CN104531708B - Cotton fibre specific expression promoter TB17P1 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and in particular relates to a cotton fibre specific expression promoter TB17P1 and application thereof. The technical problem to be solved is to provide a new choice for improving cotton fibre quality. The cotton fibre specific expression promoter TB17P1 provided by the invention has a nucleotide sequence shown in SEQ ID NO.1. The invention further provides an expression vector of the promoter. The invention further provides a host containing the expression vector. According to the invention, the TB17P1 promoter of a specifically expressed GhTUB17 gene in cotton fibre is successfully separated; the fact that a TB17P1 promoter sequence has the fibre expression specificity in cotton is proved; and the possibility and an effective way are provided for improving the yield and the quality of cotton fibre in genetic engineering.

Description

Cotton fiber specifically-expressed promoter TB17P1 and its application

Technical field

The invention belongs to gene engineering technology field, specifically relate to a kind of cotton fiber specifically-expressed promoter TB17P1 and its Using.

Background technology

China is Cotton Production and consumption big country, and Cotton Production occupies an important position in Chinese national economy.Utilize Over traditional breeding method once achieves larger success, but nearly 20 years in cotton variety improvement, the product of World cotton kind Amount has reached plateau.Therefore, it is difficult to increase substantially Cotton Gossypii product again using existing genetic resourcess and traditional breeding method means Amount.The advantages of there is gene engineering method offspring to be easy to stable, breeding cycle is short, can break the genetic block between species, real The orientation transfer of existing excellent genes of interest.It is to solve having for this difficult problem to improve output of cotton and fiber quality using genetic engineering Effect approach.But effect of the genetic engineering in crop improvement plays the progress extent for depending on three aspects：The work(of target gene The specific promoter that energy, the molecule mechanism of goal of regulation and control character and control targe gene are expressed in specific part.In cotton fiber In the genetic engineering improvement of quality and yield, it is often necessary to excess or suppress the expression of some genes to reach in fibrocyte To the purpose for improving yield or improvement quality.Simultaneously the important indicator of cotton fiber yield is Cotton Gossypii fertility, and pollen fertility is The important indicator of Cotton Gossypii fertility, is directly connected to cotton boll forming rate, percentage of fertile fruit and yield, it is therefore desirable to have fiber pollen-specific Promoter carrys out the expression of control targe gene.In the molecular mechanism research of cotton fiber development, it is also desirable to which fibrocyte is special The expression for expressing promoter to raise or lower target gene, and then analyze its function in fibrocyte.To greatest extent Reduce harmful effect of the target gene to its hetero-organization and organ.But, reported at present with cotton fiber specific Promoter is very limited.Therefore, the clone of cotton fiber specific promoter, to the functional study of cotton fiber development related gene and The genetic engineering improvement of cotton fiber, tool is of great significance.

In recent years, the research of plant tissue organ and development-specific expression become one of molecular biology of plants it is important Research field, particularly in the practical stage of plant genetic engineering, in order that genes of interest in specific histoorgan and Specific stage of development precedence table reaches, and the research to tissue specific expression even more becomes focus.Due to the development of cotton fiber Directly affect the quality and yield of fiber.Therefore, the promoter of research and screen fibre specifically expressing not only facilitates parsing base Because of the molecular mechanism of expression regulation, and useful controlling element can be provided for plant genetic engineering, there is important theory significance And using value.

The content of the invention

The technical problem to be solved in the present invention is to provide a kind of new selection to improve the fiber quality of Cotton Gossypii.

Promoter TB17P1 that cotton fiber specific is expressed is the technical scheme is that, with such as SEQ ID NO.1 Shown nucleotide sequence.

Present invention also offers the expression vector of also described promoter.

Preferably, described expression vector is plant expression vector.

Present invention also offers the host containing the expression vector.

Specifically, described host is Agrobacterium tumefaciems.

Present invention also offers application of the described TB17P1 promoteres in prepare transgenosis plant.

Present invention also offers application of the above-mentioned expression vector in prepare transgenosis plant.

Specifically, described plant is Cotton Gossypii.

