CN105693834A - Application of soybean protein GmVPS9a2 to regulation over separation of protein stored in plants - Google Patents
Application of soybean protein GmVPS9a2 to regulation over separation of protein stored in plants Download PDFInfo
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- 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)
- C12N15/8241—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
Abstract
The invention discloses application of soybean protein GmVPS9a2 to regulation over separation of protein stored in plants. The GmVPS9a2 is protein B1 with the amino acid sequence being SEQ ID No.3, protein B2 with the amino acid sequence being the (1-465)<th> in the SEQ ID No.3, protein B3 derived from B1 or B2 and fusion protein B4, wherein the protein B3 is obtained by substituting and/or missing and/or adding one or more amino acid residues in the (1-465)<th> in the amino acid sequence SEQ ID No.3 or SEQ ID No.3 and has the same function, and the fusion protein B4 is obtained by connecting a label to the end N or/and the end C of B1 or B2 or B3. Experiments prove that the gap-associated protein in the plant stored protein separation influences separation of the protein stored in the plants and can be used for cultivating transgenic plants with normal stored protein separation.
Description
Technical field
The present invention relates to soybean protein GmVPS9a2 application in regulation and control plant storage protein sorting in biological technical field。
Background technology
Semen sojae atricolor is important industrial crops and cereal crops, containing substantial amounts of unsaturated fatty acid and multiple essential amino acids, stable yield, has plantation in China various places。The protein resource accounting for cosmopolitian plant albumen supply market about 70% is provided, by the important component part of the bean product of soybean processing mankind's ordinary meal especially for the mankind。In nutritive value, its aminoacid composition is close with milk protein, it is in close proximity to the requirement of ideal protein, can be equal to animal proteinum, closest to human amino acid on gene structure, so being the phytoprotein of most nutrition, by the beverage of its making, it is described as " green milk " by nutritionist。U.S. food and the healthy of drug administration (FDA) notice display, eat the food containing 25g soybean protein every day, help minimizing cardiovascular morbidity。Storage protein, as the big class nutrient substance of first in soybean kernel, determines the nutritive value of Semen sojae atricolor and every Protein Index to a great extent。
Soybean storage protein accounts for the 70%-80% of seed total protein content, wherein the highest with 11S and 7S storage protein content, accounts for the 40% and 30% of Seed Storage Protein respectively, they rich in nutritive value, and food processing characteristic good。The sorting approach of cytological research also tentative confirmation 7S and 11S storage protein be one with endoplasmic reticulum be starting point, middle via Golgi body, the transhipment circuit being finally deposition site with protein storage vacuole。For 11S globulin, in endoplasmic reticulum, first synthesize a precursor molecule, after entering storage vacuole, acidic and alkaline subunit can be cut into by vacuolar processing enzyme。
Summary of the invention
The present invention is to provide soybean protein GmVPS9a2 application in regulation and control plant storage protein sorting。
In storage protein provided by the present invention sorting associated protein GmVPS9a2 application in regulation and control plant storage protein sorting；Described storage protein sorting associated protein GmVPS9a2 is following B1) or B2) or B3) or protein B4):
B1) aminoacid sequence is the protein of SEQIDNo.3；
B2) aminoacid sequence is the protein of the 1-465 amino acids of SEQIDNo.3；
B3) in the 1-465 amino acids sequence of SEQIDNo.3 or SEQIDNo.3 through replacement and/or disappearance and/or add that one or several amino acid residue obtains have identical function by A1) or A2) derivative protein；
B4) at B1) or B2) or B3) N end or/and C end connects the fused protein that obtains of label。
Wherein, sequence 1 is made up of 466 aminoacid。
In order to make B1) and B2) in protein be easy to purification, label that can be as shown in table 1 on the amino terminal of the protein shown in the 1-465 amino acids of SEQ ID No .3 or SEQIDNo.3 or carboxyl terminal connect。
Table 1, label sequence
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (is generally 5) | RRRRR |
