CN102465128B - Anther specific expression promoter and application thereof - Google Patents
Anther specific expression promoter and application thereof Download PDFInfo
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- CN102465128B CN102465128B CN201110360123.2A CN201110360123A CN102465128B CN 102465128 B CN102465128 B CN 102465128B CN 201110360123 A CN201110360123 A CN 201110360123A CN 102465128 B CN102465128 B CN 102465128B
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Abstract
The invention relates to an anther specific expression promoter and an application thereof. The promoter which can express the specificity in the plant anther is separated at the first time. The promoter can direct the specific high expression of a target gene in the plant anther and direct low expression or no expression in other tissues of the plant.
Description
Technical field
The invention belongs to biotechnology and phytology field; More specifically, the present invention relates to a kind of anther specific expression promoter and application thereof.
Background technology
The mankind, by the selection and utilization to spontaneous mutation gene and recombinant chou, improve the history of existing nearly one thousand years to the kind of crop.In the last hundred years, usually adopt the method for artificial hybridization, New Crop Varieties is cultivated in the importing of the restructuring and foreign gene that realize excellent genes.Crossbreeding technology can realize kind of an interior transgenosis, but the transgenosis between the kind that sibship is far away then exists obstacle; On the other hand, certain gene can not accurately be selected and be handled to crossbreeding technology, therefore, adds the complicacy of breeding result.
Carried out the seed selection of crop varieties by genetically modified means, it is advantageous that transgenosis not by the restriction of sibship between organism, from genetically modified technical standpoint, the gene source that candidate transforms is restricted hardly; Therefore, the present inventor can be polymerized various effective gene by genetically modified means, cultivates high yield, high-quality, degeneration-resistant New Crop Varieties.On the other hand, transgenic technology gene that is operated and transfer is the gene of definite functions, control objectives proterties, and therefore, the phenotype of transgenic progeny has more predictable.At present, transgenic technology has become the important means of improvement of crop cultivar, and the cultivated area of genetically engineered soybean, corn, cotton and rape has reached 25% (James, 2005 of 4 kinds of total cultivated areas in the crops whole world; Sankula et al., 2005).
The element that two required is needed, i.e. promotor and goal gene in the transgenosis of plant.Promotor is the cis-regulatory sequence being positioned at gene transcription start site upstream, is combined with the recognition factor of trans-acting, by the interaction of recognition factor and RNA polymerase, and transcribing of promotor gene; Therefore, the expression pattern of promoter regulation gene, the i.e. Space-time speciality of destination gene expression and the intensity of expression are one of key factors of transgenosis success or failure or effect height.
Different for genetically modified object, the expression pattern that the gene Selection of conversion is different, mainly comprises composing type high expression level, tissue specific expression and inducible expression etc.As proceeded to bacillus thuringiensis (Bacillus thuringiensis in the farm crop such as cotton, corn, paddy rice, the mode of what insect resistance protein Bt) adopted is composing type high expression level, in crop, the advantage of high dosage expression Bt insect resistance protein is that high dosage can guarantee the effect of desinsection, is conducive to the validity period extending Bt insect resistance protein simultaneously.In the initiative process of golden paddy (Golden rice), its target is the route of synthesis by introducing vitamin A precursor in paddy endosperm, thus in rice enhanced vitamin A, solve vitamin A nutritional lack problem; Therefore, the promotor adopted during transgenosis is the glutelin promoter of specifically expressing in paddy endosperm.In the conversion of plant disease resistance genes, the normal promotor adopting pathogenic bacterium inducing to express.
Along with transgenic crop is at the spread in the whole world, the importance of promotor in transgenosis is known together widely, and the present inventor needs dissimilar promotor to help the present inventor to provide the solution of transgene expression pattern.Such as, in the research field of rice tissue specific promoter, the promotor that what people paid close attention to the most is flower pesticide is special.Rice Anther is the organ that rice male gamete pollen produces, closely related with the output of the growth of Rice Panicle, breeding and paddy rice etc.
Therefore, the promotor that this area needs research plants flower pesticide specificity further to express, for functional genes more specific expressed in flower pesticide or structure gene, reaches the object of breed improvement.
Summary of the invention
The object of the present invention is to provide a kind of anther specific expression promoter and application thereof.
In a first aspect of the present invention, provide a kind of polynucleotide (promotor) of separation, described polynucleotide:
A () is positioned at 5 ' end and upstream thereof of osigcfa001g12 gene (GenBank accession number: CT833313);
B () bases longs is 200-2000;
C () has the necessary site and transcripting start point of causing and transcribing; And
D () has specifically expressing function in plant anther.
In a preference of the present invention, described osigcfa001g12 gene source is in grass; More preferably derive from paddy rice (Oryza Sativa).
In a preference of the present invention, in (a), be positioned at osigcfa001g12 upstream region of gene-1000 to coding region the 100th.
In another preference of the present invention, described polynucleotide are:
(1) polynucleotide of the nucleotide sequence shown in 1-521 position in SEQ ID NO:1;
(2) polynucleotide of the nucleotide sequence shown in 1-552 position in SEQ ID NO:1;
(3) polynucleotide of (1) or (2) arbitrary restriction, wherein 1-22 bit base disappearance (that is: the polynucleotide of the nucleotide sequence shown in 23-552 position in SEQ ID NO:1; Or: the polynucleotide of the nucleotide sequence shown in 23-521 position in SEQ ID NO:1);
(4) polynucleotide be made up of the nucleotide sequence shown in (1-22) ~ (521-552) position in SEQ ID NO:1;
(6) nucleotide sequence can be hybridized with the polynucleotide sequence of the arbitrary restriction in (1)-(4) and have the polynucleotide instructing goal gene specifically expressing function in plant anther under strict conditions;
(7) polynucleotide sequence of nucleotide sequence and the arbitrary restriction in (1)-(4) has more than 80% (preferably more than 85%; More preferably more than 90%; More preferably more than 95%; More preferably more than 99%; ) homology and there are the polynucleotide instructing goal gene specifically expressing function in plant anther; Or
(8) polynucleotide of the polynucleotide sequence complete complementary of nucleotide sequence and the arbitrary restriction in (1)-(4).
