CN106282209B - Application of the plant alpha amylase in pollen abortion is caused - Google Patents
Application of the plant alpha amylase in pollen abortion is caused Download PDFInfo
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- CN106282209B CN106282209B CN201610794944.XA CN201610794944A CN106282209B CN 106282209 B CN106282209 B CN 106282209B CN 201610794944 A CN201610794944 A CN 201610794944A CN 106282209 B CN106282209 B CN 106282209B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- 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
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2422—Alpha-amylase (3.2.1.1.) from plant source
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
Abstract
The present invention provides a kind of purposes of plant alpha amylase in pollen abortion is caused, and belongs to gene engineering technology field.Polynucleotides of the invention by importing 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 can not be provided, pollen germination is contained, causes abortion, and then obtain the transfer-gen plant of transgenic pollen abortion.The plant alpha amylase of the present invention may be from the grass gene such as endogenous rice, millet, sorghum, barley, wheat as pollen abortion gene, highly beneficial to crop gene engineering and the genetically modified plants for preparing pollen abortion.Plant alpha amylase gene expression dose of the present invention is accurate, can control transgenosis diffusion；Available for the holding and breeding of holding male sterile line, while artificial emasculation step is saved during hybrid seeding, had a extensive future.
Description
Technical field
The invention belongs to molecular biology of plants and gene engineering technology field, and in particular to a kind of plant alpha amylase exists
Cause the application in pollen abortion, further relate to combine the nutrition, using modern biotechnology, applied to hybrid seed production technology
System, ensure seed production quality, and improve seed production efficiency；It can also be applied to prevent transgenosis from spreading.
Background technology
Plant hybrid use of advantage is one of approach for improving grain yield the most cost-effective, but it is still suffered from
Limiting factor, such as combine excellent difficulty is larger, the proportion shared by hybrid crop is smaller etc..Although almost all of crops are all
It is maximum possibly also with hybrid vigour, but still have enormous potentialities.Therefore many scholars are studied, especially water
Rice, its hybrid vigour main path are improved the combo free degree, are improved rice heterosis by " three line method " to " two line method "
It is horizontal.But the temp-sensing sterile line of " two line method " is influenced by temperature excessive, the production of hybrid seeds can be caused to fail, loss is serious.Due to male
The resource-constrained of sterile line, it is serious to restrict paddy rice cross breeding technology.
The beginning of the nineties, Mariani et al. utilizes the specific promoter (TA29) and ribose of the anther tapetum from tobacco
Nuclease gene (Barnase) recombinant expression cassettes transformation of tobacco and rape, and male sterile line is successfully obtained, start artificial system
New way (Denis M, Delourme R, Gourret J P, the et al.Expression of of standby male sterile line
engineered nuclear male sterility in Brassica napus(genetics,morphology,
cytology,and sensitivity to temperature)[J].Plant Physiology,1993,101(4):
1295-1304.).Currently, many researchs show that transgenosis may also be built by gene engineering method by analyzing other functional genes
Male sterile plants.
Plants male sterility dominant phenotype, is embodied in pollen abortion, it be related to the generation of stamen in floral organ and development,
The numerous links or factor such as carpet Rotating fields, Microsporogenesis, anther dehiscence and external ecology environment, wherein understanding fully that pollen is sent out
The overall process and molecule mechanism educated are to study basis and the key point of plants male sterility.And pollen development is related to numerous bases
The expression regulation of cause, wherein recognizing in the pollen development later stage, starch can be that pollen germination and pollen tube extend energy reserve.Cause
This, when the starch in pollen grain is degraded by alpha-amylase in advance so that pollen grain energy source is disintegrated, so as to contain pollen
, there is lopsided pollen grain, causes plants male sterility in growth.