| Poly-His | 2-10 (is generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned B3) in GmVPS9a2 can synthetic, it is possible to first synthesize its encoding gene, then carry out biological expression and obtain。Above-mentioned B3) in the encoding gene of GmVPS9a2 can by the codon that will lack one or several amino acid residue in the DNA sequence shown in the 1-1395 position nucleotide of SEQ ID No .4 or SEQIDNo.4, and/or carry out the missense mutation of one or several base pair, and/or connect the coded sequence of the label shown in table 1 at its 5 ' end and/or 3 ' ends and obtain。
For solving above-mentioned technical problem, present invention also offers the biomaterial relevant to GmVPS9a2 application in regulation and control plant storage protein sorting。
In the biomaterial relevant to GmVPS9a2 provided by the present invention application in regulation and control plant storage protein sorting, described biomaterial is following A 1) to A14) in any one:
A1) nucleic acid molecules of GmVPS9a2 is encoded；
A2) containing A1) expression cassette of described nucleic acid molecules；
A3) containing A1) recombinant vector of described nucleic acid molecules；
A4) containing A2) recombinant vector of described expression cassette；
A5) containing A1) recombinant microorganism of described nucleic acid molecules；
A6) containing A2) recombinant microorganism of described expression cassette；
A7) containing A3) recombinant microorganism of described recombinant vector；
A8) containing A4) recombinant microorganism of described recombinant vector；
A9) containing A1) the transgenic plant cells system of described nucleic acid molecules；
A10) containing A2) the transgenic plant cells system of described expression cassette；
A11) containing A1) Transgenic plant tissue of described nucleic acid molecules；
A12) containing A2) Transgenic plant tissue of described expression cassette；
A13) containing A1) the transgenic plant organ of described nucleic acid molecules；
A14) containing A2) the transgenic plant organ of described expression cassette。
In the application in regulation and control plant storage protein sorting of the above-mentioned biomaterial relevant to GmVPS9a2, A1) described nucleic acid molecules can be following a1) a2) or a3) or gene a4):
A1) nucleotide sequence is cDNA molecule or the DNA molecular of SEQ ID No .4；
A2) nucleotide sequence is cDNA molecule or the DNA molecular of the 1-1395 position nucleotide of SEQ ID No .4；
A3) and a1) or a2) nucleotide sequence that limits has 75% or more than 75% homogeneity, and the cDNA molecule of coding GmVPS9a2 or genomic DNA molecule；
A4) under strict conditions with a1) or the a2) nucleotide sequence hybridization that limits, and the cDNA molecule of coding GmVPS9a2 or genomic DNA molecule。
Wherein, described nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；Described nucleic acid molecules can also be RNA, such as mRNA or hnRNA etc.。
Wherein, SEQIDNo.4 is made up of 1401 nucleotide, encodes the protein shown in SEQIDNo.3。
Those of ordinary skill in the art can adopt known method easily, for instance the method for orthogenesis and point mutation, and the nucleotide sequence of the coding GmVPS9a2 of the present invention is suddenlyd change。Those are through manually modified, there is the nucleotide of the nucleotide sequence 75% or higher homogeneity separating the GmVPS9a2 obtained with the present invention, as long as encoding GmVPS9a2 and there is GmVPS9a2 function, all it is derived from the nucleotide sequence of the present invention and is equal to the sequence of the present invention。
Term used herein " homogeneity " refers to the sequence similarity with native sequence nucleic acid。" homogeneity " includes having 75% or higher with the nucleotide sequence of the protein of the aminoacid sequence composition shown in the coding GmVPS9a2 of the present invention, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher homogeneity。Homogeneity can with the naked eye or computer software be evaluated。Using computer software, the homogeneity between two or more sequences can use percentage ratio (%) to represent, it can be used to the homogeneity evaluating between correlated series。
In the application in regulation and control plant storage protein sorting of the above-mentioned biomaterial relevant to GmVPS9a2, described stringent condition is at 2 × SSC, in the solution of 0.1%SDS, hybridize at 68 DEG C and wash film 2 times, each 5min, again in the solution of 0.5 × SSC, 0.1%SDS, hybridize at 68 DEG C and wash film 2 times, each 15min；Or, 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS solution in, under 65 DEG C of conditions hybridization and wash film。
Above-mentioned 75% or more than 75% homogeneity, can be the homogeneity of 80%, 85%, 90% or more than 95%。
