CN105693834B - Application of the soybean protein GmVPS9a2 in regulation plant storage protein sorting - Google Patents
Application of the soybean protein GmVPS9a2 in regulation plant storage protein sorting Download PDFInfo
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Abstract
The invention discloses application of the soybean protein GmVPS9a2 in regulation plant storage protein sorting.GmVPS9a2 provided by the present invention are as follows: B1) amino acid sequence be SEQ ID No.3 protein；B2) amino acid sequence is the 1-465 protein of SEQ ID No.3；B3) in the 1-465 amino acids sequence of SEQ ID No.3 or SEQ ID No.3 through substitution and/or be deleted and/or added one or several amino acid residues obtain it is with the same function as B1) or B2) derived from protein；B4) in B1) or B2) or B3) N-terminal or/and the obtained fused protein of C-terminal connection label.It is demonstrated experimentally that plant storage protein sorting GAP-associated protein GAP of the invention influences the sorting of storage protein in plant, it can be used for cultivating storage protein and sort normal genetically modified plants.
Description
Technical field
The present invention relates to the answering in regulation plant storage protein sorting of soybean protein GmVPS9a2 in field of biotechnology
With.
Background technique
Soybean is important industrial crops and cereal crops, contains a large amount of unsaturated fatty acid and a variety of required amino
Acid, stable yield have plantation throughout our country.The egg for accounting for cosmopolitian plant albumen supply market 70% or so is provided for the mankind
White matter resource is even more the important component of mankind's ordinary meal by the bean product of soybean processing.On nutritive value, amino
Acid composition is close with milk protein, is in close proximity to the requirement of ideal protein, can be equivalent to animal protein, on gene structure
Closest to human amino acid, so being the phytoprotein of most nutrition, by the drink that it makes, it is described as by nutritionist " green
Color milk ".The health notice display of United States Food and Drag Administration (FDA), eats the food containing 25g soybean protein daily
Product help reduction cardiovascular morbidity.Storage protein is as the first major class nutriment in soybean kernel, largely
Determine the nutritive value and every Protein Index of soybean.
Soybean storage protein accounts for the 70%-80% of seed total protein content, wherein most with 11S and 7S storage protein content
Height accounts for the 40% and 30% of Seed Storage Protein respectively, their nutritive values are abundant, and food processing characteristic good.It is cytological
It using endoplasmic reticulum is starting point, centre via Gorky that the sorting approach of research also tentative confirmation 7S and 11S storage protein, which is one,
Body, finally using protein storage vacuole as the transhipment route of deposition site.By taking 11S globulin as an example, synthesize in endoplasmic reticulum first
One precursor molecule can be cut into acidic and alkaline subunit by vacuolar processing enzyme into after storage vacuole.
Summary of the invention
The present invention is to provide application of the soybean protein GmVPS9a2 in regulation plant storage protein sorting.
Storage protein sorting GAP-associated protein GAP GmVPS9a2 provided by the present invention is in regulation plant storage protein sorting
In；Storage protein sorting GAP-associated protein GAP GmVPS9a2 is following B1) or B2) or B3) or protein B4):
B1) amino acid sequence is the protein of SEQ ID No.3；
B2) amino acid sequence is the protein of the 1-465 amino acids of SEQ ID No.3；
B3) by replacing and/or lacking in the 1-465 amino acids sequence of SEQ ID No.3 or SEQ ID No.3
And/or one or several amino acid residues of addition obtain it is with the same function as A1) or A2) derived from protein；
B4) in B1) or B2) or B3) N-terminal or/and the obtained fused protein of C-terminal connection label.
Wherein, sequence 1 is made of 466 amino acid.
In order to make B1) and B2) in protein convenient for purifying, can in sequence table SEQ ID No.3 or SEQ ID No.3
1-465 amino acids shown in protein amino terminal or carboxyl terminal connect upper label as shown in Table 1.
The sequence of table 1, label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned B3) in GmVPS9a2 can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain
It arrives.Above-mentioned B3) in the encoding gene of GmVPS9a2 can be by by the of SEQ ID No.4 in sequence table or SEQ ID No.4
Lack the codon of one or several amino acid residues in DNA sequence dna shown in 1-1395 nucleotide, and/or carry out one or
The missense mutation of several base-pairs, and/or obtained in the coded sequence that its 5 ' end and/or 3 ' ends connect label shown in table 1.