Amylase is hydrolysis starch and the enzyme general name of glycogen, is mainly distributed on animals and plants, bacterium and fungi, and people pass through
Using technique for gene engineering, cDNA sequence (Cui Jin, horse (2009) amylase eastwards of various amylase genes have been cloned
Diversity Zhengzhou Husbandry Engineering Higher Training School journal of gene, 29 (2):21-23).The diversity of amylase is entered simultaneously
Research is gone, has shown except in addition to the diversity of source, diversity is all presented in terms of its gene structure and function.According to it to starch
The mode of action difference of enzyme is classified, and alpha-amylase belongs to endo-type amylase, can be cut from the internal random of starch molecule
α-Isosorbide-5-Nitrae glycosidic bond is cut, makes Starch Hydrolysis into 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, sprouting, the growth of normal development and pollen tube for pollen provide energy
Amount.If on the contrary, amylase is overexpressed during pollen formation and silence is expressed, pollen energetic supersession level can be all reduced, is led
Starch accumulation amount deficiency is caused, produces abortive pollen.And the research in terms of paddy pollen abortion gene, fresh understatement road.
These abortion genes are the acquisition that rice realizes sterile line transfer-gen plant by genetic engineering, are particularly being controlled
Fertility and transgenosis drift etc. provide new selection.
The content of the invention
It is an object of the invention to provide a kind of plant alpha amylase to cause pollen abortion and prepare transgenosis pollen abortion to be planted
Application in strain.
In order to reach object above, the nucleotides sequence of plant alpha amylase provided by the invention 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 nucleotides and
The nucleotide sequence as derived from 1) with equal function；Or
3) hybridize under strict conditions with sequence shown in SEQ ID NO.1 and express the nucleotides sequence of identical function protein
Row, the stringent condition are in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C
Hybridization, and wash film with the solution；Or
4) there is more than 90% homology with nucleotide sequence 1), 2) or 3) and expresses the nucleosides of identical function protein
Acid sequence.
The present invention also provides the DNA molecular complementary with plant alpha-amylase nucleotide sequence.Those skilled in the art according to
Identical purpose can easily be identified and divided using with the complementary DNA of plant pollen abortion gene alpha-amylase nucleotide sequence
Son, therefore, there is promoter activity and hybridize under strict conditions with abortion gene alpha-amylase sequence of the present invention or its fragment
Separation sequence be included in the invention.Wherein, the nucleotide sequence is complementary, and referring under strict conditions can be with alpha-amylase
DNA sequence dna hybridizes.
Stringent condition refers to that probe will be hybridized to detectable degree with its target sequence and exceed with the hybridization of other sequences (as at least
2 times of backgrounds) condition.Stringent condition has a sequence dependent, and different and different because of environment.By control hybridization and/
Or the stringency of wash conditions, the target sequence (same to source detection) complementary with probe 100% can be identified.Selectively, can adjust
Stringent condition is saved to allow some sequence mismatch so that detect the similitude (heterologous detection) of lower degree.Generally, probe is grown
Degree is shorter than about 1000 nucleotides, is preferably shorter than 500 nucleotides.
Typically, stringent condition is that salinity is less than about 1.5M Na ions under pH value 7.0-8.3, typically about
0.01-1.0M Na ion concentrations (or other salts), temperature are right at least about 30 DEG C of short probe (such as 10-50 nucleotides)
At least about 60 DEG C of long probe (such as more than 50 nucleotides).Strict bar can be also obtained by adding destabilizing agent such as formamide
Part.Low stringency condition, it may for example comprise molten in 30-35% formamides, 1M NaCl, l%SDS (dodecyl sodium sulfate) buffering
37 DEG C of hybridization in liquid, 1 × the 50-55 DEG C of washing into 2 × SSC (20 × SSC=3.0M NaCl/0.3M trisodium citrates).In
Spend stringent condition, it may for example comprise 40-45% formamides, 1.0M NaCl, l%SDS cushioning liquid in 37 DEG C hybridization,
0.5 × 55-60 DEG C of the washing into 1 × SSC.High stringency, it may for example comprise in 50% formamide, 1M NaCl, l%SDS
Cushioning liquid in 37 DEG C hybridization, in 0.1 × SSC 60-65 DEG C washing.Optionally, lavation buffer solution can contain about
0.1%-1% SDS.Hybridization time is generally less than about 24 hours, generally about 4-12 hours.
