CN106906212B - One sesame anther specific expression promoter PSicwinv1 and its application - Google Patents
One sesame anther specific expression promoter PSicwinv1 and its application Download PDFInfo
- Publication number
- CN106906212B CN106906212B CN201610528610.8A CN201610528610A CN106906212B CN 106906212 B CN106906212 B CN 106906212B CN 201610528610 A CN201610528610 A CN 201610528610A CN 106906212 B CN106906212 B CN 106906212B
- Authority
- CN
- China
- Prior art keywords
- promoter
- anther
- sesame
- psicwinv1
- gene
- 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.)
- Expired - Fee Related
Links
Classifications
-
- 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
-
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Reproductive Health (AREA)
- Pregnancy & Childbirth (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to field of plant genetic project technology, and in particular to a sesame anther specific expression promoter PSicwinv1 and its application.The nucleotide sequence for the promoter that the present invention separates is as shown in sequence table SEQ ID NO:1.Biological function verifying is carried out to the promoter, GUS dyeing detection shows, the specific promoter PSicwinv1 driving reporter gene that the present invention clones is not expressed in the root of arabidopsis, stem, leaf and pod, only the specifically expressing in anther, and its expression quantity is related with the developmental stage of anther.In situ hybridization shows that Sicwinv1 is expressed in the tetrad a large amount of sesame anther development, and mainly expresses 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 project technology.More particularly to a sesame anther specific expression promoter
PSicwinv1 and its application.Separation, identification obtain the promoter of a specifically expressed Sicwinv1 gene from sesame, will
It is named as PSicwinv1.The promoter specifically expressing in anther, 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 technique
Promoter is positioned at gene 5 ' end upstream, guide RNA polymerase and transcription factor specific bond, and then promotor gene
Specifically expressed one section of specific dna sequence is the important cis-acting elements of controlling gene expression.Promoter is in plant gene work
It is played an important role in journey, is the important component of gene engineering expression carrier, regulates and controls the transcriptional efficiency of foreign gene
And spatial and temporal expression profile.
According to the regulating and expressing mode 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, does not have Space-time speciality.Typically answer
Constitutive promoter for plant genetic engineering has cauliflower mosaic virus (CaMV) 35S promoter, rice actin Act
Promoter and maize ubiquitin Ubi promoter (close beautiful English etc., the effective expression of foreign gene and its safety are commented in genetically modified plants
Valence Capital Normal University journal, 2002,23 (2): 52-56).But efficient, the non-specific expression of constitutive promoter is easily made
It is consumed excessively at cellular material, and target gene cannot be expressed by specific spatiotemporal database, some negative effects is caused to generate.
Thus, specific promoter is receive more and more attention in recent years.Specific promoter includes tissue-specific promoter
And inducible promoter.Tissue-specific promoter drives target gene to express in specific tissue or organ, usually adjoint
There is the characteristic of growth adjustment.Generally there are some relevant to tissue specificity specific motifs (He Hongxia etc., groups for such promoter
Knit progress China agronomy notification of the specificity promoter in crop gene engineering, 2014,30 (9): 225-231).Group
It knits specific promoter and effectively avoids 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 are 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 character
research,agricultural,and environmental applications:Promoters used in plant
transformation.In Vitro Cell Dev Biol Plant,40:1-22).It is opened using anther or pollen-specific expression
Mover initiative male sterile line is applied to crossbreeding and has made some progress.Utilize tobacco anther Tapetum-specific expression
Promoter PTA29Merge the carrier genetic transformation receptor of the ribonucleic acid hydrolase Bar gene nase building of bacillus amyloliquefaciens
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).Rice RTS gene is in subtrahend
It is expressed in the anther tapetum of division, merge the antisense strand of barnase or RTS using the promoter of the gene and converts water
Rice, arabidopsis lead to transgenic 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 utilizes, and utilizes male
Sterile line initiative cenospecies has great importance for sesame genetic improvement.Thus, excavate the starting of sesame anther specifically expressing
Son has important application value for sesame male sterility related gene functional verification and male sterile line initiative.
