CN106906212A - One sesame anther specific expression promoter PSicwinv1 and its application - Google Patents
One sesame anther specific expression promoter PSicwinv1 and its application Download PDFInfo
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- CN106906212A CN106906212A CN201610528610.8A CN201610528610A CN106906212A CN 106906212 A CN106906212 A CN 106906212A CN 201610528610 A CN201610528610 A CN 201610528610A CN 106906212 A CN106906212 A CN 106906212A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/823—Reproductive tissue-specific promoters
- C12N15/8231—Male-specific, e.g. anther, tapetum, pollen
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
- C12N15/8289—Male sterility
Abstract
The invention belongs to field of plant genetic, and in particular to a sesame anther specific expression promoter PSicwinv1 and its application.The nucleotide sequence such as sequence table SEQ ID NO of the promoter that the present invention is separate：Shown in 1.Biological function checking is carried out to the promoter, GUS dyeing detections show, the specific promoter PSicwinv1 of present invention clone drives reporter gene not expressed in the root of arabidopsis, stem, leaf and pod, only the specifically expressing in flower pesticide, and its expression quantity is relevant with the developmental stage of flower pesticide.In situ hybridization shows that Sicwinv1 is expressed in the tetrad a large amount of sesame anther development, and the main expression in tapetum and tetrad.It is anther specific expression promoter the invention discloses promoter PSicwinv1, research and the heterosis utilization of sesame male sterility gene engineering can be used for.
Description
Technical field
The invention belongs to field of plant genetic.Specifically related to one sesame anther specific expression promoter
PSicwinv1 and its application.Separated from sesame, identification obtains a promoter for the Sicwinv1 genes of specifically expressing, will
It is named as PSicwinv1.The promoter specifically expressing in flower pesticide, without expression in its hetero-organization.Present invention clone
Promoter can be applied in sesame or other plant anther development and male sterility gene engineering and breed improvement.
Background technology
Promoter is to be located at gene 5 ' end upstream, guide RNA polymerase and transcription factor specific bond, and then promotor gene
One section of specific dna sequence of specifically expressing, is the important cis-acting elements of controlling gene expression.Promoter is in plant gene work
Played an important role in journey, be the important component of gene engineering expression carrier, regulate and control the transcriptional efficiency of foreign gene
And spatial and temporal expression profile.
According to the regulating and expressing pattern of promoter, promoter can be divided into constitutive promoter and specific promoter.Group
Constitutive promoter drives foreign gene in each tissue and stage of development high efficient expression, without Space-time speciality.Typically should
There are cauliflower mosaic virus (CaMV) 35S promoter, rice actin Act for the constitutive promoter of plant genetic engineering
Promoter and maize ubiquitin Ubi promoters (close beautiful English etc., the effective expression of foreign gene and its safety are commented in genetically modified plants
Valency Capital Normal Universitys journal, 2002,23 (2)：52-56).But, efficient, the non-specific expression of constitutive promoter is easily made
Consumed excessively into cellular material, and target gene can not be expressed by specific spatiotemporal database, cause some negative effects to produce.
Thus, specific promoter is of increased attention in recent years.Specific promoter includes tissue specific promoter
And inducible promoter.Tissue specific promoter drives target gene to be expressed in specific tissue or organ, generally adjoint
There is the characteristic of Growth adjustment.Such promoter generally there are some specific motifs related to tissue specificity (He Hongxia etc., group
Knit progress China agronomy circular of the specificity promoter in crop gene engineering, 2014,30 (9)：225-231).Group
Knit specific promoter and effectively avoid the negative effects such as the wasting of resources and cytotoxic, have in transgenic breeding important
Application value.
In recent years, some tissue specific promoters are separated and identified in succession, such as chlorenchyma specific promoter, fruit
And seed-specific expression promoter, root-specific promoter, vascular-specific promoter and floral organ specific promoter, and it is applied to correlation
Genetic improvement (Potenza the et al., 2004, Targeting transgene expression in of proterties
research,agricultural,and environmental applications:Promoters used in plant
transformation.In Vitro Cell Dev Biol Plant,40:1-22).Opened using flower pesticide or pollen-specific expression
Mover initiative male sterile line is applied to crossbreeding and has made some progress.Using the special table of tobacco anther tapetum
The promoter P for reachingTA29The carrier genetic transformation for merging the ribonucleic acid hydrolase Bar gene nase structures of bacillus amyloliquefacienses is received
Body plant, has formulated male sterile line (Mariani et al., 1990, Induction of male sterility in
plants by a chimaeric ribonuclease gene.Nature,347:737-741).Paddy rice RTS genes are in subtrahend
Expressed in the anther tapetum of division, merge the antisense strand of barnase or RTS using the promoter of the gene and convert water
Rice, arabidopsis cause transfer-gen plant male sterility (Luo et al., 2006, RTS, a rice anther-specific
gene is required for male fertility and its promoter sequence directs tissue-
specific gene expression in different plant species.Plant Mol Biol,62:397-
408)。
Sesame has stronger hybrid vigour, and male sterility is the important channel that crop heterosis are utilized, using male
Sterile line initiative cenospecies has great importance for sesame genetic improvement.Thus, excavate sesame flower pesticide specifically expressing and start
Son has important application value for sesame male sterility related gene functional verification and male sterile line initiative.