Especially it is typically the function of post-hybridization washing, key factor is the ionic strength and temperature of final wash solution.
For DNA-DNA crossbreds, Tm can be from Meinkoth and Wahl (1984) Anal.Biochem.138:267-284 equation
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 are the percentage of guanylic acid and cytidylic acid in DNA, and %form is that formamide is hybridizing
Percentage in solution, L are length of the crossbred in base-pair.Tm is 50% complementary target sequence and pairing probe hybridization completely
Temperature (under defined ionic strength and pH).Every 1% mispairing needs Tm to reduce about l DEG C；Therefore, Tm hybridizes and/or washed
The condition of washing can be conditioned to hybridize with the sequence of required homogeneity.If for example, the sequence sought have >=90% homogeneity,
Tm can reduce by 10 DEG C.Usually, the stringent condition of selection is less than about 5 DEG C of the thermal melting point (Tm) of particular sequence, and
It is complementary under defined ionic strength and pH.But high stringency can be applied and be less than thermal melting point (Tm) 1,2,3
Or 4 DEG C of hybridization and/or washing；Moderate stringency can be applied less than thermal melting point (Tm) 6,7,8,9 or 10 DEG C miscellaneous
Hand over and/or wash；Low stringency conditions can be applied less than thermal melting point (Tm) 11,12,13,14,15 or 20 DEG C of hybridization
And/or washing.Using this equation, hybridization and cleaning compositions and required Tm, those of ordinary skill in the art can understand hybridization
And/or the condition of wash solution changes with the change of stringency.If required extent of mismatch makes Tm (water-soluble less than 45 DEG C
Liquid) or 32 DEG C (formamide solutions), preferably increase SSC concentration so that higher temperature can be used.The guide of nucleic acid hybridization is seen
Tijssen (1993) biochemistries and the nucleic acid probe hybridization of Molecular Biology Lab's technology one, part i, the 2nd chapter
(Elsevier, New York)；With Ausubel et al. editor's (1995) Current Protocols method the 2nd chapter (Greene
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).
The stringent condition is preferably in 6 × SSC (sodium citrate), 0.5%SDS (dodecyl sodium sulfate) solution
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 reported on alpha-amylase polynucleotide sequence in this area, in gene database (The
GenBank) open sequence, occur the website about analysis, identification 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..For example, respectively in each website
Alphalise starch Gene Name and sequence number such as table 1 below：
Table 1
Further, the amino acid sequence of plant alpha amylase of the present invention is：
(a) albumen being made up of the amino acid sequence shown in SEQ ID NO.4；Or
(b) by substitution of the SEQ ID NO.4 amino acid sequence by one or several amino acid residues and/or missing
And/or add as derived from SEQ ID NO.4 and keep the albumen of the protein function shown in SEQ ID NO.4.
The present invention provides a kind of application of the importing plant alpha amylase in pollen abortion is caused in plant, including：
A) one section of nucleotide fragments, comprising plant alpha amylase gene order, the sequence is closely coupled to pollen or flower pesticide
Specificity promoter；
B) by a) the plant alpha amylase gene transfered plant tissue；
The 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 nucleotides and
The nucleotide sequence as derived from 1) with equal function；Or
3) hybridize under strict conditions with sequence shown in SEQ ID NO.1 and express the nucleotides sequence of identical function protein
Row, the stringent condition are in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C
Hybridization, and wash film with the solution；Or
4) there is more than 90% homology with nucleotide sequence 1), 2) or 3) and expresses the nucleosides of identical function protein
Acid sequence.
