CN102002501A - Promoter of cotton KCS 12 gene and application thereof - Google Patents

Abstract

The invention discloses a promoter of a cotton KCS 12 gene and application thereof. The cotton KCS 12 gene provided by the invention comprises any deoxyribonucleic acid (DNA) molecules from 1) to 3), namely 1) DNA molecules shown by a sequence 1 in a sequence table; 2) DNA molecules which are hybridized with the DNA molecules in 1) and have the same functions as the DNA molecules in 1) under the strict condition; and 3) DNA molecules which have homology of over 90 percent with the DNA molecules in 1) or 2) and have the same function as the DNA molecules in 1) or 2). In the promoter, a drive glucuronidase (GUS) gene expression vector of a promoter fragment (1145bp) of the KCS 12 gene is constructed. GUS histochemistry dyeing confirms that the promoter fragment can guide a reporter gene to express in a cotton fiber normally. Therefore, the KCS 12 promoter can be used for studying the development and quality improvement on the cotton fiber and the specific expression of an exogenous gene in the cotton fiber.

Description

The promotor and the application thereof of cotton KCS12 gene

Technical field

The present invention relates to the promotor and the application thereof of cotton KCS12 gene in the biological technical field.

Background technology

China is the maximum Cotton Production state in the whole world, and cotton plays a part very important in Chinese national economy as primary fibre crops and important oil crops.Because the people are for improving constantly that standard of living requires, the textile industry fast development is so that more and more higher to the requirement of cotton fibre quality.Cotton in China fibrous quality deviation, specific tenacity is also lower, and the cotton fibre quality problem has become the major obstacle of restriction Cotton in China industry Sustainable development.

Cotton fiber development can be divided into four overlapped periods: fibrocellular initial (blooming preceding 1 day to blooming back 2-3 days), elongation (blooming back 2-3 days to 25 days), secondary wall deposition (blooming back 16 days to 45 days) and ripening stage (blooming back 45 days to 50 days).For the quick elongation fiber in period, the peak rate of its elongation can reach 2 millimeters/day.The length and the intensity of fiber have determined quality of fibre, and the molecule mechanism of research cotton fiber development is the basis of cotton genetic improvement.

Promotor is positioned at the section of DNA sequence of gene 5 ' end upstream (non-translational region), " switch " as regulate gene expression, the time of origin that controlling gene is expressed and the degree of expression, therefore, promotor is the dna sequence dna of decision specific gene expression, is subjected to the genetic expression of its regulation and control to present the stage of tangible tissue specificity and growth.In the cotton fibre genetic improvement, fiber specific promoter is the important function of gene engineering element, utilizes this type of promotor to drive the useful gene of fibrous quality improvement is realized specific expression in fiber.Therefore, separating the important content that the promotor strong with clone-specific, that activity is high is the improvement of cotton fibre genetically engineered, is the basis of setting up the efficient genetic conversion system of cotton.

Promotor is not only determining the spatial and temporal expression of gene, has also determined expression of gene intensity.At present, in plant genetic engineering, the CaMV35S promotor is widely used.The CaMV35S promotor is a composition type expression promoter, drives foreign gene and expresses in the growth course of each tissue of plant.But, in the process of utilizing molecular biology method that plant is improved, people more wish the foreign gene that inserts can be in specific tissue specifically expressing, thereby when obtaining good character, do not influence other characteristics.

Summary of the invention

An object of the present invention is to provide a kind of dna molecular.

Dna molecular provided by the present invention derives from cotton (Gossypium spp.), is following 1)-3) in arbitrary described dna molecular:

1) dna molecular shown in the sequence 1 in the sequence table;

2) under stringent condition with 1) dna molecule hybridize and have the dna molecular of identical function;

3) with 1) or 2) dna molecular have 90% above homology and have the dna molecular of identical function.

The expression cassette, recombinant expression vector, transgenic cell line or the reorganization bacterium that contain described dna molecular also belong to protection scope of the present invention.

Described recombinant expression vector is for replacing the recombinant expression vector that the 35S promoter on pCAMBIA 1305 carriers obtains with described dna molecular.