The content of the invention
It is an object of the invention to provide the anther specific expression promoter that separates clone from sesame.It is of the invention
Promoter belongs to a kind of endogenesis promoter, and the endogenous gene for showing promoter driving table in flower pesticide is detected by RT-PCR
Reach, without expression in its hetero-organization.Show that the gene that promoter drives only exists by the expression characteristic of examining report gene GUS
Expressed in flower pesticide, and its expression quantity is relevant with the developmental stage of flower pesticide.In situ hybridization shows the endogenous base that the promoter drives
Because of the tetrad expression quantity highest in sesame anther development.The present invention not only enriches sesame anther specific expression promoter
Resource, while imply that the promoter has quite wide application prospect in male sterility research.The promoter is used
In the functional verification and the initiative of male sterile line of male sterility related gene, for heterosis utilization provide technical foundation and
Genetic resources.
Realize that the technical scheme of the object of the invention is as described below：
In order to obtain the present invention, inventor compares the fertile plant and not of sesame genic male sterile system 95ms-5
A plant tetrad flower pesticide transcript profile is educated, the gene Sicwinv1 of isolated significant difference expression from sesame, to this
The tissue expression analysis checking of gene shows, the gene only specifically expressing in the flower pesticide of sesame, in root, stem, leaf, petal, female
(Fig. 1) is not expressed in stamen, column cap.The promoter PSicwinv1 for obtaining the gene is expanded by PCR, its nucleotide sequence is such as
Sequence table SEQ NO:Shown in 1.
Promoter of the invention derives from sesame, by 1205 base compositions.By building by the report of the promoter regulation
Accuse the plant expression vector PCAMBIA1381Xb-Pro of gene GUSSicwinv1(the B figures in Fig. 2), and the conversion carrier is converted
To in Columbia ecotype arabidopsis, the transgenic positive Arabidopsis plant of the promoter is obtained.Sent out by GUS staining analysis
Existing, the promoter only drives gus gene to be expressed in flower pesticide, and gus gene is can't detect in root, stem, leaf, pod, petal
Expression (Fig. 3).And in the different development stage of flower pesticide, PSicwinv1 drives the expression of Reporter gene GUS to have differences
(Fig. 4).Shown by situ Analysis, PSicwinv1 drives endogenous gene in the tetrad scale high of sesame flower pesticide
Reach, and the main expression (Fig. 5) in tapetum and tetrad.These researchs show that PSicwinv1 is a special table of flower pesticide
Up to promoter, can be applied in plants male sterility genetic engineering and plant hybrid use of advantage and breed improvement.
Concrete operation step of the invention is as follows：
(1) according to the promoter amplimer of Sesame group sequences Design Sicwinv1 genes, with applicant's early stage work
Sesame genic male sterile system 95ms-5 (Zhao et al., 2013, Characterization and that work is obtained
genetic mapping of a novel recessive genic male sterile gene in sesame
(Sesamum indicum L.).Mol Breeding,32:
DNA 901-908) is template, expands promoter sequence, the nucleotide sequence such as SEQ ID NO of the promoter:1
It is shown.
Primer sequence for expanding Sicwinv1 genes is as follows：
PSicwinv1-F：5'-CCATATCATTTTCTGGCCTTTC-3'
PSicwinv1-R：5'-GGTTGAGGAAAACCTCATGG-3'
PSicwinv1-MF：5'-GAGAATTC(EcoRI)CTGGCCTTTCTATTAATTGA-3'
PSicwinv1-MR：5'-GCAAGCTT(HindIII)CTCCAAGATTGAGACTACAC-3'
(2) by step 1) amplification obtained DNA fragmentation and PCAMBIA1381Xb plasmids (Fig. 2A), carry out digestion company
It is reversed to answer, the promoter is connected on carrier PCAMBIA1381Xb, then by heat-shock transformed competent escherichia coli cell
In TOP10, the recombinant vector containing the promoter is obtained, this recombinant vector is named as plant recombinant vector by applicant
PCAMBIA1381Xb-ProSicwinv1(Fig. 2 B).Using agriculture bacillus mediated transgenic method, described vector introduction is intended into south
In mustard, transformed plant is obtained.