A further object of the present invention is to provide a kind of method of utilization TB17P1 promoteres prepare transgenosis plant, including Following steps：

(1) promoter is operably inserted in expression vector, builds plant expression vector；

(2) plant expression vector conversion host will be obtained, transformant will be obtained；

(3) transformant is converted into plant, Jing cultures obtain transgenic plant.

Further, the invention provides a kind of method of utilization TB17P1 promoteres prepare transgenosis Cotton Gossypii, including with Lower step：

(1) promoter is operably inserted in expression vector, builds plant expression vector；

(2) plant expression vector is transformed in the wound healing regenerating tissues of Cotton Gossypii；

(3) the cotton callus regenerating tissues are cultivated, Jing screenings and induction obtain the promoter containing cotton fiber specific Cotton plants.

In the present invention, because cotton fiber is that the elongation of ovule external integument epidermis cell Jing polarity is formed, therefore fibrocyte It is a part for ovule epidermis cell.In order to screen the promoter of cotton fiber specific or predominant expression, inventor have detected cotton 12 alpha-tubulin genes and β -19 microtubule protein gene are in cotton plants different tissues organ and fiber in flower genome The expression characterization of development different times.The result of expression analysis shows that a 'beta '-tubulin gene (GhTUB17) is fine in Cotton Gossypii Specifically expressing in dimension.For the expression characterization of the promoter in Cotton Gossypii of clear and definite GhTUB17 genes, cloned the gene 5 '-on Trip regulating and controlling sequence 962bp, is named as TB17P1.By Cloned culturing, build the expression of promoter Analysis plant and Cotton Gossypii Genetic transformation, and it is active using transgene cotton analysis GUS.Specify that expression characterizations of the TB17P1 in Cotton Gossypii：In fiber Specifically expressing in cell, is a fiber specific expression promoter.

Beneficial effects of the present invention：

The invention provides the promoter (TB17P1) of microtubule protein gene GhTUB17 and application, the promoter is one Cotton fiber cell specific expression promoter；The present invention has been had successfully been isolated cotton fiber specifically-expressed using technique for gene engineering GhTUB17 genes promoter, the TB17P1 promoteres fragment containing 962bp；The present invention also demonstrates the TB17P1 and opens Promoter sequences have fiber expression specificity in Cotton Gossypii, and reporter gene can be instructed specific expressed in cotton fiber, are The quality and yield for improveing cotton fiber using genetic engineering provides the promoter of fiber-specific expression.The present invention is into one Step research plant gene function provides new selection, while providing effective way to improve cotton variety and fiber quality.

Description of the drawings

Fig. 1, A are GhTUB17 genes in Cotton Gossypii Different Organs and tissue and fiber and ovule different development stage Expression characterization；B is the expression characterization of fiber and ovule different development stage；

Wherein, error bar represents the standard deviation that 3 secondary pollutants repeat.

FO-0DPA～FO-4DPA:The Post flowering ovule fiber of 0 day is to the Post flowering ovule fiber of 4 days；F-6DPA～F- 20DPA:The Post flowering fiber of 6 days is to the Post flowering fiber of 20 days；O-6DPA～O-20DPA:The Post flowering ovule of 6 days is to blooming The ovule of 20 days afterwards.

Fig. 2 shows the cis-regulating element having on TB17P1 promoter sequences；

Wherein, sequence analysis carry out (http in PlantCare data bases:// bioinformatics.psb.ugent.be/webtools/plantcare/html/)。

Fig. 3 is pBI121-TB17P1::GUS plant expression vector collection of illustrative plates；

Wherein, LB:T-DNA section left margins；RB：T-DNA section right margins；Kan^r, kalamycin resistant gene；GUS： β-gluconic acid glycoside enzyme gene；Nos-T:Opines synthase gene terminator.

Fig. 4 is the digestion verification of promoter Analysis plant expression vector；

Wherein, M15：DNA Marker 15(MBI)；1 and 2：The empty plasmid of TB17P1 fragments is not accessed；3:Represent The pBI121-TB17P1 of TB17P1 fragments is accessed::GUS plasmids.

