CN106282209A - The application in causing pollen abortion of the plant alpha amylase - Google Patents
The application in causing pollen abortion of the plant alpha amylase Download PDFInfo
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
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Abstract
The present invention provides a kind of plant alpha amylase purposes in causing pollen abortion, belongs to gene engineering technology field.The present invention is by importing the polynucleotide of plant alpha amylase, build recombinant vector and genetic transformation, pollen specific promoter drives alpha amylase in the pollen development phase, degradable starch in advance, pollen germination energy cannot be provided, pollen germination is contained, causes abortion, and then obtains the transfer-gen plant of transgenic pollen abortion.The plant alpha amylase of the present invention may be from the grass genes such as Oryza sativa L. is endogenous, Semen setariae, Sorghum vulgare Pers., Fructus Hordei Vulgaris, Semen Tritici aestivi as pollen abortion gene, highly beneficial to the transgenic plant of crop gene engineering and preparation pollen abortion.Plant alpha amylase gene expression dose of the present invention is accurate, can control transgenic diffusion；Can be used for keeping holding and the breeding of male sterility line, during hybrid seeding, save artificial emasculation step simultaneously, have a extensive future.
Description
Technical field
The invention belongs to molecular biology of plants and gene engineering technology field, be specifically related to a kind of plant alpha amylase and exist
Cause the application in pollen abortion, further relate to combine this nutrition, utilize modern biotechnology, be applied to hybrid seed production technology
System, it is ensured that seed production quality, and improve seed production efficiency；Can also be applied to prevent transgenic from spreading.
Background technology
Plant hybrid use of advantage is one of approach of the most cost-effective raising grain yield, but it still suffers from
Limiting factor, such as, combine that excellent difficulty is relatively big, proportion shared by hybrid crop is less.Although almost all of crops are all
Maximum possibly also with hybrid vigor, but still have enormous potentialities.The most many scholars study, especially water
Rice, its hybrid vigor main path to " two line method " by " three line method ", is improve combo degree of freedom, improves rice heterosis
Level.But the temp-sensing sterile line of " two line method " is influenced by temperature excessive, can cause production of hybrid seeds failure, loss is serious.Due to male
The resource-constrained of sterile line, serious restriction paddy rice cross breeding technology.
At the beginning of the nineties, Mariani et al. utilizes specific promoter (TA29) and the ribose of the anther tapetum from Nicotiana tabacum L.
Nuclease gene (Barnase) recombinant expression cassettes transformation of tobacco and Brassica campestris L, and it is successfully obtained male sterility line, start artificial system
New way (Denis M, Delourme R, Gourret J P, the et al.Expression of of standby male sterility line
engineered nuclear male sterility in Brassica napus(genetics,morphology,
cytology,and sensitivity to temperature)[J].Plant Physiology,1993,101(4):
1295-1304.).Currently, a lot of researchs show that analyzing other functional genes is likely to build transgenic by gene engineering method
Male sterile plants.
Plants male sterility dominant phenotype, is embodied in pollen abortion, it relate to the generation of stamen and growth in floral organ,
Numerous links or the factors such as tapetum structure, Microsporogenesis, anther dehiscence and external ecology environment, wherein understand fully that pollen is sent out
The overall process educated and molecule mechanism are basis and the key points of research plants male sterility.And pollen development relates to numerous base
The expression regulation of cause, wherein recognizes in the pollen development later stage, and starch can be pollen germination and pollen tube extension energy reserve.Cause
This, when the starch in pollen grain is degraded by α-amylase in advance so that pollen grain energy source is disintegrated, thus contains pollen
, there is malformed flower powder, causes plants male sterility in growth.
Amylase is hydrolysis starch and the enzyme general name of glycogen, is mainly distributed on animals and plants, antibacterial and fungus, and people pass through
Utilize technique for gene engineering, clone cDNA sequence (Cui Jin, horse (2009) amylase eastwards of various amylase gene
The multiformity of gene. Zhengzhou Husbandry Engineering Higher Training School's journal, 29 (2): 21-23).Diastatic multiformity is entered simultaneously
Go research, shown except, in addition to the multiformity of source, its gene structure and function aspects all present multiformity.According to it to starch
The model of action difference of enzyme is classified, and α-amylase belongs to endo-type amylase, can cut from the internal random of starch molecule
Cut α-Isosorbide-5-Nitrae glycosidic bond, make Starch Hydrolysis become maltose, glucose etc., release energy (Morris G P, Beck P L,
Herridge M S,et al.Hapten-induced model of chronic inflammation and
ulceration in the rat colon[J].Gastroenterology,1989,96(3):795-803.).Therefore, exist
In mature pollen, appropriate amylase hydrolyzable starch, normal development and the sprouting of pollen tube, growth for pollen provide energy
Amount.On the contrary, if amylase process LAN and reticent expression during pollen formation, all can reduce pollen energy metabolism level, lead
Cause starch accumulation amount not enough, produce abortive pollen.And the research in terms of paddy pollen abortion gene, fresh understatement road.
