrange and IbOr-Ins gene and the wild type organized is in contrast compared.
Figure 14 is illustrated under the NaCl condition of 150mM, the result that the relative moisture of the arabidopsis that transforms IbOrange and IbOr-Ins gene and the wild type organized is in contrast measured.
Embodiment
In order to realize purpose of the present invention, the invention provides a kind of IbOr-Ins gene mutation body that in being derived from the IbOrange gene of sweet potato, inserts specific base sequence artificially, described IbOr-Ins gene mutation body is made of the base sequence of SEQ ID No.2.In the present invention, though prepare the IbOr-Ins gene mutation body by inserting KSQNPNL amino acid, so long as can increase the mutant of carotenoid content or salt resistance, just be not particularly limited.
In addition, the invention provides a kind of recombinant vector that contains described gene mutation body.
Term " reorganization " refers to the cellular replication heterologous nucleic acid or expresses above-mentioned nucleic acid or express cell by peptide, xenogenesis peptide or heterologous nucleic acid encoded protein matter.Recombinant cell can will be in the natural form of above-mentioned cell undiscovered gene or gene fragment expression be in sense primer or the antisense primer form one.And recombinant cell can be expressed the gene of finding from the cell of native state, but said gene deforms, and is to be directed into intracellular gene again by artificial means.
Above-mentioned IbOr-Ins gene mutation body sequence of the present invention can be inserted in the recombinant expression carrier.Term " recombinant expression carrier " refers to bacterial plasmid, phage, saccharomycete plasmid, plant cell virus, mammalian cell virus or other carrier.Substantially, plasmid and carrier all can use as long as can copy and stabilisation in the host arbitrarily.The key property of above-mentioned expression vector is to have replication origin, promotor, marker gene and translation control module (translation control element).
Comprise IbOr-Ins protein-DNA sequences encoding and suitable transcribing/the translate expression vector of conditioning signal by well known to a person skilled in the art that method can make up.Said method comprises in vitro recombinant DNA technology, DNA synthetic technology and recombinant technique etc. in vivo.In order to guide the synthetic of mRNA, above-mentioned dna sequence dna can be connected on the interior suitable promotor of expression vector effectively.And expression vector can comprise as the ribosome binding site at translation initiation position and transcription terminator.
The preference of recombinant vector of the present invention is the Ti-plasmid vector, when it is present in the suitable hosts such as Agrobacterium tumefaciems, can be with the part of himself, and so-called T-zone-transfer is in plant cell.The Ti-plasmid vector of other type (with reference to EP0116718B1 number) is used for the hybrid DNA sequence is transferred to plant cell or protoplast at present, described protoplast can be by suitably being inserted into hybrid DNA in the genome of plant, thereby produce new plant.The most preferred form of Ti-plasmid vector is claimed so-called double base (binary) carrier in No. the 4th, 940,838, EP0120516B1 number and the United States Patent (USP).DNA of the present invention can be imported to other carrier that is fit in the plant host can be selected from and (for example be derived from double-stranded plant virus, cauliflower mosaic virus (CaMV)) and the viral vectors of strand virus, Geminivirus etc., for example can be selected from non-integrality plant viral vector.The use of these carriers is especially favourable when being difficult to suitably transform plant host.
Expression vector preferably contains more than one selected marker.Above-mentioned mark comprises all genes that transformant can be distinguished as the nucleotide sequence of the characteristic with the enough conventional chemical method screenings of energy from non-transformed cell.Glyphosate (glyphosate) or careless fourth phosphine herbicide resistance genes such as (phosphinothricin), kanamycin (kanamycin), G418, bleomycin (Bleomycin), hygromycin (hygromycin), chloramphenicol antibiotic resistance genes such as (chloramphenicol) are for example arranged, but be not limited to this.
In recombinant vector of the present invention, promotor can be CaMV35S, actin, ubiquitin, pEMU, MAS or histone promotor, but is not limited thereto.Term " promotor " refers to start from the dna upstream zone of structural gene, is that RNA polymerase is transcribed and the dna molecular of combination in order to start." plant promoter " refers to start the promotor of transcribing in plant cell." composing type (constitutive) promotor " refers to tool promoters active under most environmental condition and growth conditions or cell differentiation condition.Because the screening of transformant can be in each stage, form by various tissues, so the preferred constitutive promoter of the present invention.And, do not limit the selection possibility of constitutive promoter.
