CN105368845A - GTB-HM gene of echinochloa colona and application of GTB-HM gene - Google Patents

GTB-HM gene of echinochloa colona and application of GTB-HM gene Download PDF

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CN105368845A
CN105368845A CN201510922400.2A CN201510922400A CN105368845A CN 105368845 A CN105368845 A CN 105368845A CN 201510922400 A CN201510922400 A CN 201510922400A CN 105368845 A CN105368845 A CN 105368845A
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gtb
gene
protein
seqidno
controlling element
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陈宏伟
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis

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  • Life Sciences & Earth Sciences (AREA)
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  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

The invention belongs to the technical field of bioengineering, and in particular relates to GTB-HM gene for encoding high-methionine protein of echinochloa colona, and an application of the GTB-HM gene. With the GTB-HM gene, the synthesis of the protein can be greatly promoted, the content of the non-protein nitrogen can be reduced, growth of crops can be promoted, the quality of agricultural products can be improved, and the GTB-HM gene has a relatively high practical application value.

Description

The GTB-HM gene of a kind of bare headed barnyard grass and application thereof
Technical field
The present invention relates to a kind of grass---the GTB-HM gene of bare headed barnyard grass coding homomethionin albumen, belongs to technical field of molecular biology.
Background technology
The shortage of the short especially quality protein of albumen, be 21 century global serious problems, while the protein content improving crop varieties as early as possible, actively improve the amino acid composition of protein, improve the content of limiting amino acid to strengthen its nutritive value, significant.Plant seed has stored rich in protein, carbohydrate and lipid, the storage protein be wherein stored in aleuroplast is the main component of plant seed, its function is not only confined to as the normal sprouting of seed and growth of seedling provide nutrition supply, and take part in the regulation and control (Zhao Wenming, 1995) of seed germination and growth course.
Sulphur is required the 4th large nutritive element of plant being only second to nitrogen, phosphorus, potassium, and during a lack of sulfur, protein synthesis is obstructed, and causes non-protein nitrogen(NPN) to be accumulated, not only affects plant growth, and reduces quality of agricultural product; Plant sulphur content is 0.1% ~ 0.5%, and its luffing is obviously by floristics, kind, organ and the impact of breeding time.Cress needs sulphur maximum, and pulse family, liliaceous plant are taken second place, grass less (Lu Jingling, 1994).Sulfur-containing amino acid such as methionine(Met) (also referred to as methionine(Met), methionine, Met), Gelucystine, halfcystine (cysteine, Cys) etc. are the important amino acids of synthetic protein; In protein, the content of sulfur-containing amino acid and seed germination, growth and crop quality all have substantial connection.Wherein, Met is as one of the indispensable amino acid of human body and single cud animal, paddy rice, wheat, barley, support wheat, especially in the food crop that beans and peanut etc. are main and cash crop, content is extremely low, it is main limiting amino acid, therefore the Met content improving these crops seems particularly important (Zhao Wenming, 1995; M ü ntzK, 1998).
Along with the development of modern biotechnology, the goal gene of more how high sulfur-containing amino acid is obtained by expanding screening, the method of proteomics is utilized to disclose the new function (Wang Wenjun etc. of high Met seed storage protein, 2005), means Crop Improvement quality, the growth of adjustment seed growth etc. in conjunction with plant genetic engineering will have more practical significance (HaftBK, 2005).
Having separated in the world at present can be few in number for the high Met plant seed storage protein gene being used for improving crop Met content, its main source is corn (AltenbachSB, 1990) and paddy rice (Masum-uraT, 1989), in addition small part is also had from Brazilian chestnut (SaalbachI, 1994), peanut (BashaSM, 1994) etc., China is backwardness especially, thus the germ plasm resource of China's abundant is made full use of, rationally and effectively excavate and utilize plant resources, the valuable high Met plant seed storage protein gene of separation and Extraction be by improve quality-improving genetically engineered field for the purpose of crop Met content in the urgent need to basic work, also new field is opened up in the research for high Met plant seed storage protein.
Summary of the invention
The invention provides a kind of bare headed barnyard grass gene GTB-HM of homomethionin albumen of encoding and the application of coding homomethionin albumen thereof; This gene can improve the expression of associated protein, especially can improve the content of methionine(Met) in plant.
The present invention is by designing primer, and with bare headed barnyard grass DNA for template, pcr amplification obtains this full length gene sequence, clones and check order through carrier T, then proceeds to soybean and carry out strongly expressed, shows that this is the gene of a coding homomethionin albumen.This gene coding region is 405bp, 135 amino acid of encoding, and wherein containing 13 methionine(Met), and the molar content of methionine(Met) reaches 9.62% in this albumen.
Therefore, the present invention's first object is to provide a kind of bare headed barnyard grass gene GTB-HM of homomethionin albumen of encoding, and it belongs to new coding homomethionin protein gene, has or be selected from the sequence of SEQIDNO:1.Or with the nucleotide sequence that SEQIDNo:1 limits, there is the nucleotide sequence of more than 90% homology; The nucleotide sequence that the DNA sequence dna that can limit with SEQIDNo:1 under high high stringency conditions is hybridized.
The present invention's second object is to provide the functional protein of described coded by said gene, and it has or is selected from the protein sequence of SEQIDNO:2, or has or be selected from the protein sequence coded by gene order of SEQIDNO:1.Or to growing of plant, there is the protein of regulating and controlling effect with the amino acid residue sequence of SEQIDNo:2 through the replacement of one to ten two amino-acid residue or disappearance or interpolation.
The present invention's the 3rd object is to provide described gene or functional protein, the purposes in the bare headed barnyard grass homomethionin protein expression of regulation and control; Utilize this gene, develop the functional type food protein with homomethionin albumen and even the healthcare products in more efficient, nontoxic bare headed barnyard grass source.