These abortion genes are that Oryza sativa L. realizes the acquisition of sterile line transfer-gen plant by genetic engineering, are particularly controlling
The aspects such as fertility and transgenic drift provide new selection.
Summary of the invention
It is an object of the invention to provide a kind of plant alpha amylase causing pollen abortion and preparing transgenic pollen abortion and plant
Application in strain.
In order to reach object above, the nucleotides sequence of the plant alpha amylase that the present invention provides is classified as:
1) nucleotide sequence shown in SEQ ID No.1, or
2) be substituted, lack or add in the nucleotide sequence shown in SEQ ID No.1 one or several nucleotide and
Have equal function by 1) derivative nucleotide sequence；Or
3) under strict conditions with sequence hybridization shown in SEQ ID NO.1 and express identical function protein nucleotides sequence
Row, described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C
Hybridization, and wash film with this solution；Or
4) with 1), 2) or 3) nucleotide sequence there is more than 90% homology and express the nucleoside of identical function protein
Acid sequence.
The present invention also provides for and the DNA molecular of plant alpha-amylase nucleotide sequence complementary.Those skilled in the art according to
Identical purpose can easily identify and utilize the DNA with plant pollen abortion gene α-amylase nucleotide sequence complementary to divide
Son, therefore, has promoter activity and hybridizes with abortion gene α-amylase sequence of the present invention or its fragment under strict conditions
Separation sequence be included in the invention.Wherein, described nucleotide sequence complementary, referring under strict conditions can be with α-amylase
DNA sequence hybridizes.
Stringent condition refers to that probe will be hybridized to detectable degree with its target sequence and exceed with other sequence hybridization (as at least
2 times of backgrounds) condition.Stringent condition has sequence dependent, and different because of the difference of environment.By control hybridization and/
Or the stringency of wash conditions, can identify and the target sequence of probe 100% complementation (same to source detection).Selectively, can adjust
Joint stringent condition is to allow some sequence mismatch so that detect the similarity (allos detection) of lower degree.Generally, probe is long
Degree is shorter than about 1000 nucleotide, is preferably shorter than 500 nucleotide.
Typically, stringent condition is that salinity is less than about 1.5M Na ion, the most about under pH value 7.0-8.3
0.01-1.0M Na ion concentration (or other salt), temperature is to short probe (such as 10-50 nucleotide) at least about 30 DEG C, right
Long probe (such as more than 50 nucleotide) at least about 60 DEG C.Also strict bar can be obtained by adding destabilizing agent such as Methanamide
Part.Low stringency condition, it may for example comprise molten in the buffering of 30-35% Methanamide, 1M NaCl, l%SDS (dodecyl sodium sulfate)
37 DEG C of hybridization in liquid, 1 × to 50-55 DEG C of washing in 2 × SSC (20 × SSC=3.0M NaCl/0.3M trisodium citrate).In
Degree stringent condition, it may for example comprise 40-45% Methanamide, 1.0M NaCl, l%SDS buffer solution in 37 DEG C of hybridization,
0.5 × to 55-60 DEG C of washing in 1 × SSC.High stringency, it may for example comprise at 50% Methanamide, 1M NaCl, l%SDS
Buffer solution in 37 DEG C of hybridization, 60-65 DEG C of washing in 0.1 × SSC.Optionally, lavation buffer solution can be containing about
The SDS of 0.1%-1%.Hybridization time is generally less than about 24 hours, generally about 4-12 hour.
The most typically function of post-hybridization washing, key factor is ionic strength and the temperature of final wash solution.