In the application in regulation and control plant storage protein sorting of the above-mentioned biomaterial relevant to GmVPS9a2, the expression cassette (GmVPS9a2 expression casette) of the nucleic acid molecules containing coding GmVPS9a2 described in A2), refer to express the DNA of GmVPS9a2 in host cell, this DNA not only can include the promoter starting GmVPS9a2 genetic transcription, may also include the terminator terminating GmVPS9a2 genetic transcription。Further, described expression cassette may also include enhancer sequence。Can be used for the promoter of the present invention to include but not limited to: constitutive promoter, the promoter that tissue, organ and growth are special, and inducible promoter。The example of promoter includes but not limited to: the constitutive promoter 35S of cauliflower mosaic virus: from the wound-inducible promoter of Fructus Lycopersici esculenti, leucine aminopeptidase (" LAP ", Chao et al. (1999) PlantPhysiol120:979-992)；From Nicotiana tabacum L. chemical inducible promoter, pathogeny be correlated with 1 (PR1) (by salicylic acid and BTH (diazosulfide-7-carbothioic acid S-methyl ester) induction)；Fructus Lycopersici esculenti protease inhibitor II promoter (PIN2) or LAP promoter (all 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 specific promoters, such as Millet Seed specificity promoter pF128 (CN101063139B (Chinese patent 200710099169.7)), the special promoter of seed storage protein matter is (such as, the promoter (Beachy et al. (1985) EMBOJ.4:3047-3053) of phaseollin., napin, oleosin and Semen sojae atricolor betaconglycin)。They can be used alone or be combined use with other plant promoter。All references cited herein all quotes in full。Suitable transcription terminator includes but not limited to: Agrobacterium nopaline syntase terminator (NOS terminator), cauliflower mosaic virus CaMV35S terminator, tml terminator, Semen Pisi sativi rbcSE9 terminator and nopaline and octopine synthase terminator (referring to, for instance: Odell et al. (I985) Nature313:810；Rosenberg et al. (1987) Gene, 56:125；Guerineau et al. (1991) Mol.Gen.Genet, 262:141；Proudfoot (1991) Cell, 64:671；Sanfacon et al. GenesDev., 5:141；Mogen et al. (1990) PlantCell, 2:1261；Munroe et al. (1990) Gene, 91:151；Ballad et al. (1989) NucleicAcidsRes.17:7891；Joshi et al. (1987) NucleicAcidRes., 15:9627)。
Available existing expression vector establishment contains the recombinant vector of described GmVPS9a2 expression casette。Described plant expression vector includes double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment。Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company) etc.。Described plant expression vector also can comprise 3 ' end untranslated regions of exogenous gene, namely comprises polyadenylation signals and the DNA fragmentation of any other participation mRNA processing or gene expression。The bootable polyadenylic acid of described polyadenylation signals joins 3 ' ends of mRNA precursor, as Agrobacterium crown gall nodule induces the untranslated region that (Ti) plasmid gene (such as rouge alkali synthetase gene Nos), plant gene (such as soybean storage protein gene) 3 ' end is transcribed to be respectively provided with similar functions。When using the gene constructed plant expression vector of the present invention, it be also possible to use enhancer, including translational enhancer or transcriptional enhancer, these enhancer regions can be ATG initiation codon or neighboring region start codon etc., but must be identical with the reading frame of coded sequence, to ensure the correct translation of whole sequence。The source of described translation control signal and start codon is widely, it is possible to be natural, it is also possible to be synthesis。Translation initiation region can come from transcription initiation region or structural gene。For the ease of transgenic plant cells or plant being identified and screening, plant expression vector used can be processed, the enzyme of color change or the gene (gus gene of luminophor can be produced as added the coding can expressed in plant, luciferase genes etc.), antibiotic marker gene is (as given the nptII gene to kanamycin and associated antibiotic resistance, give the bar gene to herbicide phosphinothricin resistance, give the hph gene to antibiotic hygromycin resistance, with the dhfr gene given methotrexate resistance, give EPSPS gene to glyphosate) or anti-chemical reagent marker gene etc. (such as anti-herbicide gene), the mannose-6-phosphate isomerase gene of metabolism mannose ability is provided。From the security consideration of transgenic plant, any selected marker can be not added with, directly screen transformed plant with adverse circumstance。
In the application in regulation and control plant storage protein sorting of the above-mentioned biomaterial relevant to GmVPS9a2, described carrier can be plasmid, glutinous grain, phage or viral vector。