(3) method counted by hygromycin selection and segregation ratio obtains transgenic positive plant, and by GUS dyeing point
Analysis, detection promoter PSicwinv1Expression.
(4) by the method for in situ hybridization, detection promoter PSicwinv1 drives table of the endogenous gene in sesame flower pesticide
Expression patterns.
The advantage of the invention is that：
1. the promoter PSicwinv1 of present invention clone is anther specific expression promoter, and target gene can be driven only to exist
Flower pesticide is expressed, and without being expressed in its hetero-organization, therefore the present invention can effectively overcome the negative effect that constitutive promoter is caused
Should.
2. can be used in studying male sterility with the sesame anther specific expression promoter PSicwinv1 of present invention clone
The functional verification of related gene, it is also possible to utilize promoter creating plants male sterility system of the invention or breed improvement.
Brief description of the drawings
Sequence table SEQ ID NO:1 is the nucleosides of the promoter PSicwinv1 that the present invention separates clone's Sicwinv1 genes
Acid sequence, sequence length is 1205bp.
Fig. 1：The tissue expression pattern for detecting Sicwinv1 genes using the method for RT-PCR is analyzed.Description of reference numerals：
In Fig. 1, first swimming lane is root (being labeled as Ro) from left to right, and second swimming lane is stem (being labeled as St), and the 3rd swimming lane is leaf (mark
It is Le to note), the 4th swimming lane is capsule (being labeled as Ca), and the 5th swimming lane is bud (being labeled as Bu), and the 6th swimming lane is flower
Hat (being labeled as Co), the 7th swimming lane is gynoecium (being labeled as Pi), and the 8th swimming lane is stamen (being labeled as Sta), and SiUBQ6 is
Sesame ubiquitin protein (number of logging in is JP631638), it is whether consistent to detect applied sample amount as reference gene.
Fig. 2：Build the carrier used by promoter expression vector.Description of reference numerals：Fig. 2A is used expression vector
The collection of illustrative plates of PCAMBIA1381Xb, Fig. 2 B are plant recombinant vector PCAMBIA1381Xb-Pro prepared by the present inventionSicwinv1Collection of illustrative plates.
Fig. 3：A kind of histochemical stain result schematic diagram of the gus gene that PSicwinv1 promoters drive.Reference
Explanation：A figures in Fig. 3 are the bud of transgenic arabidopsis, and the B figures in Fig. 3 are the flower pesticide of transgenic arabidopsis, the C figures in Fig. 3
It is the inflorescence of transgenic arabidopsis, the D figures in Fig. 3 are transgenic arabidopsis seedling, the E figures in Fig. 3 are transgenic arabidopsis
Pod.GUS coloration results are shown, blueness is only detected in the flower pesticide of arabidopsis, and without blue aobvious in its hetero-organization
Show, show promoter PSicwinv1 drive gus gene only in arabidopsis flower pesticide express, and root, stem, leaf, pod and
Do not expressed in petal and filigree.
Fig. 4：Expression of the gus gene that promoter PSicwinv1 drives in the flower pesticide of the different development stage of arabidopsis
Situation schematic diagram.Description of reference numerals：Bud size increases successively from left to right in Fig. 4, shows that promoter PSicwinv1 drives
Gus gene expression quantity height it is relevant with Anther stage.
Fig. 5：Expression of the Sicwinv1 genes in the flower pesticide of the different development stage of sesame is identified in situ hybridization.It is attached
Figure description of symbols：A-C figures in Fig. 5 are results of hybridization of the mRNA antisense probes of Sicwinv1 genes in sesame flower pesticide, Fig. 5
In D-F figures be positive-sense strand probe hybridization after negative control.A-C figures in Fig. 5 are respectively pollen mother cell period, tetrad
Period and mature pollen period；When D-F figures in prominent 5 are respectively pollen mother cell period, tetrad and mature pollen
Phase.
Specific embodiment
Following examples define the present invention, and according to following description and these embodiments, those skilled in the art can be with
Determine essential characteristic of the invention, and without departing from the spirit and scope of the invention, the present invention can be made respectively
Plant and change and change, so that it is applicable different purposes and condition.
