CN109517837A

CN109517837A - A kind of rice alpha-amylase and its encoding gene and application

Info

Publication number: CN109517837A
Application number: CN201811212062.3A
Authority: CN
Inventors: 唐晓艳; 王梦龙; 彭小群; 严维; 尹峰; 陈竹锋
Original assignee: Shenzhen Guangsan Agricultural Technology Co Ltd; Shenzhen Institute of Molecular Crop Design
Current assignee: Shenzhen Guangsan Agricultural Technology Co Ltd; Shenzhen Institute of Molecular Crop Design
Priority date: 2018-10-18
Filing date: 2018-10-18
Publication date: 2019-03-26

Abstract

The present invention provides a kind of purposes of rice alpha-amylase and its encoding gene in pollen fertility regulation, belongs to field of biotechnology.The present invention passes through the nucleotide sequence of cloning rice OryzasativaLcv.Nipponbare alpha-amylase, building recombinant expression carrier and progress genetic transformation.Under the driving of PG47 promoter, alpha-amylase is specific expressed in the pollen development later period；Simultaneously under the guidance of BT1 encoding transport signals peptide, starch in the degradable pollen grain of the alpha-amylase causes Transgenic Rice pollen sterility, and accuracy is high, effectively prevents genetically modified crops by pollen and transgenic element is broadcast to other crop varieties.Alpha-amylase of the invention can be used for keeping the homozygous recessive condition of male sterile plants, to save artificial emasculation step during hybrid seeding, and it can largely expand numerous sterile line, reduce the investment of labour and the influence to yield, make its crop-planting resource improvement and in terms of have broad application prospects.

Description

A kind of rice alpha-amylase and its encoding gene and application

Technical field

The invention belongs to field of biotechnology, and in particular to a kind of rice alpha-amylase and its encoding gene are in pollen fertility Application in control further relates to that the alpha-amylase is combined to answer the hydrolysis function of starch with the male fertility for influencing rice With.By utilizing modern biotechnology, it is applied to hybrid seed production technology system, not only can guarantee seed production quality, but also can Improve seed production efficiency；It also can effectively prevent transgenosis diffusion.

Background technique

Breeding of hybridized rice is the main path for effectively improving yield and quality of rice.Due to the application of hybridization technique, make It must increase production, is disease-resistant, is pest-resistant, drought resisting, the polymerization of antiweed, waterlogging, cold-resistant, salt tolerant and the various characters such as resistant to lodging all become It may.The application of hybridization technique provides the male parent of pollen dependent on to maternal, is then obtained by male parent and maternal hybridizing method Obtain cenospecies.Although paddy rice cross breeding use of advantage is one of the approach for improving yield and quality of rice the most cost-effective, It still has more limiting factor, for example, the polymerization difficulty of various advantage characters is larger, specific gravity shared by hybrid crop compared with It is small etc..Although most crops all utilize hybrid vigour as far as possible, but still there are huge potentiality.

There are " three line method " and " two line method " applied to the main crossbreeding technology in Rice Production at present." three line method " by It is restricted in by Rescued virus, limits the utilization of Rice Germplasm Resources and the breeding of influential point set；And " two line method " due to Its sterile line fertility is influenced regulation by environmental condition factor, the under cover biggish risk in practical production of hybrid seeds production.Thus may be used See, there is certain technological deficiency in traditional " three line method " and " two line method ", limit to a certain extent hybrid rice into The development of one step and popularization and application, and male sterile line resource is the serious key factor for restricting the development of paddy rice cross breeding technology.In addition, " three line method " and " two line method " breeding method period length, slow effect, it is difficult to meet the urgent need of production development.

In order to solve the problems, such as the breed males sterile material such as " three line method " and " two line method ", successively there are numerous scientists to think By solving the problems, such as this using molecular design method.1993, PLANT GENETIC SYSTEM company proposed patent Shen Please (Williams M, Leemans J..Maintenance of male-sterile plants.1993.Patent No.WO93/25695.), i.e., be transferred in male sterile plants chain restoring gene, pollen inactivation (abortion) gene and For the marker gene of screening, the holding system of the sterile line plant can be obtained, sterile line and holding are then realized by selfing The breeding of system.In addition, Perez-Prat in 2002 et al. proposition is extensive by being transferred to chain fertility in male sterile plant Two sets of elements of multiple genes and riddled basins are achieved with the holding system of male sterile plants, and can further breed into infertility It is (Perez-Prat, E., and van Lookeren Campagne, M.M..Hybrid seed production and the challenge of propagating male-sterile plants.Trends Plant Sci.2002.7,199- 203.).The it is proposed of these conceptions provides new think of accurately to carry out the crossbreeding of crop molecule using Protocols in Molecular Biology Road.Currently have a lot research shows that other functional genes may also construct the plant of Transgenic male infertility by biological technique method Strain, however, type and cultivated area recently as genetically modified crops is continuously increased, Transgene-safty problem is also by more Come the concern and attention of more people.The foreign gene carried in genetically modified plants can be integrated into non-turn by way of natural hybrization In gene kind or other wild relatives, to cause Transgene-safty problem.Hybridizing between affine floristics, flower The genetic drift that powder mediates is inevitable.

The male sterility of plant is mainly manifested in pollen abortion, it is related to male organs missing, sporogenous cell exception, subtrahend Abnormal division, callose metabolic disorder, tapetal development exception, pollen wall dysplasia, anther dehiscence exception and pollen germination Each processes such as failure.Therefore, the overall process and molecule mechanism for understanding pollen development be study plants male sterility basis and Key point.The growth course of pollen is complex, it is related to the expression regulation of many genes, wherein after pollen development Phase, starch can be the sprouting of pollen and the extension energy reserve of pollen tube.Therefore, when the starch in pollen grain is in advance by alphalise starch Enzyme degradation, so that pollen grain energy source is disintegrated, there is lopsided pollen grain in the final growth for containing pollen, causes plant male Property infertility.