In order to solve the above technical problems, the present invention also provides biomaterials relevant to GmVPS9a2 in regulation plant storage
Hide the application in albumen sorting.
Application of the biomaterial relevant to GmVPS9a2 provided by the present invention in regulation plant storage protein sorting
In, the biomaterial is following A 1) any one of to A14):
A1 the nucleic acid molecules of GmVPS9a2) are encoded；
A2) contain A1) expression cassettes of the nucleic acid molecules；
A3) contain A1) recombinant vectors of the nucleic acid molecules；
A4) contain A2) recombinant vector of the expression cassette；
A5) contain A1) recombinant microorganisms of the nucleic acid molecules；
A6) contain A2) recombinant microorganism of the expression cassette；
A7) contain A3) recombinant microorganism of the recombinant vector；
A8) contain A4) recombinant microorganism of the recombinant vector；
A9) contain A1) the transgenic plant cells systems of the nucleic acid molecules；
A10) contain A2) the transgenic plant cells system of the expression cassette；
A11) contain A1) Transgenic plant tissues of the nucleic acid molecules；
A12) contain A2) Transgenic plant tissue of the expression cassette；
A13) contain A1) the genetically modified plants organs of the nucleic acid molecules；
A14) contain A2) the genetically modified plants organ of the expression cassette.
Above-mentioned biomaterial relevant to GmVPS9a2 is in the application in regulation plant storage protein sorting, A1) it is described
Nucleic acid molecules can be following a1) or a2) or a3) or gene a4):
A1) nucleotide sequence is the cDNA molecule or DNA molecular of SEQ ID No.4 in sequence table；
A2) nucleotide sequence is that the cDNA molecule of the 1-1395 nucleotide of SEQ ID No.4 or DNA divide in sequence table
Son；
A3) and a1) or a2) nucleotide sequence that limits has 75% or 75% or more identity, and encodes GmVPS9a2
CDNA molecule or genomic DNA molecule；
A4) the nucleotide sequence hybridization limited under strict conditions with a1) or a2), and the cDNA for encoding GmVPS9a2 divides
Son or genomic DNA molecule.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also
To be RNA, such as mRNA or hnRNA.
Wherein, SEQ ID No.4 is made of 1401 nucleotide, encodes protein shown in SEQ ID No.3.
Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation
Method is mutated the nucleotide sequence of coding GmVPS9a2 of the invention.Those have and the present invention by manually modified
The nucleotide sequence 75% of isolated GmVPS9a2 or the nucleotide of higher identity, as long as coding GmVPS9a2 and tool
There is GmVPS9a2 function, is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair
The nucleotide sequence of the protein of amino acid sequence composition shown in bright coding GmVPS9a2 has 75% or higher or 85%
Or higher or 90% or higher or 95% or higher identity nucleotide sequence.Identity can with the naked eye or computer is soft
Part is evaluated.Using computer software, the identity between two or more sequences can be indicated with percentage (%), can
To be used to evaluate the identity between correlated series.
Above-mentioned biomaterial relevant to GmVPS9a2 is described stringent in the application in regulation plant storage protein sorting
Condition is hybridized at 68 DEG C and washes film 2 times, each 5min in 2 × SSC, the solution of 0.1%SDS, and in 0.5 × SSC,
In the solution of 0.1%SDS, hybridizes at 68 DEG C and wash film 2 times, each 15min；Or, 0.1 × SSPE (or 0.1 × SSC),
In the solution of 0.1%SDS, hybridizes under the conditions of 65 DEG C and wash film.
Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90% or 95% or more identity.