In another preference of the present invention, described polynucleotide are: based on nucleotide sequence shown in 1-552 position in 1-521 position in SEQ ID NO:1 or SEQ ID NO:1,436-442 position, 146-153 position, 278-283,305-311 position and/or 287-312 position (26bp) nucleotide sequence are constant, and the polynucleotide sequence of other site and the arbitrary restriction in (1)-(4) has 50% (preferably 60%; More preferably 70%; More preferably 80%; More preferably 90%; More preferably 95%; More preferably 99%) above homogeny, and instruct the polynucleotide of goal gene specifically expressing function in plant anther.
In another preference of the present invention, described polynucleotide are: based on nucleotide sequence shown in 1-552 position in 1-521 position in SEQ ID NO:1 or SEQ ID NO:1,99-104 position, 115-120 position and/or 131-138 position nucleotide sequence are constant, and the polynucleotide sequence of other site and the arbitrary restriction in (1)-(4) has 50% (preferably 60%; More preferably 70%; More preferably 80%; More preferably 90%; More preferably 95%; More preferably 99%) above homogeny, and there are the polynucleotide instructing goal gene specifically expressing function in plant anther.
In another preference of the present invention, described plant is monocotyledons.
In another preference of the present invention, described plant includes, but is not limited to: grass, amrallid, liliaceous plant, irides or Dioscoreaceae plant etc.
Preferred, described plant is grass.Such as described plant includes but not limited to: paddy rice, wheat, barley, corn, Chinese sorghum etc.
In another aspect of this invention, provide the purposes of described polynucleotide, be used to guide goal gene specifically expressing in plant anther.
In another aspect of this invention, provide a kind of carrier, described carrier contains described polynucleotide, as promoter element.
In a preference of the present invention, described carrier is also containing the goal gene be connected with described polynucleotide manipulation.
In another preference of the present invention, described goal gene is structure gene.
In another preference of the present invention, described goal gene codified has the albumen of specific function.
In another preference of the present invention, described goal gene is foreign gene.
In another preference of the present invention, described goal gene includes, but is not limited to: cytotoxic group is because of (Cytotoxic gene), and rnase (Barnase) gene or connection as come from bacillus amyloliquefaciens (Bacillus amyloliquefaciens) come from ribonuclease T1 (RNase T1) gene of aspergillus tubigensis (Aspergillus oryzae); The DYT1 gene of flower pesticide suede adhesion coating function and pollen formation is affected, ABORTED MICROSPORE gene (AMS) and MALE STERILITY 1 gene (MS1) etc. in Arabidopis thaliana; The MULTIPLE SPOROCYTE1 gene (MSP1) of suede adhesion coating function and pollen formation is affected in paddy rice, OsTDL1A gene, UNDEVELOPED TAPETUM gene (OsUDT1) and TAPETUMDEGENERATION RETARDATION gene (OsTDR) etc.
In another preference of the present invention, described goal gene is positioned at the downstream of described polynucleotide, and and the interval of described polynucleotide be less than 1000bp.Preferably, 500bp is less than; Preferred, be less than 100bp; Most preferred, be less than 50bp.
In another aspect of this invention, a kind of genetically engineered host cell is provided, described cell:
Containing described carrier; Or
The described polynucleotide of external source are integrated with in its genome.
In another aspect of this invention, provide a kind of method making goal gene specifically expressing in plant anther, described method comprises:
By construction transformed plant cells, the goal gene that described construction contains described polynucleotide and is connected with described polynucleotide manipulation;
Filter out the vegetable cell having proceeded to and be integrated with described construction in described construction or karyomit(e); With
Described Plant cell regeneration is become plant.
In another preference of the present invention, described method comprises:
A () provides the Agrobacterium of carrying expression vector, containing construction in described expression vector, and the goal gene that described construction contains described polynucleotide and is connected with described polynucleotide manipulation;
B vegetable cell, tissue or organ contact with the Agrobacterium in step (a) by (), thus make described construction proceed to vegetable cell.
In another preference of the present invention, described method also comprises:
C () selects the vegetable cell, tissue or the organ that have proceeded to described construction; And
D vegetable cell in step (c), tissue or neomorph are become plant by ().
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1 shows pcr amplification Totomycin sequence (for the gene of resistance screening in transgene carrier), the electrophoresis result whether inserted containing T-DNA in qualification transgenic paddy rice seedling.M, DNA MarkerDL2000 (DNA molecular amount standard); 1-12 representative comes from the qualification result of the different transfer-gen plants of different transgenic lines (lines).
Fig. 2 shows the variant result organizing gus reporter gene to dye in promotor transgenic paddy rice.Wherein:
A, the GUS dyeing in rice young panicle;
B, paddy rice grain husk spend in GUS dyeing;
C is same stage with figure B, two panels grain husk shell (inner glume with the coetonium) strip off of grain husk flower is come, arrow indication be the special anther tissue dying blueness;
D, paddy rice grain husk spend in GUS dyeing; A, flower pesticide; S, column cap; O, ovary.
E, the GUS dyeing of the grain husk flower of Post flowering;
F is same stage with figure E, is peeled off by the two panels grain husk shell of grain husk flower, the dyeing of display stamen and gynoecium; A, flower pesticide; S, column cap; O, ovary.
G is same stage with figure E, the flower pesticide part of amplification, the dyeing of display flower pesticide;
H is same stage with figure E, display pollen staining;
I, the GUS dyeing of root;
J, the GUS dyeing of blade;
K, the GUS dyeing of leaf sheath, the tip of a leaf, auricle;
L, the GUS dyeing of stem;
In figure, the scale in G, H is 100 μm, and the scale in other figure is 1mm.
Fig. 3 shows GUS coloration result in flower pesticide section, and blueness shows that GUS dyeing is for positive.A, flower pesticide; T, suede adhesion coating; L: clever shell; E, epidermis; MC, maiotic cell.
Fig. 4 shows the column diagram of gus gene quantitative result of special high expression level in fringe.
The variant result organizing gus reporter gene to dye in Fig. 5, M1 promotor transgenic paddy rice.
A, the GUS dyeing of root; B, the GUS dyeing of blade; C, the GUS dyeing of leaf sheath, the tip of a leaf, auricle; D, the GUS dyeing of stem; E, the GUS dyeing of flower.Scale in figure is 1mm.
The variant result organizing gus reporter gene to dye in Fig. 6, M2 promotor transgenic paddy rice.