Summary of the invention
The purpose of the present invention is to provide one to separate the anther specific expression promoter of clone from sesame.Of the invention
Promoter belongs to a kind of endogenesis promoter, and the endogenous gene for showing promoter driving table in anther is detected by RT-PCR
It reaches, without expression in its hetero-organization.Show that the gene of promoter driving only exists by the expression characteristic of examining report gene GUS
It is expressed in anther, and its expression quantity is related with the developmental stage of anther.In situ hybridization shows the endogenous base of promoter driving
Because of the tetrad expression quantity highest in sesame anther development.The present invention not only enriches sesame anther specific expression promoter
Resource, while implying that the promoter has quite wide application prospect in male sterility research.The promoter is used
In the functional verification of male sterility related gene and the initiative of male sterile line, for heterosis utilization provide technical foundation and
Genetic resources.
Realize that the technical solution 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 anther transcript profile is educated, the gene Sicwinv1 of isolated significant difference expression from sesame, to this
The tissue expression analysis verifying of gene shows the gene only specifically expressing in the anther of sesame, in root, stem, leaf, petal, female
(Fig. 1) is not expressed in stamen, column cap.The promoter PSicwinv1 of the gene is obtained by PCR amplification, nucleotide sequence is such as
Shown in sequence table SEQ NO:1.
Promoter of the invention derives from sesame, by 1205 base compositions.By constructing the report by the promoter regulation
Accuse the plant expression vector PCAMBIA1381Xb-Pro of gene GUSSicwinv1(Fig. 2 B), and the conversion carrier is transformed into brother's human relations
Than the transgenic positive Arabidopsis plant in sub- Arabidopsis thaliana ecotype, obtaining the promoter.It is found by GUS staining analysis, it should
Promoter only drives gus gene to express in anther, can't detect the expression of gus gene in root, stem, leaf, pod, petal
(Fig. 3).And (Fig. 4) is had differences in the expression of the different development stage of anther, PSicwinv1 driving Reporter gene GUS.It is logical
It crosses situ Analysis and shows that PSicwinv1 driving endogenous gene is expressed in the tetrad a large amount of sesame anther, and is main
(Fig. 5) is expressed in tapetum and tetrad.These studies have shown that PSicwinv1 is an anther specific expression promoter,
It can be applied in plants male sterility genetic engineering and plant hybrid use of advantage and breed improvement.
It is of the invention specific steps are as follows:
(1) according to the promoter amplimer of Sesame group sequence design Sicwinv1 gene, with applicant's work early period
Make sesame genic male sterile system 95ms-5 (Zhao the et al., 2013, Characterization and obtained
genetic mapping of a novel recessive genic male sterile gene in sesame
(Sesamum indicum L.) .Mol Breeding, 32:901-908) DNA be template, expand promoter sequence, this is opened
The nucleotide sequence of mover is as shown in SEQ ID NO:1.
Primer sequence for expanding Sicwinv1 gene 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) step (1) is expanded into DNA fragmentation and PCAMBIA1381Xb plasmid (Fig. 2A) obtained, carries out digestion company
It is reversed to answer, which is connected on carrier PCAMBIA1381Xb, then pass through 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 the transgenic method of mediated by agriculture bacillus, by the quasi- south of the vector introduction
In mustard, transformed plant is obtained.
(3) transgenic positive plant is obtained by the method that hygromycin selection and segregation ratio count, and passes through GUS dyeing point
Analysis detects the expression of promoter PSicwinv1.
(4) by the method for in situ hybridization, table of the promoter PSicwinv1 driving endogenous gene in sesame anther is detected
Expression patterns.
The present invention has the advantages that
1. the promoter PSicwinv1 that the present invention clones is anther specific expression promoter, target gene can be driven only to exist
Anther expression, without expressing in its hetero-organization, therefore the present invention can effectively overcome and negatively imitate caused by constitutive promoter
It answers.
2. can be used in studying male sterility with the sesame anther specific expression promoter PSicwinv1 that the present invention clones
The functional verification of related gene also can use promoter creating plants male sterility system or breed improvement of the invention.
Detailed description of the invention
Sequence table SEQ ID NO:1 is the nucleosides of the promoter PSicwinv1 of present invention separation clone's Sicwinv1 gene
Acid sequence, sequence length 1205bp.