For DNA-DNA crossbred, Tm can be from the equation of Meinkoth and Wahl (1984) Anal.Biochem.138:267-284
Estimation: Tm=81.5 DEG C+16.6 (logM)+0.41 (%GC)-0.61 (%form)-500/L；Wherein M is univalent cation
Molar concentration, %GC is guanylic acid and cytidylic acid percentage ratio in DNA, and %form is that Methanamide is in hybridization
Percentage ratio in solution, L is crossbred length in base pair.Tm is 50% complementary target sequence and pairing probe hybridization completely
Temperature (regulation ionic strength and pH under).The mispairing of every 1% needs Tm to reduce about l DEG C；Therefore, Tm hybridizes and/or washes
The condition of washing can be conditioned with the sequence hybridization with required homogeneity.Such as, if the sequence sought has >=the homogeneity of 90%,
Tm can reduce by 10 DEG C.Usually, the stringent condition of selection is less than the thermal melting point of particular sequence (Tm) about 5 DEG C, and
It is complementary under the ionic strength and pH of regulation.But, high stringency can be applied less than thermal melting point (Tm) 1,2,3
Or the hybridization of 4 DEG C and/or washing；It is miscellaneous that moderate stringency can be applied less than thermal melting point (Tm) 6,7,8,9 or 10 DEG C
Hand over and/or washing；Low stringency conditions can apply the hybridization less than thermal melting point (Tm) 11,12,13,14,15 or 20 DEG C
And/or washing.Applying this equation, hybridization and cleaning compositions and required Tm, those of ordinary skill in the art will understand that hybridization
And/or the condition of wash solution changes with the change of stringency.If required extent of mismatch make Tm be less than 45 DEG C (water-soluble
Liquid) or 32 DEG C (formamide solution), preferably increase SSC concentration so that higher temperature can be used.The guide of nucleic acid hybridization sees
Tijssen (1993) biochemistry and Molecular Biology Lab's technology one nucleic acid probe hybridization, part i, the 2nd chapter
(Elsevier, New York)；(1995) Current Protocols method the 2nd chapter (Greene is edited with Ausubel et al.
Publishing and Wiley-Interscience,New York).See Sambrook et al. (1989) molecular cloning: experiment
Room handbook (second edition, Cold Spring Harbor Laboratory Press, Plainview, NewYork).
Described stringent condition is preferably at 6 × SSC (sodium citrate), the solution of 0.5%SDS (dodecyl sodium sulfate)
In, hybridize at 65 DEG C, then respectively wash film 1 time with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS.
Wherein, it is in the news in this area about α-amylase polynucleotide sequence, at gene database (The
GenBank) open sequence, occurs the website about analyzing, identify gene successively, including: NCBI (http:// www.ncbi.nlm.nih.gov/)、Gramene(http://gramene.org/)、UniProt(http:// And EMBL-EBI www.uniprot.org/)(http://www.ebi.ac.uk/) etc..Such as, respectively in each website
Alphalise starch Gene Name and sequence number such as table 1 below:
Table 1
Further, the aminoacid sequence of plant alpha amylase of the present invention is:
A albumen that () is made up of the aminoacid sequence shown in SEQ ID NO.4；Or
B the aminoacid sequence of SEQ ID NO.4 is passed through replacement and/or the disappearance of one or several amino acid residue by ()
And/or add the albumen that is derivative and that keep the protein function shown in SEQ ID NO.4 by SEQ ID NO.4.
The present invention provides a kind of and imports the application in causing pollen abortion of the plant alpha amylase in plant, including:
A) one section of nucleotide fragments, comprises plant alpha amylase gene order, and described sequence is closely coupled to pollen or flower pesticide
Specificity promoter；
B) by a) described plant alpha amylase gene transfered plant tissue；
Described plant alpha amylase gene has:
1) nucleotide sequence shown in SEQ ID No.1, or
2) be substituted, lack or add in the nucleotide sequence shown in SEQ ID No.1 one or several nucleotide and
Have equal function by 1) derivative nucleotide sequence；Or
3) under strict conditions with sequence hybridization shown in SEQ ID NO.1 and express identical function protein nucleotides sequence
Row, described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C
Hybridization, and wash film with this solution；Or
4) with 1), 2) or 3) nucleotide sequence there is more than 90% homology and express the nucleoside of identical function protein
Acid sequence.
The invention provides above-mentioned plant alpha amylase or its encoding gene or the biomaterial containing this encoding gene is being led
Cause the application in plant pollen abortion.
The invention provides above-mentioned plant alpha amylase or its encoding gene or the biomaterial containing this encoding gene in system
Application in standby pollen abortion transfer-gen plant.