A, the GUS dyeing of root; B, the GUS dyeing of blade; C, the GUS dyeing of leaf sheath, the tip of a leaf, auricle; D, the GUS dyeing of stem; E, the GUS dyeing of flower.Scale in figure is 1mm.
The variant result organizing gus reporter gene to dye in Fig. 7, M3 promotor transgenic paddy rice.
A, the GUS dyeing of root; B, the GUS dyeing of blade; C, the GUS dyeing of leaf sheath, the tip of a leaf, auricle; D, the GUS dyeing of stem; E, the GUS dyeing of flower.Scale in figure is 1mm.
The variant result organizing gus reporter gene to dye in Fig. 8, M4 promotor transgenic paddy rice.
A, the GUS dyeing of root; B, the GUS dyeing of blade; C, the GUS dyeing of leaf sheath, the tip of a leaf, auricle; D, the GUS dyeing of stem; E, the GUS dyeing of flower.Scale in figure is 1mm.
The GUS quantitative result of Fig. 9, promotor and clipped form thereof.
Embodiment
The present inventor through extensive and deep research, be separated to first one can be specific expressed in plant anther promotor.Described promotor can instruct goal gene specificity overexpression in plant anther, and low expression or do not express in other tissue of plant.
Term
As used herein, described " plant " mainly refers to monocotyledons, includes, but is not limited to: grass, amrallid, liliaceous plant, irides or Dioscoreaceae plant etc.Preferred, described plant is grass, includes but not limited to: paddy rice, wheat, barley, corn, Chinese sorghum etc.
As used herein, " separation " refers to that material is separated from its primal environment (if natural substance, namely primal environment is natural surroundings).As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials existed separately, then for separation and purification.
As used herein, described " being operably connected " or " operability connection " refers to functional spatial disposition of two or more nucleic acid region or nucleotide sequence.Such as: promoter region is placed in the specific position relative to goal gene nucleotide sequence, what make nucleotide sequence transcribes the guiding being subject to this promoter region, thus promoter region is " operably connected " on this nucleotide sequence.
As used herein, described " promotor " or " promoter region (territory) " refers to a kind of nucleotide sequence, and it is present in the upstream (5 ' end) of goal gene encoding sequence usually, can be transcribed into mRNA by guiding nucleus acid sequence.Usually, promotor or promoter region provide the recognition site of RNA polymerase and the necessary other factors of correct initiation transcription.In this article, described promotor or promoter region comprise the variant of promotor, and it, by inserting or delete regulation and control region, carries out random or rite-directed mutagenesis etc. and obtain.
As used herein, term " specific expressed " refers to that goal gene is at specific time and/or specific tissue expression.
As used herein, " tissue-specific promoter ", also known as " organ specific promoters ", under this kind of promoter regulation, gene is often only expressed at some specific organ or tissue position, and shows the characteristic of Growth adjustment.In the present invention, described " tissue-specific promoter " is plant anther specific expression promoter.
Usually, if mRNA is with than height at least 10 times in other tissue or organ in certain tissue or organ, preferably at least high 100 times, more preferably at least high 1000 times of levels are expressed, then this promotor is considered to tissue or organ specific.
As used herein, " external source " or " allos " refers to from the relation between the two or more pieces nucleic acid of different sources or protein sequence.Such as, if the combination of promotor and goal gene sequence is not naturally occurring usually, then promotor is external source for this goal gene.Particular sequence is " external source " for its cell inserted or organism.
As used herein, " cis-regulating element " refers to the conservative property base sequence played regulatory role transcription initiation and the transcriptional efficiency of gene.
As used herein, " goal gene " refers to the gene that can be started by promotor of the present invention or instruct and express.Suitable goal gene includes but not limited to: the gene that improvement plant quality, proterties or metabolism are relevant.Suitable goal gene includes but not limited to: cytotoxic group is because of (Cytotoxic gene), and rnase (Barnase) gene or connection as come from bacillus amyloliquefaciens (Bacillus amyloliquefaciens) come from ribonuclease T1 (RNase T1) gene of aspergillus tubigensis (Aspergillus oryzae); The DYT1 gene of flower pesticide suede adhesion coating function and pollen formation is affected, ABORTED MICROSPORE gene (AMS) and MALE STERILITY 1 gene (MS1) etc. in Arabidopis thaliana.The MULTIPLE SPOROCYTE1 gene (MSP1) of suede adhesion coating function and pollen formation is affected in paddy rice, OsTDL1A gene, UNDEVELOPEDTAPETUM gene (OsUDT1) and TAPETUM DEGENERATION RETARDATION gene (OsTDR) etc.
As used herein, described " containing ", " having " or " comprising " include " comprising ", " primarily of ... form ", " substantially by ... form " and " by ... form "; " primarily of ... form ", " substantially by ... form " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".
As used herein, namely described " in SEQ ID NO:1 the nucleotide sequence shown in (1-22) ~ (521-552) position " also refer to that described sequence can originate in the base of arbitrary in SEQ ID NO:1 in the 1st to the 22nd, ends at the base of arbitrary of in SEQ ID NO:1 the 521st to the 552nd.
Promotor
The invention provides a kind of anther-specific expression promotor, described promotor has following characteristics: (a) is positioned at 5 ' end and upstream thereof of osigcfa001g12 gene; B () bases longs is 200-2000; C () has the necessary site and transcripting start point of causing and transcribing; And (d) has specifically expressing function in plant anther.
Promotor of the present invention has the necessary site and transcripting start point of causing and transcribing, and the present inventor have studied the critical sites of the performance function of this promotor particularly, comprising:
box (TATAAAT),
(CCAATGCA),
sequence and 26bp sequence
these sites are that promotor of the present invention plays promotor gene expressive function and plays the critical area of anther-specific expression function.
As one embodiment of the present invention, described promotor has 1-521 position, 1-552 position, the nucleotide sequence shown in 23-552 position in SEQ ID NO:1.
The hybridization of polynucleotide is technology well known to those skilled in the art, their similarity of hybrid trait instruction of specific a pair nucleic acid or identity.Therefore, the invention still further relates to and aforementioned nucleotide sequence hybridization of specifying and have at least 50% between two sequences, preferably at least 70%, the more preferably polynucleotide of at least 80% (such as 85%, 90%, 95%, 96%, 97%, 98% or 99%) homogeny.The present invention be more particularly directed to polynucleotide interfertile with polynucleotide of the present invention under strict conditions.