Fig. 5 is the gus gene in amplification checking transgene cotton；

Wherein, M15：DNA Marker 15(MBI)；+：Positive control (the pBI121-TB17P1 for boiling::GUS agriculture bars Bacterium solution)；W：Water is the amplified production of template；-：Negative control (non-transgene cotton)；1-5：The Kan resistances regenerated after transgenic Seedling, represents that T-DNA sections have been incorporated in transgene cotton genome.

Fig. 6 is expression of the TB17P1 promoteres in transgene cotton seedling；

Wherein, A:Whole strain transgene cotton seedling；B：Hypocotyls are crosscutting；C:Seminal root and hypocotyls rip cutting.This figure shows TB17P1 promoteres are not expressed in seminal root, hypocotyls and growing point.

Fig. 7 is expression of the TB17P1 promoteres in transgene cotton root；

Wherein, WT:Wild type cotton root；TB17P1:pBI121-TB17P1::The root of GUS vector transgene Cotton Gossypiis；35S: The root of pBI121 vector transgene Cotton Gossypiis；ROOT：Root middle part；ROOT TIP:The tip of a root.This figure shows TB17P1 promoteres in Cotton Gossypii Do not express in root.

Fig. 8 is expression of the TB17P1 promoteres in transgenic cotton scape；

Wherein, WT:Wild type cotton stem；TB17P1:pBI121-TB17P1::The stem of GUS vector transgene Cotton Gossypiis；35S: The stem of pBI121 vector transgene Cotton Gossypiis；The left, center, right of young stem is respectively the rip cutting of young stem, crosscutting and stipes rip cutting；Old stem Left, center, right is respectively the rip cutting of old stem, crosscutting and stipes rip cutting.This figure shows that TB17P1 promoteres are not expressed in Cotton Gossypii stem.

Fig. 9 is expression of the TB17P1 promoteres in transgenic cotton floral leaf；

Wherein, WT:Wild type cotton blade and petiole；TB17P1:pBI121-TB17P1::GUS vector transgene Cotton Gossypiis Blade and petiole；35S:The blade and petiole of pBI121 vector transgene Cotton Gossypiis；The left, center, right of spire is respectively the leaf of spire Handle rip cutting and crosscutting and blade；The left, center, right of climax leaves is respectively the petiole rip cutting of climax leaves and crosscutting and blade.This figure Show that TB17P1 promoteres are not expressed in cotton leaf.

Figure 10 is that TB17P1 is bloomed the expression of same day floral organ each several part in transgene cotton；

Wherein, WT:The floral organ each several part of wild type cotton；TB17P1:pBI121-TB17P1::GUS vector transgenes The floral organ each several part of Cotton Gossypii；35S:The floral organ each several part of pBI121 vector transgene Cotton Gossypiis；1：The ovary and ovule of Cotton Gossypii； 2：Fruit stem is crosscutting；3：Fruit stem rip cutting；4：Petal；5：Bract；6：Flower pesticide (including filigree)；7：Stigma；8：Pollen.This figure shows TB17P1 promoteres are not expressed substantially in Cotton Gossypii floral organ each several part, only there is trace expression in pollen.

Figure 11 is expressions of the TB17P1 during transgene cotton ovule and Fibre Development；

Wherein, WT:Wild type cotton fiber and ovule；TB17P1:pBI121-TB17P1::GUS vector transgene Cotton Gossypiis Fiber and ovule；35S:PBI121 vector transgenes cotton fiber and ovule；0DPA：Blooming, (there is fiber on surface for the ovule on the same day Cell)；8DPA：The Post flowering ovule of 8 days and fibrocyte；12DPA：The Post flowering ovule of 12 days and fibrocyte；16DPA： The Post flowering ovule of 16 days and fibrocyte；20DPA：The Post flowering ovule of 20 days and fibrocyte.This figure shows that TB17P1 is opened Mover stretches long-term expression higher (8DPA～16DPA) in cotton fiber cell, and in fibrocyte later stage, namely fibrocyte are extended The initial phase expression of secondary wall synthesis reduces (20DPA), does not express on (0DPA) ovule blooming the same day, seed development each Period does not express substantially.Illustrate that TB17P1 promoteres are a fiber specific expression promoters.