Biomaterial of the present invention refers to recombinant expression carrier, expression cassette, Host Strains or host cell.
The recombinant expression carrier that the present invention contains above-mentioned plant alpha-amylase can pass through agrobacterium-mediated transformation, particle gun
The conventional biology methods such as method, pollen tube passage method converts plant cell or tissue obtains independent transgenic cell or tissue,
Obtain male sterile transgenic line.
Described transgenic plant is exogenous gene transgenic plant of specifically expressing in pollen, is preferably pollination/fertilization
The transgenic plant that ability strengthens/weakens, more preferably male sterility transgenic plant.
Biomaterial of the present invention is possibly together with transduction peptide and male gamete type of priority promoter.
The invention provides the plant expression vector of the effective catenation sequence that can make pollen abortion, comprise and effectively connect regulation and control
Expression cassette, wherein said contain by the DNA fragmentation institute shape shown in SEQ ID No.1-3 at the expression cassette effectively connecting regulation and control
Become.This expression cassette is to connect in the upstream of pollen abortion gene α-amylase to have PG47 promoter and the SBE signal peptide can be effective
Starch accumulation and accumulation expression casette in interference pollen grain.
Wherein, the sequence length shown in SEQ ID No.1 be 1278bp be pollen abortion gene α-amylase forward DNA
Fragment；The transduction peptide that sequence length is 216bp shown in SEQ ID No.2；Sequence length shown in SEQ ID No.3 is
Male gamete type of priority promoter PG47 of 2732bp.
The present invention further provides the plant pollen of alpha-amylase gene expression cassette containing above-mentioned pollen abortion gene.
The present invention also provides for the method driving exogenous gene to cause pollen abortion to be expressed, and comprises the steps: to lose pollen
Educate gene α-amylase and outside purpose exogenous gene imports to obtain in carrier containing pollen abortion gene α-amylase and purpose
The expression cassette recombinant expression carrier of source gene, and it is introduced into Plant Genome, obtain exogenous gene specifically expressing in pollen
Transgenic plant.
The invention provides a kind of regulate and control Plant Pollen Development method, it is characterised in that make plant express above-mentioned plant α
Amylase gene.
Described plant is grass.
Preferably, described plant includes, without being limited to Oryza sativa L., Semen Maydis, Sorghum vulgare Pers., Fructus Hordei Vulgaris, Herba bromi japonici, Semen Tritici aestivi, foxtail millet, Caulis Sacchari sinensis, big
Bean, Brassica species, Cotton Gossypii, Flos Carthami, Nicotiana tabacum L., Herba Medicaginis and Helianthi.
Described regulation and control are amylase or induction plants male sterilities in degrading plant pollen.
The invention provides a kind of method of exogenous gene diffusion in degrading plant pollen, will be containing above-mentioned plant α starch
The expression cassette transformed calli of enzyme gene, the callus after converting carries out induction differentiation and obtains transgenic pollen abortion
Transfer-gen plant, render transgenic plant pollen cannot normally be pollinated, and then exogenous gene diffusion in degrading plant pollen.
The pollen abortion gene α-amylase advantage that the present invention provides is as follows:
1) plant alpha-amylase may be from Oryza sativa L., Sorghum vulgare Pers., Fructus Hordei Vulgaris, Semen Tritici aestivi etc. as pollen abortion gene, wherein from water
Rice α-amylase is endogenous gene, favourable to paddy gene engineering.
2) experiment of plant alpha-amylase gene pollen grain iodine dye shows, α-amylase can be under promoter PG47 drives, energy
Accurately act on pollen grain in flower pesticide so that the ratio of fertile pollen and abortive pollen meets for 1:1.
3) plant alpha-amylase gene expression dose of the present invention is accurate, can control transgenic diffusion；Can be used for keeping male
The holding of sterile line and breeding, save artificial emasculation step during hybrid seeding simultaneously, have a extensive future.
4) present invention provides a kind of new method for obtaining Transgenic male sterile plant.
Accompanying drawing explanation
Fig. 1 is that the recombinant expression carrier DX2182-α-Amylase of pollen abortion gene α-amylase in embodiment 2 builds
Flow chart.