Another object of the present invention provides described dna molecular and make the application of goal gene in expressing in cotton fiber cell.

Described cotton fiber cell is the cotton fiber cell in the cotton ovule.

Described cotton is wild-type upland cotton Xuzhou-142.

The present invention made up 1 KCS12 gene promoter fragment (1,145bp) drive the gus gene expression vector.The GUS histochemical stain confirms that this promoter fragment can instruct reporter gene normal expression in cotton fiber.Therefore, the KCS12 promotor can be used for studying growth, quality-improving and the foreign gene specifically expressing in cotton fiber of cotton fiber.

Description of drawings

Fig. 1 detects the expression of KCS12 gene at the cotton different tissues for real-time quantitative qRT-PCR.

Fig. 2 is the expression that cotton ovule transient expression system anlysis KCS12 promoter-pCAMBIA1305 carrier starts gus gene.

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

Embodiment 1, clone upland cotton KCS12 gene promoter sequence

One, obtains upland cotton KCS12 gene promoter sequence

1, obtains the promoter sequence of gene KCS12 upstream of coding region with the chromosome walking method

Because cotton gene group information is still imperfect, with the promoter sequence of chromosome walking method (Genomic Walking) with acquisition gene coding region upstream.The chromosome walking method is based on a technology of PCR and flush end restriction endonuclease.Genomic dna links to each other with Adaptor with the postdigestive fragment of flush end restriction endonuclease, as template, uses primer and the inner primer of gene at the Adaptor design to carry out PCR with this, can seek close unknown gene group dna sequence dna by one section known sequences.

The promoter sequence of the fiber expression specificity gene KCS12 upstream of coding region that obtains with the chromosome walking method is 1,145bp, and its nucleotide sequence is shown in sequence in the sequence table 1.This sequence is carried out the cis-regulating element analysis, and the sequential analysis website is Http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/By to KCS12 gene A TG upstream 1, the sequence cis-regulating element of 145bp is analyzed, and has determined transcription initiation site TATA box and CAAT box.

2, detect of the expression of the promoter sequence of acquisition goal gene KCS12 upstream of coding region at the cotton different sites

Extract (The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute National Cotton germplasm storehouse in mid-term, wild-type upland cotton Xuzhou-142, ZM-01345) root, stem, spire, climax leaves, flower, 10DPA fiber (the 10DPA fibrocyte is in elongating stage) and 10 DPA ovule tissues (used 10DPA ovule is organized as the 10DPA ovule tissue that removes the 10DPA cotton fibre in this experiment), and there is not fiber mutant fl (this mutant does not produce cotton fibre) (The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute National Cotton germplasm storehouse in mid-term, ZM-30188) the no blank pearl RNA of 10DPA, behind reverse transcription cDNA first chain as real-time quantitative qRT-PCR template, primer is KCS12_RT_SP (SP-sense primer) and KCS12_RT_AP (AP-antisense primer), and primer sequence is as follows:

KCS12_RT_SP:5 '-ACCTTCGTTCTCTCCCAACTATCTCG-3 ' and

KCS12_RT_AP：5′-CCAGGGTGCACAACAGGTAATAGGA-3’。

With no fiber mutant fl is contrast, detects the expression of this gene at the cotton different sites.Detected result is followed successively by root, stem, spire, climax leaves, flower, 10DPA fiber and 10DPA ovule tissue as shown in Figure 1 from left to right, and the no blank pearl that does not have the 10DPA of fiber mutant fl.No blank pearl with the 10DPA of no fiber mutant fl is contrast, and KCS12 expression of gene amount is represented with known house-keeping gene GhUBQ7 relative expression quantity in the cotton.As seen from Figure 1, KCS12 gene specifically expressing in the 10DPA fiber.