Amylase can hydrolyze starch, be mainly distributed in animals and plants, bacterium and fungi, and people, which pass through, utilizes biological skill Art has cloned various amylase genes (the diversity Zhengzhou animal husbandry work of Cui Jin, Ma Xiangdong (2009) amylase gene Journey higher junior college journal, 29 (2): 21-23), while correlative study has been carried out to the diversity of amylase.Studies have shown that Amylase gene is in addition to being all presented diversity other than the diversity of source in terms of gene structure and function.Amylase can be divided into α- Amylase, beta amylase and λ-amylase, wherein alpha-amylase belongs to Endoglucanases, can be from starch chain internal random α-Isosorbide-5-Nitrae glycosidic bond is cut, makes Starch Hydrolysis at maltose and glucose etc., and 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, at In ripe pollen, suitable amylase hydrolyzable starch, generated energy is provided to normal development and the pollen of pollen The sprouting and growth of pipe.However, can all reduce pollen energy if amylase is overexpressed during pollen formation or silencing is expressed Metaboilic level causes starch accumulation amount insufficient, to generate abortive pollen.Existing research report, corn alpha-amylase gene with Pollen development later period specific expressing promoter recombination, can render transgenic plant express alpha-amylase, cause transgenosis flower Powder abortion, thus be effectively reduced the genetic drift of pollen-mediated risk (Zhang, D., Wu, S., An, X., Xie, K., Dong, Z.,Zhou,Y.,Xu,L.,Fang,W.,Liu,S.,Zhu,T.,Li,J.,Rao,L.,Zhao,J.,and Wan,X.(2017) .Construction of a multi-control sterility system for a maize male-sterile line and hybrid seed production based on the ZmMs7 gene encoding a PHD-finger transcription factor.Plant Biotechnol.J.16,459-471.).These alpha-amylase genes are logical for rice The acquisition that biotechnology realizes sterile line transgenic plant is crossed, is especially provided in terms of controlling fertility and transgenosis New selection.To utilize above-mentioned technology, the alpha-amylase gene of separate sources, such as important crops rice are needed, but related Study also fresh understatement road.

Summary of the invention

The object of the present invention is to provide a kind of rice alpha-amylase genes to lead to pollen abortion and prepare transgenosis pollen Application in abortion plant, the i.e. new alpha-amylase gene of from rice, to can effectively avoid transgenic pollen Pollution, to obtain new male sterile line and its application.

To achieve the above object, the present invention provides a kind of rice alpha-amylase genes, comprising:

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 The nucleotide sequence as derived from a) with same function；Or

3) hybridize under strict conditions with sequence shown in SEQ ID NO:1 and the nucleotide of identical function protein can be expressed Sequence；Or

4) there is 90% or more homology with nucleotide sequence 1), 2) or 3) and the core of identical function protein can be expressed Nucleotide sequence.

The present invention also provides the DNA moleculars complementary with rice alpha-amylase nucleotide sequence.Those skilled in the art can hold It easily identifies and utilizes the DNA molecular complementary with paddy pollen abortion gene alpha-amylase nucleotide sequence, therefore, in stringent condition The lower sequence hybridized with pollen abortion gene alpha-amylase sequence of the invention or its segment is included in the invention.Wherein, institute Nucleotide sequence complementation is stated, referring to can hybridize under strict conditions with the DNA sequence dna of alpha-amylase.

It is more than to hybridize with other sequences that the stringent condition, which refers to that probe is hybridized to detectable degree with its target sequence, Condition.Stringent condition has sequence dependent, and can change because of the difference of environment.Hybridized by strict control or is washed Condition, it is possible to identify go out the target sequence complementary with probe 100%.Can by selectivity adjusting stringent condition to allow, there are some Sequence mismatch, to may detect the similitude of lower degree.

Common, stringent condition is that salinity is lower than about 1.5M sodium ion at pH value 7.0-8.3, typically about 0.01-1.0M Na ion concentration (or other salts), temperature are right at least about 30 DEG C of short probe (such as 10-50 nucleotide) At least about 60 DEG C of long probe (such as more than 50 nucleotide).It also can get stringent item by adding destabilizing agent such as formamide Part.Low stringency condition, it may for example comprise molten in 1M NaCl, 30-35% formamide, the buffering of l%SDS (dodecyl sodium sulfate) 37 DEG C of hybridization in liquid, the 50-55 DEG C of washing in 1 to 2 × SSC (20 × SSC=3.0M NaCl/0.3M trisodium citrate).Moderate Stringent condition, it may for example comprise 1M NaCl, 40-45% formamide, l%SDS buffer solution in 37 DEG C hybridization, 0.5 to 1 55-60 DEG C of washing in × SSC.High stringency, it may for example comprise molten in 1M NaCl, 50% formamide, the buffering of l%SDS 37 DEG C of hybridization in liquid, the 60-65 DEG C of washing in 0.1 × SSC.Non-essential, washing buffer contains about 0.1%-1%'s SDS.Hybridization time about 4-12 hours.

Especially typical is the washing after hybridization, and wherein key factor is the ionic strength and temperature of final washing solution. For DNA-DNA hybrid, Tm=81.5 DEG C+16.6 (logM)+0.41 (%GC) -0.61 (%form) -500/L；Wherein Tm 50% complementary target sequence and the temperature (under defined ionic strength and pH) that hybridize of pairing probe completely, M be unit price it is positive from The molar concentration of son, %GC is the percentage of guanylic acid and cytidylic acid in DNA, and %form is that formamide exists Percentage in hybridization solution, L are length of the hybrid in base-pair.Tm reduces about l DEG C, and mispairing is increased by 1%；Cause This, Tm hybridization or wash conditions can be conditioned to hybridize with the sequence of required identity.For example, if the sequence sought have >= 85% identity, Tm can reduce by 10 DEG C.Generally, 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 defined ionic strength and pH.But high stringency can be using lower than pyrolysis The hybridization or washing that 1,2,3 or 4 DEG C of chain temperature (Tm)；Moderate stringency can using lower than thermal melting point (Tm) 6,7,8, 9 or 10 DEG C of hybridization or washing；Low stringency conditions can be using lower than thermal melting point (Tm) 11,12,13,14,15 or 20 DEG C hybridization or washing.If required extent of mismatch makes Tm lower than 45 DEG C (aqueous solutions) or 32 DEG C (formamide solution), preferably Increase SSC concentration to be able to use higher temperature.The guide of nucleic acid hybridization sees Tijssen (1993) biochemistry and molecule The nucleic acid probe hybridization of biology laboratory technology one, part i, the 2nd chapter (Elsevier, New York)；With Ausubel etc. People edit the 2nd chapter of (1995) Current Protocols method (Greene Publishing and Wiley-Interscience, New York).See Sambrook et al. (1989) molecular cloning: laboratory manual (second edition, Cold Spring Harbor Laboratory Press,Plainview,NewYork)。

The stringent condition is preferably the solution in 0.5%SDS (dodecyl sodium sulfate), 6 × SSC (sodium citrate) In, hybridize at 65 DEG C, is then respectively washed film 1 time with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS.