Fig. 2 is DX2182-α-Amylase transgenic paddy rice pollen iodine dye photo in embodiment 4.It is right that A figure is that Oryza sativa L. can educate
Taking pollen IKI dye picture, whole pollen can be educated and can stained with color with iodine.B figure be the specific expressed α of paddy pollen-
Amylase pollen staining, wherein half pollen abortion cannot be dyeed, white arrow point to can stained pollen, black arrow point to dye
Do not paint pollen.
Fig. 3 is transgenic plant hygromycin selection T1 of embodiment 5DX2182-α-Amylase plant recombination expression vector
For seed, it is thus achieved that T2 is for plant.A figure is that non-transgenic rice paddy seed hygromycin is germinateed.B figure is transgenic α-amylase rice seed
Sub-hygromycin is germinateed.C figure is that non-transgenic Mature Embryos of Rice hygromycin is germinateed.D figure is transgenic α-amylase Mature Embryos of Rice
Hygromycin is germinateed.Arrow indication is for can germinate for transgenic α-amylase mature embryo.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.Without departing substantially from present invention spirit
In the case of essence, the amendment that the inventive method, step or condition are made or replacement, belong to the scope of the present invention.If
Do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art.
Embodiment 1 rice alpha-amylase gene obtains
1, the extraction of the fine RNA of Oryza sativa L. Japan
Biozol Reagent method is utilized to extract the fine RNA of Oryza sativa L. Japan: to weigh the fine fresh children fringe tissue 1ml of 0.1g Japan
The ratio mixing of Biozol Reagent, room temperature stands 5min；0.2ml chloroform is added, firmly by every 1ml Biozol Reagent
Concussion 15s, after solution is fully emulsified, then room temperature stands 5min, 12000rmp, 4 DEG C of centrifugal 15min；From centrifuge carefully
Taking out centrifuge tube, Aspirate supernatant is transferred in the centrifuge tube that another is new；In supernatant, add isopyknic isopropanol, run up and down
After the centrifuge tube that falls fully mixes, at room temperature stand 10min, 12000rmp, 4 DEG C of centrifugal 10min；Abandoning supernatant, tube wall can show in vain
Color precipitates, and adds 0.5ml 75% ethanol (with DEPC water configuration after sterilizing) washing, and reverse mixing, 10000rmp, 4 DEG C are centrifuged
5min；It is repeated once, cold drying, makes ethanol volatilize；Add the Rnase-free water dissolution precipitation of 50ul, be placed in-80 DEG C of preservations.
2, the cDNA that Japan is fine is obtained
With Japanese fine RNA as template, utilizing reverse transcriptase M-MLV reverse transcription, concrete grammar is as follows: (1) configures on ice, mixed
Even.
RNA 5-10μl
Olig(dT) 2μl
RNase-free H2O to 14.5 μ l
(2) 70 DEG C, 5 minutes, it is placed at once on ice, opens secondary structure.
(3) reverse transcription etc. is added, mixing.
(4) 42 DEG C, extend 90 minutes.70 DEG C, 15 minutes.Obtain the fine cDNA subpackage of Japan and be stored in-40 DEG C.
Note: all experimental articles are RNase-free.
3, the primer of design synthesis cloning rice α-amylase
The nucleotide sequence of fine α-amylase (OS01G0715400) gene of Oryza sativa L. Japan is obtained by ncbi database
(as shown in SEQ ID NO.1 in sequence table), its aminoacid sequence (as shown in SEQ IDNO.4 in sequence table).OsAmylase
Nucleotide sequence is (as in sequence table, SEQ ID NO.5-6 clones from the fine cDNA of Japan and obtains by design primer sequence.
Design of primers uses Gibson Assembly method, and amplified production is inserted into DX2182 carrier MluI and Sac1 single endonuclease digestion site.
Amplification alpha-amylase gene primer is to as shown in sequence SEQ ID NO:5-6.Wherein upstream and downstream primer 5 ' end all has about 15
Nucleotide sequence link position corresponding to carrier repeats, in order to Gibson Assembly connects.Amplification system and program are as follows:
PCR program: 94 DEG C of 3min of denaturation, 94 DEG C of 30s of degeneration, anneal 55-65 DEG C of 40s, extends 68 DEG C of 1min20s, and 35
Individual circulation, extends 68 DEG C of 10min.
Embodiment 2 builds the recombinant expression carrier DX2182-α-Amylase of rice alpha-amylase
Build flow process and see Fig. 1, embodiment 1 amplified production is inserted DX2182 carrier Mlu1 and Sac1 restriction enzyme site.