In recombinant vector of the present invention, can use normally used terminator, terminator of octopine (Octopine) gene of nopaline synthase (NOS), paddy rice α-Dian Fenmei RAmy1A terminator, phaseoline (phaseoline) terminator, Agrobacterium tumefaciems (Agrobacterium tumefaciens) etc. for example, but be not limited to this.About the necessity of terminator, be generally considered to be these zones and can promote certainty and the efficient of transcribing in the plant cell.Therefore, it is fit closely using terminator in content of the present invention.
In addition, the invention provides a kind of host cell that transforms recombinant vector of the present invention.Can be stable in the prokaryotic, and clone and express the host cell of carrier of the present invention serially, can use any host cell well known in the art, for example Bacillus strains such as Escherichia coli (E.coli) JM109, e. coli bl21, Escherichia coli RR1, Escherichia coli LE392, Escherichia coli B, Escherichia coli X1776, Escherichia coli W3110, Bacillus subtillis, thuringiensis can also be enterobacteriaceae bacterial strains such as salmonella typhimurium, serratia marcescens and multiple pseudomonas etc.
In addition, carrier of the present invention is transformed under the eukaryotic situation, can use yeast (saccharomyces cerevisiae (Saccharomyce cerevisiae)), insect cell, human cell's (for example, Chinese hamster ovary celI system (Chinese hamster ovary cell (Chinese hamster ovary)), W138, baby hamster kidney cell (BHK), African green monkey kidney cell (COS-7), 293, HepG2,3T3, RIN and dog kidney (MDCK) cell-line) and plant cell etc. as host cell.Host cell preferred plant cell.
Host cell is under the situation of prokaryotic, can pass through the CaCl2 method, breathe out and take sweat (Hanahan) method (Hanahan, D., J.Mol.Biol., 166:557-580(1983) (Hanahan, D., molecular biology magazine, 166:557-580(1983))) and method such as electroporation, carrier transport just of the present invention is to host cell.In addition, be under the eukaryotic situation at host cell, can pass through methods such as microinjection, calcium phosphate precipitation method, electroporation, liposome-mediated infection protocol, diethylaminoethanol (DEAE)-glucan infection protocol and particle gun blast technique, carrier is injected in the host cell.
The conversion of plant refers to DNA is transferred to any means of plant.These method for transformation not necessarily need through regeneration and (or) the tissue culture period.At present, the conversion of plant species comprises that not only for dicotyledon the plant species of monocotyledon quantum is also very general.In principle, method for transformation all can be used to hybrid DNA of the present invention is imported in the CFU-GM arbitrarily.Described method can be selected from calcium/polyethylene glycol method (Krens, F.A.et al., 1982, Nature296, the 72-74 for protoplast; Negrutiu I.et al., June1987, Plant Mol.Biol.8,363-373(Krens, people such as F.A., 1982, nature 296,72-74; People such as Negrutiu I., in June, 1987, molecular biology of plants 8,363-373)), the electroporation of protoplast (Shillito R.D.et al., 1985Bio/Technol.3, people such as 1099-1102(Shillito R.D., 1985 biologies/technology 3,1099-1102)), microinjection (Crossway A.et al. as the plant element, 1986, Mol.Gen.Genet.202, people such as this prestige of 179-185(clo A., 1986, molecular genetics and genome 202,179-185)), (DNA or RNA-coating) microprojectile bombardment methods (Klein T.M.et al., 1987 of various plant elements, Nature327, people such as 70(Klein T.M., 1987, nature 327,70)), transform according to the infiltration of plant or mature pollen or microspore, in the Agrobacterium tumefaciens mediated gene transfer (non-integrality) according to the infection (EP0301316 number) of virus etc.The preferred method of the present invention comprises agriculture bacillus mediated DNA and shifts.More preferably utilize EPA120516 number and the method for the so-called binary vector technology that United States Patent (USP) is put down in writing for the 4th, 940, No. 838.