Biomaterial the invention provides above-mentioned plant alpha amylase or its encoding gene or containing the encoding gene is being led
Cause the application in plant pollen abortion.
Biomaterial the invention provides above-mentioned plant alpha amylase or its encoding gene or containing the encoding gene is being made
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.
Recombinant expression carrier of the present invention containing above-mentioned plant alpha-amylase can pass through agrobacterium-mediated transformation, particle gun
The conventional biology methods such as method, pollen tube passage method convert plant cell or tissue obtains independent transgenic cell or tissue,
Obtain male sterile transgenic line.
The genetically modified plants be foreign gene specifically expressing in pollen genetically modified plants, preferably pollination/fertilization
The genetically modified plants of ability enhancing/weakening, more preferably male sterility genetically modified plants.
Biomaterial of the present invention also contains transduction peptide and male gamete type of priority promoter.
The invention provides the plant expression vector for the effective catenation sequence that can make pollen abortion, includes effective connection regulation and control
Expression cassette, wherein the expression cassette in effective connection regulation and control contains as the DNA fragmentation institute shape shown in SEQ ID No.1-3
Into.The expression cassette is is connected with PG47 promoters and SBE signal peptides can be effective in the upstream of pollen abortion gene alpha-amylase
Disturb starch accumulation and accumulation expression casette in pollen grain.
Wherein, the sequence length shown in SEQ ID No.1 is that 1278bp is pollen abortion gene alpha-amylase forward direction DNA
Fragment；The transduction peptide that sequence length shown in SEQ ID No.2 is 216bp；Sequence length shown in SEQ ID No.3 is
2732bp male gamete type of priority promoter PG47.
The present invention further provides the plant pollen of the alpha-amylase gene expression cassette containing above-mentioned pollen abortion gene.
The present invention also provides the method that driving foreign gene causes pollen abortion to be expressed, and comprises the following steps：Pollen is lost
Educate gene alpha-amylase and purpose foreign gene imported into carrier and obtained outside containing pollen abortion gene alpha-amylase and purpose
The expression cassette recombinant expression carrier of source gene, and Plant Genome is introduced into, obtain foreign gene specifically expressing in pollen
Genetically modified plants.
The invention provides a kind of method of regulation and control Plant Pollen Development, it is characterised in that plant is expressed above-mentioned plant α
Amylase gene.
Described plant is grass.
Preferably, the plant includes but not limited to rice, corn, jowar, barley, oat, wheat, grain, sugarcane, big
Beans, Brassica species, cotton, safflower, tobacco, clover and sunflower.
Described regulation and control are amylase or induction plants male sterility in degrading plant pollen.
The invention provides the method that foreign gene in a kind of degrading plant pollen spreads, above-mentioned plant α starch will be contained
The expression cassette transformed calli of enzyme gene, the callus after conversion is subjected to induction differentiation and obtains transgenic pollen abortion
Transfer-gen plant, render transgenic plant pollen can not normally pollinate, and then foreign gene spreads in degrading plant pollen.
Pollen abortion gene alpha-amylase advantage provided by the invention is as follows：
1) plant alpha-amylase may be from rice, sorghum, barley, wheat etc. as pollen abortion gene, wherein from water
Rice alpha-amylase is endogenous gene, favourable to paddy gene engineering.
2) experiment of plant alpha-amylase gene pollen grain iodine dye shows, alpha-amylase can be under promoter PG47 drivings, 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 transgenosis diffusion；Available for holding male
The holding and breeding of sterile line, while artificial emasculation step is saved during hybrid seeding, have a extensive future.
4) present invention provides a kind of new method to obtain Transgenic male sterile plant.
Brief description of the drawings
Fig. 1 is the recombinant expression carrier DX2182- α-Amylase structures of pollen abortion gene alpha-amylase in embodiment 2
Flow chart.