Above-mentioned biomaterial relevant to GmVPS9a2 is in the application in regulation plant storage protein sorting, A2) it is described
Containing coding GmVPS9a2 nucleic acid molecules expression cassette (GmVPS9a2 expression casette), be to refer in host cell
The DNA of GmVPS9a2 is expressed, which may include not only the promoter for starting GmVPS9a2 genetic transcription, may also include termination
The terminator of GmVPS9a2 genetic transcription.Further, the expression cassette may also include enhancer sequence.It is for use in the present invention to open
Mover includes but is not limited to: constitutive promoter, organizes, the promoter and inducible promoter that organ and development are special.Starting
The example of son includes but is not limited to: the constitutive promoter 35S of cauliflower mosaic virus: the Wound-inducible from tomato opens
Mover, leucine aminopeptidase (" LAP ", Chao et al. (1999) Plant Physiol 120:979-992)；From tobacco
Chemical inducible promoter, pathogenesis correlation 1 (PR1) is (by salicylic acid and BTH (diazosulfide -7- carbothioic acid S- first
Ester) induction)；Tomato protease inhibitors II promoter (PIN2) or LAP promoter (available methyl jasmonate induction)；
Heat-shock promoters (United States Patent (USP) 5,187,267)；Tetracycline inducible promoter (United States Patent (USP) 5,057,422)；Seed is special
Specific Promoters, such as Millet Seed specificity promoter pF128 (CN101063139B (Chinese patent 200710099169.7)),
The special promoter of seed storage protein matter is (for example, phaseolin, napin, oleosin and soybean beta conglycin
Promoter (Beachy et al. (1985) EMBO is J.4:3047-3053)).They can be used alone or open with other plants
Mover is used in combination.All references cited herein is cited in full text.Suitable transcription terminator includes but is not limited to: agriculture
Bacillus nopaline syntase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea
RbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g.: Odell et al. (I985)Nature
313:810；Rosenberg et al. (1987) Gene, 56:125；Guerineau et al. (1991) Mol.Gen.Genet, 262:
141；Proudfoot(1991)Cell,64:671；Sanfacon et al. Genes Dev., 5:141；Mogen et al. (1990)
Plant Cell,2:1261；Munroe et al. (1990) Gene, 91:151；Ballad et al. (1989) Nucleic Acids
Res.17:7891；Joshi et al. (1987) Nucleic Acid Res., 15:9627).
The recombinant vector of the GmVPS9a2 expression casette can be contained with existing expression vector establishment.The plant table
It include double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment up to carrier.As pAHC25, pBin438,
PCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or
PCAMBIA1391-Xb (CAMBIA company) etc..The plant expression vector also may include 3 ' end non-translational regions of foreign gene
Domain, i.e., comprising polyadenylation signals and any other DNA fragmentation for participating in mRNA processing or gene expression.The polyadenylic acid letter
Number bootable polyadenylic acid is added to 3 ' ends of mRNA precursor, as Agrobacterium crown gall nodule induces (Ti) plasmid gene (such as nopaline
Synthase gene Nos), plant gene (such as soybean storage protein gene) 3 ' end transcription non-translational region all have similar functions.
When using gene constructed plant expression vector of the invention, enhancer, including translational enhancer or transcriptional enhancer also can be used,
These enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must read with coded sequence
Frame is identical, to guarantee the correct translation of entire sequence.The source of the translation control signal and initiation codon be it is extensive,
Can be it is natural, be also possible to synthesis.Translation initiation region can come from transcription initiation region or structural gene.In order to just
In transgenic plant cells or plant are identified and screened, plant expression vector used can be processed, it can as being added
The coding expressed in plant can produce the enzyme of color change or gene (gus gene, luciferase genes of luminophor
Deng), the marker gene of antibiotic (if assigned the nptII gene to kanamycins and associated antibiotic resistance, assigns to herbicide
The bar gene of phosphinothricin resistance assigns the hph gene to antibiotic hygromycin resistance, and assigns to methotrexate resistance
Dhfr gene is assigned to the EPSPS gene of glyphosate) or (such as anti-herbicide base such as anti-chemical reagent marker gene
Cause), provide metabolism mannose ability mannose-6-phosphate isomerase gene.It, can not from the security consideration of genetically modified plants
Add any selected marker, transformed plant is directly screened with adverse circumstance.
Above-mentioned biomaterial relevant to GmVPS9a2 is in the application in regulation plant storage protein sorting, the carrier
It can be plasmid, sticking grain, bacteriophage or viral vectors.
Above-mentioned biomaterial relevant to GmVPS9a2 is in the application in regulation plant storage protein sorting, micro- life
Object can be yeast, bacterium, algae or fungi, such as Agrobacterium.The Agrobacterium can be Agrobacterium EHA105.
Above-mentioned biomaterial relevant to GmVPS9a2 is described to turn base in the application in regulation plant storage protein sorting
Because plant cell, Transgenic plant tissue and genetically modified plants organ do not include propagation material.