" stringent condition " (or " stringent condition ") refers to: (1) compared with the hybridization under low ionic strength and comparatively high temps and wash-out, as 0.2 × SSC, 0.1%SDS, 60 DEG C; Or be added with denaturing agent during (2) hybridization, and as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.; Or (3) homogeny only between two sequences is at least 50%, preferably more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% or more than 90%, just hybridize when being more preferably more than 95%.Further, interfertile polynucleotide also have the function instructing goal gene specific expressed in flower pesticide.
The present invention also comprises and to have 50% or more with any one promoter sequence of the present invention (preferably more than 60%, more than 70%, more than 80%, more preferably more than 90%, most preferably more than 95%, as 98%, 99%) nucleic acid of homogeny, described nucleic acid also has and instructs goal gene specific expressed function in flower pesticide." homogeny " refers to according to the identical per-cent in position, the similar level (i.e. sequence homology, similarity or identity) between two or more pieces nucleic acid.
Should understand, although provide this promotor and function thereof of deriving from paddy rice in example of the present invention, but, the information that those skilled in the art provide according to the application after having read the application derives from other monocotyledonous promotor with this promotor with certain homogeny (conservative property) to be also included within scope of the present invention, as long as can be separated easily from other plant obtain this promotor.
Start destination gene expression
Promotor of the present invention can be operatively connected on goal gene, and this goal gene can be external source (allos) for promotor.The nucleotide sequence of described goal gene is had no particular limits (as a kind of structural nucleic acid sequence), described goal gene optimized encoding has the albumen of specific function, and such as some has the albumen of key property or function in agricultural or plant improvement.
Promotor of the present invention can also be operably connected in the goal gene sequence that is modified, and this goal gene is external source (allos) relative to promotor.Described goal gene can be modified the characteristic producing various expectation.Such as, goal gene can be modified the content increasing indispensable amino acid, improves the translation of aminoacid sequence, change the modification (as phosphorylation site) after translation, by outside translation product transporte to cells, improve the stability of albumen, insert or delete cell signal etc.
In addition, promotor and goal gene can be designed to lower specific gene.This is generally realize by promotor being connected in goal gene sequence, and this sequence is oppositely directed with antisense.Those of ordinary skill in the art is familiar with this antisense technology.Any nucleotide sequence can be conditioned by this way.
The aforesaid promotor of any one and/or goal gene sequence can be comprised in recombinant vectors.
As a kind of mode, described recombinant vectors comprises promotor of the present invention, comprises multiple clone site or at least one restriction enzyme site in the downstream of described promotor.When goal gene expressed by needs, goal gene is connected in applicable multiple clone site or restriction enzyme site, thus goal gene is operably connected with promotor.
Alternatively, described recombinant vectors comprises in (from 5 ' to 3 ' direction): promotor, and goal gene.If needed, described recombinant vectors can also comprise 3 ' transcription terminator, 3 ' polymerized nucleoside acidifying signal, other untranslated nucleotide sequence, transhipment and target nucleotide sequence, resistance selective marker, enhanser or operator.
Method for the preparation of recombinant vectors is well known in the art.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral or other carriers.In a word, as long as it can copy and stablize in host, any plasmid and carrier are all can be adopted.
Method well-known to those having ordinary skill in the art can be used for the expression vector built containing promotor of the present invention and/or goal gene sequence.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.Expression vector also comprises ribosome bind site and the transcription terminator of translation initiation.
In addition, expression vector preferably comprises one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as Tetrahydrofolate dehydrogenase, neomycin resistance, hygromycin resistance and green fluorescent protein (GFP) etc.
Except containing promotor of the present invention in recombinant vectors, also one or more other promotors can be contained.Other described promotor is such as: tissue-specific, composing type or induction type.Cauliflower mosaic virus 19S and 35S (CaMV19S CaMV35S) of such as mannopine synthase, CaMV, the tobacco RB7 etc. of enhancing.
Comprise the carrier of above-mentioned suitable promotor and goal gene, may be used for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell is as yeast; Vegetable cell etc.Persons skilled in the art all know how to select suitable carrier and host cell.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host be prokaryotic organism as intestinal bacteria time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaCl
2method process, step used is well-known in this area.Another kind method uses MgCl
2.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical methods is as microinjection, electroporation, liposome packaging etc.Conversion of plant also can use the method such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, such as leaf disk method, rataria conversion method, bud infusion method etc.Can ordinary method regeneration plant be used for the vegetable cell transformed, tissue or organ, thus obtain genetically modified plant.
As a kind of mode, the method preparing transgenic plant is: the carrier carrying promotor and goal gene (both are operably connected) is proceeded to Agrobacterium, and the carrier segments containing promotor and goal gene is incorporated on the karyomit(e) of plant by Agrobacterium again.The transgene receptor plant related to is such as paddy rice, wheat, Arabidopis thaliana, tobacco, fruit tree etc.
In example of the present invention, described recombinant vectors is pCambia carrier, it carries beta-glucosidase (GUS) gene, promotor of the present invention is building up to the upstream of gus gene in this carrier, transformed plant, promotor will activate the expression of gus gene, and described startup is subject to the regulation and control of each cis-acting elements in promoter region, simulate gene and be activated in vivo the situation of transcribing.The a series of beta-glucoside of beta-glucosidase (GUS) energy catalytic pyrolysis, produces the material with chromophoric group or fluorescence, and the methods such as available spectrophotometer, photofluorometer or histological chemistry are carried out quantitatively and spatial positioning analysis GUS activity.In the art, gus gene has been widely used as the reporter gene of transgenic plant, bacterium and fungi, and particularly it can be used to the concrete biological cells and tissues position of Study of Exogenous genetic expression.
Application
Anther specific promoter of the present invention has important using value in theoretical investigation and agronomy improvement.The present invention can be widely used in plant genetic engineering: promotor can merge with target gene as an important tool in plant genetic engineering, and by transgene carrier, conversion of plant also obtains transfer-gen plant.