Fig. 1: it is analyzed using the tissue expression pattern of the method detection Sicwinv1 gene of RT-PCR.Description of symbols:
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 third swimming lane is leaf (mark
Note is Le), 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
It is preced with (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
Whether consistent sesame ubiquitin protein (number of logging in is JP631638), detect applied sample amount as reference gene.
Fig. 2: carrier used in building promoter expression vector.Description of symbols: Fig. 2A is used expression vector
The map of PCAMBIA1381Xb, Fig. 2 B are plant recombinant vector PCAMBIA1381Xb-Pro prepared by the present inventionSicwinv1Map.
A kind of Fig. 3: histochemical stain result schematic diagram of the gus gene of PSicwinv1 promoter driving.Appended drawing reference
Illustrate: the A figure in Fig. 3 is the bud of transgenic arabidopsis, and the B figure in Fig. 3 is the anther of transgenic arabidopsis, the C figure in Fig. 3
For the inflorescence of transgenic arabidopsis, the D figure in Fig. 3 is transgenic arabidopsis seedling, and the E figure in Fig. 3 is transgenic arabidopsis
Pod.GUS coloration result is shown, blue is only detected in the anther of arabidopsis, and aobvious without blue in its hetero-organization
Show, show promoter PSicwinv1 driving gus gene only expressed in arabidopsis anther, and root, stem, leaf, pod and
It is not expressed in petal and filigree.
Expression of the gus gene of Fig. 4: promoter PSicwinv1 driving in the anther of the different development stage of arabidopsis
Situation schematic diagram.Description of symbols: bud size is sequentially increased from left to right in Fig. 4, shows that promoter PSicwinv1 drives
Gus gene expression quantity level it is related with Anther stage.
Fig. 5: expression of the Sicwinv1 gene in the anther of the different development stage of sesame is identified in situ hybridization.It is attached
Figure description of symbols: the A-C figure in Fig. 5 is results of hybridization of the mRNA antisense probe of Sicwinv1 gene in sesame anther, Fig. 5
In D-F figure be positive-sense strand probe hybridization after negative control.A-C figure in Fig. 5 is respectively pollen mother cell period, tetrad
Period and mature pollen period；When D-F figure in Fig. 5 is respectively pollen mother cell period, tetrad and mature pollen
Phase.
Specific embodiment
Following embodiment defines the present invention, and according to description below and these embodiments, those skilled in the art can be with
It determines essential characteristic of the invention, and without departing from the spirit and scope of the invention, the present invention can be made respectively
Kind changes and modification, so that it is applicable in different purposes and condition.
The separation clone of the promoter sequence of 1 Sicwinv1 gene of embodiment 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, 32:901-908) fertile plant and sterile plant tetrad anther transcript profile in isolate one section of est sequence,
TBLASTx comparison is carried out in NCBI gene pool to this sequence, it is found that this sequence may be cell wall invertase gene.This
Est sequence does not include complete opening code-reading frame, and applicant uses routine RACE (rapid-amplification of
CDNA ends) method by PCR carry out the end cDNA quick clone technology to obtain the complete coded sequence of this gene.By this
The complete coded sequence of item is compared with Sesame group, obtains the genome sequence of the gene, is drawn according to genome sequence design
Object expands promoter sequence.
A. the extraction of Sesame group DNA
DNA (the extracting method reference Uzun of sesame genic male sterile 95ms-5 is extracted using conventional CTAB method
and Cagirgan,2009,Identification of molecular markers linked to determinate
The method of growth habit in sesame.Euphytica, 166,379-384 report).
The acquisition of B.Sicwinv1 gene promoter sequence
Promoter amplimer is designed according to the genome sequence of Sicwinv1, primer is as follows: upstream primer
PSicwinv1-F5'-CCATATCATTTTCTGGCCTTTC-3', downstream primer PSicwinv1-R5'-
GGTTGAGGAAAACCTCATGG-3' utilizes the promoter sequence of PCR amplification Sicwinv1 using the DNA of 95ms-5B as template.
PCR reaction system are as follows: 10 × buffer, 2 μ l；dNTP 0.4μl；Each 0.2 μM of upstream and downstream primer；0.2 μ l of Taq polymerase, adds
ddH2O complements to 20 μ l systems.PCR condition is 94 DEG C of initial denaturation 3min；94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 1min,
28 circulations；72 DEG C of extension 7min.The PCR product that amplification obtains is connected into pGEM-T carrier (purchased from Promega company, beauty
State), screening positive clone is simultaneously sequenced.