In addition, the invention provides the preparation method of the conversion plant corpus that a kind of carotenoid content obtains increasing, described preparation method comprises recombinant vector of the present invention is transformed in the plant cell, crosses and expresses the step of IbOr-Ins gene mutation body.
In addition, the invention provides a kind of conversion plant corpus and seed thereof that is obtained increasing by the carotenoid content of method for preparing.Above-mentioned plant corpus can be dicotyledon, but is not limited to this.
Above-mentioned dicotyledon can be Diapensiaceae (fringebell section; Diapensiaceae); alder Ye Shu section (Clethraceae); Pyrolaceae (Pyrolaceae); Ericaceae (Ericaceae); Myrsinacea (Myrsinaceae); Primulaceae (Primulaceae); lumbaginaceae (Plumbaginaceae); Ebenaceae (Ebenaceae); Styracaceae (Styracaceae); Symplocaceae; ash Ochnaceae (Symplocaceae); Oleaceae (sweet-scented osmanthus; Oleaceae); Loganiaceae (Loganiaceae); Gentianaceae (Gentianaceae); Menyanthaceae (Menyanthaceae); Apocynaceae (Asia trachelospermum jasminoide; Apocynaceae); Asclepiadaceae (Asclepiadaceae); Rubiaceae (Rubiaceae); Polemoniaceae (Polemoniaceae); Convolvulaceae (Convolvulaceae); Boraginaceae (Boraginaceae); Verenaceae (Verbenaceae); Labiatae (Labiatae); Solanaceae (Solanaceae); Scrophulariaceae (Scrophulariaceae); Bignoniaceae (Bignoniaceae); Acanthaceae (Acanthaceae); Pedaliaceae (Pedaliaceae); Orobanchaceae (Orobanchaceae); Gesneriaceae (Gesneriaceae); Lentibulariaceae (Lentibulariaceae); Phrymaceae (Phrymaceae); Plantaginaceae (Plantaginaceae); Caprifoliaceae (Caprifoliaceae); Adoxaceae (Adoxaceae); Valerianaceae (Valerianaceae); Dipsacaceae (Dipsacaceae); Campanulaceae (Campanulaceae); composite family (Compositae); Myruca ceas (Myricaceae); Juglandaceae (Juglandaceae); Salicaceae (Salicaceae); Betulaceae (Betulaceae); Fagaceae (Fagaceae; Fagaceae); Ulmaceae (Ulmaceae); Moraceae (Moraceae); Urticaceae (Urticaceae); Santalaceae (Santalaceae); Loranthaceae (Loranthaceae); polygonaceae (knotweed; Polygonaceae); Phytolaccaceae (Phytolacca acinosa; Phytolaccaceae); Nyctaginaceae (Nyctaginaceae); Aizoaceae (Aizoaceae); Portulacaceae (Portulacaceae); Caryophyllaceae (Caryophyllaceae); Chenopodiaceae (Chenopodiaceae); Amaranthaceae (Amaranthaceae); Cactaceae (Cactaceae); Magnoliaceae (Magnoliaceae); Winteraceae (Illiciaceae); Lauraceae (Lauraceae); Cercidiphyllaceae (Cercidiphyllaceae); Ranunculaceae (Ranunculaceae); Berberidaceae (Berberidaceae); Lardizabalaceae (Lardizabalaceae); Menispermaceae (stem rattan; Menispermaceae); Nymphaeceae (Nymphaeaceae); Ceratophyllaceae (Ceratophyllaceae); Cabombaceae (Cabombaceae); Saururaceae (Saururaceae); Piperaceae (Piperaceae); Chloranthaceae (Chloranthaceae); Aristolochiaceae (Aristolochiaceae); Actinidiaceae (Actinidiaceae); Theaceae (Theaceae; Theaceae); Guttiferae (Guttiferae); Droseraceae (Droseraceae); Papaveraceae (Papaveraceae); Capparidaceae (Capparidaceae); Cruciferae (cruciate flower; Cruciferae); ocean paulownia Ochnaceae (Platanaceae; Platanaceae); (gold is engraved plum to Hamamelidaceae; Hamamelidaceae); Crassulaceae (red-spotted stonecrop; Crassulaceae); Saxifragaceae (Saxifragaceae); Eucommiaceae (Eucommiaceae); Pittosporaceae (Pittosporaceae); the rose family (Rosaceae); pulse family (Leguminosae); Oxalidaceae (Oxalidaceae); ox