In the application in regulation and control plant storage protein sorting of the above-mentioned biomaterial relevant to GmVPS9a2, described microorganism can be yeast, antibacterial, algae or fungus, such as Agrobacterium。Described Agrobacterium can be Agrobacterium EHA105。
In the application in regulation and control plant storage protein sorting of the above-mentioned biomaterial relevant to GmVPS9a2, described transgenic plant cells system, Transgenic plant tissue and transgenic plant organ all do not include propagating materials。
In an embodiment of the invention, the encoding gene of GmVPS9a2 is imported in Agrobacterium tumefaciems EHA105 by the recombinant vector of the expression cassette of the encoding gene containing GmVPS9a2。Described recombinant vector is the DNA molecular shown in the nucleotide of 1-1401 position inserting SEQ ID No .4 between the XbaI recognition sequence of carrier pCAMBIA1300-221, and other sequences are constant, the recombinant vector pCAMBIA1300-221-GmVPS9a2 obtained。PCAMBIA1300-221-GmVPS9a2 and pCAMBIA1300-221 differs only in: pCAMBIA1300-221-GmVPS9a2 inserts the recombinant vector that the DNA molecular shown in the nucleotide of 1-1401 position of SEQ ID No .4 obtains between the XbaI recognition sequence of pCAMBIA1300-221。PCAMBIA1300-221-GmVPS9a2 expresses the protein (namely storage protein sorting associated protein GmVPS9a2, is called for short GmVPS9a2 albumen) shown in SEQIDNo.3, and the expression of GmVPS9a2 albumen is started by 35S。
Wherein, SEQIDNo.3 is made up of 466 aminoacid, DNA molecular (namely storage protein sorting associated protein GmVPS9a2 gene, the is called for short GmVPS9a2 gene) coding shown in SEQIDNo.4。
For solving above-mentioned technical problem, present invention also offers a kind of method cultivating the storage protein normal transgenic plant of sorting。
A kind of method cultivating the storage protein normal transgenic plant of sorting provided by the present invention, obtains the step of transgenic plant including the encoding gene importing GmVPS9a2 in recipient plant；The sorting of described recipient plant storage protein is abnormal, and the sorting of described transgenic plant storage protein is normal。
In said method, the encoding gene of described GmVPS9a2 is A1) described nucleic acid molecules。
In an embodiment of the present invention, the encoding gene of described GmVPS9a2 is imported in purpose plant by the GmVPS9a2 gene recombinant vectors containing GmVPS9a2 expression casette。
In said method, wherein said GmVPS9a2 gene can first be modified as follows, then imports in receptor seed plant, to reach better expression effect:
1) carry out according to actual needs modifying and optimizing, so that gene efficient expression；Such as, the codon can having a preference for according to recipient plant, while keeping the aminoacid sequence of GmVPS9a2 gene of the present invention, change its codon to meet plant-preference；In optimization process, it is desirable that the coded sequence after optimization keeps certain G/C content, with the high level expression being best implemented with in plant quiding gene, wherein G/C content can be 35%, more than 45%, more than 50% or more than about 60%；
2) gene order of contiguous initial methionine is modified, so that translation is effectively initial；Such as, effective sequence known in plant is utilized to modify；
3) it is connected with the promoter of various expression of plants, is beneficial to its expression in plant；Described promoter can include composing type, induction type, sequential adjustment, Growth adjustment, Chemical Regulation, tissue preferably and tissue-specific promoter；The selection of promoter will change along with expression time and space requirement, and also depend on target species；The such as specific expressing promoter of tissue or organ, receptor in what period grown is determined as required；Although it is operational for demonstrating the many promoteres deriving from dicotyledon in monocotyledon, vice versa, but it is desirable to select dicot promoters is for the expression in dicotyledon, monocotyledonous promoter is for the expression in monocotyledon；
4) it is connected with the transcription terminator being suitable for, it is also possible to improve the expression efficiency of gene of the present invention；Such as derive from the tml of CaMV, derive from the E9 of rbcS；Any known available terminator worked in plant can be attached with gene of the present invention；
5) enhancer sequence is introduced, such as intron sequences (such as deriving from Adhl and bronzel) and viral leader sequence (such as deriving from TMV, MCMV and AMV)。