Fig. 2 is that DX2182- α-Amylase transgenic paddy rice pollen iodine contaminates photo in embodiment 4.A figures are that rice is fertile right
Pollen iodine-potassium iodide dye picture is taken, whole pollen are fertile and can stained with color with iodine.B figures be the specific expressed α of paddy pollen-
Amylase pollen staining, wherein half pollen abortion can not dye, white arrow point to can stained pollen, black arrow point to dye
Do not paint pollen.
Fig. 3 is the genetically modified plants hygromycin selection T1 of embodiment 5DX2182- α-Amylase plant recombination expression vectors
For seed, T2 is obtained for plant.A figures are the germination of non-transgenic rice paddy seed hygromycin.B figures are transgenosis alpha-amylase rice seed
Sub- hygromycin germination.C figures are the germination of non-transgenic Mature Embryos of Rice hygromycin.D figures are transgenosis alpha-amylase Mature Embryos of Rice
Hygromycin germinates.Signified arrow is that can be germinateed for transgenosis alpha-amylase mature embryo.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention
In the case of essence, the modifications or substitutions made to the inventive method, step or condition belong to the scope of the present invention.If
Do not specialize, the conventional meanses that technological means used is well known to those skilled in the art in embodiment.
The rice alpha-amylase gene of embodiment 1 obtains
1st, rice Nipponbare RNA extraction
Utilize Biozol Reagent methods extraction rice Nipponbare RNA：Weigh the fresh young fringe tissue 1ml of 0.1g Nipponbares
Biozol Reagent ratio mixes, and is stored at room temperature 5min；Add 0.2ml chloroforms by every 1ml Biozol Reagent, firmly
15s is shaken, after solution is fully emulsified, then is stored at room temperature 5min, 12000rmp, 4 DEG C of centrifugation 15min；It is careful from centrifuge
Centrifuge tube is taken out, Aspirate supernatant is transferred in another new centrifuge tube；Isometric isopropanol is added into supernatant, up and down top
After centrifuge tube fully mixes, 10min, 12000rmp, 4 DEG C of centrifugation 10min are stood at room temperature；Supernatant is abandoned, tube wall can be now white
Color precipitates, and adds the ethanol of 0.5ml 75% (being configured with DEPC water after sterilizing) to wash, overturns and mix, 10000rmp, 4 DEG C of centrifugations
5min；It is repeated once, low temperature drying, ethanol is volatilized；Add 50ul Rnase-free water to dissolve precipitation, be placed in -80 DEG C of preservations.
2nd, the cDNA of Nipponbare is obtained
It is as follows using reverse transcriptase M-MLV reverse transcriptions, specific method using Nipponbare RNA as template：(1) configure, mix on ice
It is 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 transcriptase etc. is added, is mixed.
(4) 42 DEG C, extend 90 minutes.70 DEG C, 15 minutes.The cDNA packing for obtaining Nipponbare is stored in -40 DEG C.
Note：All experimental articles are RNase-free.
3rd, the primer of design synthesis cloning rice alpha-amylase
The nucleotide sequence of alpha-amylase (OS01G0715400) gene of rice Nipponbare is obtained by ncbi database
(as shown in SEQ ID NO.1 in sequence table), its amino acid sequence (as shown in SEQ IDNO.4 in sequence table).OsAmylase
Nucleotide sequence is that (SEQ ID NO.5-6 are cloned from the cDNA of Nipponbare and obtained in such as sequence table by designing primer sequence.
Design of primers uses Gibson Assembly methods, and amplified production is inserted into DX2182 carrier MluI and Sac1 single endonuclease digestions site.
Expand alpha-amylase gene primer pair such as sequence SEQ ID NO:Shown in 5-6.The wherein end of upstream and downstream primer 5 ' has 15 or so
Nucleotide sequence link position corresponding to carrier repeats, so as to Gibson Assembly connections.Amplification system and program are as follows：
PCR programs:94 DEG C of 3min of pre-degeneration, be denatured 94 DEG C of 30s, anneal 55-65 DEG C of 40s, extend 68 DEG C of 1min20s, 35
Individual circulation, extend 68 DEG C of 10min.