seedling section (Geraniaceae); Tropaeolaceae (Tropaeolaceae); zygophyllaceae (Zygophyllaceae); flax family (Linaceae) (Linaceae); Euphorbiaceae (Euphorbiaceae); Callitrichaceae (Callitrichaceae); Rutaceae (Rutaceae); Simarubaceae (Simaroubaceae); Meliaceae (Meliaceae); Polygalaceae (Polygalaceae); Anacardiaceae (Anacardiaceae); Aceraceae (Aceraceae; Aceraceae); Sapindaceae (Sapindaceae); Hippocastanaceae (hippocastabaceae); Sabiaceae (Sabiaceae); Balsaminaceae (Shui Fengxian; Balsaminaceae); Aquifoliaceae (Aquifoliaceae); Celastraceae (is defended Mao Ke; Celastraceae); Stachyuraceae (Staphyleaceae); Buxaceae (Buxaceae); Empetraceae (Empetraceae); Rhamnaceae (Rhamnaceae); Vitaceae (Vitaceae); Elaeocarpaceae (Elaeocarpaceae); Tiliaceae (Tiliaceae); Malvaceae (Malvaceae); Sterculiaceae (Sterculiaceae); Thymelaeceae (auspicious spray nose section; Thymelaeaceae); Elaeangnaceae (Elaeagnaceae); Flacourtiaceae (Flacourtiaceae); Violaceae (Violaceae); Passifloraceae (Passifloraceae); suspend (Tamaricaceae); Elatinaceae (Elatinaceae); Malus spectabilis section (Begoniaceae); Curcurbitaceae (Cucurbitaceae); Lythraceae (Lythrum salicaria; Lythraceae); Punicaceae (Punicaceae); Oenotheraceae (Onagraceae); Haloragaceae (Haloragaceae); Alangiaceae (Alangiaceae); Cornaceae (Dendronenthamia japonica var.chinensis section; Cornaceae); five Solanaceaes (five Coegas; but be not limited to this Araliaceae) or Umbelliferae (Carrot family) (Umbelliferae (Apiaceae)).
In addition, the invention provides the preparation method of the conversion plant corpus that a kind of salt stress patience obtains increasing, it comprises recombinant vector of the present invention is transformed in the plant cell, crosses and expresses the step of IbOr-Ins gene mutation body.
In addition, the invention provides a kind of conversion plant corpus and seed thereof that is obtained increasing by the salt stress patience of method for preparing.Above-mentioned plant corpus can be dicotyledon, but is not limited to this.Above-mentioned dicotyledon as mentioned above.
In addition, the invention provides the preparation method of the conversion plant corpus that a kind of salt stress patience obtains increasing, it comprises that the recombinant vector that will contain the IbOrange gene that is derived from sweet potato is transformed in the plant cell, cross the step of expressing the IbOrange gene, the described IbOrange gene that is derived from sweet potato is made of the base sequence of SEQ ID No.1.
In addition, the invention provides conversion plant corpus and the seed thereof that the salt stress patience by method for preparing obtains increasing.Above-mentioned plant corpus can be dicotyledon, but is not limited to this.Above-mentioned dicotyledon as mentioned above.
In addition, the invention provides a kind of composition for increasing the plant corpus carotenoid content, it contains the IbOr-Ins gene mutation body that inserts specific base sequence in being derived from the IbOrange gene of sweet potato artificially.Described IbOr-Ins gene mutation body is made of the base sequence of SEQ ID No.2.Composition of the present invention contains IbOr-Ins gene mutation body that the base sequence by SEQ ID No.2 constitutes as active ingredient, by the said gene mutant is transformed in the plant, can increase the carotenoid content of plant corpus.