Described GmVPS9a2 gene recombinant vectors can pass through to use Ti-plasmids, plant virus carrying agent, directly delivered DNA, microinjection, the standard biologic technical method such as electroporation import plant cell (Weissbach, 1998, MethodforPlantMolecularBiologyVIII, AcademyPress, NewYork, pp.411-463；GeisersonandCorey, 1998, PlantMolecularBiology (2ndEdition) .)。
In said method, described transgenic plant is interpreted as not only comprising the first generation transgenic plant obtained by described GmVPS9a2 gene transformation purpose plant, also includes its filial generation。For transgenic plant, it is possible to breed this gene in these species, it is also possible to this gene transfer is entered other kind of same species by traditional breeding method, in commercial variety。Described transgenic plant includes seed, callus, whole plant and cell。
For solving above-mentioned technical problem, present invention also offers GmVPS9a2 or described biomaterial in the application cultivating the storage protein normal transgenic plant of sorting。
For solving above-mentioned technical problem, present invention also offers the application in cultivating the storage protein normal transgenic plant of sorting of the described biomaterial。
For solving above-mentioned technical problem, present invention also offers GmVPS9a2 or described biomaterial。
In the present invention, described plant can be monocotyledon or dicotyledon。Described monocotyledon can be Oryza sativa L.。Described Oryza sativa L. concretely Oryza sativa L. maturation glutelin content reduces mutant glup6。
In the present invention, described storage protein can be the glutelin of described plant, the glutelin in described plant seed。The abnormal concretely glutelin precursor abnormal accumulation of described storage protein sorting, the normal concretely glutelin precursor Normal accumulation of described storage protein sorting。
Experiment proves, the glutelin associated protein GmVPS9a2 of the present invention and encoding gene thereof can make ripe glutelin content reduce ripe glutelin content in mutant glup6 and rise to normal level: rice mutant glup6 and T0 generation turns the basic zero difference of glutelin content in empty carrier plant seed, in rice mutant glup6 seed two-story valley amyloid protein precursor intensity respectively T0 generation, turns GmVPS9a2 plant difference strain L1, 2.80 times of L2 and L3, 4.08 times and 3.55 times, in T0 generation, turns GmVPS9a2 plant difference strain L1, ripe acidic and the alkaline subunit intensity of L2 and L3 all have lifting。It is essentially identical with the seed outward appearance of rice mutant glup6 that in T0 generation, turns empty carrier plant, and T0 generation turns the seed of GmVPS9a2 plant difference strain and occurs in that Transparency Phenomenon。It is demonstrated experimentally that the storage protein sorting associated protein GmVPS9a2 of the present invention and encoding gene thereof can be used to cultivate a kind of storage protein sorts normal transgenic plant。
Accompanying drawing explanation
Fig. 1 is qualification result T0 generation turning GmVPS9a1 plant。Wherein, swimming lane 1 is positive control, and swimming lane 2 is negative control, and swimming lane 3-7 is T0 for turning GmVPS9a1 plant。
Fig. 2 is qualification result T0 generation turning GmVPS9a2 plant。Wherein, swimming lane 1 is positive control, and swimming lane 2 is negative control, and swimming lane 3-7 is T0 for turning GmVPS9a2 plant, and swimming lane 8 turns empty carrier plant for T0 generation。
Fig. 3 is the seed outward appearance and the cross section that T0 generation turn GmVPS9a1 plant。Wherein, TC65 represents in platform that 65, glup6 represent that rice mutant glup6, L1-L3 respectively T0 generation turns the different strains of GmVPS9a1 plant。
Fig. 4 is the seed outward appearance and the cross section that T0 generation turn GmVPS9a2 plant。Wherein, TC65 represents in platform that 65, glup6 represent that rice mutant glup6, L1-L3 respectively T0 generation turns the different strains of GmVPS9a2 plant。
Fig. 5 is electrophoresis result T0 generation turning the seed-protein of GmVPS9a1 plant。Wherein, TC65 represents in platform that 65, glup6 represent that rice mutant glup6, L1-L3 respectively T0 generation turns the different strains of GmVPS9a1 plant。
Fig. 6 is electrophoresis result T0 generation turning the seed-protein of GmVPS9a2 plant。Wherein, TC65 represents in platform that 65, glup6 represent that rice mutant glup6, L1-L3 respectively T0 generation turns the different strains of GmVPS9a2 plant。
Fig. 7 is glutelin precursor intensity results T0 generation turning in the seed of GmVPS9a1 plant。Wherein, TC65 represents in platform that 65, glup6 represent that rice mutant glup6, L1-L3 respectively T0 generation turns the different strains of GmVPS9a1 plant。