In an embodiment of the invention, the encoding gene of GmVPS9a2 passes through the encoding gene containing GmVPS9a2
Expression cassette recombinant vector import Agrobacterium tumefaciems EHA105 in.The recombinant vector is in carrier pCAMBIA1300-221
XbaI identification sequence between be inserted into sequence table in SEQ ID No.4 1-1401 nucleotide shown in DNA molecular, other sequences
Arrange constant, to obtain recombinant vector pCAMBIA1300-221-GmVPS9a2.PCAMBIA1300-221-GmVPS9a2 with
The difference of pCAMBIA1300-221 is only that: pCAMBIA1300-221-GmVPS9a2 is the XbaI in pCAMBIA1300-221
The recombination that DNA molecular shown in the 1-1401 nucleotide of SEQ ID No.4 obtains in sequence table is inserted between identification sequence to carry
Body.PCAMBIA1300-221-GmVPS9a2 expresses protein shown in SEQ ID No.3, and (i.e. storage protein sorts GAP-associated protein GAP
GmVPS9a2, abbreviation GmVPS9a2 albumen), the expression of GmVPS9a2 albumen is started by 35S.
Wherein, SEQ ID No.3 is made of 466 amino acid, (the i.e. storage egg of the DNA molecular as shown in SEQ ID No.4
White sorting GAP-associated protein GAP GmVPS9a2 gene, abbreviation GmVPS9a2 gene) coding.
In order to solve the above technical problems, the present invention also provides a kind of cultivation storage proteins to sort normal genetically modified plants
Method.
A kind of method cultivated storage protein and sort normal genetically modified plants provided by the present invention, including planted to receptor
The step of encoding gene of importing GmVPS9a2 obtains genetically modified plants in object；The recipient plant storage protein sorting is abnormal,
The genetically modified plants storage protein sorting is normal.
In the above method, the encoding gene of the GmVPS9a2 is A1) nucleic acid molecules.
In an embodiment of the present invention, the encoding gene of the GmVPS9a2 is by containing GmVPS9a2 expression casette
GmVPS9a2 gene recombinant vectors import in purpose plant.
In the above method, wherein the GmVPS9a2 gene can be modified first as follows, then import in receptor seed plant,
To reach better expression effect:
1) it modifies and optimizes according to actual needs, so that gene efficient expression；For example, can be according to recipient plant institute partially
The codon of love changes its codon while keeping the amino acid sequence of GmVPS9a2 gene of the present invention to meet and plant
Object preferences；In optimization process, it is desirable that keep certain G/C content in the coded sequence after optimization, planted with being best implemented with
The high level expression of quiding gene in object, wherein G/C content can be 35%, be more than 45%, more than 50% or more than about 60%；
2) gene order of neighbouring initial methionine is modified, so that translation effectively starting；For example, using in plant
The effective sequence known is modified；
3) it is connect with the promoter of various plants expression, in favor of its expression in plant；The promoter may include
Composing type, induction type, timing adjusting, growth adjustment, Chemical Regulation, tissue are preferably and tissue-specific promoter；Promoter
Selection will need with expression time and space and be changed, and also depend on target kind；Such as the specificity of tissue or organ
Promoter is expressed, receptor as needed is depending on what period of development；Although demonstrating many from dicotyledon
Promoter can act in monocotyledon, and vice versa, but it is desirable to select dicot promoters are used for
Expression in dicotyledon, monocotyledonous promoter is for the expression in monocotyledon；
4) it is connect with suitable transcription terminator, can also be improved the expression efficiency of gene of the present invention；Such as from
The tml of CaMV, from the E9 of rbcS；Any known available terminator to work in plant can be with the present invention
Gene is attached；
5) enhancer sequence is introduced, such as intron sequences (such as from Adhl and bronzel) and viral leader sequence
(such as from TMV, MCMV and AMV).
The GmVPS9a2 gene recombinant vectors can be by using Ti-plasmids, plant virus carrying agent, and direct DNA turns
Change, microinjection, the standard biologics technical method such as electroporation imports plant cell (Weissbach, 1998, Method for
Plant Molecular Biology VIII,Academy Press,New York,pp.411-463；Geiserson and
Corey,1998,Plant Molecular Biology(2nd Edition).)。
In the above method, the genetically modified plants are interpreted as not only including by the GmVPS9a2 genetic transformation purpose plant
Obtained first generation genetically modified plants also include its filial generation.For genetically modified plants, the gene can be bred in the species,
The gene transfer can also be entered to other kinds of same species with traditional breeding techniques, particularly including in commercial variety.It is described
Genetically modified plants include seed, callus, intact plant and cell.
In order to solve the above technical problems, the present invention also provides GmVPS9a2 or the biomaterial to cultivate storage protein
Sort the application of normal genetically modified plants.