The expression vector of GTB-HM gene and controlling element thereof in a particular embodiment.
The transgenic cell line of GTB-HM gene and controlling element thereof in a particular embodiment.
The engineering bacteria of GTB-HM gene and controlling element thereof in a particular embodiment.
The primer pair of arbitrary fragment in GTB-HM gene and controlling element thereof in a particular embodiment.
Application in a particular embodiment in GTB-HM gene and controlling element thereof in growth and development of plants.
GTB-HM gene and albumen thereof can be applicable to plant and comprise monocotyledons and dicotyledons in a particular embodiment.
Accompanying drawing explanation
Fig. 1 is pcr gene amplification (1 is bare headed barnyard grass pcr amplification product, and M is DNAMarker).Fig. 2 is that enzyme is cut result (1 is the recombinant plasmid extracted, and 2 is restructuring plasmid enzyme restriction product, and M is DNA
Marker)。
Connection diagram (L.JaponicusUbiquitinpromoterpCAMBIA1390) in Fig. 3 pCAMBIA1390 recombinant plasmid, wherein GTB-HM is bare headed barnyard grass homomethionin gene.
Technique effect
1, utilize GTB-HM genetic expression of the present invention to obtain containing the protein of 13 methionine(Met), contribute to the content of methionine(Met) in soybean.
2, utilize GTB-HM gene of the present invention and albumen, can be used in the investigation and application of existing homomethionin albumen, and develop the protein of functional type and even the healthcare products of efficient, nontoxic homomethionin albumen.
Embodiment
In following embodiment of the present invention, experiment material used is bare headed barnyard grass (EchinochloacrusgalliL.Beauv.).
Embodiment 1, bare headed barnyard grass Total RNAs extraction
Utilize RNA extraction and isolation test kit (Invitrogen company provides) to extract total serum IgE in bare headed barnyard grass bract, its concrete grammar is: collect bare headed barnyard grass bract 100mg, be placed in liquid nitrogen immediately and grind, add 1mlTrizol reagent, fully mix; Room temperature places 5min; Add the fresh chloroform of 0.2ml, violent jolting 15s, incubation at room temperature 3min; 4 DEG C, the centrifugal 15min of 12000g; Supernatant water phase transition, in a new 1.5ml centrifuge tube, adds 0.5ml isopropanol precipitating RNA; Be dissolved in appropriate DEPC treated water after RNA precipitation 1ml75% washing with alcohol ,-70 DEG C save backup.
Embodiment 2, bare headed barnyard grass total serum IgE purifying
In the bare headed barnyard grass total serum IgE of embodiment 1 acquisition, 6 μ lDEPC process water, 10 μ lRNA, 2 μ lDNaseIBuffer, 2 μ lDNaseI are added successively according to DNaseI test kit (ThermoScientific company provides), after mixing, 37 DEG C of 30min, add EDTA2 μ l, 65 DEG C of 10min.
Embodiment 3, bare headed barnyard grass RNA reverse transcription
First chain synthesis: the user manual according to PlantRT-PCRKit2.01 (TaKaRa, Japan) carries out.1-2ng total serum IgE (about 1-2 μ l), and the various Reverse Transcription mixing (MgCl of Kit 24 μ l; 10XRNAPCRBuffer2 μ l; RNaseInhibitor0.5 μ l; RNasefreeWater8.5 μ l; DNTPMixture2 μ l; ReverseTranscriptase1 μ l; OligodT-Adaptor1 μ l).After mixing, 42 DEG C of 30min; 99 DEG C of 5min; 5 ° of DEG C of 5min, complete reverse transcription reaction.
Second chain synthesis: design following primer according to the sequence data that information biology provides:
5 ' end primer (SEQIDNO:3): 5'-CG gGATCCcGATGGCAGCCAAGATGCTTGC-3';
3 ' end primer (SEQIDNO:4): 5'-C gAGCTCgCTAGAATGCAGCACCAACAAAGGG-3'.
Draw 1 μ l reverse transcription product, mix (reverse transcription product 1 μ l, upstream primer (SEQIDNO:3) 0.5 μ l, downstream primer (SEQIDNO:4) 0.5 μ l, Taq0.5 μ l, 10XBuffer2 μ l, Mg with DNATaq enzyme as template 2+0.5 μ l, ddH 2o15 μ l) carry out PCR reaction: enter amplification program after 94 DEG C of 5min: 94 DEG C of 1min, 57 DEG C of 1min, 72 DEG C of 1min, after 30 circulations, 72 DEG C of 5min.
Embodiment 4, TA clone
By the pcr amplification product obtained in embodiment 3, target DNA fragment is separated through agarose electrophoresis (Fig. 2), adopt glue to reclaim DNA test kit (purchased from Shanghai Sheng Gong biotechnology company limited) method and carry out recovery DNA fragmentation, by the second chain synthetic product of recovery and DNA clone reagent mix (the second chain product 6 μ l, PEG40001 μ l, carrier T 1 μ l, 10Xligationbuffer1 μ l, ligase1 μ l), spend the night in 16 DEG C of connections.
Embodiment 5, transformation of E. coli and target DNA fragment check order
Get and connect product 5 μ l and 200 μ l bacillus coli DH 5 alpha competent cells (purchased from Shanghai Sheng Gong biotechnology company limited) and mix, place 30min on ice, 42 DEG C of 90s, ice bath 1-2 minute, add 800 μ lLB substratum, in 37 DEG C, 45min is cultivated in 250rpm concussion, the centrifugal 5min of 4000rpm, supernatant leaves about 150 μ l, add 7 μ lIPTG, 40 μ lx-gal mix, coat add Amp (penbritin) in advance LB solid medium on overnight incubation, picking white colony shakes and spends the night in the LB liquid nutrient medium being added with Amp, get 1ml bacterium liquid and deliver to the order-checking of Shanghai Sheng Gong biotechnology company limited.Sequencing result shows: the sequence SEQIDNO:1 that obtains of increasing from initiator codon to terminator codon overall length 390 base, encode 129 amino acid, supposition molecular weight is 14.2kD, iso-electric point 8.74, coding methionine(Met) 17, the percentage molar content of methionine(Met) is 13.18%.
Embodiment 6, target DNA fragment imported in soybean and carries out strongly expressed
Plasmid is extracted containing the bacterium liquid of GTB-HM gene from above-mentioned, double digestion is carried out with Xba I and BamH I, digestion products, after reclaiming, connects with the same pCAMBIA1390 plasmid reclaimed through Xba I and BamH I double digestion, construction recombination plasmid (as Fig. 3); By recombinant plasmid transformed agrobacterium tumefaciens AGL1, filtered out by Kan and Rif and transform successful AGL1 bacterial strain, and infected soybean (purchased from country of Agricultural University Of Nanjing Soybean Germplasm center) cotyledonary node, hygromycin selection is utilized to obtain the successful soybean seedling of GTB-HM gene transfection, and obtain soybean seeds through cultivating, soybean seeds Met content is detected by amino acidanalyser, find that its Met content is increased to 3.968% by contrast 0.651%, show that GTB-HM gene obtains strongly expressed in soybean.
SEQIDNO:1 gene order table is as follows:
SEQIDNO:2 aminoacid sequence table is as follows:

Claims (9)

1. the GTB-HM gene of bare headed barnyard grass and an application thereof, is characterized in that the gene for one of following nucleotide sequence:
(1)SEQIDNo:1；
(2) nucleotide sequence limited with SEQIDNo:1 has the nucleotide sequence of more than 90% homology;
(3) nucleotide sequence that the DNA sequence dna that can limit with SEQIDNo:1 under high high stringency conditions is hybridized.
2. the protein of GTB-HM genetic expression described in claim 1, is characterized in that for one of following amino acid residue sequences:
(1)SEQIDNo:2；
(2) amino acid residue sequence of SEQIDNo:2 has the protein of regulating and controlling effect through the replacement of one to ten two amino-acid residue or disappearance or interpolation to growing of plant.
3. contain the expression vector of GTB-HM gene according to claim 1 and controlling element thereof.
4. contain the transgenic cell line of GTB-HM gene according to claim 1 and controlling element thereof.
5. contain the engineering bacteria of GTB-HM gene according to claim 1 and controlling element thereof.
6. the primer pair of the arbitrary fragment in GTB-HM gene according to claim 1 and controlling element thereof of increasing.
7. the application in GTB-HM gene according to claim 1 and controlling element thereof in growth and development of plants.
8. application according to claim 7, described plant comprises monocotyledons and dicotyledons.
9. application according to claim 1 and 2, the purposes in the bare headed barnyard grass homomethionin protein expression of regulation and control; Utilize this gene, develop the functional type food protein with homomethionin albumen and even the healthcare products in more efficient, nontoxic bare headed barnyard grass source.
CN201510922400.2A 2015-12-11 2015-12-11 GTB-HM gene of echinochloa colona and application of GTB-HM gene Pending CN105368845A (en)

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Application publication date: 20160302