It is left that primer SEQ ID NO:5 and SEQ ID NO:6 amplification PCR primer 1% agarose gel electrophoresis reclaims 1300bp
Right product.Mlu1 and Sac1 enzyme action carrier DX2182, reclaims linear enzyme action carrier.
It is as follows to DX2182,10ul system that 2X connects test kit connection abortion gene:
2.5 μ 1 α-amylase PCR primer (50ng), 2.5 μ 1 enzyme action carrier (100ng), 5 μ 1Ligation Mix, connect
Program: 50 DEG C 60 minutes.
Convert: take the electroporated competent escherichia coli cell of above-mentioned connection product 2 μ 1.Select positive colony order-checking.Life
Entitled DX2182-α-Amylase.DX2182-α-Amylase carrier contains hygromycin gene, its sequence such as SEQ ID
Shown in NO.7, available hygromycin selection, it is thus achieved that resistant plant, simultaneously can also be with screening transgenic T0 for seed, it is thus achieved that turn base
Because plant T1 is for resistant plant.
The acquisition of embodiment 3 transgenic rice plant
Take-70 DEG C of Agrobacterium EHA105 preserved in containing Kan (50 μ g/ml)+Rif (25 μ g/ml)+streptomycin (50 μ g/
Ml) plate streaking, 28 DEG C of cultivations.Picking list colony inoculation in 50ml YEB fluid medium, 28 DEG C of shaken cultivation of 220rpm
12-16hr.Taking 2ml bacterium solution and transfer in 100ml (containing antibiotic) YEB fluid medium, 28 DEG C of 220rpm shaken cultivation are extremely
OD600=0.5.Pre-cooling on ice 10 minutes, 5000rpm 10min (refrigerated centrifuger is pre-chilled to 4 DEG C).Sterile deionized water washes 2
Secondary (each 10ml), 10% glycerol is washed 1 time and is dissolved in 3ml 10% glycerol.Take 100 μ l competent cells to add in 1 μ l embodiment 1
DX2182-α-Amylase the plasmid obtained, 2.5KV is electroporated.On the YEB culture plate containing kanamycin and rifampicin
28 DEG C of cultivations, select positive colony, verify with DX2182-α-Amylase vector-specific primers SEQ ID NO.8-9PCR.
Verify correct clone, infected by Agrobacterium-mediated genetic transformation method and Oryza sativa L. is spent 11 (Hiei Y Ohta
S,Komari T,Kumashiro T(1994)Efficient transformation of rice(Oryza sativa L.)
mediated by Agrobacterium and sequence analysis of the boundaries of the T-
DNA.The Plant Journal6:271-282).T0 is obtained for transgenic children through links such as co-culturing, screen, break up, take root
Seedling, extracts DNA, and through the transgenic positive plant of PCR checking, self-fertility obtains T1 generation.Take T1 plant and carry out subsequent analysis.
Embodiment 4 transgenic paddy rice potassium iodide staining analysis
Preparation potassium iodide dyeing liquor holds liquid and (takes 2g KI to be dissolved in 5-10mL distilled water, be subsequently adding 1g I2 (with appropriate
Anhydrous alcohol solution), after all dissolving, then add distilled water and be settled to 300mL.Store in brown bottle standby).Oryza sativa L. is turned base
Because the mature pollen of plant carries out microscopy, being analyzed the pollen grain fertility of transfer-gen plant, concrete grammar is as follows:
1, pollen collection: take the flower pesticide that abundant maturation will be bloomed, divests grain husk shell, takes out flower pesticide.
2. microscopy: by potassium iodide: the dilution proportion liquor kalii iodide of deionized water=1:1, the diluent taking 70ul is placed in
Microscope slide, takes a flower pesticide and is placed on microscope slide, fully smashed to pieces by flower pesticide with tweezers, make pollen grain discharge, and covered, in low
Observe under power microscope.All pollen grains being dyed to blueness are fertile pollen, in the filemot pollen grain for abortion.
The pollen grain of transfer-gen plant shows fertile pollen through potassium iodide staining analysis, pollen grain iodine dye result: abortion is spent
Powder meets the segregation ratio of 1:1, is partial pollen and shows as male sterility (50%), all has half (50%) can not dye black
Blue (as shown in the B of Fig. 2 schemes, 50% normal pollen staining).And wild type (or other do not carry turning of pollen abortion gene
Gene plant) be then holandry can educate (as Fig. 2 A scheme shown in, 100% normal pollen staining).Show Oryza sativa L. α-shallow lake
Powder enzyme can in paddy pollen degradable starch, therefore at pollen grain in growth course, energy supply deficiency, cause pollen to lose
Educate.