Embodiment 2 builds the recombinant expression carrier DX2182- α-Amylase of rice alpha-amylase
Structure flow is shown in Fig. 1, and the amplified production of embodiment 1 is inserted into DX2182 carrier Mlu1 and Sac1 restriction enzyme sites.
Primer SEQ ID NO:5 and SEQ ID NO:6 amplification PCR primer 1% agarose gel electrophoresis recovery 1300bp are left
Right product.Mlu1 and Sac1 digestion carrier DX2182, reclaim linear digestion carrier.
2X connections kit connection abortion gene is as follows to DX2182,10ul systems：
The alpha-amylase PCR primers (50ng) of 2.5 μ 1, the digestion carriers (100ng) of 2.5 μ 1,5 μ 1Ligation Mix, connection
Program：50 DEG C 60 minutes.
Conversion：Take above-mentioned 2 μ of connection product, 1 electroporated competent escherichia coli cells.Select positive colony sequencing.Life
Entitled DX2182- α-Amylase.DX2182- α-Amylase carriers contain hygromycin gene, its sequence such as SEQ ID
Shown in NO.7, hygromycin selection can be used, obtains resistant plant, while screening transgenic T0 can also be used to turn base for seed, acquisition
Because plant T1 is for resistant plant.
The acquisition of the transgenic rice plant of embodiment 3
The Agrobacterium EHA105 of -70 DEG C of preservations is taken in containing Kan (50 μ g/ml)+Rif (25 μ g/ml)+streptomysins (50 μ g/
Ml) plate streaking, 28 DEG C of cultures.Picking single bacterium colony is inoculated in 50ml YEB fluid nutrient mediums, 28 DEG C of shaken cultivations of 220rpm
12-16hr.2ml bacterium solutions are taken to transfer in 100ml (containing antibiotic) YEB fluid nutrient mediums, 28 DEG C of 220rpm shaken cultivations are extremely
OD600=0.5.Precooling on ice 10 minutes, 5000rpm 10min (refrigerated centrifuge is pre-chilled to 4 DEG C).Aseptic deionized water washes 2
Secondary (each 10ml), 10% glycerine are washed 1 time and are dissolved in the glycerine of 3ml 10%.100 μ l competent cells are taken to add in 1 μ l embodiments 1
Obtained DX2182- α-Amylase plasmids, 2.5KV are electroporated.On the YEB culture plates containing kanamycins and rifampin
28 DEG C of cultures, select positive colony, are verified with DX2182- α-Amylase vector-specific primers SEQ ID NO.8-9PCR.
Checking is correctly cloned, and is infected by Agrobacterium-mediated genetic transformation method and 11 (Hiei Y Ohta are spent in rice
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).Through co-culturing, screening, breaking up, the link such as take root obtains T0 for transgenosis children
Seedling, DNA is extracted, the transgenic positive plant verified through PCR, self-fertility obtains T1 generations.T1 plant are taken to carry out subsequent analysis.
The transgenic paddy rice KI staining analysis of embodiment 4
KI dyeing liquor appearance liquid is prepared (to take 2g KI to be dissolved in 5-10mL distilled water, then add 1g I2 and (use in right amount
Absolute ethyl alcohol dissolves), after all dissolving, then add distilled water to be settled to 300mL.Store in standby in brown bottle).Base is turned to rice
Because the mature pollen of plant carries out microscopy, the pollen grain fertility of transfer-gen plant is analyzed, specific method is as follows：
1st, pollen collection：The flower pesticide for taking abundant maturation to bloom, glume is divested, take out flower pesticide.