Fig. 8 is glutelin precursor intensity results T0 generation turning in the seed of GmVPS9a2 plant。Wherein, TC65 represents in platform that 65, glup6 represent that rice mutant glup6, L1-L3 respectively T0 generation turns the different strains of GmVPS9a2 plant。
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention being further described in detail, the embodiment provided is only for illustrating the present invention, rather than in order to limit the scope of the present invention。
Experimental technique in following embodiment, if no special instructions, is conventional method。
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain。
Carrier pCAMBIA1300-221 (Xiao-BaoYingetal. in following embodiment, RNA-DependentRNAPolymerase1fromNicotianatabacumSuppresse sRNASilencingandEnhancesViralInfectioninNicotianabentham iana.PlantCell.2010) public can obtain from applicant, this biomaterial only attach most importance to duplicate invention related experiment used by, can not use as other purposes。
Rice mutant glup6 (Tianetal. in following embodiment, Geneanalysisofnew57Hmutantgene, glup6, inrice.R.G.Newslett., 2001,18:48-50.) public can obtain this biomaterial from applicant, this biomaterial only attach most importance to duplicate invention related experiment used by, can not use as other purposes。
Embodiment 1, storage protein sorting associated protein can regulate and control the sorting of storage protein
Present embodiments provide two and derive from Semen sojae atricolor Williams82 (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) .TheCompositionandOriginsofGenomicVariationamongIndividu alsoftheSoybeanReferenceCultivarWilliams82.PlantPhysiolo gy155, 645-655. national genebank is numbered: I2A12645, Unified number: WDD00587) storage protein sorting related protein gene, it is storage protein sorting associated protein GmVPS9a1 gene and storage protein sorting associated protein GmVPS9a2 gene respectively。
1, the preparation of recombinant vector and recombinant bacterium
Inserting the DNA molecular shown in the nucleotide of 1-1407 position of SEQ ID No .2 between the XbaI recognition sequence of carrier pCAMBIA1300-221, other sequences are constant, obtain recombinant vector, by this recombinant vector called after pCAMBIA1300-221-GmVPS9a1。PCAMBIA1300-221-GmVPS9a1 and pCAMBIA1300-221 differs only in: pCAMBIA1300-221-GmVPS9a1 inserts the recombinant vector that the DNA molecular shown in the nucleotide of 1-1407 position of SEQ ID No .2 obtains between the XbaI recognition sequence of pCAMBIA1300-221。Expressing the protein (namely storage protein sorting associated protein GmVPS9a1, is called for short GmVPS9a1 albumen) shown in SEQIDNo.1 in pCAMBIA1300-221-GmVPS9a1, the expression of GmVPS9a1 albumen is started by 35S promoter。
Wherein, SEQIDNo.1 is made up of 468 aminoacid, DNA molecular (namely storage protein sorting associated protein GmVPS9a1 gene, the is called for short GmVPS9a1 gene) coding shown in SEQIDNo.2。
Inserting the DNA molecular shown in the nucleotide of 1-1401 position of SEQ ID No .4 between the XbaI recognition sequence of carrier pCAMBIA1300-221, other sequences are constant, obtain recombinant vector, by this recombinant vector called after pCAMBIA1300-221-GmVPS9a2。PCAMBIA1300-221-GmVPS9a2 and pCAMBIA1300-221 differs only in: pCAMBIA1300-221-GmVPS9a2 inserts the recombinant vector that the DNA molecular shown in the nucleotide of 1-1401 position of SEQ ID No .4 obtains between the XbaI recognition sequence of pCAMBIA1300-221。Expressing the protein (namely storage protein sorting associated protein GmVPS9a2, is called for short GmVPS9a2 albumen) shown in SEQIDNo.3 in pCAMBIA1300-221-GmVPS9a2, the expression of GmVPS9a2 albumen is started by 35S promoter。
Wherein, SEQIDNo.3 is made up of 466 aminoacid, DNA molecular (namely storage protein sorting associated protein GmVPS9a2 gene, the is called for short GmVPS9a2 gene) coding shown in SEQIDNo.4。
Respectively carrier pCAMBIA1300-221, pCAMBIA1300-221-GmVPS9a1 and pCAMBIA1300-221-GmVPS9a2 are imported in Agrobacterium EHA105 bacterial strain (English Weihe River victory base (Shanghai) trade Co., Ltd), the recombinational agrobacterium containing pCAMBIA1300-221, pCAMBIA1300-221-GmVPS9a1 and pCAMBIA1300-221-GmVPS9a2 respectively obtained, is respectively designated as EH-pCAMBIA1300-221, EH-pCAMBIA1300-221-GmVPS9a1 and EH-pCAMBIA1300-221-GmVPS9a2。
2, the preparation of transgenic plant
The recombinational agrobacterium EH-pCAMBIA1300-221, EH-pCAMBIA1300-221-GmVPS9a1 and the EH-pCAMBIA1300-221-GmVPS9a2 that are utilized respectively step 1 carry out transgenic experiments, specifically comprise the following steps that