C. the 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 is using Trizol kit (being purchased from Invitrogen company, the U.S.), using sesame 95ms-5 as material
Material extracts the RNA of root, stem, leaf, capsule, bud, corolla, gynoecium and each tissue sample of stamen, utilizes reverse transcriptase
Its reverse transcription is synthesized cDNA, reaction condition are as follows: 65 DEG C by Superscript III (being purchased from Invitrogen company, the U.S.)
5min, 50 DEG C of 60min, 70 DEG C of 10min.Using the cDNA of above-mentioned reverse transcription synthesis as template, detected using RT-PCR
The expression pattern of Sicwinv1, the primer are as follows: Sicwinv1-F:(5'-ATGGAGTTTCGGGCCAAGAG-3') and
Sicwinv1-R:(5'-GATTAAGAGGTGCAGGCAGGGAC-3').Primer SiUbiquitin6-F:(5'- is used simultaneously
CACCAAGCCGAAGAAGATCAAG-3') and SiUbiquitin6-R:(5'-CCTCAGCCTCTGCACCTTTC-3') to sesame
SiUbiquitin6 (the GenBank number of logging in: JP631638) gene does specific amplified, compares as internal reference and carries out relative quantification point
Analysis.The result shows that: the promoter driving endogenous gene that the present invention clones is expressed in anther, do not expressed in its hetero-organization (see
Fig. 1).
Embodiment 2: the building containing Sicwinv1 gene promoter PSicwinv1 expression vector
It is used for construction of expression vector according to obtained promoter PSicwinv1 primers, method to be in primer two
End adds restriction enzyme site respectively, and the primer sequence is respectively PSicwinv1-MF (5'-GAGAATTC (EcoRI)
) and PSicwinv1-MR (5'-GCAAGCTT (HindIII) CTGGCCTTTCTATTAATTGA-3'
CTCCAAGATTGAGACTACAC-3'), PCR amplification, PCR reaction system are carried out by template of T-Sicwinv1 plasmid are as follows: 10 ×
buffer 2μl；dNTP 0.4μl；Each 0.2 μM of upstream and downstream primer；0.2 μ l of pfu polymerase, adds ddH2O complements to 20 μ l systems.
PCR reaction condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 1min, totally 28 recycle；72℃
7min is extended to, PCR product (is purchased from Axygen company, beauty by detected through gel electrophoresis, and using DNA gel QIAquick Gel Extraction Kit
State) recycling purpose PCR product.PCR product after the recovery is by HindIII and EcoRI (purchased from the limited public affairs in precious bioengineering Dalian
Department) carry out double digestion, endonuclease reaction system are as follows: 10 × Fastdigest Buffer 2 μ l, DNA 1 μ g, HindIII and EcoRI
Each 1 μ l, adds ddH2O to 20 μ l after mixing well, places 1h in 37 DEG C of insulating boxs, digestion products is then passed through PCR product
Purification kit (being purchased from Bioflux company, Romania) is purified.Vector plasmid is passed through into HindIII and EcoRI simultaneously
Double digestion is carried out, endonuclease reaction system is same as above, and then by digestion products by detected through gel electrophoresis, and is recycled using DNA gel
Kit (be purchased from Axygen company, the U.S.) recycling purpose carrier segments (target fragment is large fragment), after by mesh after the recovery
Carrier segment be attached and react with the digestion PCR product of purifying, coupled reaction system are as follows: press purpose carrier segments 100ng,
10 × T4 ligase Buffer, 2 μ l, T4 ligase, 1 μ l (being purchased from Thermo company, the U.S.) is added in target PCR fragment 50ng,
Sterile water is supplemented to 20 μ l and mixes, after 22 DEG C of 10min, in 4 DEG C of connection reactions overnight.Then big by conventional heat shock method conversion
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' it) carries out PCR detection and carrys out picking positive colony, positive colony is sequenced.PCR reactant
System are as follows: 10 × buffer, 2 μ l；dNTP 0.4μl；Each 0.2 μM of upstream and downstream primer；0.2 μ l of Taq polymerase, adds ddH2O is complemented to
20 μ l systems.PCR reaction condition are as follows: 94 DEG C of initial denaturation 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.Being determined as positive clone is the recombinant vector PCAMBIA1381Xb- obtained for conversion
ProSicwinv1(Fig. 2 B).
