CN101974081B - PDF1 gene for controlling plant pollen fertility, and preparation method and application thereof - Google Patents
PDF1 gene for controlling plant pollen fertility, and preparation method and application thereof Download PDFInfo
- Publication number
- CN101974081B CN101974081B CN 201010190495 CN201010190495A CN101974081B CN 101974081 B CN101974081 B CN 101974081B CN 201010190495 CN201010190495 CN 201010190495 CN 201010190495 A CN201010190495 A CN 201010190495A CN 101974081 B CN101974081 B CN 101974081B
- Authority
- CN
- China
- Prior art keywords
- gene
- pdf1
- pro
- plant
- thr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention discloses a protodermal factor 1 (PDF1) gene for controlling plant pollen fertility, and a preparation method and application thereof. A separated protein is obtained from cabbage, cabbage type rape and turnip type rape respectively, wherein sequences of the proteins are nucleotide sequences shown by SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 respectively. The nucleotide sequences are coding sequences (CDS) which are cloned by taking cDNA as a template. A separated polypeptide is obtained from the cabbage, the cabbage type rape and the turnip type rape respectively, wherein the sequences of the separated polypeptides are amino acid sequences shown by SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 respectively. The invention discloses the application of the PDF1 gene for controlling the plant pollen fertility, and relates to isolation and cloning, expression analysis and application of the PDF1 homologous gene for controlling microspore development and anther fertility. ThePDF1 is a programmed cell death (PCD) suppression factor; and the gene has obvious expression advantage in rape anther. By weakening the expression of the PDF1 gene through gene engineering technology, the microspore can be aborted, the fertility of the pollen can be controlled, a male sterile material and a restorer material can be created, and the gene can be used for development research of androgamete and improvement on quality of farm crops.
Description
Technical field
The present invention relates to plant genetic engineering and biological technical field.Be specifically related to a kind of PDF1 gene of controlling plant pollen fertility, also relate to simultaneously a kind of preparation method who controls the PDF1 gene of plant pollen fertility, also relate to a kind of purposes of controlling the PDF1 gene of plant pollen fertility.
Background technology
PDF1 (protodermal factor 1) full length gene 1.421kp is present on the Arabidopis thaliana second karyomit(e), has about 57 to be proline(Pro) in 306 amino acid of PDF1 genes encoding, is the albumen of a proline rich.Common this albumen can form a kind of special quaternary structure, with other protein interactions, thereby plays regulating and controlling effect.Have report to point out that the expression of the proline rich albumen (proline-richproteins, PRPs) of some cottons has etap property and tissue specificity, the expression of these genes also is subject to the impact of pathogenic bacterial infection and external environment factor.It is relevant with the structure of the primary wall of specific cells that their function is considered to, also may in development of plants and defence, work by (yellow Geng Qing cotton GhPRP3, GhPRP4, GhPRP5, GhPRP6 and GhPRP11 gene clone and expression analysis Central China Normal University journal (natural science edition) 2005,73).Simultaneously, also someone finds also the encode albumen of proline rich of paddy rice flower development genes involved RA68 and OsUBP1, they are specifically expressing in meristematic tissue, and working in the flower development process, (Wu's filial piety Chinese scholartree paddy rice flower development genes involved RA68 and OsUBP1 separate and functional analysis Postgraduate School, Chinese Academy of Sciences (Institute of Zoology) 2004 paper numbers: 28887086).But the function about the PDF1 gene does not also have relevant report at present.
Programmed cell death (programmed cell death, PCD) is phalangeal cell orderly death initiatively, is a kind of biological phenomena, the growth of regulating plant in higher plant, but understand very few to the mechanism of this regulation and control.The type of plant PCD is very complicated, and different PCD has different periods and unique regulatory factor and dead form.In the middle of plant, PCD is plant life from cradle to grave an important development program (BeersEP.Programmed cell death during plant growth and development.Cell Death andDifferentiation in the cycle, Vol 4,649-661,1997).Identical with the PCD that occurs in the zooblast, the PCD of plant also often occurs in and runs into pathogen (anaphylaxis) and (Pennell RI when coercing, Lamb C.Programmedcell death in plants.The Plant Cell 9,1157-1168,1997.).The visible death of nearly all vegetable cell has the generation of PCD, so PCD is easy to obscure mutually with other dead form.Evaluation to cell death way, and what is looked for is the determinative of necrocytosis, to clear and definite PCD is very important (Lockshin RA, Zakeri Z..Apoptosis, autophagy, and more.TheInternational Journal of Biochemistry and Cell Biology 36,2405-2419,2004).Present research has obtained the evidence that programmed cell death occurs in some plants.For example, although in Arabidopis thaliana, do not find Capases (a kind of important PCD associated protein of animal kind), but isolated the Caspase-like molecule, shearing (Warthmann N has occured in these molecules when PCD occurs plant, Chen H, Ossowski S, Weigel D, Herv é P..Highly specific gene silencing by artificial miRNAsin rice.PLoS ONE.3:e1829., 2008); In addition, also found some genes relevant with programmed death such as shearing (Danon A of DNA in the plant, Delorme V, Mailhac N, Gallois P.Plantprogrammed cell death:a common way to die.Plant Physiology and Biochemistry 38,647-655.2000).Therefore, Translation of Programmed Cell Death in Plants and animal have similarity, but also exist difference.
The PCD of plant is divided into three types at least: autophagy, AL-PCD (apoptotic-like PCD) and downright bad PCD.In some cases, also can cause the indirectly necrocytosis of non-physiologic when PCD can cause direct necrocytosis: spontaneous death occurs in some cells when PCD, and other cells are to cause indirect death (David L. Vaux owing to the reasons such as nutritive substance shortage that cause after the PCD generation, StanleyJ.Korsmeyer.Cell death in development.Cell, Vol.96,245-254,1999).The degraded of DNA and the startup of proteolytic enzyme are the main phenomenons that PCD occur for most of higher plants, and the generation of apoptotic body (DNALaddering) is not can both see in a organized way in institute.Caspase-like mechanism also has report in some document, but is not enough to explain apoptotic all phenomenons.The generation of two kinds of the Explanation plant PCD is arranged at present, and a kind of is the mechanism of bursting apart of vacuole skin, and another kind is the generation mechanism of active oxygen.Front a kind of mechanism gone back solve seldom, but the generation of active oxygen (AOS) is very general phenomenon.In two kinds of different mechanism, the degraded of organizing non-compatibility and tapetum is to be commonly referred to be a reason (Hilary J.Rogers.Cell death and organ development in plants.Current topics indevelopmental biology.Vol.71,2005) that causes cytoplasmic male sterility.2007 to a kind of wall shape weeds Brachiaria research in report, during typical PCD phenomenon offspring's when the species hybridization of Brachiaria sporule occurs, the pollen mother cell of abortion a large amount of after births occurs and bubbles, tenuigenin is gathered in the cell periphery, nucleus chromatin degraded occurs and finally causes the degraded of whole nuclear, tenuigenin shrinks, the phenomenons such as necrocytosis (V.A.Fuzinatto, M.S.Pagliarini and C.B.Valle.Evidence of programmed cell death duringmicrosporogenesis in an interspecific Brachiaria hybrid.Genetics and MolecularResearch 6 (2): 308-315,2007).Although the male sterile that at present research causes about PCD has a lot of reports, too early generation such as the mutation induction PCD of MS1 gene, the vacuolization that causes tapetum, the degraded of sporule behind tetrad, final pollen (the Zoe A.Wilson that does not have fecundity that produces, Shaun M.Morroll, Janet Dawsonl Ranjan Swarup and Paddy J.Tighe.The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology tothe PHDfinger family of transcription factorsThe plant journal.28 (1), 27 ± 39,2001).MMD1 (male meiocyte death1) expresses specifically at the sporule Meiosis, and its mutant causes the death of sporule.The meiotic cell of mmd1 mutant shows obvious apoptotic phenomenon, unusual such as: chromosome behavior, tenuigenin shrinks, fragmentation appears in karyomit(e), then the quantity that is accompanied by cell kinase increases, death (the Xiaohui Yang that finally causes cell, Christopher A.Makaroff, and Hong Ma. (2003) .The ArabidopsisMALE MEIOCYTE DEATH1Gene EncodesaPHD-Finger Protein That Is Required for Male Meiosis.The Plant Cell, Vol.15,1281-1295,2003).Arabidopis thaliana fbr12 (fumonisin B1-resistant12) mutant shows the defective of plant strain growth and growth, cause agennetic gynoecium and stamen cell, improper division and growth appear in sporule, and the silence of this gene causes the unusual generation of PCD.The differentiation of FBR12 gene pairs plant cell, growth and mortality plays important regulating and controlling effect (Haizhong Feng, Qingguo Chen, Jian Feng, JianZhang, Xiaohui Yang, and Jianru Zuo..Functional Characterization of the ArabidopsisEukaryotic Translation Initiation Factor 5A-2 That Plays a Crucial Role in PlantGrowth and Development by Regulating Cell Division, Cell Growth, and Cell Death.Plant Physiology, Vol.144, pp.1531-1545., 2007).In paddy rice, Yoshikazu Tanaka etc. is cloned into the homologous gene of the cDNA of human DAD1.The homogenic mutant of in paddy rice this can be caught the dead cell that apoptosis causes, show that this gene is a supressor (Yoshikazu Tanaka of apoptosis, Tomoko Makishima, Michiko Sasabe, Yuki Ichinose, TomonoriShiraishi, Takeharu Nishimoto and Tetsuji Yamada.dad-1, A Putative Programmed CellDeath Suppressor Gene in Rice.Molecular biology.Vol.38, No.3 379-383,1997).But PCD is occurred in the male sterile mechanism that causes in the Microsporogenesis process also to be understood seldom.
There are some researches prove that yeast Ost2 (for oligosaccharyltransferase-2) gene can suppress the generation of the PCD of hamster cell, simultaneously also has identical function (Sugimoto at Arabidopis thaliana with homologous sequence in the paddy rice, A., Hozak, R.R., Nakashima, T., Nishimoto, T., and Rothman, J.H. Dad-1, anendogenous programmed cell death suppressor in Caenorhabditis elegans andvertebrates.EMBO is J.14,4434-4441,1995), therefore it is believed that the homologous gene of Ost2 has participated in the PCD signal pathway too, and this approach is relatively conservative.But in the other plant species, also do not find to participate in so far other genes of PCD approach.This may be lower because participate in the gene transcripts expression level of PCD, or these participate in very poor (the Roger I.Pennell and Chris Lamb.Programmed Cell death in plants.The plant cell of conservative property of the gene of PCD, Vol.9,1157-1168.1997).PCD works in the growing of control fertility and plant.
In recent years, artificial microRNA (artificial miRNA, the amiRNA) technology that develops based on microRNA (microRNA, miRNA) action principle is a kind of novel RNA perturbation technique.It utilizes the stem sequence (being generally 21-22nt) of the particular sequence displacement Mirnas of plant of target gene, makes up the expression that the amiRNA expression vector comes the jamming target gene.This technology not only can be disturbed reticent all target sequences or gene simultaneously as traditional RNAi technology, also can disturb accurately simultaneously the single special objective gene in gene family or the allelotrope, specificity and high efficiency with height, overcome gene metabolism compensating action, and (effector molecule is siRNA can to overcome traditional RNAi technology, small interfering RNA, siRNA) problem (Ossowski S such as " missing the target " of in the experimental implementation process, occurring, Schwab R, Weigel D.Genesilencing in plants using artificial microRNAs and other small RNAs.Plant J.53:674-690., 2008), effectively raise the efficient that gene disturbs.
Rape is extensively planted in the Yangtze valley as the main oil crops of China, and national economy is had material impact.Rape heterosis is obvious, and utilizing the combination of nuclear male sterility hybridization advantage is the important channel of heterosis utilization.Found at present polytype nuclear male sterility material both at home and abroad, and obtained impressive progress in theoretical and application facet, and the discovery of new kind of male sterility source and research are the bases of Study on Heterosis always, and create more natural and artificial sterile material with this, by breed and production is used.
Therefore, the present invention has developed a kind of gene relevant with pollen fertility with PCD (programmed cell death).Express by this gene of fluorescence quantitative PCR detection a large amount in the flower pesticide of fertile plant.The expression level that suppresses this gene by using microRNA interference means.The experimental result confirmation, the PDF1 gene is a new PCD supressor, its correction has guaranteed proper splitting, differentiation and the growth of cell; And the expression that suppresses it can cause the PCD of cell abnormal, and nucleus is degraded in advance, and cell development is obstructed.In male sex-cell, just show as Microspore Abortion.Applicant's result is indicating this gene and homologous sequence thereof at the control plant pollen fertility, creates in the male sterile transgenic material to have suitable application prospect.
Summary of the invention
The objective of the invention is to be to provide a kind of PDF1 gene of controlling plant pollen fertility, the PCD process in this gene pairs plant and the cell development process is inhibited.By suppressing the expression of this gene, impel sporule in growth PCD to occur in advance in early days, cause pollen abortion, reduce the fertility of plant pollen, thereby obtain male sterile plants, be applied to cross-breeding.
Another object of the present invention is to be to provide a kind of preparation method who controls the PDF1 gene (rape belongs to PDF1 homologous gene in three species wild cabbages, swede type rape and the turnip type rapes) of plant pollen fertility.Carry out the gene fragment order information that wild cabbage, swede type rape and turnip type rape gene order-checking result provide according to this laboratory, est database by BLAST comparison Arabidopis thaliana, wild cabbage, swede type rape and turnip type rape, after obtaining the electronic cloning of wild cabbage, swede type rape and turnip type rape full length sequence, use the sequence that simple PCR method namely can obtain this gene.Wild cabbage PDF1 gene has nucleotide sequence shown in SEQ.ID.No.1 and the aminoacid sequence shown in the SEQ.ID.No.4; Swede type rape PDF1 gene has nucleotide sequence shown in SEQ.ID.No.2 and the aminoacid sequence shown in the SEQ.ID.No.5; Turnip type rape PDF1 gene has nucleotide sequence shown in SEQ.ID.No.3 and the aminoacid sequence shown in the SEQ.ID.No.6.And the albumen with identical function of the albumen of above-mentioned dna fragmentation coding or process transformation modification.
A further object of the invention is to be to provide a kind of application of PDF1 gene in the control pollen fertility of controlling plant pollen fertility.By the expression of reticent this gene, the contriver can manually create male sterile material, uses in cross-breeding is produced.The PDF1 gene is a kind of PCD (programmed cell death) the supressor gene that can control Plant Pollen Development, flower pesticide fertility.PDF1 controls plant anther growth, pollen fertility by the PCD process of regulating cell.Adopt genetically modified mode, with the artificial microRNA interference carrier arabidopsis thaliana transformation of this gene of making up, the transgenic arabidopsis that can obtain to have the male sterile phenotype.