Embodiment 2, upland cotton KCS12 gene promoter Function Identification

One, construction of expression vector KCS12 promoter-pCAMBIA 1305

KCS12 gene promoter sequence according to obtaining among the embodiment 1 has designed primer 5 '-KCS12 pt-KpnI and 3 '-KCS12 pt-NcoI, is template with the genomic dna of the 10DPA fiber in wild-type upland cotton Xuzhou-142, carries out pcr amplification.KpnI and NcoI restriction enzyme site and protection base have been added at primer 5 ' end.Primer sequence is as follows:

5 '-KCS12 pt-KpnI:GGggtaccAGTGTCCAATTGAACCATTTGAGAC, lowercase are the KpnI restriction enzyme site,

3 '-KCS 12pt-NcoI:CATGccatggCTTGCTGCTGCCTTTACTACTCT, lowercase are the NcoI restriction enzyme site.

Carrier pCAMBIA 1305 (available from Australian CAMBIA company) carries out enzyme by KpnI and NcoI enzyme and cuts evaluation, removes the 35S promoter sequence, reclaims big fragment.Amplified production through KpnI be connected with carrier after the NcoI enzyme is cut, replace 35S promoter, be built into and drive gus gene expression vector KCS12 promoter-pCAMBIA1305, in this carrier, do not have other any promotor in the gus gene upstream, have only the dna fragmentation of insertion.

Change expression vector KCS12 promoter-pCAMBIA 1305 over to e.colistraindh5 (available from the Beijing Quanshijin Biotechnology Co., Ltd, catalog number is D871010), resistance screening, the picking positive colony, positive colony is carried out liquid culture, extract plasmid, check order and detect KCS12 gene promoter sequence among the expression vector KCS12 promoter-pCAMBIA1305, sequencing result shows, the gene order of inserting between the KpnI of carrier pCAMBIA 1305 and NcoI restriction enzyme site is consistent with sequence 1 in the sequence table.

Two, particle bombardment transforms ovule

1, experiment material

Cultivate in the greenhouse in wild-type upland cotton Xuzhou-142, collected the whole flower of cotton after blooming one day.

2, experiment reagent

Bronze: diameter is 1.0 μ m

CaCl ₂：2.5M/L

Spermidine: 0.1M/L

The particle gun agents useful for same is all purchased the company in Bio-Rad.

3, laboratory apparatus

Ultrasonic Cell Disruptor

Particle gun is purchased the company in Bio-Rad, model PDS-1000/H ₂Biolistic

4, experimental article

The 1100p pressure membrane, carrier film, copper mesh, a complete set of equipment is all purchased the company in Bio-Rad.

5, solid medium

Solid medium is on the basis of cotton group training liquid liquid nutrient medium, and other adds 0.6% agar powder, and the sterilization back is fallen dull and stereotyped.Consisting of shown in the table 1 of cotton group training liquid liquid nutrient medium.

The composition of table 1 cotton group training liquid liquid nutrient medium

6, experimental procedure

A) bronze is suspended in 100% ethanol, final concentration is 50mg/ml; Ultrasonication 1min, interval 1min, broken 5 times; 13, the centrifugal 10sec of 000g.

B) wash twice with 100% ethanol ethanol again; 13,000g notes allowing bronze be deposited in the centrifuge tube surface during centrifugal collection.

C) the tens of sec of vortex are centrifugal again, and it is inferior to give a baby a bath on the third day after its birth with distilled water.

D) in the 1.5ml centrifuge tube, add following reagent, make the particle gun bullet

Bronze particle suspension 20 μ L

Plasmid DNA (2mg/ml) 2 μ L

CaCl2 20μL

Spermidine 8 μ L

According to institute's upgrading grain concentration, the adjustable ratio of bronze particle suspension and plasmid DNA joint.

E) place 20min on ice, every number min vortex 1min.

F) add 80 μ L100% ethanol, vortex, 13,000 leave heart 20sec, remove supernatant.

G) wash 3～4 times with 200 μ L100% alcohol;

Add 20～30 μ L ethanol earlier, violent vortex adds remaining ethanol again, and is centrifugal more at every turn.

H) suspend with 20 μ L ethanol at last.

I) get the ovule of the cotton that tissue culture obtains, concentrate to be placed on the plate culture medium central position.Each flat board is put the ovule of three flowers.

J) soak pressure membrane with 100% Virahol, carrier film, copper mesh and carrier film steel ring are used 75% alcohol immersion afterwards, use 100% alcohol immersion then.