Cultivate EH-pCAMBIA1300-221-GmVPS9a116 hour for (1) 28 DEG C, collect thalline, and be diluted in N6 fluid medium (Sigma company, C1416) to OD600≈ 0.5, it is thus achieved that bacterium solution；
(2) by the bacterium solution mixed infection 30min of the rice mutant glup6 mature embryo embryo callus Yu step (1) that are cultured to one month, filter paper proceeds to after blotting bacterium solution and co-cultures culture medium (N6 solid medium, Sigma company) in, 24 DEG C co-culture 3 days；
(3) callus after step (2) being co-cultured is seeded in N6 solid screening culture medium 1 (N6 solid screening culture medium 1 is add the culture medium that hygromycin concentration is 100mg/L that hygromycin obtains in N6 solid medium) upper 24 DEG C of cultivations and within 16 days, carries out first time screening；
(4) the healthy callus after picking step (3) first time screening proceeds to cultivate at 24 DEG C in above-mentioned N6 solid screening culture medium 1 and carries out programmed screening, and every 15 days subcultures are once；
(5) the healthy callus after picking step (4) programmed screening proceeds to cultivate at upper 24 DEG C of N6 solid screening culture medium 2 (N6 solid screening culture medium 2 is add the culture medium that hygromycin concentration is 50mg/L that hygromycin obtains in N6 solid medium) and carries out third time screening, and every 15 days subcultures are once；
(6) resistant calli that picking step (5) obtains proceeds to Oryza sativa L. division culture medium (West Beijing Mei Jie Science and Technology Ltd. product, article No. is C167) to break up at upper 24 DEG C, in the T0 generation obtaining seedling differentiation, turns GmVPS9a1 plant。
Method according to above-mentioned steps, EH-pCAMBIA1300-221-GmVPS9a1 is replaced with respectively EH-pCAMBIA1300-221 and EH-pCAMBIA1300-221-GmVPS9a2, other steps are all constant, respectively obtain T0 for turning empty carrier plant and T0 for turning GmVPS9a2 plant。
Utilize primer 5 '-GAAGTTTTCGGGGAGTTTCC-3 ' and 5 '-AGCAACAAGGTCATCCTTCG-3 ' that T0 generation is turned GmVPS9a1 plant to identify, with pCAMBIA1300-221-GmVPS9a1 as positive control, with Oryza sativa L. japonica rice variety Kitaake genomic DNA as negative control, result is as shown in Figure 1。Result shows, T0 generation turns GmVPS9a1 plant and all contains genes of interest GmVPS9a1 gene (in Fig. 1 swimming lane 3-7)。
Utilize primer 5 '-AGTTTTCGGGGAGTTTCCAT-3 ' and 5 '-CATCACCCTCAGGAGCAACT-3 ' that T0 generation is turned GmVPS9a2 plant and T0 generation turns empty carrier plant and identifies, with pCAMBIA1300-221-GmVPS9a2 as positive control, with Oryza sativa L. japonica rice variety Kitaake genomic DNA as negative control, result is as shown in Figure 2。Result shows, T0 generation turns GmVPS9a2 positive plant and all contains genes of interest GmVPS9a2 gene (in Fig. 2 swimming lane 3-7), and T0 generation turns empty carrier plant and do not contain GmVPS9a2 gene (in Fig. 2 swimming lane 8)。
3, the Phenotypic examination of transfer-gen plant
Experiment in triplicate, repeats specifically comprising the following steps that of experiment every time
Respectively are turned empty carrier plant the T0 of step 2 generation, in T0 generation, turns that GmVPS9a1 plant and T0 generation turn in GmVPS9a2 plant and platform 65, rice mutant glup6 is planted in experimental plot, to seed maturity。After seed maturity, collect each plant seed and shell, observing seed。
Result shows, it is essentially identical with the seed outward appearance of rice mutant glup6 that in T0 generation, turns empty carrier plant, in T0 generation, turns GmVPS9a1 plant and T0 generation and turns GmVPS9a2 plant seed and all occur in that Transparency Phenomenon (Fig. 3 and Fig. 4), and T0 generation turns GmVPS9a1 plant and T0 and has Transparency Phenomenon in various degree for the seed turning the different strain of GmVPS9a2 plant。
Extracting T0 generation respectively turns GmVPS9a1 plant and T0 generation and turns the different strain of GmVPS9a2 plant and T0 and carry out protein electrophorese (Fig. 5 and Fig. 6) for turning 65 Seed Storage Proteins in empty carrier plant, rice mutant glup6, platform, carrying out quantitatively according to protein electrophoresis result to glutelin precursor, each strain two-story valley amyloid protein precursor intensity is as shown in Figure 7 and Figure 8。
Result shows, rice mutant glup6 seed two-story valley amyloid protein precursor intensity respectively T0 generation turn GmVPS9a1 plant difference strain L1,2.28 times of L2 and L3,1.87 times and 2.47 times, T0 generation turns GmVPS9a1 plant difference strain L1, the ripe acidic of L2 and L3 and alkaline subunit intensity all lifting；Rice mutant glup6 seed two-story valley amyloid protein precursor intensity respectively T0 generation turn GmVPS9a2 plant difference strain L1,2.80 times of L2 and L3,4.08 times and 3.55 times, T0 generation turns GmVPS9a2 plant difference strain L1, the ripe acidic of L2 and L3 and alkaline subunit intensity all lifting。Showing, GmVPS9a1 and gene thereof and GmVPS9a2 and gene thereof all can make the storage protein sorting of rice mutant glup6 recover normal。