In order to solve the above technical problems, the present invention also provides the biomaterials to cultivate storage protein sorting normally
Application in genetically modified plants.
In order to solve the above technical problems, the present invention also provides GmVPS9a2 or the biomaterials.
In the present invention, the plant can be monocotyledon or dicotyledon.The monocotyledon can be rice.Institute
Stating rice, concretely rice maturation glutelin content reduces mutant glup6.
In the present invention, the storage protein can be the glutelin of the plant, such as the glutelin in the vegetable seeds.Institute
State the abnormal concretely glutelin precursor abnormal accumulation of storage protein sorting, the normal concretely paddy egg of storage protein sorting
Cynanchum glaucescens body Normal accumulation.
It is demonstrated experimentally that glutelin GAP-associated protein GAP GmVPS9a2 of the invention and its encoding gene can be such that mature glutelin contains
Amount reduces mature glutelin content in mutant glup6 and rises to normal level: in rice mutant glup6 and T0 generation, turns empty carrier
The basic indifference of glutelin content in plant seed, glutelin precursor intensity is respectively T0 generation in rice mutant glup6 seed
Turn 2.80 times, 4.08 times and 3.55 times of GmVPS9a2 plant difference strain L1, L2 and L3, it is different that T0 generation turns GmVPS9a2 plant
The mature acidic and alkaline subunit intensity of strain L1, L2 and L3 have promotion.In T0 generation, turns empty carrier plant and rice is mutated
The seed appearance of body glup6 is essentially identical, and Transparency Phenomenon occurs in the seed that T0 generation turns GmVPS9a2 plant difference strain.Experiment
It proves, storage protein sorting GAP-associated protein GAP GmVPS9a2 of the invention and its encoding gene can be used to cultivate a kind of storage protein
Sort normal genetically modified plants.
Detailed description of the invention
Fig. 1 is the qualification result for turning GmVPS9a1 plant in T0 generation.Wherein, swimming lane 1 is positive control, and swimming lane 2 is negative right
According to swimming lane 3-7 is to turn GmVPS9a1 plant in T0 generation.
Fig. 2 is the qualification result for turning GmVPS9a2 plant in T0 generation.Wherein, swimming lane 1 is positive control, and swimming lane 2 is negative right
According to swimming lane 3-7 is to turn GmVPS9a2 plant T0 generation, and swimming lane 8 is to turn empty carrier plant in T0 generation.
Fig. 3 is seed appearance and the cross section for turning GmVPS9a1 plant in T0 generation.Wherein, TC65 indicates 65, glup6 table in platform
Show that rice mutant glup6, L1-L3 are respectively the different strains for turning GmVPS9a1 plant in T0 generation.
Fig. 4 is seed appearance and the cross section for turning GmVPS9a2 plant in T0 generation.Wherein, TC65 indicates 65, glup6 table in platform
Show that rice mutant glup6, L1-L3 are respectively the different strains for turning GmVPS9a2 plant in T0 generation.
Fig. 5 is the electrophoresis result for the seed-protein for turning GmVPS9a1 plant in T0 generation.Wherein, TC65 is indicated 65 in platform,
Glup6 indicates that rice mutant glup6, L1-L3 are respectively the different strains for turning GmVPS9a1 plant in T0 generation.
Fig. 6 is the electrophoresis result for the seed-protein for turning GmVPS9a2 plant in T0 generation.Wherein, TC65 is indicated 65 in platform,
Glup6 indicates that rice mutant glup6, L1-L3 are respectively the different strains for turning GmVPS9a2 plant in T0 generation.
Fig. 7 is the glutelin precursor intensity results turned in T0 generation in the seed of GmVPS9a1 plant.Wherein, TC65 is indicated in platform
65, glup6 indicate that rice mutant glup6, L1-L3 are respectively the different strains for turning GmVPS9a1 plant in T0 generation.
Fig. 8 is the glutelin precursor intensity results turned in T0 generation in the seed of GmVPS9a2 plant.Wherein, TC65 is indicated in platform
65, glup6 indicate that rice mutant glup6, L1-L3 are respectively the different strains for turning GmVPS9a2 plant in T0 generation.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Carrier pCAMBIA1300-221 (Xiao-Bao Ying et al., RNA-Dependent in following embodiments
RNA Polymerase 1from Nicotiana tabacum Suppresses RNA Si lencing and Enhances
Viral Infection in Nicotiana benthamiana.Plant Cell.2010) public can obtain from applicant
, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other purposes and uses.