In addition, the invention provides a kind of composition for increasing plant corpus salt stress patience, its contain in being derived from the IbOrange gene of sweet potato, insert artificially specific base sequence the IbOr-Ins gene mutation body, described IbOr-Ins gene mutation body is made of the base sequence of SEQ ID No.2.Composition of the present invention contains IbOr-Ins gene mutation body that the base sequence by SEQ ID No.2 constitutes as active ingredient, by the said gene mutant is transformed in the plant, can increase the salt stress patience of plant corpus.
In addition, the invention provides a kind of composition of the salt stress patience for increasing plant corpus, it contains the IbOrange gene that is derived from sweet potato, and the described IbOrange gene that is derived from sweet potato is made of the base sequence of SEQ ID No.1.Composition of the present invention contains IbOrange gene that the base sequence by SEQ ID No.1 constitutes as active ingredient, by the said gene mutant is transformed in the plant, can increase the salt stress patience of plant corpus.
Below, explain the present invention by embodiment.But following embodiment is just for illustration the present invention, and content of the present invention is not limited to following embodiment.
The clone of embodiment 1:IbOr-Ins gene and base sequence analysis
For the preparation of the PCR primer from sweet potato clone Orange gene, primer sequence is as follows: forward primer (5'-atggtatattcaggtagaatcttgtcgctc-3'; SEQ ID No.3) and reverse primer (5'-ttaatcaaatgggtcaattcgtgggtcatg-3'; SEQ ID No.4).The IbOr that obtains thus as blank, is implemented overlapping (overlapping) PCR, thereby implement the artificial mutation of the specific base sequence of IbOr.For rising, No. 133 amino acid in the amino acid sequence that inserts the IbOrange gene is estimated to be-base sequence of KSQNPNL-, prepared the PCR primer, this moment, employed primer sequence was as follows: forward primer (5'-gaaaagcaagaaaataaacttaa atcccagaaccctaac-3'; SEQ ID No.5) and reverse primer (5'-aagatttgcggatgtcaggtt agggttctgggatttaag-3'; SEQ ID No.6).Its result obtains the PCR product of about 921bp, be cloned on the pGEM-T-carrier after, confirmed from No. 133 amino acid with-sudden change (Fig. 1) that KSQNPNL-inserts by order-checking.Use the advantage 2(advantage2 of Clonetech company) polymerase enforcement PCR, use pGEMeasy cloning vector (omega-3 polyunsaturated fatty acids and multivitamin preparation, Promega) after the clone estimates the PCR product of size, check order and confirmed whole base sequence.Called after IbOr-Ins with this cDNA gene.The total length of IbOr-Ins gene of the present invention is cDNA coding 307 amino acid (Fig. 1) of 924bp.Be respectively 8.45 and 33.74kDa with the isoelectric point (pI) of amino acid sequence prediction and molecular weight (Mw).
Passage (g ateway) expression system in order to use hero company (Invitrogen) in the base sequence of primer adds linking sequence (adapt er sequence) (using the capitalization mark) respectively on the 5' of above-mentioned primer end.Base sequence is forward primer (5'-CAAAA AAGCAGGCTNNa tggtatattcaggtagaatcttgtcgctc-3'; SEQ ID No.7) and reverse primer (5'-CAAGAAAGCTGGGTNttaatcaaatgggtcaattcgtgggtcatg-3'; SEQ ID No.8).At first, (omega-3 polyunsaturated fatty acids and multivitamin preparation Promega) behind the PCR product of the above-mentioned expectation size of clone, check order and have confirmed whole base sequence to use the pGEMeasy cloning vector.Be IbOr-Ins with this cDNA unnamed gene.Have two pGEMeasy carriers of IbOrange gene and IbOr-Ins gene to carry out BP reaction respectively to the clone, thus on the pDONR207 carrier clone gene.After this, preparation pDONR207, as the pGWB11 carrier of plant expression vector, with LR reaction clone's IbOra nge and the plant expression vector (Fig. 3) of IbOr-Ins gene.