The separation clone of the promoter sequence of the Sicwinv1 genes of embodiment 1 and expression pattern analysis
Sesame recessive karyon of the applicant in the previous work of Inst. of Oil Crops, Chinese Academy of Agriculture is male
Property sterile line 95ms-5 (Zhao et al., 2013, Characterization and genetic mapping of a
novel recessive genic male sterile gene in sesame(Sesamum indicum L.).Mol
Breeding,32:One section of est sequence is isolated in fertile plant 901-908) and sterile plant tetrad flower pesticide transcript profile,
TBLASTx comparisons are carried out in NCBI gene pools to this sequence, it is found that this sequence is probably cell membrane invertase gene.This
Bar est sequence does not include complete opening code-reading frame, and applicant is using routine RACE (rapid-amplification of
CDNA ends) method carries out the cDNA ends quick clone technology coded sequence complete to obtain this gene by PCR.By this
The complete coded sequence of bar is compared with Sesame group, obtains the genome sequence of the gene, is drawn according to genome sequence design
Thing, expands promoter sequence.
A. the extraction of Sesame group DNA
DNA (the extracting method reference Uzun of sesame genic male sterile 95ms-5 are extracted using conventional CTAB methods
and Cagirgan,2009,Identification of molecular markers linked to determinate
The method of growth habit in sesame.Euphytica, 166,379-384 reports).
The acquisition of B.Sicwinv1 gene promoter sequences
Genome sequence design promoter amplimer according to Sicwinv1, primer is as follows：Sense primer
PSicwinv1-F5'-CCATATCATTTTCTGGCCTTTC-3', anti-sense primer PSicwinv1-R5'-
GGTTGAGGAAAACCTCATGG-3', the DNA with 95ms-5B expand the promoter sequence of Sicwinv1 using PCR as template.
PCR reaction systems are：10×buffer 2μl；dNTP 0.4μl；Each 0.2 μM of upstream and downstream primer；The μ l of Taq polymerase 0.2, plus
ddH2O complements to 20 μ l systems.PCR conditions are 94 DEG C of predegeneration 3min；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 1min, 28
Individual circulation；72 DEG C of extension 7min.The PCR primer for obtaining will be expanded and is connected into pGEM-T carriers (purchased from Promega companies, the U.S.),
Screening positive clone is simultaneously sequenced.
C. expression pattern analysis of the extraction of the total serum IgE of sesame different tissues and the acquisition of cDNA and Sicwinv1
The extraction of RNA uses Trizol kits (being purchased from Invitrogen companies, the U.S.), with sesame 95ms-5 as material
Material, extracts the RNA of root, stem, leaf, capsule, bud, corolla, gynoecium and each tissue sample of stamen, using reverse transcriptase
Its reverse transcription is synthesized cDNA by Superscript III (being purchased from Invitrogen companies, the U.S.), and reaction condition is：65℃
5min, 50 DEG C of 60min, 70 DEG C of 10min.CDNA with above-mentioned reverse transcription synthesis is detected as template using RT-PCR
The expression pattern of Sicwinv1, the primer is：Sicwinv1-F：(5'-ATGGAGTTTCGGGCCAAGAG-3') and
Sicwinv1-R：(5'-GATTAAGAGGTGCAGGCAGGGAC-3').Use primer SiUbiquitin6-F simultaneously：(5'-
) and SiUbiquitin6-R CACCAAGCCGAAGAAGATCAAG-3'：(5'-CCTCAGCCTCTGCACCTTTC-3') is to sesame
SiUbiquitin6 (the GenBank numbers of logging in：JP631638) gene does specific amplified, and relative quantification point is carried out as internal reference control
Analysis.Result shows：The promoter of present invention clone drives endogenous gene to be expressed in flower pesticide, do not expressed in its hetero-organization (see
Fig. 1).
Embodiment 2：Structure containing Sicwinv1 gene promoter PSicwinv1 expression vectors
It is used for construction of expression vector according to the promoter PSicwinv1 primers for obtaining, method is in primer two
End adds restriction enzyme site respectively, and described primer sequence is respectively PSicwinv1-MF (5'-GAGAATTC (EcoRI)
) and PSicwinv1-MR (5'-GCAAGCTT (HindIII) CTGGCCTTTCTATTAATTGA-3'
CTCCAAGATTGAGACTACAC-3'), performing PCR amplification is entered by template of T-Sicwinv1 plasmids, PCR reaction systems are：10×
buffer 2μl；dNTP 0.4μl；Each 0.2 μM of upstream and downstream primer；The μ l of pfu polymerases 0.2, plus ddH2O complements to 20 μ l systems.