The using value of Rice Anther specific promoter, includes but not limited to following aspect:
A, utilize Rice Anther specific promoter formulate male sterible series of rice
In the process of paddy rice cross breeding breeding, need male sterile line (Male sterility), people utilize this characteristic of male sterile, eliminate the operating process of artificial emasculation, thus have saved a large amount of manpower and the time of cross-breeding.Therefore, male sterile plant has great economic worth.Rice Anther specific promoter is utilized to formulate male sterile line.Rice Anther specific promoter connects cytotoxic group because of (Cytotoxic gene), rnase (Barnase) gene or connection as come from bacillus amyloliquefaciens (Bacillus amyloliquefaciens) come from ribonuclease T1 (RNase T1) gene of aspergillus tubigensis (Aspergillus oryzae), Barnase and RNase T1 gene destroys the normal development of cell in its cell of expressing, therefore, by means of the specifically expressing of Barnase or RNase T1 gene in flower pesticide, the normal development of flower pesticide can be suppressed, thus acquisition male sterible series of rice.
B, utilize Rice Anther specific promoter to control the gene of transgenosis in field to spread
At present, the major issue that transgenosis field controls is the powder problem of wafing of genetically modified crops.The uncontrolled propagation of transgenic pollen can cause gene contamination to other species in surrounding environment, and Rice Anther specific promoter can help to address this problem.As mentioned above, Rice Anther specific promoter is connected Barnase, together rice transformation, and the Transgenic Rice Plants obtained like this, can not form normal pollen; Therefore, the gene contamination that the pollen avoiding transfer-gen plant causes other species in surrounding environment.
C, utilize Rice Anther specific promoter study affect Rice Panicle grow important gene, be increase crop yield, improve quality, increase resistance provide excellence provenance
Anther-specificpromoter regulatory gene specifically expressing in flower pesticide, its expressive site concentrates on flower pesticide, do not express, or expression amount is very low in its hetero-organization.Compared with constitutive promoter, the advantage of anther-specificpromoter is, it makes goal gene specific action in flower pesticide organ, decreases the impact possible on its hetero-organization of paddy rice.For the improvement being carried out rice varieties by engineered method provides important operational means.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
Embodiment 1, the screening of rice tissue specific promoter and the qualification of genetic expression
The present inventor constructs the fringe of Rise's boot period, cDNA library (the Liu of the tissue such as the over-ground part of 14 days seedling and root, XH, Lu TT, Yu SL, Li Y, Huang YC., Huang T., Zhang L., Zhu JJ, Zhao Q, Fan DL, Mu J, Shangguan YY, Feng Q, Guan JP, Ying K, Zhang Y, Lin ZX, Sun ZX, Qian Q, Lu YP, Han B. (2007) A collection of 10, 096indica rice full-length cDNAs reveals highly expressed sequence divergencebetween Oryza sativa indica and japonica subspecies.Plant Mol Biol.65:403-415).5 ' the end sequencing of 20,000 clones is carried out respectively in each library.In the process of each library cDNA redundancy statistics, the present inventor finds there is the very high cDNA clone of some copy numbers in the cDNA library of often kind of tissue.By the copy number of cDNA in each library, according to sorting from high to low, finding out cDNA copy number in each library and being positioned at the cDNA of first 20; Then, add up the copy number of these cDNA in the cDNA library of its hetero-organization of paddy rice, if these cDNA do not occur in the library of its hetero-organization, then these cDNA become the candidate gene that tissue specificity High-expression promoter is separated.Then, the present inventor, in the fringe of Rise's boot period, the over-ground part of 14 days seedling and root, is identified the tissue specificity that candidate gene is expressed by the method for Real time RT-PCR, determines candidate gene further.
The clone of Rice Anther specific promoter and the conversion of paddy rice: by the comparison of candidate gene full length cDNA sequence and Rice Genome Sequence, the upstream regulatory region sequence of gene can be obtained, i.e. promoter sequence.Then by PCR method cloned promoter, between BamHI and NcoI site promoter sequence being inserted into pCambia1305.2 (available from Cambia company) expression vector (containing gus reporter gene), conventional agrobacterium co-cultivation rice transformation, identifies the expression characterization of promotor by the expression detecting gus gene in transgenic paddy rice.
The screening of embodiment 2, Rice Anther specific promoter and the tissue-specific qualification of genetic expression
By the statistics to full-length cDNA copy number in Rice Panicle cDNA library, the present inventor obtains the candidate gene of a Rice Panicle specific expression gene, osigcfa001g12 (GenBank accession number: CT833313).
Then, the present inventor carries out the qualification of Realtime RT-PCR to the expression of osigcfa001g12 in the fringe of Rise's boot period, the over-ground part of 14 days seedling and root.Using paddy rice constitutive expression gene actin1 as reference gene, result is as table 1.In table, the expression amount of osigcfa001g12 gene is the relative expression quantity relative to reference gene actin1.
Table 1
The above results shows, the expression of osigcfa001g12 in fringe is very high, and expresses very low in rice root and seedling.Osigcfa001g12 is the gene of special high expression level in fringe.
The structure of embodiment 3, the sequential analysis of Rice Anther specific promoter, the clone of promotor and transgene carrier
The promoter region sequence of osigcfa001g12 gene and coding sequence are as SEQ ID NO:1.Wherein: box indicating be initiator codon and the terminator codon of albumen.
display be the coding region of gene,
what indicate is 5 ' UTR and 3 ' UTR district of gene.Double underline is
box (TATAAAT) and
(CCAATGCA).Italic adds mark of emphasis
and
sequence.5 ' end, 20 bases are the site of answering for the primer pair of promoter region of increasing.