It is to be to provide a kind of PDF1 gene of Translation of Programmed Cell Death in Plants that suppresses as the application of PCD (programmed cell death) supressor that the present invention has a purpose again.Adopt genetically modified mode, the artificial microRNA interference carrier arabidopsis thaliana transformation with this gene of making up can obtain to have the transgenic arabidopsis of downgrading phenotype.
In order to finish above-mentioned purpose, the present invention adopts following technical scheme:
In order to obtain the present invention, the contriver has carried out deeply and comprehensively research the genes involved in the pollen development process, found that a new gene, high efficient expression in the flower pesticide of fertile plant, and express hardly in sterile plant, so the regulation and control of the fertility of this gene and plant pollen are relevant.Transgenic experiments has also confirmed this point: the expression that suppresses this gene can cause plant anther heteroplasia, causes male sterile.Simultaneously, when the Molecular Biology Mechanism of this Gene Handling flower pesticide fertility of research, the applicant finds that further this gene is a PCD supressor.Suppress the expression of this gene, make sporule that PCD occur in advance, cause Anther Abortion, the phenotype of Microspore Abortion appears in transfer-gen plant.
According to an aspect of the present invention, above-mentioned purpose can realize by providing the efficient expressing gene of plant anther and rape to belong to homologous gene, described gene contain SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 nucleotide sequence or with they nucleotide sequences of homology in fact.
According to another aspect of the present invention, above-mentioned purpose can realize by providing the efficient expressing protein of plant anther and rape to belong to homologous protein, described albumen contain SEQ ID No.4, SEQ ID No.5, SEQ IDNo.6 aminoacid sequence or with they aminoacid sequences of homology in fact.
According to another aspect of the present invention, above-mentioned purpose is by providing the artificial microRNA that contains PDF1 gene target (AATUTUTATATUUTGTUGAG) sequence to disturb recombinant vectors (micro-PDF1) to realize inhibition to PDF1 gene expression amount in the plant materials.
According to another aspect of the present invention, above-mentioned purpose can be by providing microorganism (the agrobacterium tumefaciens EHA105 that transforms with described recombinant vectors (micro-PDF1), available from the precious biotinylated biomolecule in Dalian Products Co., Ltd, below identical) realize the conversion to Arabidopis thaliana and other plant, thereby suppress the expression amount of PDF1 gene in Arabidopis thaliana and the other plant.
According to another aspect of the present invention, above-mentioned purpose can realize regulation and control to the flower pesticide fertility obtaining the male sterile transfer-gen plant by the transgenic plant that transform with described microorganism (the agrobacterium tumefaciens EHA105 that includes the micro-PDF1 recombinant vectors) are provided.
Wild cabbage PDF1 (BoPDF1), swede type rape PDF1 (BnPDF1) and three homogenic researchs of turnip type rape PDF1 (BrPDF1), a kind of PDF1 gene of controlling plant pollen fertility the steps include:
1, wild cabbage, swede type rape and the homogenic clone of turnip type rape PDF1:
Respectively wild cabbage, swede type rape and turnip type rape young leaflet tablet sample are ground to powdery in liquid nitrogen, the extraction of total RNA is carried out in the requirement that utilizes Trirol to extract test kit (available from Invitrogen company, below identical).The total RNA that extracts is dissolved in the distilled water without RNase.DNase I (available from Promega company, below identical) remove may be residual DNA.Detect respectively RNA at 260 nanometers and 280 nanometer absorbance values with Ultraviolet Detector (DU 650BECKMAN, USA), identify purity and the concentration of RNA in conjunction with 1% (mass volume ratio) agarose gel electrophoresis.Take the RNA that obtains as template, utilize the reverse transcription test kit (the M-MLV reversed transcriptive enzyme, available from Promega company, below identical) requirement carry out reverse transcription, save backup in-20 ℃ after the eDNA packing that obtains.
According to Arabidopis thaliana PDF1 gene order (gi:4929130) among the GenBank, the NCBI website with BLASTN (
Http:// www.ncbi.nlm.nih.gov/BLAST) compare with wild cabbage, swede type rape and the turnip type rape gene fragment order of this laboratory order-checking acquisition, obtain respectively CDS (the Coding sequence with the wild cabbage of Arabidopis thaliana PDF1 gene order 5 ' end homology, swede type rape, turnip type rape, encoding sequence, below identical) sequence and and the CDS sequence of wild cabbage, swede type rape and the turnip type rape of Arabidopis thaliana PDF1 gene order 3 ' end homology.Then according to CDS sequence and Arabidopis thaliana PDF1 gene start codon and the terminator codon homology zone design primer (primer sequence sees Table) of the wild cabbage that obtains, swede type rape, turnip type rape PDF1 gene, thereby guarantee that amplified production is full length sequence.Take according to each wild cabbage that obtains noted earlier, swede type rape, turnip type rape leaf cDNA as masterplate, carry out pcr amplification.Reclaim the also PCR product of purifying amplification, be connected on the pMD18-T carrier (available from Promega company, below identical), transform intestinal bacteria (J. Pehanorm Brooker .D.W. Russell work with freeze-thaw method, Huang Peitang etc. translate molecular cloning test guide (third edition) Science Press, 96-99), coating the LB solid that contains penbritin 50 μ g/mL (fills a prescription as follows: take by weighing respectively 10 gram Tryptoness, 5 gram yeast extracts and 10 gram sodium-chlor, 8 gram agar are dissolved in the distilled water successively, and constant volume is in 1000 milliliters.Be sub-packed in 500 milliliters of triangular flasks, 121 ℃, autoclave sterilization is 15 minutes under 6.859 * 104Pa.4 ℃ of refrigerations are for subsequent use) on the flat board, 37 ℃ of overnight incubation are respectively selected three of hickies, and be bacterium colony PCR and detect: the primer sequence is: M13F 5 '-AGCGGATAACAATTTCACACAGGA-3; M13R 5 '-GTAAAACGACGGCCAGT-3, below identical), reaction system is: 10 * Taq enzyme reaction buffer solution 2ul, 25mMMgCL
21.2ul, 2mM dNTP 1.5ul, 10uM primer 0.2ul, 0.3 Taq of unit enzyme, add sterilized water to 20 μ l.Response procedures is: 94 ℃ of sex change 5min, and 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 2min 32 circulations, 72 ℃ are extended 5min (following identical).Take M13F/M13R as primer (front is stated), the pMD18T carrier that connected is carried out sequencing, analytical results, obtain rape and belonged to (wild cabbage, swede type rape and turnip type rape) PDF1 full length gene sequence that three species separate, that is: a kind of protein of separation, its sequence is the nucleotide sequence shown in the SEQ ID No.1; A kind of isolated polypeptide, its sequence are the aminoacid sequence shown in the SEQ ID No.4; A kind of protein of separation, its sequence are the nucleotide sequence shown in the SEQ ID No.2; A kind of isolated polypeptide, its sequence are the aminoacid sequence shown in the SEQ ID No.5; A kind of protein of separation, its sequence are the nucleotide sequence shown in the SEQ IDNo.3; A kind of isolated polypeptide, its sequence are the aminoacid sequence shown in the SEQ ID No.6.The artificial microRNA target sequence of PDF1 gene (AATUTUTATATUUTGTUGAG) according to above-mentioned aminoacid sequence (SEQ ID No.1, SEQ ID No.2, the SEQ IDNo.3) design that obtains, by making up micro-PDF1 expression of plants recombinant vectors, arabidopsis thaliana transformation, the applicant obtains the transgenic arabidopsis that 63 strains have the male sterile phenotype, these transfer-gen plants also have the phenotype of dwarfing simultaneously, and the height of transfer-gen plant only has about the 50-80% of contemporaneously wild-type height.Illustrate that the PDF1 gene has the function of control plant anther fertility and plant height.
The sequence of wild cabbage, swede type rape and the turnip type rape that will obtain through checking order is carried out Blast with the upper Arabidopis thaliana PDF1 gene order of NCBI and is compared.Confirm to have obtained through order-checking the total length CDS sequence of wild cabbage PDF1 gene, length is 1209bp, called after SEQ ID No.1; Obtained the total length CDS sequence of swede type rape PDF1 gene, length is 1299bp, called after SEQID No.2; Obtained the total length CDS sequence of turnip type rape PDF1 gene, length is 1335bp, called after SEQ ID No.3.Fig. 1 shows is electrophorogram by the pcr amplification after product, and the obtained product electrophoretic band all is about 1.0kb, to meet expection.
Utilize the open reading frame finder (ORF founder) of NCBI to determine the open reading frame of PDF1 gene, derive the amino acid of protein sequence coding, obtain wild cabbage, swede type rape and turnip type rape PDF1 protein sequence, respectively called after SEQ ID No.4, SEQ ID No.5, SEQ ID No.6.
The sharp online gene structure indicating system of difference (Gene Structure Display Server, GSDS,
Http:// gsds.cbi.puk.edu.cn/Chinese.php) gene structure of Arabidopis thaliana, wild cabbage, swede type rape PDF1 is compared; Utilize the online software of TCoffee (http://www.tcoffee.org/) that the tissue positioned sequence of Arabidopis thaliana, wild cabbage, swede type rape, turnip type rape PDF1 gene is compared; Use SOPMA to the secondary structure of above-mentioned four species PDF1 gene coded proteins predict, online software Pro tScale (
Http:// www.expasy.ch/tools/protscale.html) to each protein sequence carry out the hydrophilic and hydrophobic analysis, the online software of TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) is analyzed the membrane spaning domain of four PDF1 albumen.According to above-mentioned analysis and comparison, the sequence of the wild cabbage that the proved invention obtains, swede type rape, turnip type rape is the homologous gene of Arabidopis thaliana PDF1 really.With the PDF1 homologous gene of the wild cabbage, swede type rape and the turnip type rape that obtain respectively called after: BoPDF1, BnPDF1 and BrPDF1.
2, the expression pattern of rape BnPDF1 gene:
The used rape of the present invention is that dominant genic male sterile heterozygosis sterile line J03AB (original name 803AB) (Msmsrfrf*msmsrfrf) for swede type rape (Brassica napus L.).System backcrosses through for many years and hands over and breed with sister, and wherein the fertile plant code name is J03B, and sterile strain code name is J03A (following identical).The two can be considered a pair of near isogenic line, only has sterile Site discrepancy.(Hu Shengwu, Yu Chengyu, Zhao Huixian, the road is bright, the seed selection of hybrid rape dominant nuclear sterile material Shaan-GMS homozygous two-type line 803AB.Northwest agricultural journal, 2002, ii (4): 25-2).Be seeded in the land for growing field crops for the examination material, normal field management.
Get root, stem, leaf, bud, angle fruit from the identical fertile plant of same grown in field condition, anatomical isolation gynoecium and flower pesticide from bud, every kind of material is got three repetitions at least, each repeats at least one strain, with the masking foil parcel, be positioned over rapidly in the liquid nitrogen-80 ℃ of preservations after the sampling.The extraction of RNA is carried out in the requirement that utilizes Trirol to extract test kit (front is stated).Get bud from rape fertile plant and sterile plant respectively in the land for growing field crops, after taking back the laboratory, flower pesticide and gynoecium in ice chest difference picking fertile plant and sterile plant bud, every kind of material is got three repetitions at least, each repeats at least one strain, with the masking foil parcel, be positioned over rapidly in the liquid nitrogen-80 ℃ of preservations after the sampling.The extraction of RNA is carried out in the requirement that utilizes Trirol to extract test kit (front is stated).
Quantitative real time PCR Instrument is RT
TM-Cycler (Bo Ao) adopts rape Actin as internal standard gene (accession number AF111812), and the Actin gene primer is 5 '-CTGGAATTGCTGACCGTATGAG-3 ' and 5 '-ATCTGTTGGAAAGTGCTGAGGG-3 '.The BnPDF1 gene primer is 5 '-AACAAGGCTAATGAGGGAGTCTG-3 ' and 5 '-TCCAAAGCGTAGTCAGGATAGG-3 '.Quantitative fluorescent PCR reacts every group of experiment and all finishes three biology repetitions, and each biology repeats to do at least three technology and repeats.Detect respectively the expression (following identical) of BnPABP5 in Oil Rape Tissue (root, stem, leaf, flower, angle fruit, flower pesticide, gynoecium).
With the relative expression quantity that compares Ct method (Δ Δ Ct) calculating gene.By 2
-Δ Δ CtThe relative expression quantity of estimate goal gene and systematic error (Kenneth J Livak.Thomas D Schmittgen.2001, Analysis of Relative Gene Expression Data Using Real-Time QuantitativePCR and the 2
-Δ Δ CT Method.METHODS 25,402-408.).When calculating relative expression quantity, take the sample of angle fruit as reference, be about to its value and convert 1 (standard value) to, other sample again with its relatively, obtain relative expression's value.The result is shown in figure two, and PDF1 is a gene that a large amount is expressed in floral organ and stamen.
3, the structure of the artificial microRNA interference carrier of PDF1 gene (microRNA-PDF1) and agrobacterium tumefaciens bacterial strain EHA105 transform (being purchased from still bio tech ltd of Shanghai, Shanghai):
Does carrier make up (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi with reference to listed method in " 2010 plans of Arabidopis thaliana "? page=Home; Project=stdwmd).The target sequence of choosing is: AATUTUTATATUUTGTUGAG, corresponding Oligo DNA is: TTAGAGACTATAGGACAGCTC.The microRNA-PDF1 carrier structure that makes up as shown in Figure 3.
Then utilize freeze-thaw method (front is stated) to change microRNA-PDF1 over to agrobacterium tumefaciens EHA105 (available from the precious biotinylated biomolecule in Dalian Products Co., Ltd, below identical), coating the LB solid that contains 50 μ g/mL penbritins and Rifampin (50 μ g/mL) (fills a prescription as follows: take by weighing respectively 10 gram Tryptoness, 5 gram yeast extracts and 10 gram sodium-chlor, 8 gram agar are dissolved in the distilled water successively, and constant volume is in 1000 milliliters.Be sub-packed in 500 milliliters of triangular flasks, 121 ℃, autoclave sterilization is 15 minutes under 6.859 * 104Pa.4 ℃ of refrigerations are for subsequent use) on the flat board, 37 ℃ of overnight incubation are respectively selected three of hickies, and be bacterium colony PCR and detect (front is stated).And the carrier that obtains checked order to verify accuracy.