PCR reaction conditions are：94 DEG C of predegeneration 3min；94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 1min, totally 28 circulations；72℃
7min is extended to, PCR primer (is purchased from Axygen companies, U.S. by detected through gel electrophoresis using DNA gel QIAquick Gel Extraction Kit
State) reclaim purpose PCR primer.PCR primer after recovery is by HindIII and EcoRI (purchased from the limited public affairs in precious bioengineering Dalian
Department) double digestion is carried out, endonuclease reaction system is：10 × Fastdigest Buffer 2 μ l, DNA 1 μ g, HindIII and EcoRI
Each 1 μ l, plus ddH2The μ l of O to 20, after fully mixing, place 1h in 37 DEG C of insulating boxs, and digestion products then are passed through into PCR primer
Purification kit (being purchased from Bioflux companies, Romania) is purified.Vector plasmid is passed through into HindIII and EcoRI simultaneously
Double digestion is carried out, endonuclease reaction system ibid, then by digestion products by detected through gel electrophoresis, and is reclaimed using DNA gel
Kit (be purchased from Axygen companies, the U.S.) reclaims purpose carrier segments (target fragment is large fragment), after by the mesh after recovery
Carrier segment with purifying digestion PCR primer be attached reaction, coupled reaction system is：By purpose carrier segments 100ng,
Target PCR fragment 50ng, adds the μ l of 10 × T4 ligases Buffer, 2 μ l, T4 ligases 1 (being purchased from Thermo companies, the U.S.),
Sterilized water is supplemented to 20 μ l mixings, after 22 DEG C of 10min, in 4 DEG C of overnight coupled reactions.Then converted by conventional heat shock method big
Enterobacteria competent cell TOP10, with the special primer PSicwinv1-MF (5'-GAGAATTC (EcoRI) of Sicwinv1
) and PSicwinv1-MR (5'-GCAAGCTT (HindIII) CTGGCCTTTCTATTAATTGA-3'
CTCCAAGATTGAGACTACAC-3') enter performing PCR detection and carry out picking positive colony, positive colony is sequenced.PCR reactants
It is to be：10×buffer 2μl；dNTP 0.4μl；Each 0.2 μM of upstream and downstream primer；The μ l of Taq polymerase 0.2, plus ddH2O is complemented to
20 μ l systems.PCR reaction conditions are：94 DEG C of predegeneration 3min；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 1min, 25 are followed
Ring；72 DEG C of extension 7min.The clone for being defined as the positive is the recombinant vector PCAMBIA1381Xb- obtained for converting
ProSicwinv1(Fig. 2 B).
The PCAMBIA1381Xb-Pro that will be builtSicwinv1Carrier conversion agrobacterium strains GV3101 (Roger et al.,
2000,A guide to Agrobacterium binary Ti vectors.Trends in Plant Sci,5,1360-
1385), picking single bacterium colony is connected in the LB fluid nutrient mediums of rifampin containing 50mg/L and 100mg/L hygromycin in 150rpm, and 28
DEG C 48h is shaken, by bacterium solution and glycerine volume ratio 1：1 adds 1.5mL centrifuge tubes to mix, -70 DEG C of preservations.Again by agriculture bacillus mediated
Method for transformation arabidopsis thaliana transformation.
It is related to LB culture medium prescriptions to be conventional medium in this specification：Peptone 10g/L, yeast extract 5g/L,
NaCl 10g/L；The pH=7.2 of culture medium is adjusted before sterilizing with 5mM NaOH；1L is settled to distilled water；In 121-125 DEG C of high pressure
Sterilizing 15-20min.The solid medium of LB is the agar of addition 8g/L in every liter of culture medium.
The genetic transformation of embodiment 3 and Screening and Identification
A. the preparation of arabidopsis material
Material to be tested is that wildtype Arabidopsis thaliana (Arabidopsis thaliana L.Columbia ecotype) is ability
The conventional material in domain.Wildtype Arabidopsis thaliana seed is sowed at arabidopsis plantation conventional nutrient soil and is put into people by point after vernalization treatment
The arabidopsis such as work culturing room (illumination in 16 hours, 22 DEG C) grow to 4 leaves or so carries out final singling, to control the stand density of arabidopsis.
Converted by when arabidopsis growth starts to bloom for 6 weeks or so, conversion the previous day pours water to arabidopsis.