By the PCAMBIA1381Xb-Pro of buildingSicwinv1Carrier conversion agrobacterium strains GV3101 (Roger et al.,
2000,A guide to Agrobacterium binary Ti vectors.Trends in Plant Sci,5,1360-
1385), picking single colonie is connected in the LB liquid medium of rifampin containing 50mg/L and 100mg/L hygromycin in 150rpm, and 28
DEG C 48h is shaken, 1.5mL centrifuge tube is added by bacterium solution and glycerol volume ratio 1:1 and mixes, -70 DEG C of preservations.Pass through mediated by agriculture bacillus again
Method for transformation arabidopsis thaliana transformation.
LB culture medium prescription involved in this specification is conventional medium: peptone 10g/L, yeast extract 5g/L,
NaCl 10g/L；With the pH=7.2 of 5mM NaOH tune culture medium before sterilizing；1L is settled to distilled water；In 121-125 DEG C of high pressure
Sterilize 15-20min.The solid medium of LB is the agar that 8g/L is added in every liter of culture medium.
3 genetic transformation of embodiment 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 point after vernalization treatment is sowed at arabidopsis plantation conventional nutrient soil and is put into people
The arabidopsis such as work culturing room (illumination in 16 hours, 22 DEG C) grow to 4 leaves or so and carry out final singling, to control the stand density of arabidopsis.
It can be converted when arabidopsis grows 6 weeks or so and starts to bloom, conversion the previous day is watered with water to arabidopsis.
B. the activation of Agrobacterium
(i.e. the present invention clones the target promoter PSicwinv1 that contains that taking-up saves out of ultra low temperature freezer
Sicwinv1 gene promoter) the glycerol tube of GV3101 bacterial strain melt on ice, after in rifampin containing 50mg/L and 100mg/L
It crosses on the LB solid medium of hygromycin, 28 DEG C of dark culture 36-48h, to grow clearly single colonie, picking single colonie in ware
(26.5 DEG C, 100rpm), OD are incubated overnight in the LB liquid medium for adding 50mg/L rifampin and 100mg/L hygromycin600
It can be used to convert when=0.8-1.0；
Bacterium solution is transferred to 5000rpm in centrifuge tube and is centrifuged 5min, abandons supernatant culture medium.It is 5% that 100ml concentration, which is added,
(W/V) Agrobacterium GV3101, the recovery 1-2h in 28 DEG C of shaking tables is resuspended in sucrose solution.0.05% (V/ of surfactant is added
V) Silwet L-77, concussion are shaken to mixed even.
C. the inflorescence method arabidopsis thaliana transformation of mediated by agriculture bacillus and the screening of transgenic arabidopsis
The methods and procedures of the floral organ leaching libation at an ancient wedding ceremony method arabidopsis thaliana transformation of mediated by agriculture bacillus referring to (Zhang et al., 2006,
grobacterium-mediated transformation of Arabidopsis thaliana using the floral
dip method.Nature Protocols,1,641-646).Specific step is as follows:
(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 is protected from light processing for 24 hours；
(2) polybag is gradually opened to ventilative, normal culture after；
(3) operation of above-mentioned (1) is repeated after a week；
(4) can stop watering and harvesting seed, i.e. T to seed maturation1For seed；
(5) by the seed disinfection of harvest: first being impregnated 1 minute with 70% (V/V) ethyl alcohol, to be made every now and then in above-mentioned processing
Seed suspension；Then with sterile washing four times；
(6) treated seed is uniformly coated on quasi- containing hygromycin with Top agar (0.1% (W/V) aqueous agar solution)
Southern mustard growth medium (the conventional 1/2MS culture medium containing 100mg/L hygromycin) surface；
(7) it by transgenic arabidopsis seed vernalization 3 days in 4 DEG C of refrigerators, then moves into culturing room and cultivates 10 days, choose tool altogether
There are 17 plants of hygromycin resistance plant；
(8) by 17 plants of transgenosis T1It is transplanted to soil incubation for Arabidopsis plant, collects seed by single plant after maturation, i.e.,
For T2For seed；
(9) T that will be collected2Operation 1 time is carried out by operating procedure (5)-(6) for seed；
(10) by transgenic arabidopsis seed vernalization 3 days in 4 DEG C of refrigerators, normal culture calculates after 10 days has hygromycin
The segregation ratio of resistant plant and non-resistance plant, and it is for statistical analysis；
(11) meeting the segregation ratio of resistance and non-resistance plant for the strain of 3:1 is considered as single copy strain, transplanting soil
Earth culture collects seed, i.e. T by single plant after maturation3For seed.