In addition, the amino acid sequence of rice alpha-amylase of the present invention are as follows:

1) protein that the amino acid sequence shown in SEQ ID NO:4 forms；Or

2) by the amino acid sequence of SEQ ID NO:4 by one or several amino acid residues substitution and/or missing and/ Or add as derived from SEQ ID NO:4 and keep the albumen of protein function shown in SEQ ID NO:4.

Replacing, missing or adding for amino acid sequence is the ordinary skill in the art in the present invention, preferably this amino acid Variation are as follows: small characteristic changing, i.e., the folding and/or active conserved amino acid for not significantly affecting albumen replace；Small missing, The missing of normally about 1-30 amino acid；Small amino or c-terminus extend, such as aminoterminal extends a methionine residues； Small link peptide, for example, about 20-25 residue are long.

The example of conservative substitution is the substitution occurred in following amino acid group: basic amino acid (such as arginine, lysine And histidine), it is acidic amino acid (such as glutamic acid and aspartic acid), polar amino acid (such as glutamine, asparagine), hydrophobic Acidic amino acid (such as leucine, isoleucine and valine), ArAA (such as phenylalanine, tryptophan and tyrosine), with And small molecule amino acid (such as glycine, alanine, serine, threonine and methionine).Do not change given activity usually Those amino acid substitutions are well-known in the art, and by for example, N.Neurath and R.L.Hill are 1979 It is described in " Protein " published by year new york academic publishing house (AcademicPress).The most common exchange has Ala/ Ser, Val/Ile, Asp/Glu, Thu/Ser, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly and their opposite exchanges.

For a person skilled in the art it should be evident that this substitution can play an important role to molecular function Region except occur, and still generate active peptides.For by polypeptide of the invention, activity is required and therefore selects not Substituted amino acid residue can reflect according to methods known in the art, such as direct mutagenesis or alanine scanning mutagenesis Determine (such as referring to Cunningham and Wells, 1989, Science 244:1081-1085).Latter technique is every in the molecule Mutation, the influence male fertility activity of detection gained mutating molecule, so that it is determined that right are introduced at one positively charged residue Important amino acid residue for the molecular activity.Substrate-enzyme interacting site can also pass through the analysis of its three-dimensional structure It measures, this three-dimensional structure can measure by technologies such as nuclear magnetic resonance spectroscopy, crystallography or photoaffinity labeling (referring to such as deVos Deng 1992, Science255:306-312；Smith etc., 1992, J.Mol.Biol224:899-904；Wlodaver etc., 1992, FEBSLetters309:59-64).

Therefore, with amino acid sequence shown in SEQ ID NO:4 there is the amino acid sequence of certain homology to be also included within In the present invention.These sequences and sequence similarities of the present invention/phase same sex are typically larger than 60%, preferably greater than 75%, more excellent Choosing is greater than 80%, is even more preferably greater than 90%, and can be greater than 95%.It can also be according to the phase same sex particularly And/or similarity range defines preferred polynucleotides and protein of the invention.Such as have with the exemplary sequence of the present invention 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the 98% or 99% phase same sex and/or similarity.

The present invention provides a kind of application of rice alpha-amylase in preparation pollen abortion transgenic plant, the water The amino acid sequence of rice alpha-amylase are as follows:

1) albumen that the amino acid sequence shown in SEQ ID NO:4 forms；Or

The encoding gene of rice alpha-amylase includes

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 The nucleotide sequence as derived from 1) with same function；Or

The present invention provides a kind of expression cassettes, which is characterized in that the water under the regulating and controlling sequence regulation effectively connected The gene of rice alpha-amylase, with adjusting and controlling rice male fertility.

Heretofore described regulating and controlling sequence include but is not limited to promoter, transit peptides, terminator, enhancer, leader sequence, Introne and other adjusting sequences for being operably connected to the gene for influencing male fertility.

Specifically, regulating and controlling sequence protein transduction containing the amyloplast peptide and pollen development later period specific expressing promoter. More specifically, the peptide of protein transduction containing amyloplast is BT1 (TP) signal peptide, nucleotide sequence such as SEQ ID NO:3 institute Show；The pollen development later period specific expressing promoter is PG47 promoter, nucleotide sequence such as SEQ ID NO:2 institute Show.

The present invention provides the DNA construct of a kind of gene comprising above-mentioned rice alpha-amylase or expression cassette, and comprising The recombinant expression carrier of the DNA construct.

The recombinant expression carrier that the present invention contains above-mentioned rice alpha-amylase gene can be by agrobacterium-mediated transformation or base Because the conventional biology methods rice transformation cell such as marksmanship method or callus obtain independent transgenic cell or tissue, thus Obtain the transgenic line of male sterility of rice.

The transgenic paddy rice is foreign gene in pollen later period specifically expressed transgenic paddy rice, preferably pollinate/by The genetically modified plants of smart ability enhancing/weakening, more preferably male sterility transgenic paddy rice.

Biomaterial the present invention provides above-mentioned rice alpha-amylase or its encoding gene or containing the encoding gene exists The method of starch in degrading plant pollen.

Biomaterial the present invention provides above-mentioned rice alpha-amylase or its encoding gene or containing the encoding gene exists The method of adjusting and controlling rice pollen development.The regulation is amylase or inducing paddy rice male sterility in degrading rice pollen.

Biomaterial the present invention provides above-mentioned rice alpha-amylase or its encoding gene or containing the encoding gene exists The male sterile method of inducing paddy rice, to generate part male sterile plants.Shown in plant be gramineae plant.

Biomaterial the present invention provides above-mentioned rice alpha-amylase or its encoding gene or containing the encoding gene exists The method of rice male fertility is influenced, to generate part male sterile rice.

Biomaterial the present invention provides above-mentioned rice alpha-amylase or its encoding gene or containing the encoding gene exists The method for blocking foreign gene diffusion in plant pollen, which is characterized in that the expression of above-mentioned rice alpha-amylase gene will be contained Box converts plant callus, and the callus after conversion is carried out induction differentiation and culture of rootage and is lost to obtain transgenic pollen The genetically modified plants educated, render transgenic plant pollen can not normally pollinate, and then foreign gene is spread in degrading plant.