SEQ ID NO：1：
ACCTCAGCCAAAACCGAAGACAGTACCGCCGAAGGACCTTATTTAA
ACGGTTTTGTTAAGTTGGGGGACCCATCGTACCCGGTTTTGCGACC
GGGGACGAAAATCGGACTAGGTGATAAATAGAGGGACCCAAAGTG
AACTTATA
ATTTCATATCAAACAGTCACGGATGGGCTT
TAGGAAAGCAGAACTGGGCCCGGCCCAGTAGATCACATAGCCCAA
CAAGATCTAAACCGCATGCTCTCGTTTCAACAAATTATCACACCGA
TTG
CATCTGCTGCACAGGCTAAAT
GTAGCCATGA
ACCATTCACCTCACAAGTCACAAGCATTGCATTTCTATGGTTACCA
GTGCAGGACGAAATGCTCAACTAGCCCAAGCAAGAATGGAGCATG
ACAACCTCAGGCACCAAAAGCTC
ATTTGCATTGCACAAA
TCAAAAGTTTCC
GTGAGCAGGCTTCA
CTTGATTGGCCACACTGATCACCTGGGTAAGAACTGACAATAACAC
GCCGGCTCCTCTGGATTGCGACTTCCAGTGGGGCCCAGGTGTCGGT
GTCAGCTGGGTAAGTTGGTGTCATCTGGTATGGGAAACTTTGATGT
TTTGTACATGTGGTGGTGCTCTTGTCAACATTATATAGATAAGGAG
AGAATAGGACAAACATGACGCTTGAATAGCAGAAGAGAAGTTCTA
TCCAGCAATATCAAAAGTACAGCTGTCATTTTCAAAAAATGTAAAA
CAAAAATAAAACGAAAAACCCACTATACTAGTGCCAATAAAAGAA
GAAACAAAAGAGAACTACATTATTGTCAATGCCATTTGTTGATGTT
ATTATTACTTTTGTTTATAAGAATAACAATTGTACTTATAAGGCCTA
CGATTTATTACAG
The transgene carrier that the present inventor selects is pCambia1305.2, and the restriction enzyme site of clone selects BamHI and NcoI.PCambia1305.2 is cut by BamHI and NcoI enzyme, is cut away by 35S promoter original in carrier, and rubber tapping recovery obtains carrier segments.
Without BamHI point of contact in the PCR fragment of amplification, but there is NcoI point of contact.Therefore the present inventor adds BsaI point of contact in the reverse primer of design, and primer sequence is as follows:
Forward primer: ggatccacctcagccaaaaccgaaga (SEQ ID NO:2);
Reverse primer: ggtctc ccatgg cagccaggctagaagacttg (SEQ ID NO:3);
The recognition site of BsaI is ggtctc, and restriction enzyme site is the 1st and the 5th base thereafter, and therefore, actual point of contact is still NcoI point of contact ccatgg.Such PCR primer, after BamHI and BsaI enzyme is cut, can be connected with the pCambia1305.2 cut through BamHI and NcoI enzyme, obtains promotor transgene carrier.
It should be noted that, after promotor is connected with pCambia1305.2, before gus protein encoding sequence, the present inventor introduces the ten amino acid encoding sequence that osigcfa001g12 gene N holds, therefore, the gus protein of expression is that N end is containing osigcfa001g12 gene 10 amino acid whose fusion roteins.Fusion rotein does not affect the tissue-specific qualification of osigcfa001g12 promoter expression.
The expression identification of gus gene in the conversion of embodiment 4, paddy rice and transgenic paddy rice
The present inventor carries out the conversion of paddy rice by the method for Agrobacterium-mediated Transformation Rice Young Embryo.Promotor transgene carrier proceeds to Agrobacterium EHA105 (see Hood, E.E., Gelvin, S.B., Melchers, and Hoekema, A., Transgenic Res. L.S., 1993,2,208-218), then transform by EHA105 the transfer-gen plant that Japan's fine (available from rice in China institute) rataria obtains promotor.Japanese fine rataria, first through hypochlorite disinfectant, is placed in the generation of evoked callus on inducing culture.The callus that rataria induction produces and Agrobacterium 26 DEG C, Dual culture 3 days in dark.Then, proceed in the screening culture medium containing 40mg/l Totomycin and 300mg/l cephamycin and cultivated for 4 week.Screening the kanamycin-resistant callus tissue obtained proceeds to containing 0.5mg/l naphthylacetic acid (NAA), 4mg/l kinetin (kinetin), 6mg/l 6-benzylaminopurine (6-BA), 40mg/l Totomycin, the regeneration culture medium of 300mg/l cephamycin, cultivates 2-3 week.Afterwards, the regeneration seedling obtained proceeds in 1/2MS root media and carries out root culture.After 2-3 week, transgenic seedling moves into phytotron and cultivates.
By the conversion of paddy rice, the transfer-gen plant that the present inventor obtains 5-10 system (lines) carries out the qualification of promoter activity.First, transgenic paddy rice carries out the qualification of T-DNA insertion.The genomic dna of extracting transgenic paddy rice seedling, with the genomic dna of transgenic paddy rice seedling for template, whether pcr amplification Totomycin sequence (for the gene of resistance screening in transgene carrier), insert containing T-DNA in qualification transgenic paddy rice seedling, see Fig. 1.The pcr amplification positive shows that T-DNA is inserted as the positive.Substantially all contain T-DNA in the transgenic paddy rice that usual screening obtains to insert.
The GUS activity identification of embodiment 5, promotor transgenic positive plant
The tissues such as the root of transfer-gen plant, blade, stem and flower are dipped in GUS staining fluid, 37 DEG C, spend the night.Second day, 70-75% ethanol decolorization, took off the chlorophyll in tissue.Then, observe under dissecting microscope and record GUS dyeing result.The component of paddy rice GUS staining fluid is:
Na
2HPO
4/NaH
2PO
4(1M，pH7.0) 5ml；
X-Gluc 100mg；
Triton X-100 0.5ml；
Methyl alcohol 10ml;
Aqua sterilisa surplus;
Cumulative volume 100ml.
The result of Reporter gene GUS dyeing:
What Fig. 2 showed is the variant result organizing gus reporter gene to dye in promotor transgenic paddy rice.
A, the GUS dyeing in rice young panicle.Children's spike length is about 7mm, and paddy rice grain husk flower is about 1mm.Now, clever shell is transparent, dye-free; Only flower pesticide specific staining is positive.Promoter regulation gene specifically expressing in flower pesticide is described.
B, paddy rice grain husk spend in GUS dyeing.Grain husk flower is about 3.78mm, now, and clever shell dye-free; Flower pesticide specific staining is positive.
C is same stage with figure B, and two panels grain husk shell (inner glume with the coetonium) strip off of grain husk flower comes by the present inventor, arrow indication be the special anther tissue dying blueness.
D, paddy rice grain husk spend in GUS dyeing, grain husk flower is about 6.53mm.Grain husk shell dye-free; The column cap of gynoecium and ovary dye-free; The flower pesticide of stamen is special dyes blueness.Promoter regulation gene specifically expressing in flower pesticide.