Thus prove that alpha-amylase gene can be degraded the starch in pollen grain, causes Transgenic Rice pollen sterility, has
Effect stops genetically modified crops, by pollen, transgenic element is broadcast to other wild crop kinds.
In sum, the present invention affects the protein α-amylase of male fertility for clone from rice varieties alphalise starch
Enzyme, and the starch in pollen grain of can degrading, cause Transgenic Rice pollen sterility, and accuracy is high, effectively stops transgenic
Transgenic element is broadcast to other wild crop kinds by pollen by crop；Can be used for keeping isozygotying of male sterile plants hidden
Sexual state；Eliminate the step of emasculation during hybrid seeding simultaneously.
Embodiment 5 to transgenic paddy rice T1 for seed hygromycin selection
By the transfer-gen plant of recombinant plant expression vector DX2182-α-Amylase, T1 is for seed for results.According to enforcement
In example 4, fertile pollen: abortive pollen meets the segregation ratio of 1:1, be i.e. half seed be non-transgenic seed, half for turn
Gene seed, carries out screening transgenic seed hence with hygromycin, and transgenic T2 is for plant in induction.Tested by two schemes
Card:
(1) 400mg/L hygromycin selection T1 is for seed
With non-transgenic ZH11 for comparison, under 400mg/L hygromycin, the screening pollen abortion gene weight containing α-amylase
The T1 of group carrier, for seed, chooses 100 respectively, screens.Its germination percentage is observed after 7d.Result is observed after showing 7d and is sprouted
Rate, adds up its survival rate and mortality rate=47:53 after 14d, substantially conform to the segregation ratio (scheme such as the A of Fig. 3 and B schemes) of 1:1
(2) 35mg/L hygromycin+MS Screening of Media T1 is for mature embryo
With non-transgenic ZH11 for comparison, in 35mg/L hygromycin+MS culture medium, the screening pollen containing α-amylase loses
Educate the T1 mature embryo for seed of gene recombined vector, choose 100 respectively, screen.Its germination percentage is observed after 7d.Result
Observe bud ratio after showing 7d, add up its survival rate and mortality rate=51:49, substantially conform to the segregation ratio of 1:1, such as the C of Fig. 3
Figure and D scheme.
With reference to the method for embodiment 2-5, the plant alpha amylase gene described in SEQ ID NO.10-19 is carried out restructuring and carries
Body builds, transgenic plant preparation and functional verification, and result is similar with embodiment 4/5 result, shows that plant alpha-amylase can be
Degradable starch in crop pollen, therefore at pollen grain in growth course, energy supply deficiency, cause pollen abortion, cause and turn
Gene crops pollen sterility, effectively stops genetically modified crops, by pollen, transgenic element is broadcast to other wild crop product
Kind.
Although, the present invention is described in detail the most with a general description of the specific embodiments, but
On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (10)
1. plant imports the application in causing pollen abortion of the plant alpha amylase, including:
A) one section of nucleotide fragments, comprises plant alpha amylase gene order, and described sequence is closely coupled to pollen or flower pesticide is special
Property promoter；
B) by a) described plant alpha amylase gene transfered plant tissue；
Described plant alpha amylase gene has:
1) nucleotide sequence shown in SEQ ID No.1, or
2) in the nucleotide sequence shown in SEQ ID No.1, it is substituted, lacks or adds one or several nucleotide and have
Equal function by 1) derivative nucleotide sequence；Or
3) under strict conditions with sequence hybridization shown in SEQ ID NO.1 and express identical function protein nucleotide sequence,
Described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, miscellaneous at 65 DEG C
Hand over, and wash film with this solution；Or
4) with 1), 2) or 3) nucleotide sequence there is more than 90% homology and express the nucleotides sequence of identical function protein
Row.
Apply the most as claimed in claim 1, it is characterised in that the aminoacid sequence of described plant alpha amylase is:
A albumen that () is made up of the aminoacid sequence shown in SEQ ID NO.4；Or
(b) by the aminoacid sequence of SEQ ID NO.4 through the replacement of one or several amino acid residue and/or disappearance and/or
Add the albumen that is derivative and that keep the protein function shown in SEQ ID NO.4 by SEQ ID NO.4.