2. microscopy：By KI：Deionized water=1:1 dilution proportion liquor kalii iodide, takes 70ul dilution to be placed in
Slide, take a flower pesticide to be placed on slide, flower pesticide is fully smashed to pieces with tweezers, discharges pollen grain, covered, in low
Observed under power microscope.All pollen grains for 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 KI staining analysis, pollen grain iodine dye result：Abortion is spent
Powder meets 1:1 segregation ratio, as partial pollen show as male sterility (50%), have half (50%) to dye black
Blue (as shown in Fig. 2 B figures, 50% normal pollen staining).And wild type (or other do not carry turning for pollen abortion gene
Gene plant) it is then that holandry is fertile (as shown in Fig. 2 A figures, 100% normal pollen staining).Show rice α-shallow lake
Powder enzyme can in paddy pollen degradable starch, therefore in pollen grain in growth course, energy supply deficiency, cause pollen to lose
Educate.
Thus the starch that alpha-amylase gene can degrade in pollen grain is proved, Transgenic Rice pollen sterility is caused, has
Effect prevents genetically modified crops that transgenic element is broadcast into other wild crop kinds by pollen.
In summary, the protein alpha-amylase that the present invention influences male fertility is to clone from rice varieties alphalise starch
Enzyme, and the starch in the pollen grain that can degrade, Transgenic Rice pollen sterility is caused, accuracy is high, effectively prevents transgenosis
Transgenic element is broadcast to other wild crop kinds by crop by pollen；Available for the homozygous hidden of holding male sterile plants
Character state；The step of eliminating emasculation during hybrid seeding simultaneously.
Embodiment 5 is to transgenic paddy rice T1 for seed hygromycin selection
By recombinant plant expression vector DX2182- α-Amylase transfer-gen plant, T1 is for seed for harvest.According to implementation
In example 4, fertile pollen：Abortive pollen meets 1:1 segregation ratio, it is that half seed is non-transgenic seed, half is to turn
Gene seed, therefore screening transgenic seed is carried out using hygromycin, transgenosis T2 is for plant for induction.Tested by two schemes
Card：
(1) 400mg/L hygromycin selections T1 is for seed
Using non-transgenic ZH11 as control, under 400mg/L hygromycin, the pollen abortion gene weight containing alpha-amylase is screened
The T1 of group carrier chooses 100, screened respectively for seed.Its germination percentage is observed after 7d.As a result budding is observed after showing 7d
Rate, its survival rate and the death rate=47 are counted after 14d:53, substantially conform to 1:1 segregation ratio (such as Fig. 3 A figures and B figures)
(2) 35mg/L hygromycin+MS Screening of Media T1 is for mature embryo
Using non-transgenic ZH11 as control, on 35mg/L hygromycin+MS culture mediums, screen the pollen containing alpha-amylase and lose
Mature embryos of the T1 for seed of gene recombined vector is educated, 100 is chosen respectively, is screened.Its germination percentage is observed after 7d.As a result
Bud ratio is observed after showing 7d, counts its survival rate and the death rate=51:49, substantially conform to 1:1 segregation ratio, such as Fig. 3 C
Figure and D figures.
With reference to embodiment 2-5 method, restructuring load is carried out to the plant alpha amylase gene described in SEQ ID NO.10-19
Body structure, genetically modified plants prepare and functional verification, as a result similar with the result of embodiment 4/5, show that plant alpha-amylase can be
Degradable starch in crop pollen, therefore in pollen grain in growth course, energy supply deficiency, cause pollen abortion, cause to turn
Gene crops pollen sterility, effectively prevent genetically modified crops that transgenic element is broadcast into other wild crop product by pollen
Kind.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. application of the Introduced into Rice alpha amylase in pollen abortion is caused in plant, the nucleotides of the rice alpha amylase gene
Sequence is as shown in SEQ ID No.1；The amino acid sequence of the rice alpha amylase gene coded protein such as SEQ ID NO.4 institutes
Show.
2. application of the rice alpha amylase in pollen abortion transfer-gen plant is prepared, the amino acid sequence of the plant alpha amylase
As shown in SEQ ID NO.4；The encoding gene of the rice alpha amylase is as shown in SEQ ID No.1.