Specific embodiment

The present invention is described in further detail below in conjunction with accompanying drawing, description below is not limited the present invention Fixed, any deformation and change to the present invention, without departing from the spirit of the present invention, all should belong to claims of the present invention Scope.

Unless otherwise stated, in present example reagent, medicine, material are commercially available, and method is referred to《Molecule Cloning experimentation guide》(Sambrook and Russell, 2001).

In following examples of the present invention, Cotton Gossypii experiment material used is (the Gossypium hirsutum of Xuzhou 142 L.cv Xuzhou 142), from the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute.Transgene receptor is Ji cotton 14, from Hebei Agriculture University.

The extraction of the Cotton Gossypii RNA of embodiment 1

(root, stem, leaf, flower, the Post flowering ovule of 0 day and fiber are to the Post flowering embryo of 6 days to choose the fresh cotton materials of about 3g Pearl and fiber, the Post flowering fiber of 6 days are to the Post flowering fiber of 20 days, the Post flowering ovule of 6 days to the Post flowering embryo of 20 days Pearl).Wear into fine powder rapidly in liquid nitrogen, load 50 milliliters of centrifuge tubes, add the RNA extracting solution of 15 milliliters of 65 DEG C of preheatings 【2% (W/V) cetyl trimethylammonium bromide (CTAB), 2% (W/V) polyvinylpyrrolidone (PVP), 100 mmoles/liters three Hydroxymethyl aminomethane-hydrochloride buffer (Tris-HCl, pH 8.0), 0.5 g/l of spermidine (Spermidine), 2.0 rub/ Rise Sodium Chloride (NaCl), 2% mercaptoethanol (V/V, using front addition)】, overturn and mix.65 DEG C of water-baths 3 minutes, period mixes 2 ～3 times.Lv Fang ︰ isoamyl alcohol (︰ 1 of volume ratio 24) is extracted 2 times (10,000 revs/min, room temperature, 5 minutes).Supernatant is taken, 1/4 is added Volume 10 is rubbed/liter lithium chloride (LiCl) solution, and 4 DEG C are placed 6 hours, with phenol (the pH4.5) ︰ Lv Fang ︰ isoamyl alcohol (︰ of 25 ︰ of volume ratio 24 And Lv Fang ︰ isoamyl alcohol (︰ 1 of volume ratio 24) respectively extracting 1 time (10,000 revs/min, room temperature, 5 minutes) 1).Plus the nothing of 2 times of volumes Water-ethanol, precipitates more than 30 minutes at -70 DEG C.12,000 revs/min, 4 DEG C are centrifuged 20 minutes, abandon supernatant.Precipitation is with 500 microlitres The water dissolution of Jing pyrocarbonic acid diethyl esters (DEPC) process.With phenol (pH4.5) ︰ Lv Fang ︰ isoamyl alcohol (︰ 1 of 25 ︰ of volume ratio 24) and chlorine Fang ︰ isoamyl alcohol (︰ 1 of volume ratio 24) respectively extracting 1 time (10,000 revs/min, room temperature, 5 minutes).Plus the anhydrous second of 2.5 times of volumes Alcohol, precipitates more than 30 minutes at -70 DEG C.12,000 revs/min, 4 DEG C of 20 molecules of centrifugation abandon supernatant.Precipitate with 70% ethanol Rinsing once, is air-dried.Plus the water dissolution of 200 microlitres of Jing pyrocarbonic acid diethyl esters (DEPC) process.Use non denatured agarose gel The quality of electrophoresis and UV spectrophotometer measuring RNA.