3. plant alpha amylase or the application in preparation pollen abortion transfer-gen plant of its encoding gene, described plant alpha amylase
Aminoacid sequence be:
A albumen that () is made up of the aminoacid sequence shown in SEQ ID NO.4；Or
(b) by the aminoacid sequence of SEQ ID NO.4 through the replacement of one or several amino acid residue and/or disappearance and/or
Add the albumen that is derivative and that keep the protein function shown in SEQ ID NO.4 by SEQ ID NO.4；
The encoding gene of plant alpha amylase is for having:
1) nucleotide sequence shown in SEQ ID No.1, or
2) in the nucleotide sequence shown in SEQ ID No.1, it is substituted, lacks or adds one or several nucleotide and have
Equal function by 1) derivative nucleotide sequence；Or
3) under strict conditions with sequence hybridization shown in SEQ ID NO.1 and express identical function protein nucleotide sequence,
Described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, miscellaneous at 65 DEG C
Hand over, and wash film with this solution；Or
4) with 1), 2) or 3) nucleotide sequence there is more than 90% homology and express the nucleotides sequence of identical function protein
Row.
4. contain the application in preparation pollen abortion transfer-gen plant of the biomaterial of plant alpha amylase gene,
Described plant alpha amylase gene is for having:
1) nucleotide sequence shown in SEQ ID No.1, or
2) in the nucleotide sequence shown in SEQ ID No.1, it is substituted, lacks or adds one or several nucleotide and have
Equal function by 1) derivative nucleotide sequence；Or
3) under strict conditions with sequence hybridization shown in SEQ ID NO.1 and express identical function protein nucleotide sequence,
Described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, miscellaneous at 65 DEG C
Hand over, and wash film with this solution；Or
4) with 1), 2) or 3) nucleotide sequence there is more than 90% homology and express the nucleotides sequence of identical function protein
Row；
Described biomaterial is recombinant expression carrier, expression cassette, recombinant bacterium or host cell.
Apply the most as claimed in claim 4, it is characterised in that described biomaterial contains transduction peptide and male gamete is preferential
Type promoter.
Apply the most as claimed in claim 5, it is characterised in that described transduction peptide is SBE signal peptide, and its nucleotide sequence is such as
Shown in SEQ ID NO.2, described male gamete type of priority promoter is PG47, and its nucleotide sequence is as shown in SEQ ID NO.3.
7. the method regulating and controlling Plant Pollen Development, it is characterised in that make plant express plant alpha amylase gene, this gene
Nucleotides sequence be classified as:
1) nucleotide sequence shown in SEQ ID No.1, or
2) in the nucleotide sequence shown in SEQ ID No.1, it is substituted, lacks or adds one or several nucleotide and have
Equal function by 1) derivative nucleotide sequence；Or
3) under strict conditions with sequence hybridization shown in SEQ ID NO.1 and express identical function protein nucleotide sequence,
Described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, miscellaneous at 65 DEG C
Hand over, and wash film with this solution；Or
4) with 1), 2) or 3) nucleotide sequence there is more than 90% homology and express the nucleotides sequence of identical function protein
Row.
8. method as claimed in claim 7, it is characterised in that described plant is grass.
9. method as claimed in claim 8, it is characterised in that described regulation and control are amylase or inductions in degrading plant pollen
Plants male sterility.
10. the method for exogenous gene diffusion in a degrading plant pollen, it is characterised in that will be containing plant alpha amylase gene
Expression cassette transformed calli, will convert after callus carry out induction differentiation obtain transgenic pollen abortion transgenic
Plant, render transgenic plant pollen cannot normally be pollinated, and then exogenous gene diffusion in degrading plant pollen,
The nucleotides sequence of described plant alpha amylase gene is classified as:
1) nucleotide sequence shown in SEQ ID No.1, or
2) in the nucleotide sequence shown in SEQ ID No.1, it is substituted, lacks or adds one or several nucleotide and have
Equal function by 1) derivative nucleotide sequence；Or
3) under strict conditions with sequence hybridization shown in SEQ ID NO.1 and express identical function protein nucleotide sequence,
Described stringent condition is in 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, miscellaneous at 65 DEG C
Hand over, and wash film with this solution；Or
4) with 1), 2) or 3) nucleotide sequence there is more than 90% homology and express the nucleotides sequence of identical function protein
Row.
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