The artificial microRNA of research PDF1 gene function used in the present invention disturbs means and recombinant plant expression vector, the target nucleotide sequence that contains described PDF1 gene, by specific target site is disturbed, thereby weaken the expression level of PDF1 gene at transcriptional level.This carrier size is fit to, in plant easily with transform, institute with weedicide marker gene Bar expression intensity high, easily detection.Can obtain the Arabidopis thaliana transfer-gen plant with male sterile phenotype of Anther Abortion by this carrier arabidopsis thaliana transformation.
4, the genetic transformation of the artificial microRNA interference carrier of microRNA-PDF1:
Method in the reference literature is carried out transformation of Arabidopsis thaliana (Zhang X.R.et al.Agrobacterium-mediated transformation of Arabidopsis thaliana usingthe floral dip method.Nature, 2006,1:1-6).Preparation contains agrobacterium tumefaciens EHA105 (front is stated) the bacterium liquid that builds carrier, changes in the LB liquid nutrient medium that contains kantlex 50 μ g/ml, Rifampin 50 μ g/ml 28 ℃ of incubated overnight over to the day before yesterday in conversion.Second day, the light absorption value with ultraviolet spectrophotometer (SPEKOL 1300) detects under the 276nm nano wave length takes out when the light absorption value of bacterium liquid reaches between 1.6-2.0.Room temperature (20-25 ℃, below identical) with the centrifugal 10min of 4000g, is abandoned supernatant, and precipitation is suspended in isopyknic 5% sucrose (mass volume ratio).The sucrose solution of muddiness is poured in the large culture dish, added the Silwet 1-77 that final concentration is 0.02% (volume ratio) (available from the Five continents, Beijing unit industry science and trade center, below identical) before transforming.The whole inflorescence of Arabidopis thaliana to be transformed being immersed in the sucrose gently, silent number 15s take out plant behind the mixing.Plant after the conversion is wrapped with a black plastic bag, is placed on the growth case and cultivates.Second day is opened plastics bag, and cultivate in the place that is placed on light intensity.Every the conversion that tries again in a week.Cultivate and gathered in the crops seed in about one month, seed under incubator or daylight dry 3-5 days.The T0 that transforms results is broadcast in the vermiculite that is mixed with the PNS nutritive medium for seed, (PNS nutritive medium composition is: every liter contains 5ml 1M KNO3,2ml 1M Ca (NO3) 24H2O, 2ml 1M MgSO47H2O, 2.5ml 20mM Fe.EDTA, 2.5ml 1M phosphoric acid buffer (pH5.5), 1ml MS trace element); 4 ℃ of one weeks of vernalization, between 21-22 ℃ growth in about the self-sow fortnight, wait the sprinkling of carrying out in early days weedicide Glufosinate ammonium (30mg/L) of the four leaf phases that arrived for the first time, carry out sprinkling second time after the week.After twice sprinkling the green seedling of survival carried out the PCR positive detection, obtain Arabidopis thaliana transgenic positive seedling.
5.BnPDF1 functional analyses of genes:
5.1, the screening of the artificial microRNA interference carrier of microRNA-PDF1 transformation seedlings and PCR identify:
According to the weedicide Glufosinate ammonium concentration gradient screening experiment that carry out in this laboratory, determine weedicide (Glufosinate ammonium, available from Bayer AG, below identical) screening concentration is 30mg/L.T1 sprays the 30mg/L weedicide for plant in seedling stage (after planting 10 days), obtains surviving plant.Adopt CTAB method (M.G.Murray and W.F.Thompson, Rapid isolation of high molecularweight plant DNA, _ Nucleic Acids Research, 1980, Vol.8, No.19 4321-4326, below identical) extract Arabidopis thaliana and the wild-type Arabidopsis leaf DNA of survival after the screening.According to (micro-PDF1) 35S on the expression vector and NOS sequence, design Mi primer carries out the PCR detection, and (sequence is F-5 '-CATGGATCAAAGATTCAAATAGAGG-3 ', R-5 '-CCCGATCTAGTAACATAGATGACACCG-3 '), the expection amplification length is 1058bp.To number and mark through the positive plant that PCR detects.
5.2, the artificial microRNA interference carrier of microRNA-PDF1 transgenic arabidopsis Phenotypic Observation:
All observed the phenotype of downgrading in for plant at numerous transgenic arabidopsis T1, so we measure to the plant height through a plurality of transgenic lines in T2 generation of obtaining behind the herbicide screening.Respectively their plant height was measured in after planting 20 days and 40 days for strain at T2, each strain is measured 20 strains, and 3 repetitions (cultivating respectively program request in the boxes at 3) have been set, sow simultaneously the wild-type Arabidopis thaliana and measure plant height at same time point (after planting 20 days and 40 days), also measure 20 strains, and set 3 repetitions.
In flowering period, choose each branch top and leaked white but unopened flower, push bud aside.At magnifying glass (3 times) and lower flower pesticide and the pollen observed of stereoscopic microscope (OLYMPUS SZ61TRC).Isolate flower pesticide with dissecting needle, with reference to Alexandria staining (Alexander, M.P.Differential staining of aborted and non-aborted pollen.Stain Technol, 44:117-122., 1969, below identical), the flower pesticide of 40 ℃ of heating in water bath Alexandria staining fluid immersions spends the night (10-15 hour), with pressed disc method Observation of Microspore under microscope (OLYMPUS IX71).Judge the abortive degree of sporule by the ratio of observing and adding up normal sporule (dying orange) and abortion sporule (dying green).The thaliana flower organ frozen section technique (Chen Dan that Arabidopis thaliana flower pesticide frozen section and brazilwood extract dyeing method are set up with reference to Chen Dan, Zhao Jie, Zhao Jie. be suitable for the frozen section technique of thaliana flower organ. Wuhan phytology research, 23 (3): 85-290,2005).
5.3 the DAPI of frozen section dyeing:
Through the Arabidopis thaliana flower pesticide frozen section that aforesaid method (step 5.2) obtains, adopt the method for DAPI dyeing that nuclear developmental condition in the flower pesticide sporule is observed, concrete grammar is as follows:
DAPI (4,6-diamino-2-phenylindone, below identical) be whether to grow normal very effective dyestuff (Park SK for the Study of Pollens nucleus, Howde n R, Twe ll D.The Arabidopsi sthaliana gametophytic mutation gemini pol lenl disrupts microsporepolarity, division asymmetry and pollen cell f ate.Development 125,3789-3799.1998), it is combined with nucleic acid specifically, sends blue-fluorescence under UV-light.1 trophonucleus and 2 spermatid nucleuses (sperm) are arranged, totally 3 nucleus in the wild-type mature pollen.Wherein trophonucleus is because being in the disperse of dyeing of transcriptional activity state.Germ nucleus is not transcribed or heterochromatin state that transcriptional activity is very low dyes fine and close bright because being in.The working concentration of DAPI is 2 micrograms/microlitre, is dissolved in the sucrose solution of 7% (mass volume ratio), and lucifuge-20 degree is preserved.The present invention uses is the DAPI dye liquor (article No. C1006 purchases in market) that green skies company configures.The at room temperature dyeing 30 minutes in the dark of viable cell and fresh material, and sliced materials at room temperature in the dark dyeing got final product in 5-10 minute.Ultraviolet band (544nm) at fluorescent microscope (BK-FLC) during observation excites, and produces blue-fluorescence.The dyeing observations as shown in Figure 7, the microspore development of transgenic arabidopsis begins to degrade to the four-strand stage nucleus, unusual PCD phenomenon occurred.
5.4DNA ladder confirms the generation of microRNA-PDF1 interference carrier transgenic line sporule PCD (programmed cell death):
1. collect pollen granule: configuration 0.5M NaCl+7% (mass volume ratio) sucrose solution, as the solvent that separates pollen.Get the EP pipe of 1.5 milliliters of semicanals, 40-50 flower maybe will opening in full bloom put into solvent, (20-25 degree, below identical) vibrated 1 minute at vortex oscillation device (MT-31/MT-51, YAMATO) under the room temperature, left standstill 1 minute, repeated 2 times.Imbitition, (front is stated) 12000g is centrifugal 1 minute under the room temperature.The cell that collection precipitates below.
2. use the cell pyrolysis liquid lysing cell: in the precipitation of collecting, add the cell pyrolysis liquid of 400 μ l, add again Proteinase K (10 μ g/ml) after fully shaking up, put 65 ℃ of water-bath digestion at least 2 hours or spend the night (front is stated).Wherein, the composition of cell pyrolysis liquid is: 20% (mass volume ratio) SDS 2.5ml; 1mol/LTris-HCl 5ml; 5mol/L NaCl 2ml; 0.5mol/L EDTA (Ph8.0) 0.2ml supplies volume to 100ml. with distilled water
3. process albumen: add 75 μ l 8mol/L KAc, 4 ℃ of lower placements 15 minutes add 750 μ l chloroforms again, fully behind the mixing, with 5000 rev/mins speed after centrifugal 10 minutes, supernatant are moved in the new EP pipe under the room temperature.
4. precipitate DNA: the dehydrated alcohol that in supernatant, adds 750 μ l, softly shake up and to see milky white precipitate, if not obvious, can place-20 ℃ spend the night after, under the room temperature with 12000 rev/mins speed after centrifugal 10 minutes, outwell liquid, collecting precipitation is washed precipitation DNA twice with the ethanol of 70% (volume volume ratio).
5. dissolving DNA: in super clean bench, precipitation is dried up, when treating the precipitation surface absence of liquid, show that alcohol has volatilized fully, adds 50 μ l aseptic double-distilled waters, 37 ℃ of dissolving DNAs in the EP pipe.
6. measure the concentration of DNA.Measure the OD value with ultraviolet-visible pectrophotometer (756MC/756CRT type, optical instrument factory, Shanghai) 260nm.
Whether 7. electrophoresis: with 2% (mass volume ratio) sepharose, electrophoresis is 2 hours under 80V voltage, have the dna ladder degree to occur with EB dyeing 5 minutes observation in gel imaging instrument (Fine-do X1, sky, Shanghai can Science and Technology Ltd.).
DNA with wild-type makes negative control, and the result shows that the sample of transgenic line extraction has the appearance of dna ladder degree, causes a series of shearing segment (Fig. 8).
6, the expression level analysis of PDF1 gene among the microRNA-PDF1 rna interference vector transgenic arabidopsis offspring:
On the basis of Phenotypic Observation, the transgenosis individual plant is pollinated individual plant results seed with the wild-type Arabidopis thaliana.Between growth normal sowing and in seedling stage spraying herbicide through the row primary dcreening operation, carrying out obtaining transgenosis T2 for the seedling alive of each strain after PCR identifies.Win the bud of these seedlings of living, quick-frozen is analyzed the expression level of target gene BnPDF1 with FQ-PCR (method is with reference to research contents two) in liquid nitrogen.
The result is as shown in Figure 9: five transgenic line mi-1, and mi-3, mi-78, mi-16, the expression amount of target gene all is starkly lower than the wild-type Arabidopis thaliana among the mi-55, less than 50% of wild-type plant expression amount.Wherein the expression level of AtPDF1 is minimum in the mi-1 strain, only has 20% of contrast, and the average suppression efficiency of all transgenic lines reaches 40%-80%.This result shows that the interference effect of microRNA is obvious, and the expression of target gene is significantly reduced.
The present invention utilizes artificial microRNA technology to suppress the expression level of PDF1 gene in Arabidopis thaliana, has studied the function of this gene.The silence of PDF1 gene can cause the pollen abortion of Arabidopis thaliana at reproductive stage in transgenic arabidopsis, be embodied in: compare with the wild-type Arabidopis thaliana, setting percentage reduces or is shaky, angle fruit quantity reduces and is not full, the flower pesticide smaller volume of RNAi transgenic arabidopsis, and pollen obviously reduces, the abortion level of pollen obviously raises, sporule quantity reduces, and shrinkage is dyed green (Fig. 6 B) by the Alexandria dye liquor.Select the higher transgenic line of abortive degree, make the observation that frozen section is observed the pollen abortion of transfer-gen plant, and examine the dyeing observation in conjunction with DAPI and find the ahead of time disappearance of sporule nuclear or do not have significantly nuclear, only district (Fig. 6 D.H) is examined in appearance.Show that the expression that suppresses or reduce the PDF1 gene can cause the plant pollen abortion, the ability of plant is descended or lose fully, obtain male sterile plants.In addition, typical PCD phenomenon has occured in DNA Ladder experiment confirm in the sporule cell of abortion.Therefore the PDF1 gene has the function of control pollen fertility, has using value for utilizing genetic engineering technique to create male sterile material.
Advantage of the present invention:
1 by this gene of clone, studies this gene in the developmental effect of rape flower pesticide, and clear and definite this gene has the function of control anther development.By disturbing the expression of this gene, the applicant can manually create male sterile material, provides new sterile gene for realizing manually operated pollination system.
2 by this gene of clone, studies the application of this gene aspect the generation of regulation and control PCD.Namely study its mechanism of action in PCD, the PCD that occurs under the PCD that the control state of nature occurs and external source (as coerce, the disease etc.) state, thereby in the Genes For Plant Tolerance disease with anti-ly coerce middle application.
The 3 artificial microRNA carrier micro-PDF1 by structure PDF1 gene, arabidopsis thaliana transformation, the transfer-gen plant of acquisition high interference efficient is for the research gene function provides an effective means.
4, identify by the Phenotypic Observation that transgenic arabidopsis is carried out, the contriver obtains the transgenic arabidopsis that 63 strains have the male sterile phenotype, and these transfer-gen plants also have the phenotype of dwarfing simultaneously.Illustrate that the PDF1 gene has the function of efficient control plant anther fertility and plant height.
Description of drawings
Fig. 1 pcr amplification product electrophoresis detection
Napus, oleraces, rapa represent respectively swede type rape, wild cabbage and turnip type rape.The arrow indication is the purpose fragment.
The research of the expression pattern of Fig. 2 PDF1 gene in rape
A: the expression amount in the rape different tissues.As reference, be made as 1 with ripe angle relative expression quantity really.B: the expression amount in rape fertile plant and sterile strain three etap.J03S: Meiosis (J03M: tetrad is to monokaryotic stage (abortion mid-term, bud length 1-3mm), J03L for abortion early stage, bud length＜1mm): and three nuclear phases (the abortion later stage, bud length＞3mm).As reference, be made as 1 with the relative expression quantity of J03A-S.C. the expression amount in the rape bud Pistil And Stamen.J03AP: sterile gynoecium, J03AS: parastamen, J03BP: can educate gynoecium, J03BS: Fertile stamen.As reference, be made as 1 with the relative expression quantity of J03AP.Can find out among the figure that PDF1 is the gene that a large amount is expressed in the fertile plant bud.