Include but is not limited to by heretofore described construct or recombinant vector importing plant, conventional transformation methods, Agrobacterium-medialed transformation, directly leads the DNA that DNA takes in protoplast, electroporation or silicon whisker mediate micro transmitting bombardment Enter.

It is heretofore described by nucleotide sequence " introducing " plant when, indicate to send out by the method directly converted Raw, the method is for example to the Agrobacterium-medialed transformation of plant tissue, corpuscular emission bombardment, electroporation etc.；Or it can pass through Plant with heterologous nucleotide sequence is hybridized with another plant to carry out, so that offspring has the core for being incorporated to their genomes Nucleotide sequence.Such breeding technique is well known to those skilled in the art.

The gene order of the rice alpha-amylase are as follows:

1) nucleotide sequence shown in SEQ ID NO:1；Or

The plant is gramineae plant.Preferably, the plant include but is not limited to rice, corn, sorghum, barley, Wheat, soybean, cotton, sunflower.

A kind of of the invention application for providing rice alpha-amylase in pollen fertility control, has the advantage that

1) plant alpha-amylase may be from rice, sorghum, barley, wheat etc. as pollen abortion gene, wherein coming from water Rice alpha-amylase is endogenous gene, advantageous to paddy gene engineering.

2) pollen abortion gene alpha-amylase OsAA is higher in the expression quantity in pollen development later period, is conducive to it to transgenosis The efficiency of hydrolysis starch can be improved, so as to effectively contain the dirt of transgenic pollen in the accurate degradation of starch in paddy pollen grain Dye；

3) experiment of rice alpha-amylase pollen iodine dye shows that alpha-amylase OsAA gene can be in pollen development later period specificity Under the driving for expressing promoter PG47, the starch in pollen grain is accurately acted on, finally making can be with pollen and abortive pollen Ratio be 1:1；

4) rice alpha-amylase gene of the invention in pollen development later period different expression gene promoter PG47 and is made Under the regulation of powder Protein transport peptide BT1 (TP), the diffusion of transgenosis can be effectively controlled；Can be used for rice keep system holding and The expansion of sterile line is numerous, and artificial emasculation step can be saved during hybrid seeding, has broad application prospects.

5) originally bright to provide a kind of new method to obtain Transgenic male sterile plant.

Detailed description of the invention

Fig. 1 is relative expression spirogram of the alpha-amylase gene OsAA in each tissue of rice.Stage indicates anther development Different phase；DAP (Day After Pollination) refers to the number of days after pollination.

Fig. 2 is the building flow chart of the recombinant expression carrier pSZYJY-02 of alpha-amylase gene OsAA.

Fig. 3 is the growth phenotype figure of the transgenic paddy rice positive plant of the pSZYJY-02 containing recombinant expression carrier.Left figure is open country Raw type control, right figure is transgenic paddy rice, scale 10cm.

Fig. 4 is the anther phenotypic map of the transgenic paddy rice of the pSZYJY-02 containing recombinant expression carrier.Left figure is wild type pair According to right figure is transgenic paddy rice, scale 1mm.

Fig. 5 is that the pollen iodine of the transgenic paddy rice of the pSZYJY-02 containing recombinant expression carrier contaminates figure.Left figure is wild type pair According to right figure is transgenic paddy rice, and wherein white arrow indicates that about half pollen is because abortion is dyed to yellowish-brown, black arrow instruction About half pollen is dyed to black and blue color because fertile, and scale is 100 μm.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Do not violating essence of the invention In the case where mind and essence, the modifications or substitutions to made by the method, step or condition used in the present invention belong to this hair Bright range.Unless otherwise specified, the routine that various technological means used in embodiment are general for those skilled in the art Means.

Embodiment 1, the acquisition of rice alpha-amylase gene OsAA and its tissue expression pattern analysis

1, the extraction of rice OryzasativaLcv.Nipponbare RNA

Utilize Trizol Reagent method extract OryzasativaLcv.Nipponbare RNA: weigh OryzasativaLcv.Nipponbare it is each tissue (root, stem, leaf, gynoecium, Lemma, glumelle, anther and endosperm) it is put into liquid nitrogen, it is clayed into power after taking-up with mortar, 1ml Trizol is then added Reagent (TransGen Biotech), acutely concussion uniformly, is subsequently added into 0.2ml chloroform, acutely shakes 15s, be stored at room temperature 3min；4 DEG C of centrifugation 15min of 12000rmp；Centrifuge tube is carefully taken out from centrifuge, absorption 0.6ml supernatant is transferred to another In new centrifuge tube；Isometric isopropanol is added into supernatant, after the centrifuge tube that turns upside down mixes well, stands at room temperature 10min；4 DEG C of centrifugation 10min of 12000rmp；Supernatant is abandoned, white gum precipitating occurs in tube bottom, adds 75% ethyl alcohol of 1ml (with sterilizing DEPC water configures afterwards) washing, acutely it is vortexed, 4 DEG C of centrifugation 5min of 10000rmp；Abandoning supernatant, room temperature dry precipitating, and (precipitating becomes It is colorless and transparent)；Add 50ul RNase-free water dissolution precipitating, 55 DEG C of -60 DEG C of incubation 10min are placed in -70 DEG C of preservations.

2, the acquisition of rice OryzasativaLcv.Nipponbare cDNA

Using each tissue RNA of rice OryzasativaLcv.Nipponbare as template, using PrimeScript RT reagent kit (DRR047A, Takara) kit carries out reverse transcription, and the specific method is as follows:

(1) gDNA is removed

5×gDNA Eraser Buffer 2ul

gDNA Eraser 1ul

RNA 1ug

Add RNase free H₂O polishing is to 10ul

42 DEG C, 2 minutes

(2) reverse transcription

(3) the cDNA packing for obtaining OryzasativaLcv.Nipponbare is stored in -20 DEG C.

Note: all experimental articles are RNase-free.