Embodiment 4: promoter expression vector PCAMBIA1381Xb-ProSicwinv1Driving GUS is expressed in arabidopsis
By T3It is sowed on conventional 1/2MS culture medium for transgenic Arabidopsis plants, by the quasi- south of transgenosis in 4 DEG C of refrigerators
Canola seed vernalization, which is placed in growth case, sprouts, and 5-7 days rear portion seedling takings of emerging carry out GUS dyeing, and another part is transplanted to
Soil incubation, to arabidopsis Post flowering, sampling carries out GUS dyeing.GUS Organization chemistry orientation method is referring 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, the Different Organs of transgenic plant will be converted, tissue is put
Into GUS dyeing liquor, vacuum suction 5min after the decoloration of 70% alcohol, is floated in 37 DEG C of heat preservation 2h to overnight with distilled water
It washes for several times, takes pictures under Stereo microscope (OLYMPUS SZX16).Following (100mL): x-gluc 90mg of GUS dye liquor ingredient,
Chloramphenicol 10.0mg, 0.1M sodium phosphate buffer (pH 7.0) 50mL, methanol 20% (v/v), supply sterile water to 100mL.
Plant is dyed to the position that blue position is exactly 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 result shows: promoter
PSicwinv1 driving gus gene is only expressed in arabidopsis anther, the equal not table in root, stem, leaf, pod and petal, filigree
Up to (Fig. 3), and expression of the PSicwinv1 driving gus gene in anther is related with the developmental stage of anther, with anther
The expression quantity of development, gus gene reduces (see Fig. 4).
The in situ hybridization of embodiment 5:Sicwinv1 gene
Fertile line 95ms-5B pollen mother cell period, tetrad and the bud of mature pollen phase is taken to fix, wrap
Bury, be sliced after hybridized, concrete operations are as follows:
A. paraffin section
(1) materials are fixed: 95ms-5B different times bud is taken, FAA fixer is added, is evacuated to tissue and is sink to tube bottom,
It fixes more than for 24 hours.
(2) it is dehydrated: being dehydrated (can stay overnight) step by step through 70% ethyl alcohol, 85% ethyl alcohol, 95% ethyl alcohol (containing 1% Yihong), every grade
1h.Dehydrated alcohol twice, each 1h.
(3) transparent: through 1/5,2/5,3/5,4/5, pure chloroform is transparent, every grade of 1-1.5h.
(4) it seeps wax: adding 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 wax cups at different levels, 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 embed.
(6) block, slice are repaired: horizontal and vertical slice being carried out respectively to sample with LEICA2150 slicer, with a thickness of 8um,
Poly-D-lysine opens up piece as bonding die agent, distilled water.
(7) it opens up piece, dry piece: on KD-H baking piece machine after 37 DEG C of exhibition pieces, being dried 2 days or more 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 is searched, avoids containing enzyme
The primers of enzyme site add restriction endonuclease sites BamHI (GGATCC) and XhoI at primer both ends
(CTCGAG).Primer sequence is as follows:
Fp:5'-CGCGGATCCTCCTCAACCTTCAATCCCTGTCC-3',
Rp:5'-CCGCTCGAGGAGCCTGACCAACAACCATACTG-3'；
(2) it using the cDNA of 95ms-5B as template, by PCR amplification target fragment, is reacted by digestion connection by target patch
Section is building up on pBluescript II KS+ carrier (purchased from Stratagene company, 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, and isometric water is added after digestion, and product is used
Isometric Tris- saturated phenol and chloroform mixed liquor (volume ratio 1:1) is stripped, and 12000rpm/min is centrifuged 10min, is inhaled
Supernatant is added chloroform and purifies 2 times into clean centrifuge tube, and 12000rpm/min is centrifuged 10min, sucts clearly to clean centrifugation
The sodium acetate of Guan Zhong, alcohol and 0.1 times of volume that 2 times of volume pre-coolings are added precipitate, and after refrigerated centrifuge, abandon supernatant.With 75%
Alcohol rinses twice, and after dry plus DEPC water dissolves.