Embodiment 2:IbOrange and IbOr-Ins transform sweet potato cultured cell phenotype and analyze
With the plant expression vector of Fig. 3 by agrobacterium mediation converted, be transformed into cream-coloured sweet potato Li Mei (
) in the cultured cell.Its result is cream-coloured no change as the Li Mei of non-transformant, and IbOrange and IbOr-Ins transform cultured cell and present buff, can confirm that with the naked eye IbOr-Ins is darker color (Fig. 4).
Embodiment 3: transform the interior carotenoid content analysis of sweet potato cultured cell of IbOrange and IbOr-Ins carrier
The sweet potato cultured cell phenotype that transforms IbOrange and IbOr-Ins carrier is observed, its result is, " Li Mei " of non-conversion cultured cell is cream-coloured, and the cultured cell of conversion IbOrange and IbOr-Ins demonstrates the phenotype (Fig. 4) of buff.The carotenoid content of analyzing each conversion sweet potato cultured cell by HPLC carries out actual quantification.Its result is that the IbOrange cultured cell increases about more than 4 times than the whole carotenoid content of " Li Mei " cultured cell, under the situation of IbOr-Ins, increase about more than 13 times.Particularly the IbOrange cultured cell increases approximately more than 4 times than the carotin of " Yulmi " cultured cell, under the situation of IbOr-Ins, increases more than 10 times.In addition, compare with the IbOrange cultured cell, the whole carotenoid of IbOr-Ins cultured cell increases more than 3 times, and carotin has also increased (Fig. 5) more than 2 times.
Embodiment 4: carotenoid biosynthesis related genes and IbOrange gene expression analysis
Transforming cultured cell with two sweet potatoes that comprise Li Mei is object, analyzes by the carotenoid biosynthesis related genes of RT-PCR and IbOrange expression of gene.The base sequence of analyzing employed primer is forward (5'-atcttgtcgctctcgtcctccacgacgccg-3'; SEQ ID No.9) reaches oppositely (5'-cgtgggtcatgctcgcttgccatagccatc-3'; SEQ IDNo.10).Its result is that under the situation of Li Mei, the expression of other carotenoid biosynthesis related genes except PSY and CHY-β is very weak or do not have expression, IbOrange conversion cultured cell also almost not to have to express.But IbOr-Ins transforms under the situation of cultured cell, and except crossing of IbOrange gene expressed, nearly all expression of gene presented the expression pattern similar to Li Mei (expression patten).But, in transforming the IbOr-Ins cultured cell, being not only the IbOrange gene, LCY-β, CHY-β are than the expression stronger (Fig. 6) in Li Mei and IbOrange conversion cultured cell.
The salt stress patience that embodiment 5:IbOrange and IbOr-Ins transform in the sweet potato cultured cell is analyzed
DAB(3', the 3-diaminobenzidine, 3' is 3-diaminobenzidine) with the interior H of cell
2O
2In conjunction with the sediment that generates brown, it is more dark that the more high cell of oxidative stress is brown.With the sweet potato cultured cell that transforms IbOrange and IbOr-Ins be immersed in contain 150 and the MS1D liquid nutrient medium of 200mM NaCl in after 24 hours, observe the oxidative stress that cultured cell is subjected to by DAB dyeing.Its result is under the situation of Li Mei, along with the heighten degree of the rising brown of NaCl concentration, under the 460nm wavelength, measure the result of DAB solution absorbency, demonstrate high oxidation and coerce, and IbOrange and IbOr-Ins conversion sweet potato cultured cell demonstration suboxides are coerced (Fig. 7).Confirm that thus IbOrange and IbOr-Ins transform the sweet potato cultured cell and show patience for NaCl.
Embodiment 6: transform the arabidopsis analysis of IbOrange and IbOr-Ins
Phenotype to the arabidopsis seed that transforms IbOrange and IbOr-Ins carrier is observed.Its result can confirm to compare with the wild type (Col-0) of non-transformant, and transformant presents yellow kind color of the leather (Fig. 8).
For whether the carotenoid content in the arabidopsis of factual survey conversion IbOrange and IbOr-Ins changes, by spectrophotometer, under absorbance 440nm, the carotene carotene content in the leaf tissue of two arabidopsis thaliana transformation plant corpuss is measured.Its result is, Zu wild type and the arabidopsis that transforms IbOrange do not assume a marked difference in contrast, and the carotene carotene content that transforms the arabidopsis of IbOr-Ins increases about more than 1.5 times (Fig. 8).But, compare in order to measure carotenoid content more accurately, think the tissue that is necessary playing infiltration (sink) effect.