3, the clone of rice alpha-amylase gene OsAA

The nucleosides of the alpha-amylase gene OsAA (LOC_Os04g33040) of rice OryzasativaLcv.Nipponbare is obtained by ncbi database Acid sequence (as shown in SEQ ID NO:1 in sequence table) and amino acid sequence (as shown in SEQ ID NO:4 in sequence table).OsAA Nucleotide sequence is by design primer sequence (as shown in SEQ ID NO:5-6 in sequence table), with the cDNA of OryzasativaLcv.Nipponbare blade It clones and obtains for template PCR, PCR product length is 1470bp, and sequence is consistent with SEQ ID NO:1 after product sequencing.Primer makes With 5 software design of Primer Premier, amplification system and program are as follows:

PCR program: 94 DEG C of 3min of initial denaturation；98 DEG C of 30s are denaturalized, anneal 55-65 DEG C of 30s, extends 72 DEG C of 2min, 30 are followed Ring；Extend 72 DEG C of 10min.

4, OsAA tissue expression pattern is analyzed

According to the cDNA sequence design primer (as shown in SEQ ID NO:7-8 in sequence table) of OsAA, while with rice Actin gene is as internal reference control design primer, using fluorescence quantifying PCR method, analyze OsAA gene rice root, stem, leaf, Expression in gynoecium, lemma, glumelle, anther (Stage 6-Stage 12) and endosperm (7DAP and 25DAP).As a result such as Fig. 1 institute Show, all there is expression in OsAA gene, and in each tissue in the expression quantity highest of anther later period (Stage 11-12), and Expression quantity is relatively low in other each tissues.As a result illustrate, high expression pattern of the OsAA gene in the anther development later period has It degrades conducive to the precise and high efficiency to starch in pollen.

The clone of embodiment 2, PG47 promoter and BT1 transhipment peptide gene

PG47 promoter (as shown in SEQ ID NO:2 in sequence table) and BT1 transhipment peptide gene (SEQ ID in such as sequence table Shown in NO:3) by PCR from pZhen18B vector plasmid (Chang, Z., Chen, Z., Wang, N., Xie, G., Lu, J., Yan, W.,Zhou,J.,Tang,X.,and Deng,X.W..Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility Gene.Proc Natl Acad Sci USA.2016.113,14145-14150.) in amplification.Wherein PG47 promoter expands Primer needed for increasing is as shown in SEQ ID NO:9-10 in sequence table, and wherein forward primer has HindIII restriction enzyme site, reversely draws Object has SacI restriction enzyme site；Primer needed for BT1 transit peptides gene magnification as shown in SEQ ID NO:11-12 in sequence table, Middle forward primer has SacI restriction enzyme site.Design of primers uses 5 software design of Primer Premier, digestion position on primer Point left side 10 bases be skeleton carrier recombinant fragment, when for connecting carrier carry out homologous recombination connection, amplification system and Program is as follows:

Embodiment 3, the OsAA of alpha-amylase gene containing rice recombinant expression carrier pSZYJY-02 building

1, the recombinant expression carrier pSZYJY-01 of the promoter of PG47 containing rice is constructed

By EcoRI the and SacI digestion position of amplified production PG47 promoter insertion pCAMBIA1300 carrier in embodiment 2 Point, building process are as shown in Figure 2.I.e. respectively with the amplified production of HindIII and SacI digestion PG47 promoter and PCAMBIA1300 carrier simultaneously recycles, and then connects recovery product, linked system is as follows:

PG47 promoter PCR product (50ng) 2ul

Digestion pCAMBIA1300 support products (50ng) 2ul

5X In-Fusion HD Enzyme Premix(TaKaRa) 1ul

50 DEG C of connection 20min.

Above-mentioned whole connection products are heat-shock transformed to competent escherichia coli cell, select positive colony sequencing, and general Plasmid contained by correct positive colony is sequenced and is named as pSZYJY-01.

2, the recombinant expression carrier pSZYJY-02 of the gene of OsAA containing rice is constructed

Firstly, amylase gene OsAA (as shown in SEQ ID NO:1 in sequence table) and BT1 is transported peptide gene (such as sequence In list shown in SEQ ID NO:3) spliced by PCR.SEQ ID NO:11 in primer needed for wherein expanding such as sequence table With shown in SEQ ID NO:6, amplification template is using amplification gained in amplification gained promoter OsAA in embodiment 1 and embodiment 2 BT1 (TP) transports peptide gene.Amplification system and program are as follows:

PCR program: 94 DEG C of 3min of initial denaturation；98 DEG C of 30s are denaturalized, anneal 55-65 DEG C of 30s, extends 72 DEG C of 2min, 30 are followed Ring；Extend 72 DEG C of 10min.Amplified production BT1-OsAA splice segment is recovered to the left side 1500bp using 1% agarose gel electrophoresis Right product.

Then, by SacI the and EcoRI restriction enzyme site of above-mentioned BT1-OsAA splice segment insertion pSZYJY-01 carrier, structure It is as shown in Figure 2 to build process.With SacI and EcoRI digestion pSZYJY-01 carrier and BT1-OsAA amplified production and recycle respectively, Then recovery product is connected, linked system is as follows:

BT1-OsAA PCR product (50ng) 2ul

Digestion pSZYJY-01 support products (50ng) 2ul

5X In-Fusion HD Enzyme Premix(TaKaRa) 1ul

50 DEG C of connection 20min.

Take above-mentioned whole connection products are heat-shock transformed to select positive colony sequencing to competent escherichia coli cell, and will Plasmid contained by correct positive colony is sequenced and is named as pSZYJY-02.

The acquisition of embodiment 4, transgenic rice plant

PSZYJY-02 plasmid is electroporated into Agrobacterium competence Agl0 through 2.5KV, and containing kanamycin and 28 DEG C of cultures, are verified with pSZYJY-02 vector-specific primers SEQ ID NO:13-14 through PCR on the YEB culture plate of rifampin And select positive colony.

Above-mentioned Agrobacterium positive colony is cultivated, and flower in rice is infected by Agrobacterium-mediated genetic transformation method 11(HieiY 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).Through co-cultivation, screening, breaks up, takes root Link obtains T₀For transgenic seedlings, DNA is extracted, the transgenic positive that the plasmid containing pSZYJY-02 is obtained after PCR is verified is planted Strain, self-fertility obtain T₁In generation, takes T₁Subsequent analysis is carried out for plant.

Embodiment 5, transgenic plant Phenotypic Observation and pollen potassium iodide staining analysis

1, transgenic paddy rice Phenotypic Observation

As shown in Figures 3 and 4, the growth phenotype and anther form of the transgenic positive plant of the plasmid containing pSZYJY-02 and open country Raw type no significant difference.