Rice mutant glup6 (Tian et al., Gene analysis of new 57H in following embodiments
Mutant gene, glup6, in rice.R.G.Newslett., 2001,18:48-50.) public can be somebody's turn to do from applicant
Biomaterial, the biomaterial are only attached most importance to used in the related experiment of duplicate invention, not can be used as other purposes and are used.
Embodiment 1, storage protein sorting GAP-associated protein GAP can regulate and control the sorting of storage protein
Present embodiments provide two from soybean Williams 82 (Haun, W.J., Hyten, D.L., Xu, W.W.,
Gerhardt,D.J.,Albert,T.J.,Richmond,T.,Jeddeloh,J.A.,Jia,G.F.,Springer,N.M.,
Vance,C.P.&Stupar,R.M.(2011).The Composition and Origins of Genomic Variation
among Individuals of the Soybean Reference Cultivar Williams 82.Plant
Physiology 155,645-655. national genebank number: I2A12645, Unified number: WDD00587) storage protein point
Related protein gene is selected, is storage protein sorting GAP-associated protein GAP GmVPS9a1 gene and storage protein sorting GAP-associated protein GAP respectively
GmVPS9a2 gene.
1, the preparation of recombinant vector and recombinant bacterium
The 1- of SEQ ID No.2 in sequence table is inserted between the XbaI of carrier pCAMBIA1300-221 identifies sequence
DNA molecular shown in 1407 nucleotide, other sequences are constant, obtain recombinant vector, which is named as
pCAMBIA1300-221-GmVPS9a1.The difference of pCAMBIA1300-221-GmVPS9a1 and pCAMBIA1300-221 only exists
In: pCAMBIA1300-221-GmVPS9a1 is to be inserted into SEQ in sequence table between the XbaI of pCAMBIA1300-221 identifies sequence
The recombinant vector that DNA molecular shown in the 1-1407 nucleotide of ID No.2 obtains.pCAMBIA1300-221-GmVPS9a1
(i.e. storage protein sorts GAP-associated protein GAP GmVPS9a1, abbreviation GmVPS9a1 egg to protein shown in middle expression SEQ ID No.1
It is white), the expression of GmVPS9a1 albumen is started by 35S promoter.
Wherein, SEQ ID No.1 is made of 468 amino acid, (the i.e. storage egg of the DNA molecular as shown in SEQ ID No.2
White sorting GAP-associated protein GAP GmVPS9a1 gene, abbreviation GmVPS9a1 gene) coding.
The 1- of SEQ ID No.4 in sequence table is inserted between the XbaI of carrier pCAMBIA1300-221 identifies sequence
DNA molecular shown in 1401 nucleotide, other sequences are constant, obtain recombinant vector, which is named as
pCAMBIA1300-221-GmVPS9a2.The difference of pCAMBIA1300-221-GmVPS9a2 and pCAMBIA1300-221 only exists
In: pCAMBIA1300-221-GmVPS9a2 is to be inserted into SEQ in sequence table between the XbaI of pCAMBIA1300-221 identifies sequence
The recombinant vector that DNA molecular shown in the 1-1401 nucleotide of ID No.4 obtains.pCAMBIA1300-221-GmVPS9a2
(i.e. storage protein sorts GAP-associated protein GAP GmVPS9a2, abbreviation GmVPS9a2 egg to protein shown in middle expression SEQ ID No.3
It is white), the expression of GmVPS9a2 albumen is started by 35S promoter.
Wherein, SEQ ID No.3 is made of 466 amino acid, (the i.e. storage egg of the DNA molecular as shown in SEQ ID No.4
White sorting GAP-associated protein GAP GmVPS9a2 gene, abbreviation GmVPS9a2 gene) coding.