The synthesis of the first chains of cDNA of embodiment 2

Take about 10 microgram total serum IgEs to be added in the amplification pipe of pyrocarbonic acid diethyl ester process, 65 DEG C of water-baths become RNA in 10 minutes After property, ice bath 3 minutes immediately.Then 2 microlitres of 10 × RNA reaction buffers are sequentially added in amplification pipe, 4 microlitre of 25 mmoles/ Rise magnesium chloride (MgCl₂), 2 microlitre of 10 mmoles/liter dideoxyribonucleotide triphosphate (dNTPs), 5 unit reverse transcriptases, 0.5 microlitre RNase inhibitor (20 unit) and 1 microlitre of 2.5 micro-/liter T repetition oligonucleotide (Oligo-dT) 3' joint that rub, add Jing cokes The water of diethyl phthalate process is to 20 microlitres of final volume.Reverse transcription reaction program is：30 DEG C, 10 minutes；50 DEG C, 45 minutes；95 DEG C, 5 minutes；5 DEG C, 5 minutes.After EP (end of program), in -20 DEG C of frozen products.

Expression of the GhTUB17 genes of embodiment 3 in Cotton Gossypii different tissues organ and fiber ovule different development stage is special Property

With the expression of real-time quantitative PCR Amplification Analysis GhTUB17, with cDNA the first chains synthetic agent box (MBI) First chain cDNA of synthesis Different Organs and total tissue RNA, operation is carried out by kit specification.Using real-time quantitative PCR Test kit (Bio-Rad) is expanded, and in 20 microlitres of reaction systems 10 microlitres of cocktail buffers are included【Including PCR bufferings Liquid, archaeal dna polymerase, dideoxyribonucleotide triphosphate (dNTPs) and magnesium chloride (MgCl₂)】, 5'- ends and 3'- ends primer it is each 1 micro- Rise (5 micro- rub/liter).Loop parameter is 94 DEG C of denaturations 3 minutes；94 DEG C, 30 seconds, 55 DEG C, 30 seconds, 72 DEG C, 30 seconds, preset loop Number is 40.Make internal standard with Cotton Gossypii Histone3 genes, the 5'- primers of Histone3 are GhHIS1 (5'- GAAGCCTCATCGATACCGTC-3') (SEQ ID NO.2), 3'- primers be GhHIS2 (5' '- CTACCACTACCATCATGGC-3')(SEQ ID NO.3).The 5'- primer sequences of amplification GhTUB17 genes are GhTUB17-1 (5'-CGGTTGGTCAGATTTTCAGAC-3') (SEQ ID NO.4), 3'- primer sequences are GhTUB17-2 (5'- GTAAGGCTCCACAACCGTATC-3')(SEQ ID NO.5)。

The total of Cotton Gossypii different tissues organ and fiber ovule different development stage is obtained by the method for above-described embodiment 1 and 2 RNA carries out real-time quantitative RT-PCR analysis.Before operation real-time quantitative PCR, adjusted in identical temperature with same primers and template Expand in control program once, by the electrophoresis of amplified production, it is ensured that amplified production is single tape, each sample is repeated 3 times.

As a result as shown in Figure 1A, GhTUB17 genes have a small amount of expression in Radix Gossypii and stem, do not express in leaf and Central China, There is very high expression in the Post flowering fiber of 8 days, hardly express in the Post flowering ovule of 8 days.Illustrate GhTUB17 Gene has obvious fibrocyte advantage of expression.And during Fibre Development, with the elongation growth of fiber, the gene Expression quickly increase, the expression highest in the Post flowering fibrocyte of 6 days and 8 days；Subsequently it is gradually lowered, is opening Spend the expression that the gene is nearly no detectable in the fibrocyte after 14 days.In whole ovule development steps, the table of the gene Up to level extremely low (Figure 1B).Thus GhTUB17 genes predominant expression in the fibre is illustrated, and in the fibrocyte rapid elongation phase Middle expression is high, does not express substantially in ovule.