Claims (10)
1. storage protein sorting associated protein GmVPS9a2 application in regulation and control plant storage protein sorting；Described storage protein sorting associated protein GmVPS9a2 is following B1) or B2) or B3) or protein B4):
B1) aminoacid sequence is the protein of SEQIDNo.3；
B2) aminoacid sequence is the protein of the 1-465 amino acids of SEQIDNo.3；
B3) in the 1-465 amino acids sequence of SEQIDNo.3 or SEQIDNo.3 through replacement and/or disappearance and/or add that one or several amino acid residue obtains have identical function by A1) or A2) derivative protein；
B4) at B1) or B2) or B3) N end or/and C end connects the fused protein that obtains of label。
2. the biomaterial relevant to storage protein sorting associated protein GmVPS9a2 described in claim 1 application in regulation and control plant storage protein sorting；Described biomaterial is following A 1) to A14) in any one:
A1) nucleic acid molecules of storage protein sorting associated protein GmVPS9a2 described in coding claim 1；
A2) containing A1) expression cassette of described nucleic acid molecules；
A3) containing A1) recombinant vector of described nucleic acid molecules；
A4) containing A2) recombinant vector of described expression cassette；
A5) containing A1) recombinant microorganism of described nucleic acid molecules；
A6) containing A2) recombinant microorganism of described expression cassette；
A7) containing A3) recombinant microorganism of described recombinant vector；
A8) containing A4) recombinant microorganism of described recombinant vector；
A9) containing A1) the transgenic plant cells system of described nucleic acid molecules；
A10) containing A2) the transgenic plant cells system of described expression cassette；
A11) containing A1) Transgenic plant tissue of described nucleic acid molecules；
A12) containing A2) Transgenic plant tissue of described expression cassette；
A13) containing A1) the transgenic plant organ of described nucleic acid molecules；
A14) containing A2) the transgenic plant organ of described expression cassette。
3. application according to claim 2, it is characterised in that: A1) described nucleic acid molecules is following a1) a2) or a3) or gene a4):
A1) nucleotide sequence is cDNA molecule or the DNA molecular of SEQ ID No .4；
A2) nucleotide sequence is cDNA molecule or the DNA molecular of the 1-1395 position nucleotide of SEQ ID No .4；
A3) and a1) or a2) nucleotide sequence that limits has 75% or more than 75% homogeneity, and the cDNA molecule of storage protein sorting associated protein GmVPS9a2 described in coding claim 1 or genomic DNA molecule；
A4) under strict conditions with a1) or the a2) nucleotide sequence hybridization that limits, and the cDNA molecule of storage protein sorting associated protein GmVPS9a2 described in coding claim 1 or genomic DNA molecule。
4. according to the arbitrary described application of claim 1-3, it is characterised in that: described plant is monocotyledon or dicotyledon。
5. cultivate the method that storage protein sorts normal transgenic plant, obtain the step of transgenic plant including importing the encoding gene of storage protein sorting associated protein GmVPS9a2 described in claim 1 in recipient plant；The sorting of described recipient plant storage protein is abnormal, and the sorting of described transgenic plant storage protein is normal。
6. method according to claim 5, it is characterised in that: described in claim 1, the encoding gene of storage protein sorting associated protein GmVPS9a2 is A1 in claim 3) described nucleic acid molecules。
7. the method according to claim 5 or 6, it is characterised in that: described plant is monocotyledon or dicotyledon。
8. storage protein sorting associated protein GmVPS9a2 application in cultivating the storage protein normal transgenic plant of sorting described in claim 1。
9. the application in cultivating the storage protein normal transgenic plant of sorting of the biomaterial described in Claims 2 or 3。
10. storage protein sorting associated protein GmVPS9a2 described in claim 1 or biomaterial described in Claims 2 or 3。
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| CN108795975A (en) * | 2018-07-04 | 2018-11-13 | 中国农业科学院油料作物研究所 | Application of the wild soybean GAP-associated protein GAP in improving plant resistance to insect |
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