Respectively by carrier pCAMBIA1300-221, pCAMBIA1300-221-GmVPS9a1 and pCAMBIA1300-221-
GmVPS9a2 is imported in Agrobacterium EHA105 bacterial strain (Invitrogen (Shanghai) Trading Co., Ltd.), and what is respectively obtained contains
The recombination agriculture bar of pCAMBIA1300-221, pCAMBIA1300-221-GmVPS9a1 and pCAMBIA1300-221-GmVPS9a2
Bacterium is respectively designated as EH-pCAMBIA1300-221, EH-pCAMBIA1300-221-GmVPS9a1 and EH-
pCAMBIA1300-221-GmVPS9a2。
2, the preparation of genetically modified plants
It is utilized respectively recombinational agrobacterium EH-pCAMBIA1300-221, EH-pCAMBIA1300-221- of step 1
GmVPS9a1 and EH-pCAMBIA1300-221-GmVPS9a2 carries out transgenic experiments, the specific steps are as follows:
(1) 28 DEG C is cultivated EH-pCAMBIA1300-221-GmVPS9a116 hours, collects thallus, and be diluted to N6 liquid
To OD in culture medium (Sigma company, C1416)600≈ 0.5 obtains bacterium solution；
(2) bacterium solution of the rice mutant glup6 mature embryo embryo callus of culture to one month and step (1) is mixed
30min is infected in conjunction, and filter paper is transferred in co-culture medium (N6 solid medium, Sigma company) after blotting bacterium solution, 24 DEG C of trainings altogether
It supports 3 days；
(3) callus after co-culturing step (2) is seeded in (the N6 solid screening and culturing of N6 solid screening and culturing medium 1
Base 1 is that the culture medium that the obtained hygromycin concentration of hygromycin is 100mg/L is added into N6 solid medium) upper 24 DEG C of cultures 16
It carries out first time screening；
(4) callus of the health after picking step (3) is screened for the first time is transferred on above-mentioned N6 solid screening and culturing medium 1
Culture carries out programmed screening at 24 DEG C, and every 15 days subcultures are primary；
(5) callus of the health after picking step (4) programmed screening is transferred to N6 solid screening and culturing medium 2 (N6 is solid
Body screening and culturing medium 2 is the culture medium that the hygromycin concentration that addition hygromycin obtains into N6 solid medium is 50mg/L) on
Culture carries out third time screening at 24 DEG C, and every 15 days subcultures are primary；
(6) resistant calli that picking step (5) obtains is transferred to rice differential medium (the outstanding science and technology of West Beijing U.S. is limited
Products, article No. C167) it is 24 DEG C upper at broken up, obtain seedling differentiation T0 generation turn GmVPS9a1 plant.
According to the method for above-mentioned steps, EH-pCAMBIA1300-221-GmVPS9a1 is replaced with into EH- respectively
PCAMBIA1300-221 and EH-pCAMBIA1300-221-GmVPS9a2, other steps are constant, respectively obtain T0 generation turn sky
In carrier plant and T0 generation, turn GmVPS9a2 plant.
Using primer 5 '-GAAGTTTTCGGGGAGTTTCC-3 ' and 5 '-AGCAACAAGGTCATCCTTCG-3 ' to T0 generation
Turn GmVPS9a1 plant to be identified, uses pCAMBIA1300-221-GmVPS9a1 as positive control, with rice japonica rice variety
Kitaake genomic DNA is as negative control, as a result as shown in Figure 1.The results show that in T0 generation, turns GmVPS9a1 plant and contains
Target gene GmVPS9a1 gene (swimming lane 3-7 in Fig. 1).
Using primer 5 '-AGTTTTCGGGGAGTTTCCAT-3 ' and 5 '-CATCACCCTCAGGAGCAACT-3 ' to T0 generation
Turn GmVPS9a2 plant and T0 generation turns empty carrier plant and identified, uses pCAMBIA1300-221-GmVPS9a2 right as the positive
According to using rice japonica rice variety Kitaake genomic DNA as negative control, as a result as shown in Figure 2.The results show that in T0 generation, turns
GmVPS9a2 positive plant contains target gene GmVPS9a2 gene (swimming lane 3-7 in Fig. 2), and T0 generation turns empty carrier plant and is free of
There is GmVPS9a2 gene (swimming lane 8 in Fig. 2).
3, the Phenotypic examination of transgenic plant
In triplicate, repeating experiment every time, specific step is as follows for experiment:
In the T0 generation of step 2, is turned into empty carrier plant respectively, T0 generation turns GmVPS9a1 plant and T0 generation turns GmVPS9a2 plant
And 65 in platform, rice mutant glup6 be planted in experimental plot, until seed is mature.After seed is mature, each plant seed is collected
And decladding, observe seed.
The results show that T0 generation turns empty carrier plant and the seed appearance of rice mutant glup6 is essentially identical, in T0 generation, turns
In GmVPS9a1 plant and T0 generation, turn GmVPS9a2 plant seed and occur Transparency Phenomenon (Fig. 3 and Fig. 4), and T0 generation turns
The seed that GmVPS9a1 plant and T0 generation turn GmVPS9a2 plant difference strain has different degrees of Transparency Phenomenon.