The artificial microRNA plant expression vector of Fig. 3 (micro-PDF1) design of graphics
The PCR checking of Fig. 4 transfer-gen plant
"-" negative contrast, "+" positive contrast are the RNAI plasmids that makes up, and 1-14 is respectively the transgenosis individual plant, and M is Marker (DL2000)
Fig. 5 transgenic arabidopsis Phenotypic Observation
A figure is the whole strain of Arabidopis thaliana wild-type and transfer-gen plant mi-1; B figure is the solid situation of wild-type and transgenosis mi-1; C figure is the angle fruit of wild-type (WT), mi-16 (half is sterile), mi-1 (fully sterile); D figure is the flower of wild-type Arabidopis thaliana under stereoscopic microscope; E figure is the flower of mi-1 (fully sterile) under stereoscopic microscope, and that Bar represents is 0.06cm.
The Alexandria dyeing of Fig. 6 transgenic arabidopsis microspore development is observed
Figure A: the ripe sporule number of wild-type (WT) is many, and primitive decision is for educating; The sporule of figure B:mi-16 (half is sterile) plant; The sporule of figure C:mi-1 (fully sterile) plant.What dye green among the figure is abortion sporule (shown in the arrow).That Bar shows is 25um.
The frozen section DAPI colored graph of Fig. 7 transgenic arabidopsis flower pesticide
A, B, E, F represents wild-type, C, D, G, H are the sections of the flower of transgenic arabidopsis.A, B and E, F are respectively sporule and are in tetrad wild-type and genetically modified section; C, D and G, H are respectively sporule and are in the wild-type of growth o. 11th and the sporule section of transgenic line.A, C, E, G are the images of observing under the daylight; B, D, F, H are that the image .Pe that observes under burst of ultraviolel represents the Petal petal; S represents the spore sporule; Vb represents Vascular bundle Vescular bundle; Ta represents tapetum Tapetum.Bar represents 50 μ m. among the figure
The DNA ladder of Fig. 8 transgenosis sporule cell detects.
WT comes from wild-type sporule DNA.1-7 comes from each transgenic line sporule DNA, is respectively mi-1, mi-4, mi-55, mi-7, mi-16, mi-44, mi-12.M is 1Kb Marker.
The expression amount of the PDF1 gene of Fig. 9 microRNA interference of transgene Arabidopis thaliana relatively
Take wild-type as reference, expression amount is made as five strains that 1,1-5 is random choose, is respectively mi-1, mi-3, mi-78, mi-16, mi-55.The expression of AtPDF1 all is subject to obvious inhibition in these five strains.
Embodiment
Embodiment 1.:
The homogenic clone of PDF1 and BnPDF1 expression pattern are analyzed:
1, the homogenic clone of PDF1:
Extract the requirement of test kit (front is stated) according to Trirol and carry out the extraction of total RNA, concrete grammar is as follows: wild cabbage, swede type rape, the turnip type rape leaf sample of getting respectively 0.05-0.1g, in liquid nitrogen, be ground to powdery, extract the requirement of test kit according to Trirol and carry out the extraction of RNA.The total RNA that extracts is dissolved in the distilled water without RNase of 60uL.DNase I (front is stated) removes the residual DNA of possibility.(DU 650 BECKMAN, USA) detect respectively the absorbance value of RNA under 260 nanometers and 280 nanometers with the Protein Detection instrument, identify purity and the concentration of RNA in conjunction with 1% (mass volume ratio) agarose gel electrophoresis.Carry out reverse transcription take the RNA of above-mentioned acquisition as template by following scheme: add 1 μ L Oligo (dT) among the 2 μ g RNA, 70 ℃ of incubation 5min, place immediately 5min on ice, of short duration centrifugal, add 5 * M-MLV Buffer, 4 μ L, dNTP (10mmol/L) 1 μ L, RNase Inhibitor 20 units, M-MLV reversed transcriptive enzyme (available from Promega company) 200 units, mend to cumulative volume 20 μ L, mixing, 42 ℃ of incubation 1h with the sterilized water that DEPC processed, 70 ℃ of water-bath 15min save backup in-20 ℃ after the cDNA packing that obtains.
According to Arabidopis thaliana PDF1 gene order (gi:4929130) among the GenBank the NCBI website with BLASTN (
Http:// www.ncbi.nlm.nih.gov/BLAST) compare with wild cabbage, swede type rape and the turnip type rape gene fragment order of this laboratory order-checking acquisition, obtain respectively CDS (the Coding sequence with the wild cabbage of Arabidopis thaliana PDF1 gene order 5 ' end homology, swede type rape, turnip type rape, encoding sequence, below identical) sequence and and the CDS sequence of wild cabbage, swede type rape and the turnip type rape of Arabidopis thaliana PDF1 gene order 3 ' end homology.Then according to CDS sequence and Arabidopis thaliana PDF1 gene start codon and the terminator codon homology zone design primer (primer sequence sees Table) of the wild cabbage that obtains, swede type rape, turnip type rape PDF1 gene, thereby guarantee that amplified production is full length sequence.Primer sequence and amplification condition are respectively shown in the table 1.
Table 1:PDF1 homologous gene clone the primer
Respectively take wild cabbage, swede type rape, turnip type rape leaf cDNA as template, according to shown in the table one, add respectively corresponding primer, on PTC-200 PCR instrument (available from Bole company, below identical), carry out pcr amplification, reaction system and condition are as follows: primer sequence is for as shown in Table 1, and reaction system is: 10 * Taq enzyme reaction buffer solution 2ul, 25mM MgCL
21.2ul, 2mM dNTP 1.5ul, 10uM primer 0.2ul, 0.3 Taq of unit enzyme, add sterilized water to 20 μ l.Response procedures is: 94 ℃ of sex change 5min, and 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 2min 32 circulations, 72 ℃ are extended 5min.
With 150 volts of voltage electrophoresis 1 hour, (front is stated) detected electrophoresis result to the PCR reaction product that obtains under the gel imaging instrument on (mass volume ratio) low melting-point agarose of 1%.On the glue 1.5ml Eppendoff centrifuge tube is put in corresponding amplified production band cutting-out, 65 ℃ of water-bath 15min, add equal-volume phenol (PH7.9), put upside down and shake up 5min, 13000 rev/mins centrifugal 8 minutes, get supernatant, add the equal-volume chloroform: primary isoamyl alcohol (volume ratio 24: 1) mixed solution is also put upside down and is shaken up 5min, then 13000 rev/mins centrifugal 8 minutes, get supernatant.3 mol/L sodium-acetate (PH5.2) solution that add 1/10 volume, 95% (mass volume ratio of 2 times of volume precoolings, below identical) ethanol, in rearmounted-20 ℃ of refrigerators of mixing more than the 20min, 13000 rev/mins centrifugal 15 minutes, use again 75% (mass volume ratio after outwelling 95% (front is stated) ethanol, below identical) ethanol embathes precipitation, natural air drying, the DNA precipitation is dissolved in 20 μ l aseptic deionized waters, obtains the PCR product that reclaims.Press pMD18-T (available from TaKaRa company) carrier specification sheets, be connected on the pMD18-T carrier reclaiming the PCR product that obtains, freeze-thaw method transforms intestinal bacteria (J. Pehanorm Brooker .D.W. Russell work, Huang Peitang etc. translate molecular cloning test guide (third edition) Science Press, 96-99).Then select positive colony by bacterium colony PCR, the primer sequence is: M13F 5 '-AGCGGATAACAATTTCACACAGGA-3; M13R 5 '-GTAAAACGACGGCCAGT-3), reaction system is: 10 * Taq enzyme reaction buffer solution 2ul, 25mM MgCL
21.2ul, 2mM dNTP 1.5ul, 10uM primer 0.2ul, 0.3 Taq of unit enzyme, add sterilized water to 20 μ l.Response procedures is: 94 ℃ of sex change 5min, and 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 2min 32 circulations, 72 ℃ are extended 5min (following identical).Each PCR product is selected respectively 3 clones and is shaken bacterium and extract plasmid, and order-checking obtains PDF1 homologous gene total length CDS sequence.The CDS sequence of swede type rape, wild cabbage and turnip type rape, a kind of protein of separation, its sequence is the nucleotide sequence shown in the SEQ ID No.1; A kind of protein of separation, its sequence are the nucleotide sequence shown in the SEQ ID No.2; A kind of protein of separation, its sequence are the nucleotide sequence shown in the SEQ ID No.3.
Utilize open reading frame finder (the ORF founder of NCBI, http://www.ncbi.nlm.nih.gov/gorf/gorf.html) determines the open reading frame of PDF1 gene, derive the aminoacid sequence of coded by said gene, obtain wild cabbage, swede type rape and turnip type rape PDF1 protein sequence, a kind of isolated polypeptide, its sequence are the aminoacid sequence shown in the SEQ ID No.4; A kind of isolated polypeptide, its sequence are the aminoacid sequence shown in the SEQ ID No.5; A kind of isolated polypeptide, its sequence are the aminoacid sequence shown in the SEQ ID No.6.
The sharp online gene structure indicating system of difference (Gene Structure Display Server, GSDS,
Http:// gsds.cbi.puk.edu.cn/Chinese.php) gene structure of Arabidopis thaliana, wild cabbage, swede type rape PDF1 is compared; Utilize the online software of TCoffee (http://www.tcoffee.org/) that the tissue positioned sequence of the PDF1 of Arabidopis thaliana, wild cabbage, swede type rape, turnip type rape is compared; Use SOPMA to the secondary structure of above-mentioned four species PDF1 gene coded proteins predict, online software Pro tScale (
Http:// www.expasy.ch/tools/protscale.html) to each protein sequence carry out the hydrophilic and hydrophobic analysis, the online software of TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) is analyzed the membrane spaning domain of four PDF1 albumen.According to above-mentioned analysis and comparison, the sequence of proved invention acquisition, wild cabbage, swede type rape, turnip type rape is the homologous gene of Arabidopis thaliana PDF1 really.With the PDF1 homologous gene of the wild cabbage, swede type rape and the turnip type rape that obtain respectively called after: BoPDF1, BnPDF1 and BrPDF1.
PDF1 homologous gene on-line analysis comparison result is shown in table 2, table 3.
Table 2PDF1 homologous gene sequence information
| Species | The CDS total length | The amino acid no of proteins encoded | The number of proline(Pro) in the albumen | The per-cent that proline(Pro) accounts for | Comparison result with PDF1 |
| Arabidopis thaliana Arabidopsis | 921bp | 307 | 62 | 20.2％ | ? |
| Wild cabbage B.o/eracea | 1209bp | 403 | 90 | 22.3％ | 92％ |
| Swede type rape B.napus | 1299bp | 433 | 18 | 4.15％ | 49％ |
| Turnip type rape B.rapa | 1335bp | 445 | 21 | 4.72％ | 42％ |
Table 3PDF1 homologous gene secondary structure relevant information
| Species species | Coded amino acid is counted numbers of aa | Molecular weight WT | α spiral Alpha helix (Hh) | Extended chain Extended strand (Ee) | β-bend Beta turn (Tt) | Irregular curling Random coil (Cc) |
| Arabidopis thaliana Arabidopsis | 307 | 298864.5 | 20.26％ | 11.44％ | 9.15％ | 59.15％ |
| Wild cabbage B.oleracea | 354 | 345592.5 | 18.41％ | 10.70％ | 5.97％ | 64.93％ |
| Swede type rape B.napus | 345 | 336506.5 | 40.28％ | 16.67％ | 9.72％ | 33.33％ |
| Turnip type rape B.rapa | 348 | 339427.0 | 37.39％ | 16.67％ | 5.18％ | 40.77％ |
By online software Pro tScale (http://www.expasy.ch/tools/protscale.html) to Arabidopis thaliana PDF1 albumen with and in Btassica the hydrophobicity of 3 homologous proteins (wild cabbage, swede type rape, turnip type rape, below identical) analyze respectively.Relatively can finding out of the hydrophilic and hydrophobic of PDF1 homologous protein: arranging of the amino acid whose hydrophilic and hydrophobic of Arabidopis thaliana and wild cabbage is more consistent, at sequence N end and middle-end a hydrophobic section is arranged respectively.The amino acid of the PDF1 homologous genes encoding of swede type rape and turnip type rape has similarity, wetting ability and hydrophobic proteins prolong the O axis and distribute more uniformly, the most amino acid of two sequences is below the O axis, and therefore most amino acid belongs to hydrophilic amino acid.Arabidopis thaliana and wild cabbage may contain two cross-film zones.By above-mentioned comparative result as can be known the wild cabbage, swede type rape, the turnip type rape PDF1 gene that obtain of the present invention have similar sequence, protein structure and clear hydrophobicity and mould location, be homologous gene really.
2, the expression pattern analysis of the efficient expressing gene BnPDF1 of a kind of rape flower pesticide:
The used rape of the present invention (Brassica napus L.) is dominant genic male sterile heterozygosis sterile line J03AB (Msmsrfrf*msmsrfrf) (material is that oil crops functional genome of institute of the Chinese Academy of Agricultural Sciences and Molecular Biology Research Lab preserve, and the front is stated).System backcrosses through for many years and hands over and breed with sister, and wherein the fertile plant code name is J03B, and sterile strain code name is J03A (following identical).The two can be considered a pair of near isogenic line, only has sterile Site discrepancy.Be seeded in the land for growing field crops for the examination material, normal field management.
Get root, stem, leaf, bud, angle fruit from the identical fertile plant of same grown in field condition, anatomical isolation gynoecium and flower pesticide from bud, every kind of material is got three repetitions at least, each repeats at least one strain, with the masking foil parcel, be positioned over rapidly in the liquid nitrogen-80 ℃ of preservations after the sampling.Extract RNA (the method front is stated).Get respectively flowering period can educate with sterile plant on get bud, go back to the laboratory after, on ice chest, take out respectively flower pesticide and gynoecium, every kind of material is got three repetitions at least, and each repeats at least one strain, wraps up with masking foil after the sampling, be positioned over rapidly in the liquid nitrogen-80 ℃ of preservations.Extract RNA (the method front is stated).
Quantitative real time PCR Instrument is RT
TM-Cycler (Bo Ao), adopt rape Actin (accession number AF111812) as internal standard gene, the Actin gene primer be forward primer 5 '-CTGGAATTGCTGACCGTATGAG-3 ' and reverse primer 5 '-ATCTGTTGGAAAGTGCTGAGGG-3 '.The BnPDF1 gene primer be forward primer 5 '-GCTCTCATCCCATTGGTGCT-3 ' and reverse primer 5 '-TGCTTTGTGGCTGCCTTGTT-3 '.