2, pollen potassium iodide staining analysis

It prepares potassium iodide and dyes liquor: 2g KI being taken to be dissolved in 5-10ml distilled water, suitable anhydrous second is then added The 1g I of alcohol dissolution₂, after all dissolution after, then plus distilled water be settled to 600ml, store in spare in brown bottle.

Microscopy is carried out to the mature pollen of Transgenic Rice Plants, and pollen grain fertility is analyzed, specific method is such as Under:

A, pollen collection: the anther for taking abundant maturation that will bloom strips glume, takes out anther；

B, it microscopy: takes the potassium iodide dyeing liquor of 100ul to be placed on glass slide, then anther is placed on glass slide, use tweezers Anther is sufficiently smashed to pieces in favor of pollen grain release, covered is placed under low-powered microscope and observes.It is in black and blue color after dyeing Pollen grain be fertile flower powder, in it is filemot for abortive pollen grain.

The pollen grain of transgenic plant shows through potassium iodide staining analysis and pollen grain iodine dye result (as shown in Figure 5): can It educates pollen and abortive pollen meets the segregation ratio of 1:1, i.e., about 50% pollen shows as male sterility, cannot dye black and blue color； About 50% pollen can dye black and blue color；And wild type (or other transgenic plants for not carrying pollen abortion gene) is then complete Male fertile, i.e., 100% pollen can be by black and blue color.As a result illustrate, rice alpha-amylase OsAA can drop in paddy pollen Starch is solved, therefore leads to pollen grain energy supply deficiency due to lacking starch in growth course, so as to cause pollen abortion.By This proves the starch that alpha-amylase gene can degrade in pollen grain, causes Transgenic Rice pollen sterility, effectively prevents to turn base Because transgenic element is broadcast to other wild crop kinds by pollen by crop.

In conclusion the protein alpha-amylase of presently disclosed influence rice male fertility derives from China Water Rice varieties OryzasativaLcv.Nipponbare, and the starch in the pollen grain that can degrade cause Transgenic Rice pollen sterility, and accuracy is high, effectively It prevents genetically modified crops that transgenic element is broadcast to other wild crop kinds by pollen, can be used for that male sterility is kept to plant The homozygous recessive condition of strain；The step of eliminating emasculation during hybrid seeding simultaneously.

Sequence table

<110>Shenzhen Crop Molecular Design Breeding Institute

Shenzhen wide three is agricultural science and technology Co., Ltd

<120>a kind of rice alpha-amylase and its encoding gene and application

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1248

<212> DNA

<213>rice (Oryza sativa)

<400> 1

atgggccaga tggtttcgga tgaaaatttt gaagagcagg ccgctcgtaa tggtggaatt 60

atcaagcacg gaagggaaat cctgttccag gcttttaact gggaatccca taagcacaac 120

tggtggagca atttggagga aaaagttgtt gacctagcac aatcggggtt tacatcagcg 180

tggctgcctc caccaacaca gtcattatct ccagaaggct atctgccgca gaacctgtac 240

tgcctcgact cttgttatgg ttccctccat gacctacaag cattgcttcg gaaaatgaaa 300

gaacacaatg taagagctat ggcagatgtt gttattaacc atcgagttgg gactactcaa 360

ggatcgaatg ggatgtataa tcgttatgat ggtatcccag tatcatggga tgaacatgct 420

gttacatctt gttctggtgg aaagggcaac gaaagtaccg gtgataactt cgatggggtt 480

cccaacatag accatacaca gccatttgta cggaaggata ttattgactg gttgatctgg 540

cttcgagaaa gcattggttt tcaagatttc cgctttgatt tcacgaaagg ttatgcagcg 600

aagtttgtga aagaatacat tgagcaatca aagcctcttt ttgcggtggg ggaatactgg 660

gatagctgtg aatatagccc ccctgattac cgtttgaact acaatcagga taaacacagg 720

cagagaatta tcaattggat ggatagcact ggaggacttt gtgctgcatt tgatttcaca 780

acaaagggta ttcttcagga agctgtcaaa ggtgagttgt ggcgtttgcg tgaccctgaa 840

ggaaagccgc ctggtgtcat gggctggtgg ccttcgagat cagtcacatt tgttgaaaat 900

catgacacag gatccactca gggtcattgg ccattcccat ctgatcatat catggaggga 960

tatgcttata tacttactca tcctggcatc cccacggtgt tctacgatca tttctatggc 1020

aaagatgatt ccttccatgg tggaattgca aaactgatgg agatcaggaa atgccaggac 1080

atacatagcc gctccgcagt caaaatcttg gaggccagct cagatctgta ctctgcaatc 1140

gttgacgata agttgtgcat gaagatcggg gacggctcct ggtgcccgag cggcccagag 1200

tggaagctgg cggcctctgg tgacagatat gctgtttggc acaagtag 1248

<210> 2

<211> 2783

<212> DNA

<213>corn (Zea mays)