The extraction of the cotton genomic dna of embodiment 4

Cotton genomic dna is extracted using cetyl trimethylammonium bromide (CTAB) method of improvement.Take 0.5 gram of Cotton Gossypii Spire, pulverizes rapidly in liquid nitrogen, and the cetyl trimethylammonium bromide (CTAB) for adding 3 milliliters of 65 DEG C of preheatings is extracted Liquid, quick oscillation is mixed.65 DEG C of water-baths 30 minutes, are subsequently adding 1 milliliter of 5/liter potassium acetate (KAc) that rubs, and ice bath is used after 20 minutes Isopyknic chloroform:Isoamyl alcohol (24:1) 1 time (12,000 revs/min, 4 DEG C are centrifuged 5 minutes) are extracted, supernatant is taken, 2/3 times is added - 20 DEG C of pre- cold isopropanols of volume, mix, and stand about 30 minutes, choose flocculent deposit, and 75% ethanol is rinsed 3 times repeatedly, then Rinsed 1 time with dehydrated alcohol, dried up, during 500 microlitres of trishydroxymethylaminomethane-disodiumedetates (TE) are dissolved in again. 1 microlitre of RNaseA (10 mg/ml) is added, 37 DEG C are processed 1 hour.Phenol is used again:Chloroform:Isoamyl alcohol (25:24:And chloroform 1): Isoamyl alcohol (24:1) each extracting 1 time (12,000 revs/min, 4 DEG C are centrifuged 5 minutes), takes supernatant, adds the anhydrous second of 2 times of volumes Alcohol precipitates DNA.- 20 DEG C are placed about 30 minutes, centrifugation (12,000 revs/min, 4 DEG C are centrifuged 5 minutes), abandon supernatant, and precipitation is used 75% ethanol rinse, air-dries, and in being dissolved in 200 microlitres of TE, -20 DEG C save backup.

Embodiment 5 clones the promoter TB17P1 sequence of GhTUB17 genes

Using the cDNA sequence of GhTUB17 genes, the Cotton Gossypii D- subgenomic sequence (http for having announced are searched for:// www.phytozome.net).The 5'- upstream regulatory sequences of GhTUB17 genes are obtained, and then at the about 1.0Kb of ATG upstreams Design special primer TB17P1-up (5'-TCCTCGCACTCTTTGCACATT-3') (SEQ ID NO.6) and in nearly ATG sites Place's design special primer TB17P1-down (5'-GGTGACTGAGAGGGATTGTTT-3') (SEQ ID NO.7), with Xuzhou 142 Genomic DNA is expanded for template, and 25 microlitres of reaction system includes about 50 nanogram cotton DNAs, and 2.5 microlitres of 10 times of amplifications are slow Rush liquid, 2 microlitre of 2.5 mmoles/liter dideoxyribonucleotide triphosphate (dNTPs), 1.5 microlitre of 25 mmoles/liter magnesium chloride (MgCl₂), 5 It is micro- rub/liter upstream and downstream primer it is each 1 microlitre, 1 unit archaeal dna polymerase (Progema).Amplification program is：94 DEG C, 5 minutes；94 DEG C, 30 seconds, 56 DEG C, 30 seconds, 72 DEG C, 1.5 minutes, 35 circulations；72 DEG C extend 10 minutes.Amplified production through electrophoresis reclaim, Connect on cloning vehicle pMD19 (TaKaRa), form pMD19-TB17P1 carriers, the carrier is through conversion large intestine bar Bacterium, checking and sequencing, as a result show that expanding fragment length is 962bp, TB17P1 are named as, as shown in SEQ ID NO.1.Sequence Analysis carries out (http in PlantCare data bases://bioinformatics.psb.ugent.be/webtools/ Plantcare/html/), analysis result is shown in Fig. 2, except a large amount of TATA frames and CAAT that have with general promoter in sequence Outer frame, also with many specificly-response elements, such as anaerobic reaction element (TTTGGT), photoreactive element (ATTAAT, TTTCAAA, AAACTTT, CCACCCAACT, TCTTAC), ethylene reaction element (ATTTCAAA), photoreaction MYB binding sites (AACCTAA), endosperm Expression element (TACTG), defence and Stress responses element (CACTTCGTTTA), salicylism reaction element (TTTTT), unknown function (CTCC), circadian rhythm controlling element (CAATTCTATC).