B. the activation of Agrobacterium
From in ultra low temperature freezer take out preserve containing target promoter PSicwinv1 (i.e. the present invention clone
Sicwinv1 gene promoters) GV3101 bacterial strains glycerol tube in thawed on ice, after rifampin containing 50mg/L and 100mg/L
Rule on the LB solid mediums of hygromycin, 28 DEG C of light culture 36-48h treat to grow clearly single bacterium colony in ware, picking single bacterium colony
Incubated overnight (26.5 DEG C, 100rpm), its OD in the LB fluid nutrient mediums of 50mg/L rifampins and 100mg/L hygromycin are added600
Can be used to convert during=0.8-1.0；
5000rpm is centrifuged 5min during bacterium solution is transferred into centrifuge tube, abandons supernatant culture medium.It is 5% to add 100ml concentration
(W/V) sucrose solution, resuspended Agrobacterium GV3101, the recovery 1-2h in 28 DEG C of shaking tables.Add (the V/ of surfactant 0.05%
V) Silwet L-77, concussion is shaken to mixed even.
The screening of C. agriculture bacillus mediated inflorescence method arabidopsis thaliana transformation and transgenic arabidopsis
The methods and procedures of agriculture bacillus mediated floral organ leaching libation at an ancient wedding ceremony method arabidopsis thaliana transformation with reference to (Zhang et al., 2006,
grobacterium-mediated transformation of Arabidopsis thaliana using the floral
dip method.Nature Protocols,1,641-646).Comprise the following steps that：
(1) arabidopsis floral organ is immersed into Agrobacterium suspension, and is gently agitated for about 30s, excessive bacterium solution is sopped up with paper handkerchief, and
Arabidopsis plant is wrapped up with black plastic bag, moisturizing lucifuge treatment 24h；
(2) polybag is gradually opened ventilative, normal culture after；
The operation of repeatedly above-mentioned (1) after (3) one weeks；
(4) treat that seed maturity can stop watering and harvesting seed, i.e. T1For seed；
(5) seed disinfection that will be harvested：First soaked 1 minute with 70% (V/V) ethanol, made every now and then in above-mentioned treatment
Seed suspension；Then with aseptic washing four times；
(6) seed after processing is uniformly coated on the plan containing hygromycin with Top agar (0.1% (W/V) aqueous agar solution)
Southern mustard growth medium (the conventional 1/2MS culture mediums containing 100mg/L hygromycin) surface；
(7) by transgenic arabidopsis seed vernalization 3 days in 4 DEG C of refrigerators, then culturing room's culture 10 days is moved into, tool is chosen altogether
There is 17 plants of hygromycin resistance plant；
(8) by 17 plants of transgenosis T1Soil incubation is transplanted to for Arabidopsis plant, after seed is collected by individual plant after maturation, i.e.,
It is T2For seed；
(9) T that will be collected2For seed operation 1 time is carried out by operating procedure (5)-(6)；
(10) by transgenic arabidopsis seed vernalization 3 days in 4 DEG C of refrigerators, normal culture is calculated after 10 days has hygromycin
The segregation ratio of resistant plant and non-resistance plant, and carry out statistical analysis；
(11) it is 3 to meet resistance with the segregation ratio of non-resistance plant:1 strain is considered as single copy strain, transplants soil
Earth culture, after collecting seed, i.e. T by individual plant after maturation3For seed.
Embodiment 4：Promoter expression vector PCAMBIA1381Xb-ProSicwinv1GUS is driven to be expressed in arabidopsis
By T3Sowed on conventional 1/2MS culture mediums for transgenic Arabidopsis plants, transgenosis is intended into south in 4 DEG C of refrigerators
It is placed in after canola seed vernalization in growth case and is sprouted, 5-7 days rear portion seedling takings of emerging carries out GUS dyeing, and another part is transplanted to
Soil incubation, treats arabidopsis Post flowering, and sampling carries out GUS dyeing.GUS Organization chemistry orientation methods are with reference to (Gus such as Jefferson
fusions:β-glucuronidase as a sensitive and versatile gene fusion marker in
higher plants.EMBO J,1987,6:3901-3907) method, will convert the Different Organs of transfer-gen plant, and tissue is put
To in GUS dyeing liquors, vacuum suction 5min is incubated 2h in 37 DEG C to overnight, after being decolourized through 70% alcohol, is floated with distilled water
Wash for several times, taken pictures under Stereo microscope (OLYMPUS SZX16).GUS dye liquors composition is following (100mL)：x-gluc 90mg、
Chloramphenicol 10.0mg, 0.1M sodium phosphate buffer (pH 7.0) 50mL, methyl alcohol 20% (v/v), supply sterilized water to 100mL.