<400> 2

agcttgcatg cctgcaggtc gactctagag gatctgcacc ggacactgtc tggtggcata 60

ccagacagtc cggtgtgcca gatcagggca cccttcggtt cctttgctcc tttgcttttg 120

aaccctaact ttgatcgttt attggtttgt gttgaacctt tatgcacctg tggaatatat 180

aatctagaac aaactagtta gtccaatcat ttgtgttggg cattcaacca ccaaaattat 240

ttataggaaa aggttaaacc ttatttccct ttcaatctcc ccctttttgg tgattgatgc 300

caacacaaac caaagaaaat atataagtgc agaattgaac tagtttgcat aaggtaagtg 360

cataggttac ttagaattaa atcaatttat acttttactt gatatgcatg gttgctttct 420

tttattttaa cattttggac cacatttgca ccacttgttt tgttttttgc aaatcttttt 480

ggaaattctt tttcaaagtc ttttgcaaat agtcaaaggt atatgaataa gattgtaaga 540

agcattttca agatttgaaa tttctccccc tgtttcaaat gcttttcctt tgactaaaca 600

aaactccccc tgaataaaat tctcctctta gctttcaaga gggttttaaa tagatatcaa 660

ttggaaatat atttagatgc taattttgaa aatataccaa ttgaaaatca acataccaat 720

ttgaaattaa acataccaat ttaaaaaatt tcaaaaagtg gtggtgcggt ccttttgctt 780

tgggcttaat atttctcccc ctttggcatt aatcgccaaa aacggagact ttgtgagcca 840

tttatacttt ctccccattg gtaaatgaaa tatgagtgaa agattatacc aaatttggac 900

agtgatgcgg agtgacggcg aaggataaac gataccgtta gagtggagtg gaagccttgt 960

cttcgccgaa gactccattt ccctttcaat ctacgactta gcatagaaat acacttgaaa 1020

acacattagt cgtagccacg aaagagatat gatcaaaggt atacaaatga gctatgtgtg 1080

taatgtttca atcaaagttt cgagaatcaa gaatatttag ctcattccta agtttgctaa 1140

aggttttatc atctaatggt ttggtaaaga tatcgactaa ttgttctttg gtgctaacat 1200

aagcaatctc gatatcaccc ctttgttggt gatccctcaa aaagtgatac cgaatgtcta 1260

tgtgcttagt gcggctgtgt tcaacgggat tatccgccat gcagatagca ctctcattgt 1320

cacataggag agggactttg ctcaatttgt agccatagtc cctaaggttt tgcctcatcc 1380

aaagtaattg cacacaacaa tgtcctgcgg caatatactt ggcttcggcg gtagaaagag 1440

ctattgagtt ttgtttcttt gaagtccaag acaccaggga tctccctaga aactgacaag 1500

tccctgatgt gctcttccta tcaattttac accctgccca atcggcatct gaatatccta 1560

ttaaatcaaa ggtggatccc ttggggtacc aaagaccaaa tttaggagtg taaactaaat 1620

atctcatgat tcttttcacg gccctaaggt gaacttcctt aggatcggct tggaatcttg 1680

cacacatgca tatagaaagc atactatctg gtcgagatgc acataaatag agtaaagatc 1740

ctatcatcga ccggtatacc ttttggtcta cggatttacc tcccgtgtcg aggtcgagat 1800

gcccattagt tcccatgggt gtcctgatgg gcttggcatc cttcattcca aacttgttga 1860

gtatgtcttg aatgtacttt gtttggctga tgaaggtgcc atcttggagt tgcttgactt 1920

gaaatcctag aaaatatttc aacttcccca tcatagacat ctcgaatttc ggaatcatga 1980

tcctactaaa ctcttcacaa gtagatttgt tagtagaccc aaatataata tcatcaacat 2040

aaatttggca tacaaacaaa acttttgaaa tggttttagt aaagagagta ggatcggctt 2100

tactgactct gaagccatta gtgataagaa aatctcttag gcattcatac catgctgttg 2160

gggcttgctt gagcccataa agcgcctttg agagtttata aacatggtta gggtactcac 2220

tatcttcaaa gccgagaggt tgctcaacat agacctattc accccatttg atcacttttt 2280

tggtccttca ggatctaata gttatgtata atttagagtc tcttgtttaa tggccagata 2340

tttctaatta atctaagaat ttatgatatt ttttaatttt ttatcatgtc tgatgagaat 2400

taacataaag gctcaattgg gtcctgaatt aataatagag tgaaaattaa tccagaggct 2460

ctattagaac cttcaattag taataccaag atatatataa gatagtagag tatagtttaa 2520

atgttggcat tgttcattct ttcttttgtt atttaattta tgctttccac ggtggttagt 2580

ggttacttct gaagggtcca aataatgcat gaagagtttg aggacaagaa gtctgcccta 2640

aaaatagcga tgcaaaggca tggtgtccaa gccatacata tagcgcacta attttatcag 2700

cagaacaatg gtatttatag gtcctagtgc ccaggcaaca agagacacga ataaagcatc 2760

gatcacgaca cgaattccgg aac 2783

<210> 3

<211> 225

<212> DNA

<213>corn (Zea mays)

<400> 3

atggcggcga caatggcagt gacgacgatg gtgacgagga gcaaggagag ctggtcgtca 60

ttgcaggtcc cggcggtggc attcccttgg aagccacgag gtggcaagac cggcggcctc 120

gagttccctc gccgggcgat gttcgccagc gtcggcctca acgtgtgccc gggcgtcccg 180

gcggggcgcg acccgcggga gcccgatccc aaggtcgtcc gggcg 225

<210> 4

<211> 415

<212> PRT

<213>corn (Zea mays)