Embodiment 6TB17P1 promoter Analysis plant expression vector pBI121-TB17P1::The structure of GUS

TB17P1 fragments are cut from pMD19-TB17P1 carriers with Hind III and the restriction endonucleases of Xba I, is connected to and is used Hind In pBI121 carriers (the excellent precious biological) fragment of III and Xba I double digestion, so that TB17P1 fragments have replaced pBI121 carriers In CaMV35S promoteres.By digestion verification (see Fig. 4), plant expression vector pBI121-TB17P1 is built into::GUS (figures 3)。

The genetic transformation of the Cotton Gossypii of embodiment 7

With electrization the plant expression carrier plasmid of structure is imported Agrobacterium LBA4404 bacterial strain and carries out Cotton Gossypii heredity and turned Change.

With reference to Bio-RAD MicroPulser instruction manual books, above-mentioned carrier is imported into Agrobacterium by Electroporation conversion LBA4404 bacterial strains.

Above-mentioned plant expression vector imports Cotton Gossypii by agriculture bacillus mediated Cotton Hypocotyl method for transformation.Concrete grammar is such as Under：

Ji cotton 14 (Agricultural University Of Hebei's offer) seed peels off shell, with 0.1% mercuric chloride (HgCl₂) sterilize 10 minutes, With a large amount of aseptic water washings 8 times.Add about 35 milliliters of sterilized water to shake overnight in 125 milliliters of triangular flasks, next day change once without Bacterium water.After seed grows hypocotyls root, it is seeded on seed germination medium, at 28 DEG C, sprouts 2～3 days under dark condition, Now seed hypocotyls initially enter the period of rapid elongation, suitably carry out genetic transformation.

Conversion containing pBI121-TB17P1::The agrobacterium strains of GUS plant expression vectors are containing 50 mg/litre OK a karaoke clubs Activate on the YEB solid mediums of mycin (Km) and 125 mg/litre streptomycins (Sm).Picking its single bacterium colony, is inoculated in 5 milliliters In YEB fluid mediums containing identical antibiotic, 28 DEG C, 200 revs/min of concussion and cultivates overnight.Agrobacterium bacterium solution after culture It is transferred in 25 milliliters of YEB fluid mediums containing identical antibiotic in the ratio of 1 ︰ 20, continues culture to OD600 values and be about Thalline is collected by centrifugation for 0.6～0.8,10,000 revs/min, 1 minute, it is resuspended standby that the isopyknic liquid of thalline co-cultures base.

During conversion, Cotton Hypocotyl is cut into into 1.5～2.0 centimetres of segment, is put in triangular flask with the agriculture bar for preparing Bacterium bacterium solution infects, and condition infects 30 minutes for 28 DEG C of 120 revs/min of shaking tables.Then bacterium solution is blotted, hypocotyls section is gone to into common training On foster base, 28 DEG C of light cultures 2-3 days.

After co-cultivation, hypocotyls section is transferred to lower embryo section screening culture medium, 28 DEG C of illumination cultivation, about 20 days subcultures once, Until there are a large amount of calluss.Calluss are transferred to embryo callus subculture inducing culture, about 15 days subcultures one together with lower embryo section It is secondary, until there is substantial amounts of light yellow embryo callus subculture.Embryo callus subculture is chosen in embryo callus subculture suspension medium, 28 DEG C, 120 turns/ Minute shaking table culture one week or so.The body embryo for drawing fine sand shape with 1.0 milliliters of pipette tips for deducting tip is laid in body embryo elongation training , there is substantial amounts of green little body embryo in foster base after 20-30 days.The good body embryo successive transfer culture of picking growth conditions, treats that body embryo extends During to 1-2 centimetre, it is transferred into seedling culture medium and takes root to emerge.When growth of seedling to 3-5 centimetre is high, by grafting or shifting The mode of cultivation is transferred in greenhouse flowerpot and grows.Wherein, culture medium used in this experimental example is as shown in table 1.

The Agrobacterium tumefaciens mediated Cotton Hypocotyl genetic transformation used medium of table 1

Note:MS:Murashige&Skoog,1962；B5:Gamborg,1986.