(4) probe is transcribed: reaction system (10 μ l) is as follows:
37 DEG C are reacted 4 hours, and DNase (RNase-free) is added into reaction system after completing for transcription and RNasin is each
50 μ l TE (PH8.0), 5 μ l licl and 155 μ l dehydrated alcohols, -20 DEG C of mistakes after mixing is added in 0.5 μ l, 37 DEG C of reaction 25min
Night is centrifuged 20min to Precipitation, 12000rpm/min, abandons supernatant, and 10 μ l DEPC are added after dry in 75% ethanol washing 2 times
Water dissolution.1 μ l point sample is taken to detect, spectrophotometric determination production concentration.
C. in situ hybridization
(1) twice by dimethylbenzene dewaxing, each 20min.It uses dehydrated alcohol 2 times respectively, 95% ethyl alcohol, 85% ethyl alcohol,
70% ethyl alcohol, 50% ethyl alcohol, 30% ethyl alcohol, the graded ethanol rehydration of 15% ethyl alcohol, with 2 rehydrations of common DEPC water washing,
Each 1min.
(2) 37 DEG C of preheating Proteinase K liquid, Proteinase K ultimate density are 1 μ g/ml, hybridization piece in 37 DEG C of processing 40min,
1 × PBS is washed once.
(3) it is dehydrated through 15%, 30%, 50%, 70%, 85%, 95%, 100% alcohol series, then each 1min exists
Super-clean bench air-dries.
(4) 150 μ l prehybridization solutions are dripped on material, cover Para film sealed membrane, and glass slide is then placed on NaOH processing
In the centrifuge tube box crossed, 42 DEG C of prehybridization 3h.
(5) it is mixed in proportion with hybridization buffer according to concentration and probe concentration, 50 μ l of hybridization solution is added dropwise on every slide, covers Para
Film sealed membrane, 42 DEG C of hybridized overnights in wet box.
(6) slide is taken off to Para film sealed membrane in 2 × SSPE at room temperature, 42 DEG C of 2 × SSPE buffer are washed 2 times,
Each 15min；Then under the conditions of 57 DEG C, 0.2 × SSPE buffer water-bath 50r/min is washed 2 times, each 30min, and elution is remaining
Probe.
(7) under room temperature (23 DEG C), slide is placed in 40min, 50r/min in 1 × Blocking buffer.Blockade liquid
It needs now to match.It is by volume 1:2500 dilution anti-DIG-AP antibody with BSA washing buffer, used kit is
ROCHE colour reagent box (CAT.NO.11745832910), will slice in room temperature in antibody oscillation incubation 2h, 50r/min.
(8) after being immunoreacted, slide is placed in 15min in 1 × Blocking buffer, room temperature (23 DEG C) condition
Lower 50r/min elution, is washed 3 times under room temperature (23 DEG C), each 15min, 50r/min in BSA washing buffer.
(9) chromogenic reaction: slice change TNM-50 room temperature 3 times, each 5min.It is added 0.5ml's in 40ml TNM-50
The developing solution that NBT is made into.Slice is placed in one, shows 3h-5h in wet box at room temperature dark.It is developed a film 3 times after colour developing with TE,
Each 5min.
(10) material is through 30%, 70%, 95%, 100% ethyl alcohol, dimethylbenzene: ethyl alcohol (volume ratio 1:1), dimethylbenzene, two
The dehydration of toluene series is transparent, microexamination photography.
From the in situ hybridization result of Fig. 5: Sicwinv1 gene sesame anther tetrad expression quantity highest,
And it is mainly expressed in tapetum and tetrad.
Claims (4)
1. a kind of specifically expressed promoter P for being isolated from sesameSicwinv1, the promoter only in anther specifically expressing, do not exist
It is expressed in its hetero-organization, the nucleotide sequence of the promoter is as shown in SEQ ID NO:1.