<400> 4

Met Gly Gln Met Val Ser Asp Glu Asn Phe Glu Glu Gln Ala Ala Arg

1 5 10 15

Asn Gly Gly Ile Ile Lys His Gly Arg Glu Ile Leu Phe Gln Ala Phe

20 25 30

Asn Trp Glu Ser His Lys His Asn Trp Trp Ser Asn Leu Glu Glu Lys

35 40 45

Val Val Asp Leu Ala Gln Ser Gly Phe Thr Ser Ala Trp Leu Pro Pro

50 55 60

Pro Thr Gln Ser Leu Ser Pro Glu Gly Tyr Leu Pro Gln Asn Leu Tyr

65 70 75 80

Cys Leu Asp Ser Cys Tyr Gly Ser Leu His Asp Leu Gln Ala Leu Leu

85 90 95

Arg Lys Met Lys Glu His Asn Val Arg Ala Met Ala Asp Val Val Ile

100 105 110

Asn His Arg Val Gly Thr Thr Gln Gly Ser Asn Gly Met Tyr Asn Arg

115 120 125

Tyr Asp Gly Ile Pro Val Ser Trp Asp Glu His Ala Val Thr Ser Cys

130 135 140

Ser Gly Gly Lys Gly Asn Glu Ser Thr Gly Asp Asn Phe Asp Gly Val

145 150 155 160

Pro Asn Ile Asp His Thr Gln Pro Phe Val Arg Lys Asp Ile Ile Asp

165 170 175

Trp Leu Ile Trp Leu Arg Glu Ser Ile Gly Phe Gln Asp Phe Arg Phe

180 185 190

Asp Phe Thr Lys Gly Tyr Ala Ala Lys Phe Val Lys Glu Tyr Ile Glu

195 200 205

Gln Ser Lys Pro Leu Phe Ala Val Gly Glu Tyr Trp Asp Ser Cys Glu

210 215 220

Tyr Ser Pro Pro Asp Tyr Arg Leu Asn Tyr Asn Gln Asp Lys His Arg

225 230 235 240

Gln Arg Ile Ile Asn Trp Met Asp Ser Thr Gly Gly Leu Cys Ala Ala

245 250 255

Phe Asp Phe Thr Thr Lys Gly Ile Leu Gln Glu Ala Val Lys Gly Glu

260 265 270

Leu Trp Arg Leu Arg Asp Pro Glu Gly Lys Pro Pro Gly Val Met Gly

275 280 285

Trp Trp Pro Ser Arg Ser Val Thr Phe Val Glu Asn His Asp Thr Gly

290 295 300

Ser Thr Gln Gly His Trp Pro Phe Pro Ser Asp His Ile Met Glu Gly

305 310 315 320

Tyr Ala Tyr Ile Leu Thr His Pro Gly Ile Pro Thr Val Phe Tyr Asp

325 330 335

His Phe Tyr Gly Lys Asp Asp Ser Phe His Gly Gly Ile Ala Lys Leu

340 345 350

Met Glu Ile Arg Lys Cys Gln Asp Ile His Ser Arg Ser Ala Val Lys

355 360 365

Ile Leu Glu Ala Ser Ser Asp Leu Tyr Ser Ala Ile Val Asp Asp Lys

370 375 380

Leu Cys Met Lys Ile Gly Asp Gly Ser Trp Cys Pro Ser Gly Pro Glu

385 390 395 400

Trp Lys Leu Ala Ala Ser Gly Asp Arg Tyr Ala Val Trp His Lys

405 410 415

<210> 5

<211> 35

<212> DNA

<213>artificial synthesized (artificial synthesis)

<400> 5

cgtccgggcg ggccagatgg tttcggatga aaatt 35

<210> 6

<211> 41

<212> DNA

<213>artificial synthesized (artificial synthesis)

<400> 6

ggtactaatg cttaagctac ttgtgccaaa cagcatatct g 41

<210> 7

<211> 18

<212> DNA

<213>artificial synthesized (artificial synthesis)

<400> 7

ggaaagggca acgaaagt 18

<210> 8

<211> 18

<212> DNA

<213>artificial synthesized (artificial synthesis)

<400> 8

cgaagccaga tcaaccag 18

<210> 9

<211> 41

<212> DNA

<213>artificial synthesized (artificial synthesis)

<400> 9

ccggtcacgg ttcgaaagct tgcatgcctg caggtcgact c 41

<210> 10

<211> 41

<212> DNA

<213>artificial synthesized (artificial synthesis)

<400> 10

aatgcttaag ctcgaggtgt cgtgatcgat gctttattcg t 41

<210> 11

<211> 47

<212> DNA

<213>artificial synthesized (artificial synthesis)

<400> 11

atcacgacac gagctcgcca ccatggcggc gacaatggca gtgacga 47

<210> 12

<211> 35

<212> DNA

<213>artificial synthesized (artificial synthesis)

<400> 12

ccatctggcc cgcccggacg accttgggat cgggc 35

<210> 13

<211> 18

<212> DNA

<213>artificial synthesized (artificial synthesis)

<400> 13

cagcgtctcc gacctgat 18

<210> 14

<211> 18

<212> DNA

<213>artificial synthesized (artificial synthesis)

<400> 14

cttctgcggg cgatttgt 18

Claims

1. a kind of application of rice alpha-amylase in pollen fertility control, the rice alpha-amylase include

1) albumen that the amino acid sequence shown in SEQ ID NO:4 forms；Or

2) amino acid sequence of SEQ ID NO:4 by the substitution of one or several amino acid residues and/or missing and/or is added Add as derived from SEQ ID NO:4 and keep the albumen of protein function shown in SEQ ID NO:4.

2. application as described in claim 1, which is characterized in that the nucleotide sequence of the rice alpha amylase are as follows:

1) nucleotide sequence shown in SEQ ID NO:1, or

2) one or several nucleotide are substituted, lack or added in the nucleotide sequence shown in SEQ ID NO:1 and have The nucleotide sequence as derived from 1) of same function；Or

3) hybridize under strict conditions with sequence shown in SEQ ID NO:1 and the nucleotides sequence of identical function protein can be expressed Column, or

4) there is 90% or more homology with nucleotide sequence 1), 2) or 3) and the nucleotide of identical function protein can be expressed Sequence.

3. a kind of application of rice alpha-amylase in preparation pollen abortion transgenic plant, the ammonia of the rice alpha-amylase Base acid sequence are as follows:

1) albumen that the amino acid sequence shown in SEQ ID NO:4 forms；Or

4. the encoding gene of rice alpha-amylase includes

1) nucleotide sequence shown in SEQ ID NO:1, or

5. a kind of expression cassette, which is characterized in that include regulation described in the claim 2 under the regulating and controlling sequence regulation effectively connected The gene of rice male fertility.

6. expression cassette according to claim 4, which is characterized in that regulating and controlling sequence protein transduction containing the amyloplast peptide and pollen Development later stage specific expressing promoter.

7. expression cassette according to claim 4, which is characterized in that the peptide of protein transduction containing amyloplast is BT1(TP) letter Number peptide, nucleotide sequence is as shown in SEQ ID NO:3；The pollen development later period specific expressing promoter opens for PG47 Mover, nucleotide sequence is as shown in SEQ ID NO:2.

8. a kind of any one of gene or claim 4-6 comprising influencing male fertility described in claim 2 expression cassette DNA construct.

9. a kind of recombinant vector comprising DNA construct described in claim 7.

10. a kind of method of starch in degrading plant pollen, which is characterized in that including by being influenced described in expression claim 2 Any one of the gene or claim 4-6 of the male fertility expression cassette is with the starch in degrading plant pollen grain.

11. a kind of method of regulation Plant Pollen Development, which is characterized in that including the use of the flower of degrading plant described in claim 9 The method of starch is in powder to regulate and control pollen formation.

12. a kind of method for inducing plants male sterility, which is characterized in that including the use of regulating and controlling plant flowers described in claim 10 The method of powder development is to generate part male sterile plants.

13. a kind of method for influencing rice male fertility, which is characterized in that including the use of inducing paddy rice described in claim 11 Male sterile method is to generate part male sterile rice.

14. a kind of method for blocking foreign gene diffusion in plant pollen, which is characterized in that by any one of claim 4-6 institute The expression cassette conversion plant callus for stating rice alpha-amylase gene, carries out induction differentiation and life for the callus after conversion Root culture obtains the genetically modified plants of transgenic pollen abortion, and render transgenic plant pollen can not normally pollinate, and then degrade Foreign gene is spread in plant.