Extracting T0 respectively, GmVPS9a1 plant turns GmVPS9a2 plant difference strain with T0 generation and T0 generation turns unloaded for turning
Body plant, rice mutant glup6,65 Seed Storage Proteins carry out protein electrophorese (Fig. 5 and Fig. 6) in platform, according to protein electrophoresis knot
Fruit quantifies glutelin precursor, and glutelin precursor intensity is as shown in Figure 7 and Figure 8 in each strain.
The results show that glutelin precursor intensity is respectively to turn GmVPS9a1 plant in T0 generation in rice mutant glup6 seed
In 2.28 times, 1.87 times and 2.47 times of different strain L1, L2 and L3, T0 generation, turn GmVPS9a1 plant difference strain L1, L2 and L3
Mature acidic and alkaline subunit intensity have promotion；Glutelin precursor intensity is distinguished in rice mutant glup6 seed
Turn 2.80 times, 4.08 times and 3.55 times of GmVPS9a2 plant difference strain L1, L2 and L3 for T0 generation, T0 generation turns GmVPS9a2 plant
The mature acidic and alkaline subunit intensity of different strain L1, L2 and the L3 of strain has promotion.Show GmVPS9a1 and its gene
With GmVPS9a2 and its gene the storage protein of rice mutant glup6 can sort and restore normal.
Claims (3)
1. a kind of method cultivated storage protein and sort normal genetically modified plants, including storage protein is imported into recipient plant
The step of encoding gene of sorting GAP-associated protein GAP GmVPS9a2 obtains genetically modified plants；The recipient plant storage protein sorting is different
Often, the genetically modified plants storage protein sorting is normal；The plant is rice mutantglup6；The storage protein sorting
The nucleotide sequence of the encoding gene of GAP-associated protein GAP GmVPS9a2 is as shown in SEQ ID No.4 in sequence table.
2. storage protein, which sorts GAP-associated protein GAP GmVPS9a2, sorts the application in normal genetically modified plants in cultivation storage protein；
The plant is rice mutantglup6；The amino acid sequence of the storage protein sorting GAP-associated protein GAP GmVPS9a2 is such as
In sequence table shown in SEQ ID No.3.
3. following any biomaterial sorts the application in normal genetically modified plants in cultivation storage protein；
A1) the nucleic acid molecules of coding storage protein sorting GAP-associated protein GAP GmVPS9a2；
The nucleotide sequence of the nucleic acid molecules is as shown in SEQ ID No.4 in sequence table；
A2) contain A1) expression cassettes of the nucleic acid molecules；
A3) contain A1) recombinant vectors of the nucleic acid molecules；
A4) contain A2) recombinant vector of the expression cassette；
A5) contain A1) recombinant microorganisms of the nucleic acid molecules；
A6) contain A2) recombinant microorganism of the expression cassette；
A7) contain A3) recombinant microorganism of the recombinant vector；
A8) contain A4) recombinant microorganism of the recombinant vector；
The plant is rice mutantglup6；The amino acid sequence of the storage protein sorting GAP-associated protein GAP GmVPS9a2 is such as
In sequence table shown in SEQ ID No.3.
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| CN102603878A (en) * | 2012-03-26 | 2012-07-25 | 南京农业大学 | Vegetable glutelin transportation storage related protein OsVps9a as well as coding gene and application thereof |
| CN102617717A (en) * | 2012-03-26 | 2012-08-01 | 南京农业大学 | Protein OsGPA3 relevant to sorting of plant glutelin, encoding gene of protein OsGPA3 and applications of protein OsGPA3 and encoding gene |
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| CN102603878A (en) * | 2012-03-26 | 2012-07-25 | 南京农业大学 | Vegetable glutelin transportation storage related protein OsVps9a as well as coding gene and application thereof |
| CN102617717A (en) * | 2012-03-26 | 2012-08-01 | 南京农业大学 | Protein OsGPA3 relevant to sorting of plant glutelin, encoding gene of protein OsGPA3 and applications of protein OsGPA3 and encoding gene |
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| Chai C et al.."ACCESSION：XM_003522545，PREDICTED: Glycine max vacuolar protein sorting-associated protein 9A-like (LOC100792686), mRNA.《Genbank》.2014,Features和Origin部分. * |
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