The quantitative fluorescent PCR reaction system is 20 μ L: contain SYBR Mix 10 μ L, and each 0.8 μ L of forward and reverse primer (10 μ mol/L), template 2 μ L, the sterilized water that DEPC processed complements to 20 μ L.Amplification condition is: 94 ℃, and 5min:94 ℃, 15s, 60 ℃, 20s, 72 ℃, 30s, 40 circulations; Each is circulated in 72 ℃ of renaturation ends and carries out fluoroscopic examination.Reaction is heated to first 95 ℃ after finishing, and then is down to 72 ℃, slowly is warming up to 95 ℃ again, records the variation of fluorescent signal, draws the melting curve of amplified production.Every group of experiment all finished three biology and repeated, and each biology repeats to do at least three technology and repeats.Detect respectively the expression of BnPDF1 in Oil Rape Tissue (root, stem, leaf, flower, angle fruit, flower pesticide, gynoecium, stamen).
With the relative expression quantity that compares Ct method (Δ Δ Ct) calculating gene.Utilize software that instrument is with, at first optimize internal standard gene and goal gene amplification condition, measure respectively the Ct value of Actin and goal gene, choosing three results (systematic error of Ct value is less than 0.3) that approach the most in nine measured value of experiment averages, then by internal standard gene goal gene is proofreaied and correct and obtained Δ Δ Ct, at last by 2
-Δ Δ CtThe relative expression quantity of estimate goal gene and systematic error (Kenneth J Livak.Thomas D Schmittgen Analysis ofRelative Gene Expression Data Using Real-Time Quantitative PCR and the2
-Δ Δ CT Method METHODS 25,402-408 (2001)).When calculating relative expression quantity, take the sample of angle fruit as reference, be about to its value and convert 1 to, other sample again with its relatively, obtain relative expression's value.
The result shows (Fig. 2): take the expression amount of angle fruit as reference, BnPDF1 in the expression characterization result in the rape different tissues shown in Fig. 2 A.This gene all has expression in these tissues, wherein, middle expression amount is the highest spending, and expression amount takes second place in root, and expression amount is minimum in the fruit of ripe angle.Therefore, BnPDF1 be one at root, leaf with spend the gene of middle high efficient expression.
Take sterile strain small bud (＜1mm) as reference, BnPDF1 in relative expression's level results of three critical periods of rape fertile plant J03B and sterile strain J03A flower bud development shown in Fig. 2 B.BnPDF1 is downward trend after rising first appears along with developmental process in the expression amount of fertile plant bud, presents obvious bell-shaped curve.In Microspore Abortion mid-term, namely tetrad is the highest to the expression amount of monokaryotic stage.And all be significantly inhibited in the expression in each period of sterile strain flower bud development, all be lower than the expression level of identical etap of fertile plant bud.In Microspore development, especially abortion mid-term (tetrad is to monokaryotic stage) BnPDF1 expression amount be suppressed just show as sterile.
BnPDF1 is compared analysis at the gynoecium of rape J03 fertile plant and sterile strain and the expression characterization in the stamen, and the result is shown in Fig. 2 C.BnPDF1 in the fertile plant Pistil And Stamen expression level all apparently higher than sterile strain, consistent with the expression trend of B figure.Simultaneously, BnPDF1 also a large amount expression in fertile plant gynoecium (J03BP).This explanation BnPDF1 mainly plays very important effect in the growth course of stamen.
Embodiment 2.:microRNA-PDF1 vector construction, transformation of Arabidopsis thaliana and PCR detect:
1, the vector construction of microRNA-PDF1
Does carrier make up (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi with reference to listed method in " 2010 plans of Arabidopis thaliana "? page=Home; Project=stdwmd).The target sequence of choosing is: AATUTUTATATUUTGTUGAG, corresponding Oligo DNA is: TTAGAGACTATAGGACAGCTC.Carrier structure as shown in Figure 3.
Then utilize freeze-thaw method (front is stated) to change microRNA-PDF1 over to agrobacterium tumefaciens EHA105 (available from the precious biotinylated biomolecule in Dalian Products Co., Ltd, below identical), coating the LB solid that contains 50 μ g/mL penbritins and Rifampin (50 μ g/mL) (fills a prescription as follows: take by weighing respectively 10 gram Tryptoness, 5 gram yeast extracts and 10 gram sodium-chlor, 8 gram agar are dissolved in the distilled water successively, and constant volume is in 1000 milliliters.Be sub-packed in 500 milliliters of triangular flasks, 121 ℃, autoclave sterilization is 15 minutes under 6.859 * 104Pa.4 ℃ of refrigerations are for subsequent use) on the flat board, 37 ℃ of overnight incubation are respectively selected three of hickies, and be bacterium colony PCR and detect (front is stated).And the carrier that obtains checked order to verify accuracy.
2, transformation of Arabidopsis thaliana and PCR detect
According to document (Zhang X R.et al.Agrobacterium-mediated transformation ofArabidopsis thaliana using the floral dip method.Nature, 2006,1: method for transformation arabidopsis thaliana transformation 1-6).
Preparation contains agrobacterium tumefaciens EHA105 (front is stated) the bacterium liquid that builds carrier, changes in the LB liquid nutrient medium that contains kantlex 50 μ g/ml, Rifampin 50 μ g/ml 28 ℃ of incubated overnight over to the day before yesterday in conversion.Second day, the light absorption value with ultraviolet spectrophotometer (SPEKOL 1300) detects under the 276nm nano wave length takes out when the light absorption value of bacterium liquid reaches between 1.6-2.0.Room temperature (20-25 ℃, below identical) with the centrifugal 10min of 4000g, is abandoned supernatant, and precipitation is suspended in isopyknic 5% sucrose (mass volume ratio).The sucrose solution of muddiness is poured in the large culture dish, added the Silwet 1-77 that final concentration is 0.02% (volume ratio) (available from the Five continents, Beijing unit industry science and trade center, below identical) before transforming.The whole inflorescence of Arabidopis thaliana to be transformed being immersed in the sucrose gently, silent number 15s take out plant behind the mixing.Plant after the conversion is wrapped with a black plastic bag, is placed on the growth case and cultivates.Second day is opened plastics bag, and cultivate in the place that is placed on light intensity.Every the conversion that tries again in a week.Cultivate and gathered in the crops seed in about one month, seed under incubator or daylight dry 3-5 days.Idiographic flow is as follows:
1. the Rifampin preparation is become the concentrated solution of 50Mg/ml, be put in 4 degree and preserve, will blow and beat evenly before using, dilute 100 times of uses.
2. add 5 microlitre Rifampin concentrated solutions in the LB of 5ml liquid nutrient medium, shake up, choose into single bacterium colony, activation is spent the night.
3. see that when the bacterium liquid of activation was very muddy, sucking-off was no less than the bacterium liquid of 2ml activation, be added in the LB liquid nutrient medium of 200ml, 28 degree 200 turn overnight incubation to OD value 1.6-2.0.
4. centrifugal 10 minutes of 4000g room temperature (desk centrifuge SIGMA2-16K), 4500 turn, and abandon supernatant, buckle at filter paper and do.
5. prepare 200 milliliters of 5% (mass volume ratio) sucrose solutions, with the resolution of precipitate that last collection obtains, even with rifle piping and druming.
6. will dissolve good bacterium liquid and be added in the plate, then 50 ml solns/plate add SiLwet-L-77 (front the is stated) mixing of 40 microlitres.
7. the inflorescence with the plant of Arabidopis thaliana immerses in the plate, holds together together with the flower of hand with branch, is immersed gently 15 seconds.
8. package with the Arabidopis thaliana plant of opaque sack with each Plasmid Transformation, the lucifuge moisturizing got final product in one day.
9. repeat again after the week to transform once.
Weedicide (Glufosinate ammonium, the front is stated) screening: transform finish after continuation cultivate the Arabidopis thaliana plant and gather in the crops seed about one month, seed under incubator or daylight dry 3-5 days.The T0 that transforms results is broadcast in the vermiculite that is mixed with the PNS nutritive medium for seed, (PNS nutritive medium composition is: every liter contains 5ml 1M KNO3,2ml 1M Ca (NO3) 24H2O, 2ml 1M MgSO47H2O, 2.5ml 20mM Fe.EDTA, 2.5ml 1M phosphoric acid buffer (pH5.5), 1ml MS trace element); 4 degree one weeks of vernalization, between the growth of 21-22 degree in about the self-sow fortnight, wait the sprinkling of carrying out in early days weedicide Glufosinate ammonium (30mg/L) of the four leaf phases that arrived for the first time, carry out the sprinkling second time after the week.Green seedling to survival carries out the PCR positive detection, obtains the transgenic positive seedling.Obtain altogether 97 strain transgenic positive seedlings through transforming.
PCR detects the screening seedling: treated for four leaf seedling periods, get a slice true leaf (seedtime is approximately the four stars phase) and extract genomic dna with CTAB method (front is stated), carry out the PCR evaluation with doing according to the primer of the 35S promoter on the artificial microRNA carrier and NOS terminator sequences Design.The primer sequence nucleotide sequence is 5 '-AAAAGACTTACCGAGTTGAACC-3 ' and 5 '-CGCTGTCGTTGGGGCAATTC-3 '; The PCR reaction system is as follows: genomic dna template 1 μ L (approximately 50ng), 10 * Taq enzyme reaction buffer solution 2ul, 25mMMgCL
21.2ul, 2mM dNTP 1.5ul, each 0.2ul of 10uM primer, 0.3 Taq of unit enzyme, add sterilized water to 20 μ l.Response procedures is: 94 ℃ of sex change 5min, and 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 2min 32 circulations, 72 ℃ are extended 5min.Detect the PCR reaction product with 1% (mass volume ratio) agarose gel electrophoresis, the result shows that this PDF1 gene interference carrier pBI121-PDF1 has successfully changed Arabidopis thaliana (Fig. 3) over to.
Embodiment 3: the transgenic arabidopsis Phenotypic Observation:
In the flowering period of Arabidopis thaliana, choose each branch top and leaked white but unopened flower, push bud aside with dissecting needle.At magnifying glass (3 times) and lower flower pesticide and the pollen observed of stereoscopic microscope (OLYMPUS SZ61TRC).Isolate flower pesticide with dissecting needle, with reference to Alexandria staining (Alexander, M.P.Differential staining of aborted and non-aborted pollen.StainTechnol, 44:117-122., 1969), the flower pesticide of 40 ℃ of heating in water bath Alexandria staining fluid immersions spends the night (front is stated), with pressed disc method Observation of Microspore under microscope (OLYMPUS IX71).Judge the abortive degree of sporule by the ratio of observing and adding up normal sporule (dying orange) and abortion sporule (dying green).The thaliana flower organ frozen section technique (Chen Dan that Arabidopis thaliana flower pesticide frozen section and brazilwood extract dyeing method are set up with reference to Chen Dan, Zhao Jie, Zhao Jie. be suitable for the frozen section technique [J] of thaliana flower organ. Wuhan phytology research, 23 (3): 85-290,2005).
To identifying the positive transfer-gen plant that obtains through herbicide screening and PCR, carry out Phenotypic Observation and evaluation since seedling stage, find that the phenomenon of obvious dwarfing has appearred in transgenic arabidopsis T1: shown in Fig. 5 A.The plant type of transgenic arabidopsis is short and small, and angle fruit quantity is fewer.Fig. 5 .4B demonstration, transformant mi-1 is solid hardly, and the quantity of flower is also lacked than wild-type.Fig. 5 C has shown the relative size of the angle fruit of wild-type, half sterile strain mi-16, sterile strain mi-1, and wild-type angle fruit is large and full; Semisterile angle fruit median size and contain a small amount of seed, the angle fruit of complete sterility is minimum and do not have a seed-bearing generation.Fig. 5 D and E be the bud of wild-type and transgenic arabidopsis mi-1 under the display body stereomicroscope respectively, and as can be seen from the figure the stamen of wild-type Arabidopis thaliana bud is near column cap and discharge pollinia (Fig. 5 D); And filigree is very short in the transgenic arabidopsis mi-1 bud, and the shrivelled and WUHUAFEN of flower pesticide discharges (5E).
Transgenic arabidopsis is being carried out on the basis of Phenotypic Observation, the method that the applicant adopts Alexandria dyeing and compressing tablet detects and observes the fertility of microspore of all transgenic arabidopsis.Alexandria staining concrete grammar is as follows:
1, prepares slide glass, tweezers, staining fluid, pencil, pipettor and rifle head, gloves, spirit lamp, filter paper etc.;
2, get required fresh sample, i.e. the bud that just showed money or valuables one carries unintentionally of Arabidopis thaliana; The bud sample is put into the 1.5ml centrifuge tube, add Alexandria dye liquor 20ul (to prepare about 100 milliliters of staining fluids as example: 100% ethanol 10ml, 1% malachite green spirituous solution 1ml, distilled water 50ml, glycerine 25ml, the C.I. 42685 aqueous solution 5ml of 1% (mass volume ratio, below identical), 1% (mass volume ratio, below identical) orange G aqueous solution 0.5ml, Glacial acetic acid 1-4ml, phenol 5 grams, Chloral Hydrate 5 grams).
3, bud is soaked in the staining fluid, 40 ℃ of dyeing are spent the night; Take out sample with tweezers, blot moisture on the sample with filter paper, push bud aside, with dissecting needle stamen is separated, press cover glass and examine under a microscope sporule.
As can see from Figure 6, the sporule of wild-type Arabidopis thaliana and transgenic arabidopsis has obvious difference: the flower pesticide of wild-type Arabidopis thaliana is very full and be full of sporule, and sporule is all dyed orange, shows that microspore development wherein is normal, has vigor (Fig. 6 A).Fig. 5 B shows that the flower pesticide form of mi-16 is normal and be full of sporule, but wherein most sporule all presents green (abortion), only has the minority sporule to dye orange.Illustrate that abortion has occured most sporule in the mi-16 flower pesticide, it is one and half sterile strains, and abortive degree is more than 60%.The solid situation of mi-16 has also confirmed this point (Fig. 6 C).Fig. 6 C has shown that the flower pesticide form of mi-1 is normal, but less than normal.The inside only have tens sporules, and most sporule present green (abortion) almost can't see dyeing normal sporule.Illustrate that abortion has all occured sporule in the mi-1 flower pesticide, it is a complete sterility strain.The solid situation of the mi-1 that shows among Fig. 6 C has also been confirmed this point.In 97 strain transgenic arabidopsis of all acquisitions, the Alexandria coloration result shows that having 31 strains is complete sterility, and 30 strains are partly sterile, and abortive degree is more than 50%.Obtain altogether 61 strain abortion strains in transgenic arabidopsis, ratio is 63%.Therefore the applicant thinks that male sterile is the representative phenotype of transfer-gen plant.The sporule proterties of whole transfer-gen plant and the statistics of solid situation are entered shown in the following table, show that the transfer-gen plant that surpasses half can not be normally solid, sporule dyeing occurs green, angle fruit setting percentage also reduces, therefore the phenotype of transgenic line be since interference base because of expression cause, and this gene is relevant with microspore development.