3. application of the biomaterial containing rice alpha amylase gene in pollen abortion transfer-gen plant is prepared, the rice α
For the nucleotide sequence of amylase gene as shown in SEQ ID No.1, the biomaterial is recombinant expression carrier, expression cassette, again
Group bacterium or host cell.
4. application as claimed in claim 3, it is characterised in that described biomaterial contains transduction peptide and male gamete is preferential
Type promoter；The transduction peptide is SBE signal peptides, and for its nucleotide sequence as shown in SEQ ID NO.2, the male gamete is preferential
Type promoter is PG47, and its nucleotide sequence is as shown in SEQ ID NO.3.
A kind of 5. method of regulation and control Plant Pollen Development, it is characterised in that make plant express rice alpha amylase gene, the gene
Nucleotide sequence as shown in SEQ ID No.1.
6. method as claimed in claim 5, it is characterised in that described plant is grass.
7. method as claimed in claim 5, it is characterised in that described regulation and control are that starch or induction are planted in degrading plant pollen
Thing male sterility.
8. a kind of reduce the method that foreign gene spreads in plant pollen, it is characterised in that will contain rice alpha amylase gene
Expression cassette transformed calli, the transgenosis that the callus after conversion is carried out to induction differentiation acquisition transgenic pollen abortion are planted
Strain, render transgenic plant pollen can not normally pollinate, and then reduce foreign gene in plant pollen and spread, and described rice α forms sediment
The nucleotide sequence of powder enzyme gene is as shown in SEQ ID No.1.
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| CN109750059B (en) * | 2018-08-31 | 2021-03-02 | 海南波莲水稻基因科技有限公司 | Rice beta-amylase BA2 and coding gene and application thereof |
| CN110923263B (en) * | 2018-08-31 | 2021-09-21 | 海南波莲水稻基因科技有限公司 | Rice beta-amylase BA1 and coding gene and application thereof |
| CN110923262B (en) * | 2018-08-31 | 2021-09-21 | 海南波莲水稻基因科技有限公司 | Sorghum alpha-amylase and coding gene and application thereof |
| CN110872584B (en) * | 2018-08-31 | 2021-09-21 | 海南波莲水稻基因科技有限公司 | Barley alpha-amylase and coding gene and application thereof |
| CN111072759B (en) * | 2018-10-18 | 2022-10-21 | 深圳市作物分子设计育种研究院 | Powder-forming protein transport signal peptide and application thereof in pollen fertility regulation |
| CN109517837A (en) * | 2018-10-18 | 2019-03-26 | 深圳市作物分子设计育种研究院 | A kind of rice alpha-amylase and its encoding gene and application |
| CN110305895B (en) * | 2019-07-31 | 2020-07-14 | 湖南杂交水稻研究中心 | Method for preventing transgene pollen drift by using rice RAMy1A gene |
| CN110642929B (en) * | 2019-09-29 | 2022-11-01 | 深圳市作物分子设计育种研究院 | Rice amyloplast protein transport signal peptide and application thereof in pollen fertility regulation |
| CN111269914A (en) * | 2020-02-18 | 2020-06-12 | 湖南杂交水稻研究中心 | DNA molecule and method for effectively preventing pollen of transgenic plant from escaping |
| CN113615567A (en) * | 2020-05-07 | 2021-11-09 | 海南波莲水稻基因科技有限公司 | Seed production carrier for crop genetic intelligent breeding |
| CN113621642A (en) * | 2020-05-07 | 2021-11-09 | 海南波莲水稻基因科技有限公司 | Genetic intelligent breeding system for crop cross breeding seed production and application thereof |
| CN113151318B (en) * | 2021-03-17 | 2022-08-16 | 云南中烟工业有限责任公司 | Tobacco starch branching enzyme gene NtGBE1 and application thereof |
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