The Molecular of the transgene cotton of embodiment 8

Embodiment 7 is extracted with easy cetyl trimethylammonium bromide (CTAB) method or any methods known in the art The DNA of the Cotton Resistance seedling of acquisition.With two primer GUS-up (5'-TCATTGTTTGCCTCCCTGCG-3') of gus gene (SEQ ID NO.8) and GUS-down (5'-GGGGACTCTAGAGGATCCC-3') (SEQ ID NO.9) expand resistant cotton base Because of a group DNA, it is contemplated that amplified fragments about 1866bp.25 microlitres of reaction system include about 50 nanogram genomic DNAs, 2.5 microlitres 10 times of amplification buffers, 2 microlitre of 2.5 mmoles/liter dideoxyribonucleotide triphosphate (dNTPs), 1.5 microlitre of 25 mmoles/liter chlorination Magnesium (MgCl₂), 5 it is micro- rub/liter upstream and downstream primer it is each 1 microlitre, 1 unit archaeal dna polymerase.Amplification program is：94 DEG C, 5 minutes；94 DEG C, 30 seconds, 56 DEG C, 30 seconds, 72 DEG C, 2 minutes, 35 circulations；72 DEG C extend 10 minutes.Agarose gel electrophoresiies detection amplification is produced Thing.

As shown in Figure 5, the gus gene (β-glucuronidase of about 1.8kb has been amplified from 5 plants of resistant cotton plants Gene) special band, illustrate that exogenous sequences have been incorporated in the genome of transgenic cotton plant.

The detection of GUS activity in the transgene cotton of embodiment 9

Randomly selecting the positive transgenic cotton plants of 5 plants of PCR carries out comprehensive β-glucuronidase (GUS) activity inspection Survey, the expression characterization of GUS is basically identical.T2 is sprouted first for the seed of homozygous plants, seedling is carried out into GUS dyeing, as a result show Show and all there is no GUS signals (Fig. 6) in cotyledon, root, hypocotyls and growing point.Next have detected the expression of root, stem, leaf, flower, Positive signal (Fig. 7, Fig. 8 and Fig. 9) is all not detected by detection sample.Further have detected the expression feelings of GUS in floral organ Condition, as a result shows there are faint GUS signals in pollen, and in colored other parts GUS signals (Figure 10) are not detected by.Finally The GUS expressions of ovule and fiber different development stage are have detected, there is no GUS signals on the ovule on the same day of blooming, opened There are GUS signals after spending in the fiber of 8 days, 12 days, 16 days and 20 days, and without obvious GUS signals in ovule.Wherein, open GUS signals are most strong (Figure 11) in the fibrocyte of 12 days after spending.Illustrate that TB17P1 promoteres are a fibrocyte specifically expressings Promoter.

The invention provides the promoter (TB17P1) of a microtubule protein gene GhTUB17 and application, the promoter is One cotton fiber cell specific expression promoter；The present invention has had successfully been isolated cotton fiber specific using technique for gene engineering The promoter of the GhTUB17 genes of expression, the TB17P1 promoteres fragment containing 962bp；The present invention also demonstrates described TB17P1 promoter sequences have fiber expression specificity in Cotton Gossypii, can instruct reporter gene specificity in cotton fiber Expression, the quality and yield to improve cotton fiber using genetic engineering provides the promoter sequence of fiber-specific expression.

Claims (8)

1. promoter TB17P1 that cotton fiber specific is expressed, its nucleotide sequence is as shown in SEQ ID NO.1.

2. containing the expression vector of promoter described in claim 1.

3. the carrier as described in claim 2, it is characterised in that：Described expression vector is plant expression vector.

4. containing the host of expression vector described in claim 2 or 3.

5. the host as described in claim 4, it is characterised in that：Described host is Agrobacterium tumefaciems.

6. application of the TB17P1 promoteres described in claim 1 in prepare transgenosis Cotton Gossypii.

7. application of the expression vector described in claim 2 or 3 in prepare transgenosis Cotton Gossypii.

8. the method for utilizing the promoter TB17P1 prepare transgenosis Cotton Gossypii of claim 1, it is characterised in that：Including following Step：

(1) promoter is operably inserted in expression vector, builds plant expression vector；

(2) plant expression vector is transformed in the wound healing regenerating tissues of Cotton Gossypii；

(3) the cotton callus regenerating tissues are cultivated, Jing screenings and induction obtain the cotton of the promoter containing cotton fiber specific Flower plant.