2. a kind of specifically expressed promoter P for being isolated from sesame described in claim 1Sicwinv11Answering in anther regulation
With.
3. a kind of specifically expressed promoter P for being isolated from sesame described in claim 1Sicwinv11Answering in plant improvement
With.
4. a kind of specifically expressed promoter P for being isolated from sesame described in claim 1Sicwinv11In plant hybrid advantage benefit
Application in.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610528610.8A CN106906212B (en) | 2016-07-06 | 2016-07-06 | One sesame anther specific expression promoter PSicwinv1 and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610528610.8A CN106906212B (en) | 2016-07-06 | 2016-07-06 | One sesame anther specific expression promoter PSicwinv1 and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106906212A CN106906212A (en) | 2017-06-30 |
| CN106906212B true CN106906212B (en) | 2019-08-30 |
Family
ID=59206396
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610528610.8A Expired - Fee Related CN106906212B (en) | 2016-07-06 | 2016-07-06 | One sesame anther specific expression promoter PSicwinv1 and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106906212B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2016
- 2016-07-06 CN CN201610528610.8A patent/CN106906212B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| Characterization and genetic mapping of a novel recessive genic male sterile gene in sesame (Sesamum indicum L.);Zhao Yingzhong;《MOLECULAR BREEDING 》;20131231;第32卷(第4期);第901-908页 * |
| 芝麻核雄性不育系ms86-1微粉发生的细胞学观察;郭伟等;《植物学报》;20140131;第49卷(第1期);第87-97页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106906212A (en) | 2017-06-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JPH06506595A (en) | Male sterile plant, method for producing male sterile plant, and recombinant DNA used in the method | |
| CN105713077B (en) | A kind of soybean MYB class transcription factor GmMYB29 and its encoding gene and application | |
| CN103710382B (en) | Dual-35S overexpression vector for changing flower types of plants and application thereof | |
| CN102559685B (en) | Anther tapetum specific expression promoter POsABCG15, recombinant expression vector thereof and application thereof | |
| CN110777152B (en) | Transcription factor EjBZR1 for inhibiting fruit cell expansion and application thereof | |
| CN107236744A (en) | The separation and application of the early real trifoliate orange PtAGL24 genes of controllable plant early blossoming | |
| CN110386967B (en) | Plant male fertility-related protein SiMS1, and coding gene and application thereof | |
| CN113150094B (en) | EjAP2L gene related to loquat flower development and encoding protein and application thereof | |
| CN106906212B (en) | One sesame anther specific expression promoter PSicwinv1 and its application | |
| US20140020130A1 (en) | csRRM2 GENE AND ITS USE FOR IMPROVING TRAITS IN INDUSTRIAL CROPS | |
| CN103290024B (en) | A kind of plant seed absciss layer district developmental regulation gene and application thereof | |
| CN110372782B (en) | Loquat flower organ development related transcription factor EjPI protein and coding gene and application thereof | |
| CN105695461A (en) | Wheat flag leaf specific expression promoter and application thereof | |
| CN102604963B (en) | Separation, cloning and application of Poncirus trifoliata EARLYFLOWERING 5 (PtELF5) gene | |
| CN101824433A (en) | Specific gene controlling growth of Arabidopsis vascular bundle and application thereof | |
| CN103725692A (en) | Zinc finger protein gene used for controlling rice fertility | |
| CN102965374B (en) | Preparation method and applications of rape BnRabGDI3 promoter | |
| US11761017B2 (en) | Rice thermo-sensitive male sterile gene mutant tms18 and uses thereof | |
| CN101883572A (en) | Sorghum aluminum tolerance gene, sbmate | |
| CN104561061B (en) | A kind of cabbage type rape fertility correlation BnCP20 genes and its application | |
| CN101113452B (en) | Plant flower specificity promoter and its application | |
| CN103525809A (en) | Building body of mediating plant fertility and application thereof | |
| CN102115751A (en) | Rape BnPABP 5 gene and application of promoter thereof | |
| CN106480026A (en) | A kind of flower pesticide early development specific expressing promoter and its application | |
| CN104450736A (en) | Cucumber AG-like gene CsMADS24 overexpression carrier and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190830 Termination date: 20200706 |