The phenotype statistics of all transfer-gen plants of table 4
| ? | Can educate | Half is sterile | Sterile |
| The sporule proterties | Dyeing is yellow; Full; Quantity is many | Coloured portions is green; Plumpness 50%; Quantity is few than wild-type | Dyeing almost be can't see full sporule, out-of-shape for green |
| Breeding status | 95%; Fruit is similar than wild-type at the angle | About 50%; Fruit is less, not too full at the angle | ＜20%; The angle fruit is very little, or shaky; Knot needs pollination rear solid hardly |
| The strain number | 34 | 39 | 24 |
In addition, the applicant carries out the Alexandria to the T2 that obtains with wild-type Arabidopis thaliana filial generation screening for the flower pesticide of transfer-gen plant and dyes to identify that each T2 is for the fertility of individual plant, the result is as shown in table 5, by T1 and T2 two generations of generation screening, male sterile phenotype has all appearred in transgenic arabidopsis basically, male sterile is the representative phenotype of transfer-gen plant, and this phenotype can genetic stability, and and weedicide chain.
Table 5T2 is for Arabidopis thaliana screening and fertility investigation result
| Strain | The number of always emerging | Total seedling number of living | Can educate | Half is sterile | |
| 1 | 14 | 8 | 0 | 5 | 3 |
| 2 | 8 | 4 | 1 | 2 | 1 |
| 3 | 5 | 3 | 0 | 1 | 2 |
| 4 | 10 | 6 | 0 | 2 | 4 |
| 5 | 8 | 4 | 1 | 4 | 3 |
| 6 | 16 | 7 | 0 | 5 | 2 |
| 7 | 12 | 6 | 0 | 1 | 5 |
| 8 | 6 | 3 | 0 | 3 | 0 |
By
In transgenic arabidopsis T1 for the phenotype that has occurred downgrading, so we to through and the hybridization of wild-type Arabidopis thaliana and individual plant gather in the crops seed.Seed under incubator or daylight dry 3-5 days.The T1 of hybridization results is broadcast in the vermiculite that is mixed with the PNS nutritive medium for seed, (PNS nutritive medium composition is: every liter contains 5ml 1M KNO3,2ml 1MCa (NO3) 24H2O, 2ml 1M MgSO47H2O, 2.5ml 20mM Fe.EDTA, 2.5ml 1M phosphoric acid buffer (pH5.5), 1ml MS trace element); 4 degree one weeks of vernalization, in between the growth of 21-22 degree about the self-sow fortnight, wait the sprinkling of carrying out in early days weedicide Glufosinate ammonium (30mg/L) of the four leaf phases that arrived for the first time, carry out second time after one week and spray, through a plurality of transgenic lines in T2 generation of acquisition after the screening of weedicide Glufosinate ammonium.Respectively their plant height was measured in after planting 20 days and 40 days for strain at T2, each strain is measured 20 strains, and 3 repetitions (cultivating respectively program request in the boxes at 3) have been set, sow simultaneously the wild-type Arabidopis thaliana and measure plant height at same time point (after planting 20 days and 40 days), also measure 20 strains, and set 3 repetitions.The result is as described in Table 6, and transgenic line T2 is for general dwarfing phenotype having occurred, and plant height only has about the 50-80% of phase contemporaneously wild-type height.Wherein obvious with complete sterility strain mi-1, sow 40 days (flowering period) center line average and only have 57.2% of wild-type.This shows, stable dwarfing phenotype has appearred in the RNAi transgenic arabidopsis.
Table 6. transgenosis T2 is for strain plant height cartogram (unit: centimetre)
The cytological observation analysis of the artificial microRNA transgenic arabidopsis of embodiment 4:PDF1:
1, the developmental process of frozen section and DAPI dyeing Observation of Microspore.
Frozen section method concrete grammar is as follows:
Collect the whole inflorescence of transgenic arabidopsis, different big or small buds are separated with dissecting needle, and do mark.
1. draw materials and pre-treatment: sample room temperature treatment in treatment solution that will need to cut into slices is spent the night, to strengthen the paster ability of sample.Treatment solution is: 0.8mol/L sucrose adds 1.0mol/L glycerine.
2. take out and organize supporter, set level and set tissue, periphery drips upper embedding medium (available from Ling Fei company), and it is central freezing that speed is put in liquid nitrogen, then will organize together with the embedding medium that becomes solid state and together take out.-20 degree are preserved.
3. with the tissue block that freezes, be clamped in slicing machine and hold and hold on the device, start thick advance and retreat key, turning knob will be organized equating.
4. mixing up the thickness of wanting to cut, decide according to different tissues, is intensive thinly-sliced of cell in principle, and a little thick the cutting that the fiber many cells are rare is generally between 5～10um.
5. mix up anti-roll bending.Make frozen section.During section, the section that cuts out can waltz through passage between the anti-roll bending of cutter in the very first time, entirely lies on the iron plate of mes holder.At this moment just can start anti-roll bending, get a slide glass, its attached sticking is got final product.
6. should look different tissue and select different freezing degree.The height of freezing degree in the refrigerated tank is mainly decided according to different tissues, cannot treat different things as the same.General, organizing of freezing plant is all adjustable about-15～20 ℃.The section temperature of flower pesticide is-21 ℃.
The frozen section that cuts carries out DAPI dyeing subsequently, and concrete grammar is as follows:
DAPI (4,6-diamino-2-phenylindone, below identical) be whether to grow normal very effective dyestuff (Park et al. for the Study of Pollens nucleus, Development 125:3789-3799,1998), it is combined with nucleic acid specifically, sends blue-fluorescence under UV-light.1 trophonucleus and 2 spermatid nucleuses (sperm) are arranged, totally 3 nucleus in the wild-type mature pollen.Wherein trophonucleus is because being in the disperse of dyeing of transcriptional activity state.Germ nucleus is not transcribed or heterochromatin state that transcriptional activity is very low dyes fine and close bright because being in.The working concentration of DAPI is 2 micrograms/microlitre, is dissolved in the sucrose solution of 7% (mass volume ratio), and lucifuge-20 degree is preserved.The present invention uses is the DAPI dye liquor (article No. C1006) that green skies company configures.The at room temperature dyeing 30 minutes in the dark of viable cell and fresh material, and sliced materials at room temperature in the dark dyeing got final product in 5-10 minute.
In order to observe more clearly the development condition of inner its hetero-organization of flower pesticide and cell, concrete period and the phenomenon of further clear and definite transgenic arabidopsis Microspore Abortion, have or not the variation of following other positions of floral organ, the applicant adopts frozen section technique that the flower pesticide of wild-type and transgenic arabidopsis has been carried out rip cutting, then with DAPI dyeing after five minutes respectively under the light microscopic and the one-piece construction of observing flower pesticide under the fluorescence.The result as shown in Figure 7, at tetrad, the structure of transgenic arabidopsis flower pesticide and sporule be (7C) and wild-type (7A) and indifference under the white light visual field, but after the DAPI dyeing, significant difference is then arranged: transgenic line flower pesticide is at microspore development tetrad (7D), can't observe clearly nuclear in the overwhelming majority sporule (S), and in the flower pesticide (7B) of wild-type Arabidopis thaliana contemporaneity, all sporules (S) can observe clearly nuclear.The DAPI dyeing of the petal of transgenic arabidopsis and wild-type Arabidopis thaliana (Pe), tapetum (Ta) and Vascular bundle (Vb) there is no significant difference, can see clear bright nucleus (7B, D).Microspore development than late period, namely sporule is from the early stage that the flower pesticide coyote hole discharges, the structure of transgenic arabidopsis flower pesticide and sporule be (7E) and wild-type (7G) and indifference under the white light visual field.Wild-type Arabidopis thaliana flower pesticide (7D) DAPI dyes, and as seen each organize all clearly nuclear, can see the germ nucleus of two state of aggregations in indivedual lower spores.And the sporule (S) of transgenic line (7H) can't make DAPI painted, also can't see bright nucleus.And there is the part cell also not observe clear bright nuclear in the petal (Pe), Vascular bundle (Vb).
Can find out from DAPI dyeing comparing result (Fig. 7), not significantly difference of the structure of wild-type and transgenic arabidopsis flower pesticide under white light, but the nucleus dyeing of seeing RNAi transgenic arabidopsis sporule under burst of ultraviolel changes: just can't observe nucleus the tetrad sporule, nuclear begins degraded (7B, D).This phenomenon is more obvious when having arrived 11 phases of middle and advanced stage (three nuclear phases) of microspore development: sporule can't be dyeed by DAPI, can't observe nucleus.Nucleus degradable (7F, H) is described.In addition, applicant also use respectively 0.1% aniline blue solution (to parallel Zhi matter dyeing) and the DIOC of (mass volume ratio, below identical)
2(cell walls dyeing) dyes to section and compressing tablet, does not observe the unusual of parallel Zhi matter and cell walls.The above results explanation, the abortion main manifestations of the sporule of the RNAi transgenic arabidopsis of PDF1 are the nuclear ahead of time degraded of tetrad sporule.The applicant infers might be at sporule generating program in this dead (PCD) in period, thereby causes Microspore Abortion.
2, DNA ladder confirms that Microspore Abortion is relevant with PCD:
For PCD has occured in the sporule that confirms transgenic arabidopsis really, the index phenomenon that the present invention adopts the mode of DNA electrophoresis to detect whether PCD has occured in the sporule: the degraded of DNA and fragmentation.Extraction and the electrophoresis process of sporule DNA are as follows:
1. collect pollen granule: configuration 0.5M NaCl+7% (mass volume ratio) sucrose solution, as the solvent that separates pollen.Get the EP pipe of 1.5 milliliters of semicanals, 40-50 flower maybe will opening in full bloom put into solvent, (20-25 degree, below identical) vibrated 1 minute at vortex oscillation device (MT-31/MT-51, YAMATO) under the room temperature, left standstill 1 minute, repeated 2 times.Imbitition, (front is stated) 12000g is centrifugal 1 minute under the room temperature.The cell that collection precipitates below.
2. use the cell pyrolysis liquid lysing cell: in the precipitation of collecting, add the cell pyrolysis liquid of 400 μ l, add again Proteinase K (10 μ g/ml) after fully shaking up, put 65 ℃ of water-bath digestion at least 2 hours or spend the night (front is stated).Wherein, the composition of cell pyrolysis liquid is: 20%SDS 2.5ml; 1mol/L Tris-HCl 5ml; 5mol/L NaCl 2ml; 0.5mol/L EDTA (Ph8.0) 0.2ml supplies volume to 100ml. with distilled water
3. process albumen: add 75 μ l 8mol/1KAc, 4 ℃ of lower placements 15 minutes add 750 μ l chloroforms again, fully behind the mixing, with 5000 rev/mins speed after centrifugal 10 minutes, supernatant are moved in the new EP pipe under the room temperature.
4. precipitate DNA: in supernatant, add the dehydrated alcohol of 750 μ l, softly shake up and can see milky white precipitate, if precipitate not obvious, can place-20 ℃ spend the night after, after centrifugal 10 minutes, outwell liquid, collecting precipitation with 12000 rev/mins speed under the room temperature.The ethanol of (volume volume ratio, below identical) is washed precipitation DNA twice with 70%.
5. dissolving DNA: in super clean bench, precipitation is dried up, when treating the precipitation surface absence of liquid, show that alcohol has volatilized fully, adds 50 μ l aseptic double-distilled waters, 37 ℃ of dissolving DNAs in the EP pipe.
6. measure the concentration of DNA.Measure the OD value with ultraviolet-visible pectrophotometer (756MC/756CRT type, optical instrument factory, Shanghai) 260nm.
Whether 7. electrophoresis: with 2% (mass volume ratio) sepharose, electrophoresis is 2 hours under 80V voltage, have the dna ladder degree to occur with EB dyeing 5 minutes observation in gel imaging instrument (Fine-do X1, sky, Shanghai can Science and Technology Ltd.).
If obvious PCD occurs in the sporule cell, then have the fragmentation of DNA, namely electrophoresis can produce Gradient distribution.DNA with wild-type makes negative control, the result shows that the sample of transgenic line extraction has the appearance of dna ladder degree, cause a series of shearing segment, as shown in Figure 8: the sporule of wild-type Arabidopis thaliana only has a bright total DNA band, does not have the dna ladder shape to distribute; And each strain of transgenosis (swimming lane 1-7) also produces the dna degradation fragment of different sizes, i.e. DNA ladder except seeing total DNA band.This explanation programmed death occurred in the sporule of transgenic line, namely PCD.Because be the time period that shows money or valuables one carries unintentionally the period of the flower won, this period, corresponding microspore development was approximately after the tetrad before the ripening stage period, illustrated that unusual PCD has occured the transgenic line sporule cell of this time period.This result is consistent with the result of the sporule compressing tablet that carries out above and frozen section: the unusual PCD that sporule begins at tetrad has caused nuclear ahead of time degraded, finally causes male sterile.Sporule PCD's is ahead of time to cause the male sterile major reason of transgenic arabidopsis.
The detection of the artificial microRNA transgenic arabidopsis of embodiment 5.PDF1 gene jamming effectiveness:
On the basis of Phenotypic Observation, the transgenic arabidopsis individual plant is pollinated individual plant results seed with the wild-type Arabidopis thaliana.Between growth normal sowing and in seedling stage spraying herbicide through the row primary dcreening operation, carrying out obtaining transgenosis T2 for the seedling alive of each strain after PCR identifies.Win the bud of these seedlings of living, quick-frozen is analyzed the expression level of target gene PDF1 with Q-PCR in liquid nitrogen.Concrete grammar is as follows: the PCR that learnt from else's experience detects the rear T2 that obtains for the inflorescence of positive plant,, in liquid nitrogen, Young Plant leaflet tablet sample is ground to powdery, the extraction of RNA is carried out in the requirement that utilizes Trirol to extract test kit.The total RNA that extracts is dissolved in the distilled water without RNase.DNase I removes the residual DNA of possibility.Detect respectively RNA at 260 nanometers and 280 nanometer absorbance values with Protein Detection instrument (DU 650 BECKMAN, USA), identify purity and the concentration of RNA in conjunction with 1% (mass volume ratio) agarose gel electrophoresis.Carry out reverse transcription (front is stated) take the RNA that obtains as template, save backup in-20 ℃ after the cDNA packing that obtains.