E, the grain husk flower of Post flowering, grain husk flower is about 6.58mm.Grain husk shell dye-free, flower pesticide specific staining is blue.
F is same stage with figure E, is peeled off by the two panels grain husk shell of grain husk flower, the dyeing of display stamen and gynoecium.The column cap dye-free of gynoecium, has light dyeing in the zero position of style and the base portion of ovary; Flower pesticide dyeing is the very strong positive.
G is same stage with figure E, the flower pesticide part of amplification, and display flower pesticide is the strong positive.
H is same stage with figure E, and pollen staining is positive.
What A-H showed is the GUS dyeing that grain husk is spent, and in the different steps (1mm-7mm) of clever flower development, is flower pesticide specifically expressing.
I, the GUS dyeing of root is for negative.
J, the GUS dyeing of blade is for negative.
K, the GUS dyeing of leaf sheath, the tip of a leaf, auricle is for negative.
L, the GUS dyeing of stem.The GUS dyeing of internode is for negative, and the GUS dyeing of joint is the very light positive.A, anther, flower pesticide; S, stigma, column cap; O, ovary, ovary.
Conclusion: the promotor of osigcfa001g12 gene connects gus reporter gene, rice transformation, identified by the histochemical staining of gus reporter gene in transfer-gen plant, prove the promoter regulation gene of osigcfa001g12 special high expression level in the anther tissue of paddy rice grain husk flower stamen, at clever shell, do not express in gynoecium.All do not express in root, blade, leaf sheath.Do not express in the internode of stem, but only have very weak expression in the joint of stem.
The section of flower pesticide: flower pesticide section the results are shown in Figure 3, wherein blueness shows that GUS dyeing is for positive.
Figure A result shows, GUS is specifically expressing in the suede adhesion coating cell of flower pesticide, does not express in clever shell.A, anther, flower pesticide; T, Tapetum, suede adhesion coating; L, Lemma, clever shell.Figure B, the flower pesticide part of amplification, display GUS specifically expressing in suede adhesion coating.E, epidermis, epidermis; MC, meiotic cell, maiotic cell.
Quantitative result in the blade of paddy rice, leaf sheath, stem, fringe also confirms, gus gene is special high expression level in fringe specifically, sees Fig. 4.
The variant research of embodiment 6, promotor reservation function
As previously mentioned, promoter region sequence of the present invention is as shown in 1-521 position in SEQ ID NO:1, and in SEQ ID NO:1, the sequence shown in 1-552 position also has the effect of promotor.Through qualification, be arranged in SEQID NO:1 436-442 position
box (TATAAAT), 146-153 position
(CCAATGCA) for promotor plays the critical sites (the necessary site that initiation is transcribed and transcripting start point) of function.
In the promoter region of osigcfa001g12 gene, except TATA box (TATAAAT) and CAAT box (CCAATGCA), the present inventor also have found: E-box, and sequence is 5 '-CATTTG-3 ' (in SEQ ID NO:1 278-283 position).E-box is the cis regulatory assembly of the promoter region of fat translocator (Lipid transfer protein) gene family, is Binding site for transcription factor, points out it to be the critical sites regulating and controlling flower pesticide specifically expressing.
In addition, the cis regulatory assembly of the promoter region of CAAACAC sequence Shi Zhi transporter gene family is the critical sites affecting promotor intensity.
Therefore, the above-mentioned critical sites of base, present inventor has performed promoter variants research.
M1：
The present inventor has prepared the promotor based on sequence shown in 1-552 position in SEQ ID NO:1, wherein 5 ' end minimizing, 22 bases (M1).Based on the analysis of critical sites, TATA box, CAAT box, E-box and these sites as conservative property of CAAACAC sequence remain unchanged.Sequence following (single underscore is the primer sequence for increasing):
GTACCGCCGAAGGACCTTATTTAAACGGTTTTGTTAAGTTGGGGGACCCA
TCGTACCCGGTTTTGCGACCGGGGACGAAAATCGGACTAGGTGATAAAT
AGAGGGACCCAAAGTGAACTTATA
ATTTCATATCAAACAGT
CACGGATGGGCTTTAGGAAAGCAGAACTGGGCCCGGCCCAGTAGATCAC
ATAGCCCAACAAGATCTAAACCGCATGCTCTCGTTTCAACAAATTATCAC
ACCGATTG
CATCTGCTGCACAGGCTAAAT
GTAGCCATG
AACCATTCACCTCACAAGTCACAAGCATTGCATTTCTATGGTTACCAGTG
CAGGACGAAATGCTCAACTAGCCCAAGCAAGAATGGAGCATGACAACCT
CAGGCACCAAAAGCTC
ATTTGCATTGCACAAATCAAAAGTTT
CC
In promotor transgenic paddy rice, the variant result organizing gus reporter gene to dye is as Fig. 5.
The result that transgenic paddy rice GUS dyes shows, in paddy rice grain husk is spent, clever shell dyes without GUS; The flower pesticide specific staining positive (display is blue); In addition, all do not express in root, blade, leaf sheath, stem.M1 promotor has the function instructing goal gene specifically expressing in plant anther.
M2：
The present inventor has prepared the promotor compared with the short 135bp sequence of M1, and wherein 5 ' end reduces 135bp (M2).Based on the analysis of critical sites, CAAT box (CCAATGCA) lacks, and TATA box, E-box and these sites as conservative property of CAAACAC sequence remain unchanged.Sequence following (single underscore is the primer sequence for increasing):
CATATCAAACAGTCACGGATGGGCTTTAGGAAAGCAGAACTGGGCCCG
GCCCAGTAGATCACATAGCCCAACAAGATCTAAACCGCATGCTCTCGTT
TCAACAAATTATCACACCGATTG
CATCTGCTGCACAGGCTAAA
T
GTAGCCATGAACCATTCACCTCACAAGTCACAAGCATTGCA
TTTCTATGGTTACCAGTGCAGGACGAAATGCTCAACTAGCCCAAGCAA
GAATGGAGCATGACAACCTCAGGCACCAAAAGCTC
ATTTGC
ATTGCACAAATCAAAAGTTTCC
In promotor transgenic paddy rice variant organize gus reporter gene to dye the results are shown in Figure 6.