K) treat that ethanol volatilizees fully after, carrier film is contained on the carrier film steel ring, carefully bullet is blown and beaten the mixing point again in the carrier film central position.Load onto pressure membrane, the flat board that is loaded with ovule is placed on 10cm or 15cm place.The promotor gene rifle.

L) ovule that will beat particle gun is on original substratum, and 30 ℃ of thermostat containers were cultivated 2～3 days, and this moment, the ovule in wild-type upland cotton Xuzhou-142 produced cotton fiber cell, and no fiber mutant fl does not produce cotton fiber cell.

The method of utilizing the said gene rifle to transform changes carrier pCAMBIA 1305 and expression vector KCS12promoter-pCAMBIA 1305 in the ovule of wild-type upland cotton Xuzhou-142 and no fiber mutant fl over to respectively; Respectively carrier pCAMBIA 1305 and expression vector KCS12 promoter-pCAMBIA 1305 are changed in the onion epidermis cell simultaneously.

Three, GUS dyeing

1, laboratory apparatus and reagent

Anatomical lens, thermostat container

The GUS formula for dye liquor

Phosphoric acid buffer (PH7.0) 100mM

EDTA(PH8) 1mM

Triton-X-100 1％

POTASSIUM FERROCYANIDE 99 5mM

Tripotassium iron hexacyanide 5mM

X-Gluc 1mg

2, experimental procedure

In 1.5mL EP pipe, add 5 μ L N earlier, dinethylformamide dissolving 1mgX-Gluc, vortex 1min adds above-mentioned other reagent again.Mend to 1mL with distilled water at last.

Ovule and the onion epidermis cultivated in the above-mentioned steps two 2～3 days are put into the GUS dye liquor for preparing, place 37 ℃ of thermostat containers, dye 1.5h.Take pictures with anatomical lens mirror mirror, the result as shown in Figure 2 again.1 changes GUS coloration result in the ovule in wild-type upland cotton Xuzhou-142 over to for expression vector KCS12promoter-pCAMBIA 1305 among Fig. 2,2 change GUS coloration result in the ovule in wild-type upland cotton Xuzhou-142 over to for carrier pCAMBIA 1305,3 change GUS coloration result in the ovule of no fiber mutant fl over to for expression vector KCS12 promoter-pCAMBIA 1305,4 change GUS coloration result in the ovule of no fiber mutant fl over to for carrier pCAMBIA 1305,5 change GUS coloration result in the onion epidermis cell over to for expression vector KCS12 promoter-pCAMBIA 1305, and 6 change GUS coloration result in the onion epidermis cell over to for carrier pCAMBIA 1305.As can be seen from Fig. 2, there be blue the demonstration in 1, show that gus gene has expression, show that dna molecular of the present invention has promoter function.The blue demonstration arranged in 1, and in 3 and 5, do not have blue the demonstration, show that KCS12 promotor of the present invention only makes gus gene express in cotton fiber cell, in other tissue, do not express.The blue demonstration all arranged in 2,4 and 6, show that 35S promoter had both made gus gene express in cotton fiber cell, in other tissue, express again.GUS histological chemistry detected result shows that the KCS12 promotor can start GUS to be expressed, and KCS12 promotor startup GUS expression only limits in the cotton fiber cell.

Claims (5)

1. a dna molecular is following 1)-3) in arbitrary described dna molecular:

1) dna molecular shown in the sequence 1 in the sequence table;

2) under stringent condition with 1) dna molecule hybridize and have the dna molecular of identical function;

3) with 1) or 2) dna molecular have 90% above homology and have the dna molecular of identical function.

2. the expression cassette, recombinant expression vector, transgenic cell line or the reorganization bacterium that contain the described dna molecular of claim 1.

3. recombinant expression vector according to claim 2 is characterized in that: described recombinant expression vector is for replacing the recombinant expression vector that the 35S promoter on pCAMBIA 1305 carriers obtains with the described dna molecular of claim 1.

4. the described dna molecular of claim 1 makes the application of goal gene in expressing in cotton fiber cell.

5. application according to claim 4 is characterized in that: described cotton fiber cell is the cotton fiber cell in the cotton ovule.

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