In addition, confirm the expression situation of the carotenoid biosynthesis gene in these transformant by RT-PCR, the result shows that the orange expression of gene of existing carotenoid biosynthesis related genes and arabidopsis itself does not have big variation.But the expression of the IbOrange that inserts only obtains expressing in transformant, and does not express in the wild type of control group.Can confirm that thus employed primer has specificity (Fig. 9) to sweet potato among the present invention.
Stress-inducing gene NCED expression analysis in embodiment 7:IbOrange and the IbOr-Ins arabidopsis thaliana transformation
If prolong the biological approach of carotenoid, with synthetic abscisic acid as representative plant hormone, and abscisic acid is not only one of hormone important concerning development of plants such as seed sprouting, dormancy, growth of seedling, and for overcoming drying stress and low temperature stress plays an important role.The present invention is in order to confirm that whether abscisic acid induces by the carotenoid content that increases in the arabidopsis thaliana body that transforms IbOrange, analyzes the expression of NCED.Report that NCED has 9 pedigrees (family) in arabidopsis, and report there are abscisic acid content and the expression of coercing to obtain increasing (Rodrigo et al., people such as 2006(Rodrigo, 2006)).The result who implements RT-PCR is, NCED1 demonstrates high expressed in three transformant and the control group.But under the situation of NCED3, NCED6 and NCED4, the arabidopsis thaliana body surface that transforms IbOr-Ins reveals the expression pattern higher than control group.Thus, based on this result, can think that the arabidopsis thaliana body that transforms IbOr-Ins environment-stress such as coerces to high abscisic acid content and salt and dehydration and osmotic pressure and show high patience (Figure 10).
The phenotype analysis of the conversion IbOrange when embodiment 8:NaCl handles and the arabidopsis of IbOr-Ins
NaCl is the maximum reason of coercing in development of plants and the process of growth, when being subjected to NaCl and coercing, at first brings the dehydration of cell and the loss of turgescence, therefore water stress is coerced the identical mechanism that is interpreted as with NaCl.But NaCl coerces reality and shows more serious dehydrating effect at plant cell internal ratio water stress.For the arabidopsis thaliana body confirming on the plant corpus aspect whether practical manifestation goes out the NaCl stress tolerance to IbOrange, make to transform IbOrange and IbOr-Ins contain 0,50,100 and the 1/2MS solid culture medium of 150mM NaCl in about 2 weeks of growth.At inoculation of medium (seeding) afterwards, the result who observes the 7th day germination rate is that transformant not only raises than control group NaCl concentration, and shows high germination rate (Figure 11).And under the situation of Zu wild type, along with NaCl concentration raises, live body weight sharply reduces in contrast, and the arabidopsis of conversion IbOrange and IbOr-Ins is compared with control group, but shows high live body weight (Figure 12).
And, under 100mM NaCl condition, observe the arabidopsis that transforms IbOrange and IbOr-Ins and have longer primary root (primary root) than control group.Particularly, IbOr-Ins is more about more than 2 times than the contrast group leader.And under the situation of the arabidopsis that transforms IbOrange, under high NaCl condition, also observe the primary root phenotype (Figure 13) than the contrast group leader.
In addition, relative moisture under the NaCl concentration of 150mM is measured, its result shows that control group under 150mM, has the moisture about 70%, and arabidopsis thaliana body and the non-processed group of conversion IbOrange and IbOr-Ins do not show notable difference.Think thus, transform the arabidopsis thaliana body of IbOrange and IbOr-Ins under high salt condition, because of in vivo in conjunction with the ratio height of water, thereby moisture height relatively, so tolerance height (Figure 14) under the high salt condition externally.
Claims (18)
1. IbOr-Ins gene mutation body that inserts specific base sequence in the IbOrange gene that is derived from sweet potato (Ipomoea batatas) artificially, described IbOr-Ins gene mutation body is made of the base sequence of SEQ ID No.2.