The position that plant is dyed to blueness is exactly the position of gus gene expression.By detecting gus gene under different space-times
Expressive site and expression intensity explore the spatial and temporal expression profile of promoter PSicwinv1.GUS coloration results show：Promoter
PSicwinv1 drives gus gene only to be expressed in arabidopsis flower pesticide, the equal not table in root, stem, leaf, pod and petal, filigree
Up to (Fig. 3), and PSicwinv11Drive expression of the gus gene in flower pesticide relevant with the developmental stage of flower pesticide, with flower pesticide
Development, gus gene expression quantity reduction (see Fig. 4).
Embodiment 5：The in situ hybridization of Sicwinv1 genes
Fertile line 95ms-5B pollen mother cell periods, tetrad and the bud of mature pollen phase is taken to fix, wrap
Bury, cut into slices after hybridized, concrete operations are as follows：
A. paraffin section
(1) materials are fixed：95ms-5B different times buds are taken, FAA fixers are added, tissue are evacuated to and are sink to ttom of pipe,
Fixed more than 24h.
(2) it is dehydrated：(can be overnight) be dehydrated step by step through 70% ethanol, 85% ethanol, 95% ethanol (containing 1% Yihong), every grade
1h.Absolute ethyl alcohol twice, each 1h.
(3) it is transparent：Through 1/5,2/5,3/5,4/5, pure chloroform is transparent, every grade of 1-1.5h.
(4) wax is oozed：Plus broken wax, put in 37 DEG C of incubators 2 days, broken wax：Chloroform volume ratio=1：2~3
(5) waxdip and embedding：Material is through waxes at different levels cup, 50% wax cup (45 DEG C)~75% (52 DEG C) each 2h, A glasss, B glasss, C
(56~58 DEG C) difference 1h of cup (100% wax), then embedding.
(6) block, section are repaiied：Horizontal and vertical section is carried out respectively to sample with LEICA2150 slicers, thickness is 8um,
Poly-D-lysine is used as bonding die agent, distilled water exhibition piece.
(7) exhibition piece, baking piece：Dried on piece machine after 37 DEG C of exhibition pieces in KD-H, baking more than 2 days in 36 DEG C of insulating boxs.
B. prepared by situ hybridization probe
(1) target gene specific sequence is chosen, the restriction enzyme digestion sites in sequence are searched, avoids containing enzyme
The primers of enzyme site, restriction endonuclease sites BamHI (GGATCC) and XhoI is added at primer two ends
(CTCGAG).Primer sequence is as follows：
Fp：5'-CGCGGATCCTCCTCAACCTTCAATCCCTGTCC-3',
Rp：5'-CCGCTCGAGGAGCCTGACCAACAACCATACTG-3'；
(2) cDNA with 95ms-5B is as template, and target fragment is expanded by PCR, by digestion coupled reaction by target patch
Section is building up on pBluescript II KS+ carriers (purchased from Stratagene companies, the U.S.), and converts Escherichia coli, picking
Positive colony is simultaneously sequenced, and extracts the plasmid of correct positive colony.
(3) plasmid linearization：Selection BamHI and XhoI carries out digestion to plasmid, adds isometric water, product to use after digestion
(volume ratio is 1 for isometric Tris- saturated phenols and chloroform mixed liquor：1) it is stripped, 12000rpm/min centrifugation 10min inhale
In supernatant to clean centrifuge tube, chloroform is added to purify 2 times, 12000rpm/min centrifugation 10min are sucted clear to clean centrifugation
Guan Zhong, adds the alcohol and 0.1 times of sodium acetate of volume of 2 times of volume precoolings to precipitate, and after refrigerated centrifuge, abandons supernatant.With 75%
Alcohol is rinsed twice, and DEPC water dissolves are added after drying.
(4) probe transcription：Reaction system (10 μ l) is as follows：
37 DEG C are reacted 4 hours, and transcription completes to add DNase (RNase-free) and RNasin each in backward reaction system
0.5 μ l, 37 DEG C of reaction 25min, add 50 μ l TE (PH8.0), 5 μ l licl and 155 μ l absolute ethyl alcohols, -20 DEG C of mistakes after mixing
Night abandons supernatant to Precipitation, 12000rpm/min centrifugation 20min, and 75% ethanol is washed 2 times, and 10 μ l DEPC are added after drying
Water dissolves.Take the detection of 1 μ l point samples, spectrophotometric determination production concentration.
C. in situ hybridization
(1) by dimethylbenzene dewaxing twice, each 20min.Use absolute ethyl alcohol 2 times respectively, 95% ethanol, 85% ethanol,
70% ethanol, 50% ethanol, 30% ethanol, the graded ethanol rehydration of 15% ethanol, with conventional 2 rehydrations of DEPC water washings,
Each 1min.