Visible, the promotor M2 rice transformation of brachymemma, instructs gus reporter gene to express in the flower pesticide of paddy rice grain husk flower; Express in blade and leaf sheath.But, do not express in root, do not express in stem or express weak.
From above-mentioned M2 and M1 transfer-gen plant GUS coloration result relatively, simultaneously containing the critical sites of regulation and control at blade, leaf sheath transcription in the 135bp region of reducing, after this site deletion, show as and express in blade and leaf sheath.
M3：
The present inventor has prepared the promotor compared with the short 129bp sequence of M2, wherein 5 ' end minimizing, 129 bases (M3).Based on the analysis of critical sites, CAAT box (CCAATGCA), E-box lack, and TATA box and these sites as conservative property of CAAACAC sequence remain unchanged.Sequence following (single underscore is the primer sequence for increasing):
GTAGCCATGAACCATTCACCTCACA
AGTCACAAGCATTGCATTTCTATGGTTACCAGTGCAGGACGAAATGCTC
AACTAGCCCAAGCAAGAATGGAGCATGACAACCTCAGGCACCAAAAGC
TC
ATTTGCATTGCACAAATCAAAAGTTTCC
In promotor transgenic paddy rice variant organize gus reporter gene to dye the results are shown in as Fig. 7.
Visible, the promotor M3 rice transformation of brachymemma, instructs gus reporter gene to express in the flower pesticide of paddy rice grain husk flower, expresses in blade, leaf sheath, stem; But do not express in root.In addition, express in the grain husk point of grain husk flower.
From above-mentioned M3 and M2 transfer-gen plant GUS coloration result relatively, containing the critical sites of regulation and control at stem transcription in this 129bp region, after this site deletion, high expression level in stem is shown as.
M2 promotor is compared with the promotor (Pro-osigcfa001g12) of sequence shown in 1-552 position in SEQ ID NO:1, and its 5 ' end reduces 157bp; M3 reduces 129bp than M2 again; Their GUS quantitative result is as Fig. 9.Shown in figure, the intensity of M4 promotor significantly reduces compared to Pro-osigcfa001g12, but does not have significant difference with M5, the motif as seen containing regulation and control promotor expression intensity in flower pesticide in this 157bp; In this section, have CAAT box, CAAT box may affect the expression intensity of gus reporter gene.In addition, by the comparison of homologous sequence, find the motif of GTGA, the motif of GTGA may affect the expression intensity in promotor in flower pesticide.
M4：
The present inventor has prepared the promotor compared with the short 26bp sequence of M3, wherein 5 ' end minimizing, 26 bases (M4).Based on the analysis of critical sites, CAAT box (CCAATGCA), E-box, CAAACAC sequence deletion, and TATA box remains unchanged as the site of conservative property.Sequence following (single underscore is the primer sequence for increasing):
TAGCCATGAACCATTCACCTCACAAGTCACAAGCATTGCATTTCTATGGT
TACCAGTGCAGGACGAAATGCTCAACTAGCCCAAGCAAGAATGGAGCAT
GACAACCTCAGGCACCAAAAGCTC
ATTTGCATTGCACAAATC
AAAAGTTTCC
In M4 promotor transgenic paddy rice variant organize gus reporter gene to dye the results are shown in Figure 8.
Visible, the promotor M4 rice transformation of brachymemma, the result that transgenic paddy rice GUS dyes shows, root, blade, leaf sheath, the internode of stem, and the GUS dyeing of flower pesticide is feminine gender; Only the joint position GUS of stem dyes as the weak positive (aobvious light blueness).
M4 is compared with the short 26bp of M3, and from above-mentioned M4 and M3 transfer-gen plant GUS coloration result relatively, when lacking this 26bp sequence, promotor is at blade, and leaf sheath, the expression in the internode of stem and flower pesticide disappears.The GUS expression intensity of the joint of stem also has the reduction of highly significant.Therefore, this 26bp is the crucial motif of regulation and control osigcfa001g12 genetic transcription, is also the crucial motif that flower pesticide is expressed.
The sequential analysis of embodiment 7, Rice Anther specific promoter
By homology comparison, find other several cis regulatory assemblies,
GT1consensus: 99-104 position in following sequence
115-120 position
relevant to light regulate gene expression.Supposition may participate in the regulation and control of genetic expression in blade, leaf sheath.
sequence: 131-138 position in following sequence.With multiple cis regulatory assembly, there is sequence homology, as CACTFTPPCA1, DOFCOREZM, TBOXATGAPB etc., infer the regulation and control that may participate in genetic expression in blade, leaf sheath.
ACCTCAGCCAAAACCGAAGACAGTACCGCCGAAGGACCTTATTTAAAC
GGTTTTGTTAAGTTGGGGGACCCATCGTACCCGGTTTTGCGACCGGGGA
C
CGGACTAGGT
TAGAGGGACC
ACTTATA
ATTTCATATCAAACAGTCACGGATGGGCTTTAGGAAAGCA
GAACTGGGCCCGGCCCAGTAGATCACATAGCCCAACAAGATCTAAACC
GCATGCTCTCGTTTCAACAAATTATCACACCGATTG
CATCTGCT
GCACAGGCTAAAT
GTAGCCATGAACCATTCACCTCACAAGTC
ACAAGCATTGCATTTCTATGGTTACCAGTGCAGGACGAAATGCTCAACT
AGCCCAAGCAAGAATGGAGCATGACAACCTCAGGCACCAAAAGCTC
ATTTGCATTGCACAAATCAAAAGTTTCC
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (7)
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| WO2015161744A1 (en) * | 2014-04-22 | 2015-10-29 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Identification and use of promoter ptaasg048 specifically expressed by plant anther |
| CN104946649B (en) * | 2015-07-07 | 2017-10-20 | 安徽省农业科学院水稻研究所 | A kind of Rice Anther specific expression promoter OsAnth1 |
| CN111041042B (en) * | 2018-10-11 | 2022-08-30 | 内蒙古农业大学 | Method for establishing agrobacterium-mediated immediate expression system of caragana intermedia |
| CN111676229B (en) * | 2020-06-30 | 2021-07-13 | 四川农业大学 | Maize male nuclear sterility gene ms40 and molecular marker and application thereof |
| CN113416735B (en) * | 2021-03-17 | 2023-01-31 | 云南中烟工业有限责任公司 | Tobacco germ cell specific high expression gene and application thereof |
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