2. the recombinant vector that contains the gene mutation body of claim 1.
3. transform the host cell of the described recombinant vector of claim 2.
4. the preparation method of the carotenoid content conversion plant corpus that obtains increasing, described preparation method comprise the described recombinant vector of claim 2 are transformed in the plant cell, cross and express the step of IbOr-Ins gene mutation body.
5. the conversion plant corpus that is obtained increasing by the carotenoid content of the described method of claim 4 preparation.
6. conversion plant corpus according to claim 5 is characterized in that, described plant corpus is dicotyledon.
7. the seed of the described plant corpus of claim 5.
8. the preparation method of the salt stress patience conversion plant corpus that obtains increasing, it comprises the described recombinant vector of claim 2 is transformed in the plant cell, crosses and expresses the step of IbOr-Ins gene mutation body.
9. the conversion plant corpus that is obtained increasing by the salt stress patience of the described method of claim 8 preparation.
10. conversion plant corpus according to claim 9 is characterized in that, described plant corpus is dicotyledon.
11. the seed of the described plant corpus of claim 9.
12. the preparation method of the conversion plant corpus that a salt stress patience obtains increasing, it comprises that the recombinant vector that will contain the IbOrange gene that is derived from sweet potato (Ipomoea batatas) is transformed in the plant cell, crosses and expresses the step of IbOrange gene; The described IbOrange gene that is derived from sweet potato is made of the base sequence of SEQ ID No.1.
13. the conversion plant corpus that the salt stress patience that is prepared by the described method of claim 12 obtains increasing.
14. conversion plant corpus according to claim 13 is characterized in that, described plant corpus is dicotyledon.
15. the seed of the described plant corpus of claim 13.
16. composition for increasing the plant corpus carotenoid content, it contains the IbOr-Ins gene mutation body that inserts specific base sequence in the IbOrange gene that is derived from sweet potato (Ipomoea batatas) artificially, and described IbOr-Ins gene mutation body is made of the base sequence of SEQ ID No.2.
17. composition for increasing plant corpus salt stress patience, it contains the IbOr-Ins gene mutation body that inserts specific base sequence in being derived from the IbOrange gene of sweet potato artificially, and described IbOr-Ins gene mutation body is made of the base sequence of SEQ ID No.2.
18. the composition for increasing plant corpus salt stress patience, it contains the IbOrange gene that is derived from sweet potato, and the described IbOrange gene that is derived from sweet potato is made of the base sequence of SEQ ID No.1.
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| CN103917087A (en) * | 2011-08-05 | 2014-07-09 | 韩国生命工学研究院 | Method for obtaining a transformed ipomoea batatas plant body including highly accumulated carotenoid and anthocyanin, and plant body obtained by same |
| CN108834803A (en) * | 2018-07-12 | 2018-11-20 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | A method of induction sweet potato buddings |
| CN111448206A (en) * | 2017-12-19 | 2020-07-24 | 韩国生命工学研究院 | Ibor-R96H variants derived from sweet potatoes and uses thereof |
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| KR101478112B1 (en) * | 2012-10-12 | 2014-12-31 | 한국생명공학연구원 | IbOr-Ins gene increasing black rot resistance from Ipomoea batatas and uses thereof |
| KR102525540B1 (en) * | 2021-03-05 | 2023-04-25 | 한국생명공학연구원 | LCYB2 mutant from Daucus carota and uses thereof |
| CN117164686A (en) * | 2022-05-11 | 2023-12-05 | 中国农业大学 | Stress resistance related protein IbRCD1, related biological material and application thereof |
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| CN103917087A (en) * | 2011-08-05 | 2014-07-09 | 韩国生命工学研究院 | Method for obtaining a transformed ipomoea batatas plant body including highly accumulated carotenoid and anthocyanin, and plant body obtained by same |
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| CN111448206A (en) * | 2017-12-19 | 2020-07-24 | 韩国生命工学研究院 | Ibor-R96H variants derived from sweet potatoes and uses thereof |
| CN108834803A (en) * | 2018-07-12 | 2018-11-20 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | A method of induction sweet potato buddings |
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