Quantitative real time PCR Instrument is RTTM-Cycler (Bo Ao), adopts Arabidopis thaliana Actin as internal standard gene.Arabidopis thaliana PDF1 primer is 5 '-GCTCTCATCCCATTGGTGCT-3 ' and 5 '-TGCTTTGTGGCTGCCTTGTT-3 '.The quantitative fluorescent PCR reaction system is 20 μ L: contain SYBR Mix10 μ L, and each 0.8 μ L of forward and reverse primer (10 μ mol/L), template 2 μ L, the sterilized water that DEPC processed complements to 20 μ L.Amplification condition is: 94 ℃, and 5min; 94 ℃ of 15s, 60 ℃ of 20s, 72 ℃ of 30s, 40 circulations; Each is circulated in 72 ℃ of renaturation ends and carries out fluoroscopic examination.Reaction is heated to first 95 ℃ after finishing, and then is down to 72 ℃, slowly is warming up to 95 ℃ again, records the variation of fluorescent signal, draws the melting curve of amplified production.Every group of experiment all finished three biology and repeated, and each biology repeats to do at least three technology and repeats.
With the relative expression quantity that compares Ct method (Δ Δ Ct) calculating gene.Utilize software that instrument is with, at first optimize internal standard gene and goal gene amplification condition, measure respectively the Ct value of Actin and goal gene, choosing three results (Ct valve system error is less than 0.3) that approach the most in nine measured value of experiment averages, then by internal standard gene goal gene is proofreaied and correct and obtained Δ Δ Ct, at last by 2
-Δ Δ CtThe relative expression quantity of estimate goal gene and systematic error (front is stated).When calculating relative expression quantity, take the sample of wild-type Arabidopis thaliana inflorescence as reference, be about to its value and convert 1 to, other sample again with its relatively, obtain relative expression's value.The result shows that the genetically modified jamming effectiveness of artificial microRNA is very obvious, as shown in Figure 8: five transgenic line mi-1, mi 3, mi-78, mi-16, the expression amount of target gene all is starkly lower than the wild-type Arabidopis thaliana among the mi-55, less than 50% of wild-type plant expression amount.Wherein the expression level of AtPDF1 is minimum in the mi-1 strain, only has 20% of contrast, and the average suppression efficiency of all transgenic lines reaches 40%-80%.This result shows that the interference effect of microRNA is obvious, and the expression of target gene is significantly reduced.
Show that in conjunction with the result of case study on implementation 3 and 4 expression that suppresses the PDF1 gene makes sporule that PCD occur in advance, cause the pollen abortion of plant, the ability of plant is descended or lose fully, obtain male sterile plants.The PDF1 gene has the function of control pollen fertility, has using value for utilizing genetic engineering technique to create male sterile material.
Sequence table
＜110〉Inst. of Oil Crops, Chinese Academy of Agriculture
＜120〉a kind of PDF1 gene and preparation method and purposes of controlling plant pollen fertility
＜130〉a kind of PDF1 gene and preparation method and purposes of controlling plant pollen fertility
<160>6
<170>PatentIn?version?3.1
<210>1
<211>1209
<212>DNA
＜213〉wild cabbage PDF1 gene C DS sequence
<400>1
atgagaggga?tgaagagtct?taccatttgg?gctatctttg?ctgctgtact?ctcccagcag 60
ctctttgcct?ctgttgcttc?cataaagttt?gaggatgaga?aaacatacta?ctctcctcca 120
gacccaaatg?caggatctcc?tccctctgga?actcctccat?cacacggagg?atacactcca 180
actcctccaa?ctccatcaca?tactcctccc?tctaactgtg?gaagtccacc?ttacgaccca 240
actcctcctt?ctcacactcc?aactccttct?actccatctc?acactccttc?gactccttct 300
catactccaa?ctccttcgac?tccatctcac?acaccaactc?catcacacac?tagtcctcct 360
tgccactgtg?gaactccacc?ttcacaccct?tccacacctt?cacggccttc?aaggccttca 420
cacccatcta?ggccttcacg?cccttccacg?ccttcaaatc?ctccttctgg?tggatactac 480
tcatctcctc?caccaagtac?tcctgtcgtt?gtgacaccac?caacacccat?tgttgaccca 540
ggcacaccca?gtactggagg?aacccctcct?tcttctggtg?gatattactc?atctcctcca 600
ccaagtactc?cagtcgttga?gactccacca?acacctattg?ttgacccagg?cacacccatc 860
attggagaaa?ccccaccaac?tccttccggt?ggatattact?catctcctcc?accaagtact 720
cccattgttg?taactccacc?aacacccatt?gttgacccag?gcacacccat?cattggagga 780
accccaccaa?ctccttttat?tgaccctggc?actcctggaa?cccccttcct?tcctgctcct 840
tttccaccaa?tcactggaac?atgcgattac?tggagaaacc?accctacatt?gatatggggt 900
cttcttggct?ggtggggcac?tgttggagga?gcttttggtg?cagtcagtat?tcccagtagc 960
attccagggt?ttgatcctca?catgaacctt?cttcaggctc?tttccaacac?ccgcaccgat 1020
gccattggat?ctctctaccg?tgaaggcaca?gcgtcttggt?taaactcaat?ggtcaacaat 1080
cagttccctt?tcacaacctc?tcaagtcaga?gaccagtttc?tggcaggact?ttcctcaacc 1140
aaggcagcca?caaaacaggc?ccagaccttc?aagcttgcca?atgaaggtcg?tcttaagcct 1200
agaatctga 1209
<210>2
<211>1299
<212>DNA
＜213〉swede type rape PDF1 gene C DS sequence
<400>2
atgacttgtg?tgtattgtaa?ggttgttccg?accaacgaag?tcccaagtcc?gaaagagact 60
gaccttcctc?tcaacgccta?tatgcttcac?aacctcgctc?acatcgagct?caacgctatc 120
gacttggcgt?gggacactgt?tgctcgcttc?tcccctttct?tcgacgtttt?gggtcgcaag 180
ttcttcgatg?acttcgctca?tgttgctgat?gatgagagtc?ggcacttcat?gtggtatgga 240
gatatgccgg?ctaataattt?gttgatgaga?gaatgcgaga?atacatctaa?taatgtcgct 300
gctcggttag?ctgttatccc?tcttgttcag?gaggctagag?gacttgatgc?tggacctagg 360
ttggtgaaaa?ggctgatggg?gtttggagat?cataggactt?caaaaatagt?ggctaagatt 420
gctgaggagg?gaattgcaca?tgtagctgtt?ggtgtagact?ggtttctctc?cgtttgtcaa 480
aagatgaacc?gtgctccttg?tcctactttt?aaaggcactg?acaagtatga?tccatcatgt 540
gccaccgaag?ctgatgaagg?cggcaacaaa?cagggtgaca?aggagaaact?ttctgcggtg 600
tacgacaggc?tcactcactt?aatctcaatg?gagacttcaa?ggctatgtta?tggggactcc 660
tttgtgtacg?acccaagcaa?aaccttgagg?gaacgattca?agaagtggct?tcaaacccat 720
agcatcttat?atggaggaaa?agaggagtgg?atgctacggt?ttgggatata?ccaatctaat 780
ctccagctaa?tagactacat?caactctctc?cacttgccct?ttaagttaac?agacaacaga 840
tttgcagaca?tgaccaatgc?tgagtttaag?gcccattttc?tcggtttgaa?cacgtctacc 900
ttgaggttaa?acacggacca?aaggccggtt?tgcgacgcag?taggaaatgt?gccagcctct 960
gtggactgga?gaaaagaagg?tgctgtgact?cctgttagaa?accaaggaaa?atgcggaggt 1020
tgttgggcat?tcgctacagt?ggcagctatt?gaaggcatca?acaagataaa?gacagggaac 1080
ttagtatctc?tctcagagca?acagctcata?gactgtgaca?tcagcgcttt?taacaaagga 1140
tgtagcggtg?gattactgac?aacagcatat?gaatacctca?tacccaatgg?cggaatagtc 1200
accgaggctg?actatccata?cacagctata?cagggaactt?gcgaccaaga?gaagtctcaa 1260
aacaagggtt?ttgtggaaat?catcttaacc?atggagtga 1299
<210>3
<211>1335
<212>DNA
＜213〉turnip type rape PDF1 gene C DS sequence
<400>3
atggatgaag?agtacgaggt?cattgttctc?ggtaccggtc?tcaaggagtg?catcctcagc 60
ggtctcctct?ccgtcgatgg?tgtcaaggtg?cttcacatgg?acaggaatga?ttactatggt 120
ggagaatcaa?cttcgcttaa?tctcaatcag?ctgtggaaga?agttcaggag?agaagagaag 180
gctcctgagc?atctaggtgc?tagcagggat?tacaatgttg?acatgatgcc?taagtttatg 240
atgggaaatg?gcaagcttgt?tcgtaccctt?attcacactg?acgttacaaa?gtatttgtct 300
ttcaaagctg?ttgatggaag?ctatgtcttc?gttaaaggga?aggttcaaaa?ggtgccagtt 360
actcctatgg?aggccctgaa?gtctcctctc?atgggaattt?ttgagaaacg?tcgagctggc 420
aagtttttca?gttatgttca?ggattacgac?gagaaggatc?ccaaaacaca?caatgggatg 480
gacttgacca?aagttacaac?aaaggaactg?attgcgaagt?ttggtcttga?tgacaacacc 540
attgatttta?ttggtcatgc?agtggcgctt?cacacgaatg?accaacatct?caatcaaccc 600
gccttggata?ctgtaatgag?aatgaagctc?tatgccgaat?ctcttgcacg?tttccaagga 660
acctctccat?atatttatcc?tctctatggc?ttgggagaac?taccccaggc?atttgcacgg 720
cttagtgctg?tgtatggtgg?cacatatatg?ttgtgcaaac?ctgagtgcaa?ggtagagttt 780
gatgaagagg?gtaaggttat?tggtgtaaca?tctgagggag?agactgccaa?atgcaaaaag 840
attgtgtgtg?acccttccta?ccttccaaac?aaggttcgga?agattggtac?agttgctcgg 900
gccatcgcaa?ttatgagcca?ccctattcca?aacaccaatg?actctcactc?agtgcaggtc 960
atcatacctc?agaagcagct?gggccgcaaa?tcagacatgt?atgtcttctg?ctgttcctac 1020
tcccacaacg?ttgctcccaa?gggaaaattc?atcgcatttg?tatctacaga?tgcagagact 1080
gataaccctc?aaaccgaact?aaaggctggg?attgatcttt?tgggtcctgt?tgatgagata 1140
ttcttcgaca?tgtatgatag?atatgagcct?gtcaacgagc?ctgctctgga?caactgcttt 1200
atatcaacga?gctatgatgc?tacaacacac?ttcgagacaa?ctgttgctga?tgtgttgaac 1260
atgtataccc?tgatcaccgg?aaagcaactg?gacctaagtg?ttgatctgag?tgcagcaagt 1320
gctgcagagg?aatga 1335
<210>4
<211>402
<212>PRT
＜213〉aminoacid sequence of wild cabbage PDF1 albumen
<400>4
Met?Arg?Gly?Met?Lys?Ser?Leu?Thr?Ile?Trp?Ala?Ile?Phe?Ala?Ala?Val
1 5 10 15
Leu?Ser?Gln?Gln?Leu?Phe?Ala?Ser?Val?Ala?Ser?Ile?Lys?Phe?Glu?Asp
20 25 30
Glu?Lys?Thr?Tyr?Tyr?Ser?Pro?Pro?Asp?Pro?Asn?Ala?Gly?Ser?Pro?Pro
35 40 45
Ser?Gly?Thr?Pro?Pro?Ser?His?Gly?Gly?Tyr?Thr?Pro?Thr?Pro?Pro?Thr
50 55 60
Pro?Ser?His?Thr?Pro?Pro?Ser?Asn?Cys?Gly?Ser?Pro?Pro?Tyr?Asp?Pro
65 70 75 80
Thr?Pro?Pro?Ser?His?Thr?Pro?Thr?Pro?Ser?Thr?Pro?Ser?His?Thr?Pro
85 90 95
Ser?Thr?Pro?Ser?His?Thr?Pro?Thr?Pro?Ser?Thr?Pro?Ser?His?Thr?Pro
100 105 110
Thr?Pro?Ser?His?Thr?Ser?Pro?Pro?Cys?His?Cys?Gly?Thr?Pro?Pro?Ser
115 120 125
His?Pro?Ser?Thr?Pro?Ser?Arg?Pro?Ser?Arg?Pro?Ser?His?Pro?Ser?Arg
130 135 140
Pro?Ser?Arg?Pro?Ser?Thr?Pro?Ser?Asn?Pro?Pro?Ser?Gly?Gly?Tyr?Tyr
145 150 155 160
Ser?Ser?Pro?Pro?Pro?Ser?Thr?Pro?Val?Val?Val?Thr?Pro?Pro?Thr?Pro
165 170 175
Ile?Val?Asp?Pro?Gly?Thr?Pro?Ser?Thr?Gly?Gly?Thr?Pro?Pro?Ser?Ser
180 185 190
Gly?Gly?Tyr?Tyr?Ser?Ser?Pro?Pro?Pro?Ser?Thr?Pro?Val?Val?Glu?Thr
195 200 205
Pro?Pro?Thr?Pro?Ile?Val?Asp?Pro?Gly?Thr?Pro?Ile?Ile?Gly?Glu?Thr
210 215 220
Pro?Pro?Thr?Pro?Ser?Gly?Gly?Tyr?Tyr?Ser?Ser?Pro?Pro?Pro?Ser?Thr
225 230 235 240
Pro?Ile?Val?Val?Thr?Pro?Pro?Thr?Pro?Ile?Val?Asp?Pro?Gly?Thr?Pro
245 250 255
Ile?Ile?Gly?Gly?Thr?Pro?Pro?Thr?Pro?Phe?Ile?Asp?Pro?Gly?Thr?Pro
260 265 270
Gly?Thr?Pro?Phe?Leu?Pro?Ala?Pro?Phe?Pro?Pro?Ile?Thr?Gly?Thr?Cys
275 280 285
Asp?Tyr?Trp?Arg?Asn?His?Pro?Thr?Leu?Ile?Trp?Gly?Leu?Leu?Gly?Trp
290 295 300
Trp?Gly?Thr?Val?Gly?Gly?Ala?Phe?Gly?Ala?Val?Ser?Ile?Pro?Ser?Ser
305 310 315 320
Iie?Pro?Gly?Phe?Asp?Pro?His?Met?Asn?Leu?Leu?Gln?Ala?Leu?Ser?Asn
325 330 335
Thr?Arg?Thr?Asp?Ala?Ile?Gly?Ser?Leu?Tyr?Arg?Glu?Gly?Thr?Ala?Ser
340 345 350
Trp?Leu?Asn?Ser?Met?Val?Asn?Asn?Gln?Phe?Pro?Phe?Thr?Thr?Ser?Gln