(2) 37 DEG C of preheating Proteinase K liquid, Proteinase K ultimate density is 1 μ g/ml, and hybridization slice, thin piece processes 40min in 37 DEG C,
1 × PBS is washed once.
(3) through 15%, the series dehydration of 30%, 50%, 70%, 85%, 95%, 100% alcohol, each 1min, Ran Hou
Super-clean bench is air-dried.
(4) 150 μ l prehybridization solutions are dripped on material, Para film sealed membranes are covered, slide is then placed on NaOH treatment
In the centrifuge tube box crossed, 42 DEG C of prehybridization 3h.
(5) mix with hybridization buffer in proportion according to concentration and probe concentration, the μ l of hybridization solution 50 are added dropwise on every slide, cover Para
Film sealed membranes, 42 DEG C of hybridized overnights in wet box.
(6) slide is taken Para film sealed membranes in 2 × SSPE at room temperature off, 42 DEG C of 2 × SSPE buffer solutions are washed 2 times,
Each 15min；Then under the conditions of 57 DEG C, 0.2 × SSPE buffer solution water-baths 50r/min is washed 2 times, each 30min, and wash-out is remaining
Probe.
(7) under room temperature (23 DEG C), slide is positioned over 40min, 50r/min in 1 × Blocking buffer.Blockade liquid
Needs are now matched somebody with somebody.It is by volume 1 with BSA washing buffer：2500 dilute anti-DIG-AP antibody, and used kit is
ROCHE colour reagents box (CAT.NO.11745832910), will cut into slices in room temperature oscillation incubation 2h, 50r/min in antibody.
(8) after immune response terminates, slide is placed in 15min in 1 × Blocking buffer, room temperature (23 DEG C) condition
Lower 50r/min wash-outs, wash 3 times, each 15min, 50r/min under room temperature (23 DEG C) in BSAwashing buffer.
(9) chromogenic reaction：Section changes to TNM-50 room temperatures 3 times, each 5min.Add 0.5ml's in 40ml TNM-50
The nitrite ion that NBT is made into.Section is placed in one, 3h-5h is shown at room temperature dark in wet box.Colour developing is developed a film 3 times after terminating with TE,
Each 5min.
(10) material is through 30%, 70%, 95%, 100% ethanol, dimethylbenzene:(volume ratio is 1 to ethanol:1), dimethylbenzene, two
Toluene series dehydration is transparent, microexamination photography.
From the in situ hybridization result of Fig. 5：Sicwinv1 genes sesame flower pesticide tetrad expression quantity highest,
And the main expression in tapetum and tetrad.
Claims (4)
1. a kind of promoter PSicwinv1 of the specifically expressing for being isolated from sesame, the promoter only specifically expressing in flower pesticide, no
Expressed in its hetero-organization, the nucleotide sequence such as SEQ ID NO of the promoter：Shown in 1.
2. a kind of promoter PSicwinv1 of the specifically expressing of sesame that is isolated from described in claim 1 is in flower pesticide regulation and control
Using.
3. a kind of promoter PSicwinv1 of the specifically expressing for being isolated from sesame described in claim 1 is cultivating male sterility
Application in genetic resources.
4. a kind of promoter PSicwinv1 of the specifically expressing for being isolated from sesame described in claim 1 is in plant hybrid advantage
Application in utilization.
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| CN112941098A (en) * | 2019-12-10 | 2021-06-11 | 江苏师范大学 | Arabidopsis thaliana anther tapetum promoter expression vector and construction method and application thereof |
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|---|---|---|---|---|
| CN105039338A (en) * | 2014-04-22 | 2015-11-11 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Identification and application of plant anther specific-expression promoter pTaASG004 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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Non-Patent Citations (2)
| Title |
|---|
| ZHAO YINGZHONG: "Characterization and genetic mapping of a novel recessive genic male sterile gene in sesame (Sesamum indicum L.)", 《MOLECULAR BREEDING 》 * |
| 郭伟等: "芝麻核雄性不育系ms86-1微粉发生的细胞学观察", 《植物学报》 * |
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| CN112941098A (en) * | 2019-12-10 | 2021-06-11 | 江苏师范大学 | Arabidopsis thaliana anther tapetum promoter expression vector and construction method and application thereof |
| CN112941098B (en) * | 2019-12-10 | 2022-04-19 | 江苏师范大学 | Arabidopsis thaliana anther tapetum promoter expression vector and construction method and application thereof |
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