355 360 365
Val?Arg?Asp?Gln?Phe?Leu?Ala?Gly?Leu?Ser?Ser?Thr?Lys?Ala?Ala?Thr
370 375 380
Lys?Gln?Ala?Gln?Thr?Phe?Lys?Leu?Ala?Asn?Glu?Gly?Arg?Leu?Lys?Pro
385 390 395 400
Arg?Ile
<210>5
<211>432
<212>PRT
＜213〉aminoacid sequence of swede type rape PDF1 albumen
<400>5
Met?Thr?Cys?Val?Tyr?Cys?Lys?Val?Val?Pro?Thr?Asn?Glu?Val?Pro?Ser
1 5 10 15
Pro?Lys?Glu?Thr?Asp?Leu?Pro?Leu?Asn?Ala?Tyr?Met?Leu?His?Asn?Leu
20 25 30
Ala?His?Ile?Glu?Leu?Asn?Ala?Ile?Asp?Leu?Ala?Trp?Asp?Thr?Val?Ala
35 40 45
Arg?Phe?Ser?Pro?Phe?Phe?Asp?Val?Leu?Gly?Arg?Lys?Phe?Phe?Asp?Asp
50 55 60
Phe?Ala?His?Val?Ala?Asp?Asp?Glu?Ser?Arg?His?Phe?Met?Trp?Tyr?Gly
65 70 75 80
Asp?Met?Pro?Ala?Asn?Asn?Leu?Leu?Met?Arg?Glu?Cys?Glu?Asn?Thr?Ser
85 90 95
Asn?Asn?Val?Ala?Ala?Arg?Leu?Ala?Val?Ile?Pro?Leu?Val?Gln?Glu?Ala
100 105 110
Arg?Gly?Leu?Asp?Ala?Gly?Pro?Arg?Leu?Val?Lys?Arg?Leu?Met?Gly?Phe
115 120 125
Gly?Asp?His?Arg?Thr?Ser?Lys?Ile?Val?Ala?Lys?Ile?Ala?Glu?Glu?Gly
130 135 140
Ile?Ala?His?Val?Ala?Val?Gly?Val?Asp?Trp?Phe?Leu?Ser?Val?Cys?Gln
145 150 155 160
Lys?Met?Asn?Arg?Ala?Pro?Cys?Pro?Thr?Phe?Lys?Gly?Thr?Asp?Lys?Tyr
165 170 175
Asp?Pro?Ser?Cys?Ala?Thr?Glu?Ala?Asp?Glu?Gly?Gly?Asn?Lys?Gln?Gly
180 185 190
Asp?Lys?Glu?Lys?Leu?Ser?Ala?Val?Tyr?Asp?Arg?Leu?Thr?His?Leu?Ile
195 200 205
Ser?Met?Glu?Thr?Ser?Arg?Leu?Cys?Tyr?Gly?Asp?Ser?Phe?Val?Tyr?Asp
210 215 220
Pro?Ser?Lys?Thr?Leu?Arg?Glu?Arg?Phe?Lys?Lys?Trp?Leu?Gln?Thr?His
225 230 235 240
Ser?Ile?Leu?Tyr?Gly?Gly?Lys?Glu?Glu?Trp?Met?Leu?Arg?Phe?Gly?Ile
245 250 255
Tyr?Gln?Ser?Asn?Leu?Gln?Leu?Ile?Asp?Tyr?Ile?Asn?Ser?Leu?His?Leu
260 265 270
Pro?Phe?Lys?Leu?Thr?Asp?Asn?Arg?Phe?Ala?Asp?Met?Thr?Asn?Ala?Glu
275 280 285
Phe?Lys?Ala?His?Phe?Leu?Gly?Leu?Asn?Thr?Ser?Thr?Leu?Arg?Leu?Asn
290 295 300
Thr?Asp?Gln?Arg?Pro?Val?Cys?Asp?Ala?Val?Gly?Asn?Val?Pro?Ala?Ser
305 310 315 320
Val?Asp?Trp?Arg?Lys?Glu?Gly?Ala?Val?Thr?Pro?Val?Arg?Asn?Gln?Gly
325 330 335
Lys?Cys?Gly?Gly?Cys?Trp?Ala?Phe?Ala?Thr?Val?Ala?Ala?Ile?Glu?Gly
340 345 350
Ile?Asn?Lys?Ile?Lys?Thr?Gly?Asn?Leu?Val?Ser?Leu?Ser?Glu?Gln?Gln
355 360 365
Leu?Ile?Asp?Cys?Asp?Ile?Ser?Ala?Phe?Asn?Lys?Gly?Cys?Ser?Gly?Gly
370 375 380
Leu?Leu?Thr?Thr?Ala?Tyr?Glu?Tyr?Leu?Ile?Pro?Asn?Gly?Gly?Ile?Val
385 390 395 400
Thr?Glu?Ala?Asp?Tyr?Pro?Tyr?Thr?Ala?Ile?Gln?Gly?Thr?Cys?Asp?Gln
405 410 415
Glu?Lys?Ser?Gln?Asn?Lys?Gly?Phe?Val?Glu?Ile?Ile?Leu?Thr?Met?Glu
420 425 430
<210>6
<211>444
<212>PRT
＜213〉aminoacid sequence of turnip type rape PDF1 albumen
<400>6
Met?Asp?Glu?Glu?Tyr?Glu?Val?Ile?Val?Leu?Gly?Thr?Gly?Leu?Lys?Glu
1 5 10 15
Cys?Ile?Leu?Ser?Gly?Leu?Leu?Ser?Val?Asp?Gly?Val?Lys?Val?Leu?His
20 25 30
Met?Asp?Arg?Asn?Asp?Tyr?Tyr?Gly?Gly?Glu?Ser?Thr?Ser?Leu?Asn?Leu
35 40 45
Asn?Gln?Leu?Trp?Lys?Lys?Phe?Arg?Arg?Glu?Glu?Lys?Ala?Pro?Glu?His
50 55 60
Leu?Gly?Ala?Ser?Arg?Asp?Tyr?Asn?Val?Asp?Met?Met?Pro?Lys?Phe?Met
65 70 75 80
Met?Gly?Asn?Gly?Lys?Leu?Val?Arg?Thr?Leu?Ile?His?Thr?Asp?Val?Thr
85 90 95
Lys?Tyr?Leu?Ser?Phe?Lys?Ala?Val?Asp?Gly?Ser?Tyr?Val?Phe?Val?Lys
100 105 110
Gly?Lys?Val?Gln?Lys?Val?Pro?Val?Thr?Pro?Met?Glu?Ala?Leu?Lys?Ser
115 120 125
Pro?Leu?Met?Gly?Ile?Phe?Glu?Lys?Arg?Arg?Ala?Gly?Lys?Phe?Phe?Ser
130 135 140
Tyr?Val?Gln?Asp?Tyr?Asp?Glu?Lys?Asp?Pro?Lys?Thr?His?Asn?Gly?Met
145 150 155 160
Asp?Leu?Thr?Lys?Val?Thr?Thr?Lys?Glu?Leu?Ile?Ala?Lys?Phe?Gly?Leu
165 170 175
Asp?Asp?Asn?Thr?Ile?Asp?Phe?Ile?Gly?His?Ala?Val?Ala?Leu?His?Thr
180 185 190
Asn?Asp?Gln?His?Leu?Asn?Gln?Pro?Ala?Leu?Asp?Thr?Val?Met?Arg?Met
195 200 205
Lys?Leu?Tyr?Ala?Glu?Ser?Leu?Ala?Arg?Phe?Gln?Gly?Thr?Ser?Pro?Tyr
210 215 220
Ile?Tyr?Pro?Leu?Tyr?Gly?Leu?Gly?Glu?Leu?Pro?Gln?Ala?Phe?Ala?Arg
225 230 235 240
Leu?Ser?Ala?Val?Tyr?Gly?Gly?Thr?Tyr?Met?Leu?Cys?Lys?Pro?Glu?Cys
245 250 255
Lys?Val?Glu?Phe?Asp?Glu?Glu?Gly?Lys?Val?Ile?Gly?Val?Thr?Ser?Glu
260 265 270
Gly?Glu?Thr?Ala?Lys?Cys?Lys?Lys?Ile?Val?Cys?Asp?Pro?Ser?Tyr?Leu
275 280 285
Pro?Asn?Lys?Val?Arg?Lys?Ile?Gly?Thr?Val?Ala?Arg?Ala?Ile?Ala?Ile
290 295 300
Met?Ser?His?Pro?Ile?Pro?Asn?Thr?Asn?Asp?Ser?His?Ser?Val?Gln?Val
305 310 315 320
Ile?Ile?Pro?Gln?Lys?Gln?Leu?Gly?Arg?Lys?Ser?Asp?Met?Tyr?Val?Phe
325 330 335
Cys?Cys?Ser?Tyr?Ser?His?Asn?Val?Ala?Pro?Lys?Gly?Lys?Phe?Ile?Ala
340 345 350
Phe?Val?Ser?Thr?Asp?Ala?Glu?Thr?Asp?Asn?Pro?Gln?Thr?Glu?Leu?Lys
355 360 365
Ala?Gly?Ile?Asp?Leu?Leu?Gly?Pro?Val?Asp?Glu?Ile?Phe?Phe?Asp?Met
370 375 380
Tyr?Asp?Arg?Tyr?Glu?Pro?Val?Asn?Glu?Pro?Ala?Leu?Asp?Asn?Cys?Phe
385 390 395 400
Ile?Ser?Thr?Ser?Tyr?Asp?Ala?Thr?Thr?His?Phe?Glu?Thr?Thr?Val?Ala
405 410 415
Asp?Val?Leu?Asn?Met?Tyr?Thr?Leu?Ile?Thr?Gly?Lys?Gln?Leu?Asp?Leu
420 425 430
Ser?Val?Asp?Leu?Ser?Ala?Ala?Ser?Ala?Ala?Glu?Glu
435 440
Claims (6)
1. the gene of a separation, its sequence is the nucleotide sequence shown in the SEQ ID No.2.
2. isolated polypeptide, its sequence is the aminoacid sequence shown in the SEQ ID No.5.
3. the application of gene claimed in claim 1 in control flower pesticide fertility.
4. the application of polypeptide claimed in claim 2 in control flower pesticide fertility.
5. the application of gene claimed in claim 1 in the apoptosis supressor.
6. the application of polypeptide claimed in claim 2 in the apoptosis supressor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010190495 CN101974081B (en) | 2010-05-28 | 2010-05-28 | PDF1 gene for controlling plant pollen fertility, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010190495 CN101974081B (en) | 2010-05-28 | 2010-05-28 | PDF1 gene for controlling plant pollen fertility, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101974081A CN101974081A (en) | 2011-02-16 |
| CN101974081B true CN101974081B (en) | 2013-03-06 |
Family
ID=43573998
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010190495 Active CN101974081B (en) | 2010-05-28 | 2010-05-28 | PDF1 gene for controlling plant pollen fertility, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101974081B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014018B (en) * | 2012-11-27 | 2014-03-12 | 中国农业科学院油料作物研究所 | Rape bnrabgdi3 gene and application thereof |
| CN103045610A (en) * | 2012-12-01 | 2013-04-17 | 中国农业科学院油料作物研究所 | Rape bnpab5 gene and application thereof |
| AU2015321591B2 (en) * | 2014-09-26 | 2022-03-10 | Pioneer Hi-Bred International, Inc. | Wheat MS1 polynucleotides, polypeptides, and mehtods of use |
| CN109161551B (en) * | 2018-09-27 | 2022-07-26 | 西南大学 | Cabbage BoMS1 gene and application thereof in creating sterile materials |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1249133A (en) * | 1998-12-25 | 2000-04-05 | 北京大学 | Molecular method for culturing male sterile plant |
| CN1611607A (en) * | 1999-11-19 | 2005-05-04 | 独立行政法人农业生物资源研究所 | Method for reducing fertilization ability of pollen using |
-
2010
- 2010-05-28 CN CN 201010190495 patent/CN101974081B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1249133A (en) * | 1998-12-25 | 2000-04-05 | 北京大学 | Molecular method for culturing male sterile plant |
| CN1611607A (en) * | 1999-11-19 | 2005-05-04 | 独立行政法人农业生物资源研究所 | Method for reducing fertilization ability of pollen using |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101974081A (en) | 2011-02-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Gene silencing using the recessive rice bacterial blight resistance gene xa13 as a new paradigm in plant breeding | |
| Schmülling et al. | Restoration of fertility by antisense RNA in genetically engineered male sterile tobacco plants | |
| CN109576282B (en) | Chinese rose transcription factor RhMYB4 and application thereof in flower organ development regulation | |
| WO2019062895A1 (en) | Use of maize gene zmabcg20 in regulating crop male fertility and dna molecular markers associated with maize male fertility and use thereof | |
| CN103131715A (en) | Plant flavonoid synthesis regulation gene and its application | |
| WO2007030510A2 (en) | Plants modified with mini-chromosomes | |
| CN110093354A (en) | A kind of seed separation method of fixed plant hybrid advantage | |
| CN101974081B (en) | PDF1 gene for controlling plant pollen fertility, and preparation method and application thereof | |
| CN112522283A (en) | Pollen development related gene and application thereof | |
| CN102250903A (en) | Application of rice miR166 in enhancing plant drought stress tolerance | |
| CN109554373B (en) | Rice FON2 gene mutant and molecular identification method and application thereof | |
| de Groot et al. | Silencing of the pollen-specific gene NTP303 and its family members in tobacco affects in vivo pollen tube growth and results in male sterile plants | |
| CN112094862A (en) | Creation method of marked cotton male sterile line | |
| CN110229825A (en) | The brown Leaf color mutant GBL1 gene of rice ash and its application | |
| CN113088526B (en) | Heat shock related gene ZmHsf11 and application thereof in regulation and control of plant heat resistance | |
| CN101987867B (en) | Ethylene receptor NTHK1 interactive protein relevant to plant stress tolerance as well as coding gene and application thereof | |
| Li et al. | Modified fiber qualities of the transgenic cotton expressing a silkworm fibroin gene | |
| CN102115751A (en) | Rape BnPABP 5 gene and application of promoter thereof | |
| CN102718853B (en) | Upland cotton GhLFY protein and coding gene and application thereof | |
| WO2021114156A1 (en) | Petal purple spot protein and coding gene thereof | |
| CN103014018B (en) | Rape bnrabgdi3 gene and application thereof | |
| CN103923917A (en) | Application of microRNA444a or coding gene thereof in adjustment of rice salt tolerance | |
| WO2013138399A1 (en) | Improving agronomic characteristics of plants through abph2 | |
| CN103045610A (en) | Rape bnpab5 gene and application thereof | |
| US9850495B2 (en) | Nucleotide sequences encoding fasciated EAR4 (FEA4) and methods of use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |