CN100471954C - Cotton fiber specific promoter and its use - Google Patents

Abstract

The invention discloses an astopic promoter of cotton fiber with 1037bp from -634 to -543 as core area, which is characterized by the following: constructing the plant expressive carrier with promoter; transmitting the gene into tobacco and cotton; dyeing GUS tissue; displaying expressive activity of exosperm superiority through enzyme activity and GFP fluorescent fiber detection; possessing expressive astopy of fiber in the land cotton.

Description

Cotton fiber specific promoter and application thereof

Technical field

The invention belongs to plant genetic engineering field, specifically, relate to cotton fiber specific promoter, contain the plant expression vector of this promotor, and the transformant that transforms by this plant expression vector;

The invention still further relates to the preparation method of the transgenic plant that contain above-mentioned promoter sequence.

Background technology

Cotton fiber is important textile industry raw material, and China is cotton textiles consumption and big export country, and therefore, Cotton Production occupies an important position in Chinese national economy, also is the important source of finance of peasant simultaneously.Can improve the fibrous quality of Cotton in China kind rapidly, be directly connected to the survival and development of industries such as the ups and downs of Cotton in China industry and textile production processing, Weaving device manufacturing, foreign export.Though utilize traditional breeding method once in the cotton variety improvement, to obtain very big success, but over nearly 20 years, world's cotton variety has reached a plateau on output, utilize existing genetic resources and breeding technique to be difficult to increase substantially output of cotton (Meredith W.R.Better Crops 2,000 84 (4): 6～9) again.The simple quite difficulty of conventional breeding improvement cotton fiber quality that relies on.This be because: 1) quality trait gene such as cotton fiber brute force, fineness and lint yield exist and bear chainly, and the conventional breeding method is difficult to break the negative correlation in this heredity; 2) the culture of cotton kind of China mainly is a upland cotton, and good fibrous quality gene mainly comes from diplontic plucked instrument Bai Shi cotton (fibre strength), cotton unusually (fibre strength and fineness) and tetraploid sea island cotton (fibre strength and fineness) etc., and the utilization of these good character genes is subject to many limitations in conventional breeding.Plucked instrument Bai Shi cotton and unusual cotton be wild diploid species, after hybridizing with tetraploid upland cotton, problem such as the economical character that exists distant hybirdization to bring is poor, the offspring is difficult to stablize, the cycle is oversize; Even if be all the hybridization of tetraploid sea * land, exist the offspring to separate strongly equally, the negative linkage relationship of output-quality still difficulty such as breaks at problem (Pan family coltfoal 1998 cotton breedings 99～104 Beijing: Chinese agriculture press).

Cotton fiber is thickened and forms through differentiation projection, elongation and cell walls by ovule outer integument cells of superficial layer, and whole growth course can be divided into: the differentiation of fiber initiating cell, elongate fiber (primary wall formations), secondary wall formation and four periods of fiber dewatering maturation.The growth of cotton fiber occurs with unicellular form, and in its growth course, fibrocyte has homogeneity and synchronism, and after the epidermic cell projection, fibrocyte extends rapidly, the differentiation of cell can be separated fully with the elongation process of cell.Along with the carrying out that fibroblast growth is grown, cellular form changes increasing, and its length can reach 1 at last, 000-3,000.Mature fibers is mainly formed (accounting for 87%) by Mierocrystalline cellulose, contains materials such as a spot of pectin, hemicellulose and protein in addition.The physico-chemical property (quality) of the chemical constitution of cotton fibre and intermolecular arrangement mode decision cotton fibre thereof.Introducing new biomacromolecule in cotton fibre is one of Critical policies of improvement cotton fibre quality.

Utilize gene engineering method can break genetic block between species, realize that the orientation of good goal gene shifts, have the offspring simultaneously and be easy to stablize, advantage such as breeding cycle is short.Therefore, separate gene and the specific promoter thereof relevant with cotton fiber development, and then utilize engineered means that cotton fiber quality is carried out orderly improvement, be the focus of domestic and international cotton research in recent years always.Utilize fiber specific promoter, people attempt the quality by biotechnology improvement cotton fiber, as polyhydroxybutyrate synthetase series specifically expressing in cotton fiber having been improved the thermal property of cotton fiber.

The investigative test of U.S. Agracetus company reported first genetically engineered improvement cotton fibre in 1996.---poly-D--(-)--the 3 hydroxyl fourth Fu (PHB) that in the cotton fibre of genetic transformation, contain a kind of natural aliphatic polyester compound.The PHB polyester is a kind of natural degradable thermal plastic (thermoplastic), and its physico-chemical property is similar to polypropylene.PHB often is present in the body of some bacterium as carbon source with the form of inclusion body.The biosynthesizing of PHB relates to 3 kinds of enzymes: β-Tong Liuxiemei, acetyl-CoA reductase enzyme and PHB synthetic enzyme.Cotton fibre itself contains endogenic β-Tong Liuxiemei.(JohnM E such as John, Keller G.Metabolic Proc NatlAcad Sci, USA 1996,93:12768-12773.) acetyl-CoA reductase gene (phaB) is connected with FbL2A with fiber-specific promotor E6 respectively with PHB synthase gene (phaC), make up plant expression vector, the utilization gene gun technology in upland cotton cultivar DP50, successfully obtains to express simultaneously the transgene cotton of phaB and phaC gene with phaB and phaC gene cotransformation.Gus gene is expressed, Northern hybridization equimolecular detected result all proves: phaB and phaC express in transgenosis cotton plant fiber.Fluorescent microscope is consistent with transmission electron microscope observation to be shown: contain the PHB particle in the transgenic cotton fibre cell.To the GC-MC analysis revealed of transgenic cotton fibre extract, have in the fiber and the identical material of bacterium PHB mass spectrum, prove and synthesized the PHB macromolecular substance in the transgenic cotton fibre.The HPLC analytical results shows: every gram fiber dry weight contains the PHB of about 30-3440 μ g, the synthetic back 10d of blooming that starts from of PHB, and PHB content does not reduce after the fiber maturation.Utilize in the plant materials acetyl-CoA synthetic macromolecule PHB of self, cotton fiber quality such as length, intensity and mic value are not brought any influence, yet the existence of PHB has improved the thermal insulation properties of fiber really.But because of PHB content in the fiber is still lower, the amplitude that insulativity improves is still limited.

Aspect the genetically engineered improvement cotton fibre rather another focus of paying close attention to of people be the research work of the Daniell of U.S. Auburn university.Daniell (Henry Daniell, Beltwide Cotton Conferences, 1998,595～598) attempt coding is contained albumen aggressiveness (the protein-based polymers of Val-Pro-Gly-Val-Gly repeating unit, PBPs) gene genetic converting cotton makes cotton cells go out PBPs albumen by specifically expressing.PBPs albumen extensively is present in occurring in nature, and as the elastin in the Mammals reticular tissue, intramolecularly contains the repetitive sequence of being made up of a plurality of amino acid, and the PBPs molecule often shows stronger elasticity (coefficient of elasticity 10.6～10.9Pa).Daniell (Henry Daniell, Beltwide CottonConferences, 1998,595～598) think PHBs is introduced cotton fibre, be expected to improve the elasticity and the intensity of fiber on the one hand, the raising of protein content in the fiber on the other hand, the fiber water-retaining capacity strengthens, with the also corresponding enhancing of avidity of dyestuff, this is that present conventional breeding method is not accomplished.China Chinese Academy of Sciences plants the living scientist of institute and utilizes the pollen tube path for transformation, place cotton fiber specific promoter E6 control down the rabbit keratin gene, obtained to change the cotton of rabbit hair keratin gene, thereby the elongation of cotton fibre is obviously increased, fiber specific tenacity (the Zhang Zhenlin that increases, cotton journal 2004,16 (2): 72～76).

Promotor is the section of DNA molecule that RNA polymerase can specific recognition, just makes the position of transcribing beginning.In the regulation and control of genetic expression, transcribing is the first step of genetic expression, also is one step of key (Lewi, 2005 gene VIII 669～705, Beijing: Science Press) of expression regulation.Along with engineered development, usually need to make up a kind of expression vector of energy high level expression heterologous protein, promotor is very big to expressive site and the expression level influence of foreign gene in biology, is the critical elements of gene engineering expression carrier.In the genetically engineered improvement of cotton fiber quality, often need foreign gene in fibrocyte, to express specifically, thereby reduce the influence of foreign gene the cotton normal growth.In the gene regulating mechanism research of relevant cotton fiber development, adopt genetically modified means usually, with goal gene overexpression or expression silencing, thereby infer its function.Therefore, when verifying its function by the transgenosis means, the normal growth of meeting interference of transgene cotton produces unforeseen result, influences the accuracy and the experiment progress of experimental result.If the employing cotton fiber specific promoter then can make the expression of goal gene be controlled in the fiber growth course.Therefore, the clone and the expression analysis thereof of cotton fiber or kind skin specific promoter to the gene functional research of cotton fiber development and the genetically engineered improvement of fibrous quality, all have crucial value.

Summary of the invention

One object of the present invention is to provide a kind of cotton fiber specific promoter sequence, it contains the nucleotide sequence shown in the SEQ ID NO.3 at least, more preferably has the sequence shown in SEQ ID NO.2, this sequence is the cDNA sequences Design primer according to cotton GhSCFP gene, adopt YADE (Xiao Yuehua, Acta Genetica Sinica 2002,29 (1): 22～26) method, the length that obtains are 5 ' upstream sequence of the GhSCFP gene of 1037bp.The a large amount of cis-regulating element such as RY-element (CATGCATC), AT enrichment region (TAAAATAATTATT), a plurality of CAAT-box, A-box, G-box, AAGAA-motif etc. and and the TFII bonded core sequence TATAA that comprise seed specific in this sequence.25bp place, TATA-box downstream is a GhSCFP gene transcription initiation site.By GUS histochemical stain in transgene tobacco and transgene cotton, GUS enzyme activity determination and GFP fluoroscopic examination, confirm that this promoters driven gus gene and GFP gene are in the transgene tobacco seed, specificity predominant expression in kind of skin drives gus gene and expresses specifically in upland cotton fiber in transgene cotton further.In addition, in order to identify the core area of GhSCFP gene promoter, the GUS enzymic activity of the transgene tobacco that the serial deletion fragment of having analyzed GhSCFP gene 5 ' sequence merges with gus gene respectively.The result shows-634 to-543 zones (being SEQ ID NO.3 sequence) when the deletion promotor, the GUS enzymic activity reduces about 30 times in the transgene tobacco seed of blooming back 5 days, show that this zone contains an enhancer sequence,-634 to-543 zones when the deletion promotor, GUS enzymic activity in stem and the leaf is higher than seed and corolla, shows that this zone contains the element of control promotor specifically expressing in seed.

Another purpose of the present invention is to provide the plant expression vector that contains the GhSCFP gene promoter.Adopt gene engineering method, the GhSCFP gene promoter is inserted into the plant expression vector that can obtain the GhSCFP of containing gene promoter of the present invention in the suitable expression vector.

In a specific embodiments of the present invention, made up the GhSCFP::GUS plant expression vector that contains gus gene, it has structure as shown in Figure 4.

In another specific embodiments of the present invention, make up the GhSCFP::GFP plant expression vector that contains the GFP gene, it has structure as shown in Figure 5.

Another purpose of the present invention is to provide the transformant that contains the GhSCFP gene promoter sequence.Above-mentioned plant expression vector is transformed suitable host can obtain transformant of the present invention, in a specific embodiments of the present invention, adopt plant expression vector that electric shocking method will contain the GhSCFP gene promoter to be transformed into and obtain transformant in the agrobacterium tumefaciens lba4404.

Another purpose of the present invention is to provide preparation to contain the method for the transgenic plant of GhSCFP gene promoter.GhSCFP gene promoter of the present invention and reporter gene are made up plant expression vector, described plant expression vector is transformed the transformant that the host obtains to contain the GhSCFP gene promoter, and transform plant acquisition transgenic plant with described transformant.Described transgenic plant are preferably tobacco or upland cotton.

The present invention separates the promotor that has obtained the cotton fiber specific gene, and confirmed that this promoter sequence has exosper predominant expression activity in tobacco, in upland cotton, has the fiber expression specificity, for genetically engineered improvement tobacco, cotton variety provide possibility and valid approach.

Brief Description Of Drawings

Fig. 1 represents the nucleotide sequence of GhSCFP gene promoter, shade cover part wherein, and the nucleus for-634～-543, the numbering of this sequence is from 1 to 1037 in sequence table; And-634～-543 numbering be with transcripting start point as+1, the above sequence of transcripting start point is from-1; Sequence below the transcripting start point is from+1; Do not have 0 site, the whole order of sequence is from 5 ' to 3 ';

Fig. 2 represents the primer that DNA amplification is used, wherein, P-up1.0 and P-down be used to the to increase GhSCFP promotor of 1037bp, P-up947 and P-down, P-up739 and P-down, P-up530 and P-down, P-up465 and P-down, P-up258 and P-down are respectively applied for the GhSCFP promoter fragment of amplification 946bp, 739bp, 530bp, 465bp, 258bp.Pgfp-up and the Pgfp-down GFP gene that is used to increase.Pgus-up and pgus-down are used to the gus gene that increases;

Fig. 3 represents that the GhSCFP promotor is cloned into the plasmid map on the pGEM-T carrier;

Fig. 4 represents pGhSCFP1.0-GUS plant expression vector collection of illustrative plates;

Fig. 5 represents pGhSCFP1.0-GFP plant expression vector collection of illustrative plates;

Fig. 6 represents the serial analysis expression vector mode chart after GhSCFP promotor 5 ' end deletion;

Fig. 7 represent with ³²The GhSCFP of P mark and 18sRNA gene are probe, hybridize the Northern result who obtains with the total RNA in upland cotton root, stem, leaf, flower, ovule, the fiber (R, S, L, FL, O, F);

Fig. 8 represent with ³²The GhSCFP of P mark and 18sRNA gene are probe, the Northern result that the total RNA hybridization after blooming with upland cotton in (dpa) different time fiber obtains;

Fig. 9 represents that with the GhSCFP protein antibodies be probe, and the total protein of different times ovule, fiber (O, F) is hybridized after blooming with upland cotton, the Western results of hybridization that obtains, and M is a standard molecular weight albumen;

Figure 10 represents GhSCFP promotor 5 ' end deletion series, the electrophorogram behind pcr amplification, and 1,2,3,4,5 GhSCFP promoter DNAs that are respectively 946bp, 739bp, 530bp, 465bp, 258bp wherein, 6 is standard molecular weight DNA;

Figure 11 represents that GhSCFP1.0 ∷ GUS integrative gene expression vector makes up schema;

Figure 12 represents that GhSCFP1.0 ∷ GFP integrative gene expression vector makes up schema;

Figure 13 represents the restriction enzyme digestion checking agarose electrophoresis collection of illustrative plates of pGhSCFP1.0 ∷ GUS carrier, wherein 1 is standard molecular weight DNA, 2 is the electrophoresis of pGhSCFP1.0-GUS recombinant plasmid through restriction enzyme XbaI and SmaI digestion after product, shows that the GhSCFP promotor of 1037bp merges with gus gene;

Figure 14 represents the restriction enzyme digestion checking agarose electrophoresis collection of illustrative plates of pGhSCFP1.0 ∷ GFP carrier, wherein 1,2,3 is different p5-GhSCFP1.0-gfp-m recombinant plasmids digests after product through restriction enzyme XbaI and BamHI electrophoresis, the GhSCFP promotor that shows 1037bp with the GFP gene fusion, 4 is standard molecular weight DNA;

Figure 15 represents to change the PCR checking result of GhSCFP1.0 ∷ gus gene tobacco, and wherein 1 is standard molecular weight DNA, and 2-7 is different transgenic line, 8 positive contrasts, 9 negative contrasts.Show that 3,4,5,6 are transgenic positive strain system, 2,7 is the negative strain of transgenosis system;

Figure 16 represents to change the PCR checking result of GhSCFP1.0 ∷ gus gene cotton, and wherein 1 is standard molecular weight DNA, and 2-6 is different transgenic line, 7 positive contrasts, 8 negative contrasts.Show that 2,3,4,5,6 are transgenic positive strain system;

Figure 17 represents to change the GUS coloration result in the GhSCFP1.0 ∷ gus gene tobacco, and GUS is predominant expression on the transgene tobacco seed.After the commentaries on classics GhSCFP1.0-GUS genetic tobacco seed GUS dyeing of blooming back 5 days, carry out paraffin section more according to a conventional method, obtain observations at last, show that GhSCFP1.0 starts GUS and expresses on the tobacco seed exterior skin as figure;

Figure 18 represents wild-type and changes the GhSCFP1.0 ∷ GFP genetic tobacco back 5 days seed of blooming that the observations under fluorescent microscope: the seed of wild-type is inspired the genetically modified gfp of ruddiness and then is inspired green fluorescence;

Figure 19 is illustrated in seed, corolla, blade, stem, sepal and the ovary wall of transgene tobacco, the variation of GUS enzymic activity; Wherein enzyme is alive the highest in seed, and is all very low in the remaining tissue;

Figure 20 represents to change the GUS coloration result of GhSCFP1.0 ∷ gus gene upland cotton; A wherein: the ovule on the same day of blooming; B: the same day ovule surface amplification; C: the back 3 days ovule of blooming; The amplification on D:3 days ovule surfaces;

Figure 21 represents to change GhSCFP1.0 ∷ gus gene upland cotton, the rip cutting observations after the ovule GUS dyeing; Wherein, uncoloured ovule is contrast among the A, and B is the rip cutting of transgenosis ovule, and C is the enlarged view of B;

Figure 22 represents to change GhSCFP1.0 ∷ gus gene upland cotton, the paraffin section result after the ovule GUS dyeing on the same day of blooming; Wherein, B and D are respectively the enlarged view of A and B, and showing only has the fiber of ovule protrusion of surface to be colored;

After Figure 23 represents GhSCFP promotor 5 ' end disappearance, GUS enzyme activity assay result in the transgene tobacco seed; Behind-634 to-543 zones of this promotor of deletion, the GUS enzymic activity in the transgene tobacco seed reduces about 30 times, shows that this zone contains enhancer sequence.After all the other 4 the zone deletions, the GUS enzymic activity changes little in the seed;

After Figure 24 represents GhSCFP gene promoter disappearance-634 to-543 zones, GUS enzyme assay result in the transgene tobacco different tissues, U-7 is different transgenic lines with U-14, show behind-634 to-543 zones of deletion GhSCFP gene promoter, GUS enzyme work in stem and the blade is higher than seed, corolla, shows the element that also has controlling gene to express in seed in this zone.

Embodiment

The extraction of [embodiment 1] cotton fiber RNA

The same day of blooming to the cotton flower mark of listing, take away respectively and spend the same day to the back 9 days cotton boll of blooming.The extraction of cotton ovule and fiber RNA is with reference to method (the Shujun Chang of Chang Shujun etc., Plant Molecular Biology Reporter 1993 11:113～116), but changed part steps, specified operational procedure is as follows: after the sampling cotton boll is placed ice chest at once, indoor bloom back ovule, fiber on the same day and the fiber of blooming back 6 days, 8 days of stripping respectively is under liquid nitrogen in the quick grind into powder and the 50ml centrifuge tube of packing into.Extraction damping fluid (the CTAB2% that adds 10ml65 ℃ of preheating, PVP2%, Tris-HCl100mmol/L pH8.0, EDTA25mmol/L, NaCl2mol/L, Spermidine 0.5mg/ml), mixing is in 65 ℃ of water-bath 3min, add isopyknic chloroform again: primary isoamyl alcohol (24:1), centrifugal behind the mixing: 20 ℃, 10000rpm, 5min.Chloroform: primary isoamyl alcohol (24:1) repeats extracting once, and water intaking is added to the lithium chloride of the 10mol/L of 1/4 volume, and 4 ℃ of precipitations are spent the night.Abandon supernatant liquor in 4 ℃ of centrifugal 20min of 10000rpm, 500 μ lSSTE dissolving adds isopyknic phenol (pH4.5), chloroform: the primary isoamyl alcohol extracting, add 2 times of volume ethanol in aqueous phase at last, and deposit for-80 ℃.

The structure and the gene clone in [embodiment 2] cotton fiber cDNA library

The construction process in cotton fiber cDNA library mainly carries out with reference to STRATAGENE company product description, summary is for getting 100 μ g cottons bloom back ovule on the same day and total RNA of the fiber and back 6 days, the 8 days fibers of blooming, the 1st chain that synthesizes cDNA by the Super-script II reversed transcriptive enzyme of GIBCO BRL, use STRATAGENE cDNA library construction test kit (λ ZAP) then, set up the cDNA library of cotton fiber.After measured, the elementary storehouse titre that obtains is 9.8 * 10 ⁶The titre of amplifying the library is 1 * 10 ¹¹Altogether picked at random 20 clones and subclone to plasmid pBSsk, detect and show that the external source fragment length of being cloned between 0.7-2.5kb, can satisfy the needs of gene clone.

The separation of [embodiment 3] cotton genomic dna

Get about 1 gram cotton leaf material, put into mortar, quick grind into powder under the liquid nitrogen, the centrifuge tube and add the CTAB extracting solution of 3ml65 ℃ of preheating, quick oscillation mixing, 65 ℃ of water-bath 30min of packing into.In this centrifuge tube, add 1ml5mol/L KAc mixing ice bath 30min then, add the chloroform of equal-volume (4ml): primary isoamyl alcohol (24:1, v/v) put upside down mixing, 8000rpm, centrifugal 10min, get the pre-cold isopropanol that upper water is added to 2/3 times of volume, mixing is placed 2h for-20 ℃.Be deposited in the new 1.5ml centrifuge tube with sealing the cotton-shaped DNA of kapillary picking, air-dry precipitation after 70% washing with alcohol 2 times adds an amount of TE and fully dissolves phenol: chloroform: (24:23:1, v/v/v) after the extracting, it is standby to add ℃ preservation of 2 times of volume ethanol-20 for primary isoamyl alcohol.

Clone and the sequence signature analysis of [embodiment 4] cotton fiber specific gene GhSCFP

From constructed cotton fiber cDNA library, adopting at random, cloning has obtained 80 genes.The Subtilisin-like Protease gene of plants such as the nucleotide sequence of one of them gene and Arabidopis thaliana has higher homology, called after cotton kind hide fiber specific gene (Seed Coat Fiber specificProteinase, GhSCFP), this gene length 2436bp contains the open reading frame of a 2337bp, 5 ' end non-coding region 31bp, 3 ' end non-coding region 68bp.Supposition encoded protein matter is made up of 778 amino-acid residues, molecular weight 83474 dalton, and pI equals 8.244, and nucleotide sequence is shown in SEQ ID NO.1.Homology analysis shows: at protein level, be respectively 72%, 70% and 68% with the homology of the Subtilisin-likeProtease gene of potato, Arabidopis thaliana, paddy rice.

Subtilisin belongs to a kind of of serine protease, and it has a substrate binding site, a contact reacts territory that is made of D, H and three districts of S.Subtilisin all has discovery in protokaryon and eukaryote, but the research in the plant is less relatively.From plant, clone some Subtilisin-like genes at present and carried out preliminary study.Berger and Altmann (Berger D, Altmann T 6enes Dev 2000 14:1119-1131) when the sdd1-1 list cryptic mutant that research Arabidopis thaliana epidermis pore clusters, clones the SDD1 gene by mapping, the result shows that it and subtilisin gene have very high homology.Sdd1 has lost the control that epidermic cell is divided into pore, thereby has caused the phenomenon of clustering of pore owing to lack the catalytic activity that the S district may lose subtilisin.But by mutant strain is transformed the SDD1 gene, pore clusters phenomenon because the complementary action of SDD1 gene can be restored, and shows that the subtilisin gene may have the function of control table chrotoplast differentiation in plant.Tanaka (Tanaka H, Onouchi H, Kondo M, Hara-Nishimura I, Nishimura M, Machida C, Machida Y.Development.2001 128 (23): 4681-9) result of study of Denging also confirms, at Arabidopis thaliana alel (abnormal leaf

Shapel) in the mutant growth course, embryo's outside surface does not separate with remaining endosperm epidermis, and the ALE1 gene by swivel base spike clone also has very high homology with subtilisin.Further research is learnt the ALE1 gene only the tender embryo of children be close in embryo's the endosperm epidermic cell and express, in case the embryo sprouts, its expression promptly stops, and shows that the ALE1 gene may influence the formation of embryo or young tender plant epidermis.Paik-Ro application such as (Paik-Ro O.G.Seib J.C.Smith R.L.TheorAppl Genet 2002 104:236-240) difference subtracts the method for hybridization and cloned four seed development specific genes from the cDNA library of peanut, wherein Psc11 gene specifically expressing in kind of skin, and coding subtilisin-like albumen.These results of study show that the subtilisin gene may participate in plant embryos tender tissue epidermic cell and form and plant processes such as skin growth.

The expression analysis of [embodiment 5] cotton fiber specific gene GhSCFP

We have at first carried out the Northern hybridization analysis to the GhSCFP expression of gene.With its conservative region is probe, carry out the specific expressed analysis of this gene organization, find that this gene do not express at root, stem, leaf, in spending, only in ovule and fiber, express, as seen cotton GhSCFP expression of gene has the fiber development-specific, may be relevant with the growth of cotton fiber.Further use identical probe, hybridize with the RNA of different development stage fiber respectively, the result shows: the early stage GhSCFP gene at cotton fiber development begins to express, the 8-10 days expression amounts in back reach the highest to blooming, this expression of gene begins to descend subsequently, close to back 14 days these genes of blooming, promptly the formation of the formation of this expression of gene and cotton fiber and primary wall is relevant.These result's hints: cotton GhSCFP gene may participate in processes such as the differentiation of ovule epidermic cell, fibrocellular growth.

Secondly, by the expression of this gene of Western hybridization analysis at protein level, the result shows the appearance that just can detect target protein from 4DPA, reaches high expression level amount at 8DPA; Along with fibrocellular growth, GhSCFP albumen continues to exist (surpassing 30DPA) and content not to significantly decrease.

The clone and the signature analysis of [embodiment 6] cotton fiber specific promoter

According to the cDNA sequences Design dna primer of cotton GhSCFP gene, adopt the method for YADE, 5 ' end to this gene on the cotton gene group extends, and having obtained length is 5 ' upstream sequence of the GhSCFP gene of 1037bp, shown in SEQ ID NO.2.Utilize analysis software (Plant CARE, http://intra.psb.ugent.be:8080/PlantCARE/), from the plant promoter database, this sequence is carried out the promoter regulation component analysis, therefrom find a large amount of cis-regulating element and with TFII bonded core sequence TATAA, at this 25bp place, TATA-box downstream be GhSCFP gene transcription initiation site (initiator, Inr).These cis-regulating element comprise RY-element (CATGCATC), AT enrichment region (TAAAATAATTATT), a plurality of CAAT-box, A-box, G-box, the AAGAA-motif etc. of seed specific.

[embodiment 7] cotton GhSCFP gene promoter is analyzed the structure of expression vector

(P-up1.0 P-down) utilizes pcr amplification to go out this segment DNA fragment and is cloned among the TA carrier pGEM-T and is pGEM-GhSCFP1.0 primer by design, by dna sequencing this dna sequence dna is identified at last.According to the distribution of various controlling elements in this promoter sequence, the principle according to 5 ' end disappearance designs the promoter fragment that following primer (Fig. 2) amplifies 946bp, 739bp, 530bp, 464bp and 257bp respectively.The dna fragmentation of amplification is cloned among the TA carrier pUCm-T, adds their confirmation by sequencing.Owing to behind the primer of promotor 5 ' end and 3 ' end, introduced XbaI and SmaI restriction endonuclease sites respectively, by XbaI and SmaI with the promoter deletion fragment directed cloning confirmed in the pGPTV-KAN carrier with the uidA gene fusion, make up GhSCFP ∷ GUS plant expression vector (Fig. 4).

The PCR reaction conditions: the reaction cumulative volume is 25 μ l, comprises 10 * LA Taq buffer, 2.5 μ l, every kind of dNTP 100 μ mol/L, 1.5mmol/LMgCl ₂, template DNA 10ng, each 400nmol/L of upstream and downstream primer, the 1 LA Taq of unit archaeal dna polymerase (TaKaRa company).Amplification condition: 94 ℃ of sex change 4min, continue with 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ of extension 1min, totally 35 circulations, last 72 ℃ are extended 10min.

The GFP gene source is in pBINm-gfp5-ER, but removal alkaline chitinase signal peptide wherein.Increasing and be cloned among the pUCm-T by synthetic primer (P gfp-up, P gfp-down) is pUCm-gfp5, utilize restriction enzyme HindIII and EcoRI with this gene directed cloning to P ⁵In be p ⁵-gfp-m.Secondly, utilizing EcoRI and SmaI that GhSCFP1.0 is cut out and be cloned into pBS from pGEM-GhSCFP1.0 is pBS-GhSCFP 1.0; Logical again XbaI and BamHI GhSCFP1.0 is cut out from pBS-GhSCFP1.0 and directed cloning to p ⁵With the GFP gene fusion, be built into GhSCFP ∷ GFP plant expression vector (Fig. 5) in-gfp-m carrier.

At last, by electric shocking method these promoter Analysis expression vectors are transformed in the agrobacterium tumefaciens lba4404.

The genetic transformation of [embodiment 8] tobacco

1, the acquisition of tobacco aseptic seedlings and agrobacterium tumefaciens are infected the preparation of liquid

With 1% clorox sterilization 15min, aseptic water washing 4-5 time places then on the solid MS substratum and sprouts with tobacco seed, cultivates under 25 ℃, 16h illumination/8h dark photoperiod condition.

On the YEB solid medium that contains 50mg/L Km and 125mg/L Sm, the Agrobacterium that activation contains expression vector preserves bacterial strain.One of picking contains the single bacterium colony of Agrobacterium of goal gene, is inoculated in the 5ml additional phase with in the antibiotic YEB liquid nutrient medium, 28 ℃, 200rpm overnight incubation.Get this bacterium liquid of 250 μ l and be inoculated in the 25mlYEB substratum, 28 ℃, 200rpm are cultured to OD ₆₀₀About 0.6～0.8, then bacterium liquid is collected thalline in the centrifugal 5min of 6000rpm, be diluted to the original bacteria liquid volume with liquid MS medium.

2, Agrobacterium tumefaciens mediated tobacco genetic transformation

Get the healthy and strong blade of aseptic seedlings, be cut into about 0.5cm ²Big leaflet dish adds OD ₆₀₀Be 0.6～0.8 Agrobacterium bacterium liquid, after 15min was infected in vibration gently, the bacterium liquid that inclines at once changed explant on the common substratum that the surface is covered with one deck filter paper over to, and 24 ℃ of dark places are cultivated 3d altogether.

3, the screening of transformed plant and regeneration

After cultivating altogether, explant changed over to screen differentiation culture in the screening culture medium, per 4 all subcultures once.When green bud appears in callus, green bud downcut to change in the root media take root, long during up to seedling to high about 5cm, the transgenosis seedling is detected.To merging the promoter Analysis transgene tobacco of GFP, root and blade are directly carried out the GUS histochemical stain, the positive seedling replanting of GUS is buried in the alms bowl; To merging the transgene tobacco of GUS, adopt the method for PCR to identify, PCR male tobacco is transplanted and management.

The genetic transformation of [embodiment 9] cotton

1, cotton embryonic callus induction and agrobacterium tumefaciens are infected the preparation of liquid

Cotton seeds is peeled off, with 0.1% mercuric chloride (HgCl ₂) sterilization 10min, rinsed with sterile water 6 times is inoculated on the seed germination substratum, and 28 ℃, the following 5～7d that sprouts of dark condition obtain aseptic cotton seedling.Choose the aseptic seedlings hypocotyl of robust growth, be cut into the segment of about 0.4cm, be inoculated in evoked callus on cotton healing tissue's inducing culture, every 3-4 week subculture once.Choose loose callus and be inoculated on the embryonic callus induction substratum generation of induced embryonic callus.

On the YEB solid medium that contains 50mg/L Km and 125mg/L Sm, the Agrobacterium that activation contains expression vector preserves bacterial strain.Choose the single bacterium colony of Agrobacterium, be inoculated in 5mL and contain in the identical antibiotic YEB liquid nutrient medium, 28 ℃, 200r/min shake overnight incubation.Agrobacterium bacterium liquid after the cultivation is transferred to 50mL in the ratio of 1:20 and contains in the identical antibiotic YEB liquid nutrient medium, continues to cultivate 3-5hr, and behind the centrifugal 10min of 6000rpm, thalline is resuspended with liquid MSB (containing 100 μ mol/LAS) substratum, adjustment OD ₆₀₀Value is standby for 0.3-0.5.

2, dip-dye and cultivation altogether

Inhale the liquid that goes to the embryo callus surface with aseptic thieving paper, put into the Agrobacterium bacterium liquid of adjusting concentration, contaminate 20-30min.The bacterium liquid that inclines is transferred to the embryo callus subculture of contaminating on the common culture medium (containing 100 μ mol/LAS), cultivates 4d altogether under 24 ℃ of dark conditions.

3, the screening of transformant

Cultivate altogether embryo callus to be transferred to after finishing and take off bacterium and screening and culturing on the screening culture medium, per 3 all subcultures once.Most of callus browning death after 1-2 month, small part shows kalamycin resistance, grows fresh embryo callus.The callus lines subculture when every block organization breeds to 2.0-3.0g, inserts in the fluid suspension culture base, and 120r/min concussion suspension culture is to obtain a large amount of body embryos on shaking table.2 week of suspension, the suspension culture tissue was filtered with 30 eye mesh screens in the back, and throw out off the net is transferred into body embryo maturation medium.The mature embryo of sprouting changes on the body embryo elongation inducing culture, the elongation of inductor embryo, sprouting.Getting length transfers into SH substratum Cheng Miao greater than the sprouting embryo of 0.5cm.Treat that seedling is long to about 2cm when high, the grafting of clip seedling is to the cotton seedling that 3-4 sheet true leaf is arranged.Be transplanted to the greenhouse growth behind the grafting survival.

The PCR checking of [embodiment 10] transfer-gen plant

Take DNA extracting method in a small amount, extract transfer-gen plant DNA and be used for the PCR checking.Concrete steps comprise: divide individual plant to take a morsel T0 respectively for transgene tobacco and cotton leaf, put in the 1.5ml centrifuge tube, add a little liquid nitrogen and use the glass rod grind into powder.The DNA extraction damping fluid that adds 0.5ml65 ℃ of preheating, mixing, 65 ℃ of water-bath 30min.The equal-volume chloroform: primary isoamyl alcohol extracting twice, draw supernatant liquor in new centrifuge tube, add 2 times of volume dehydrated alcohols-20 ℃ precipitation 2h.The centrifugal 10min of 12000rpm under the room temperature abandons or adopts the air-dry precipitation of supernatant liquor, last 200 μ lTE damping fluids dissolving.

The transfer-gen plant DNA that extracts is carried out the checking of PCR amplification in vitro, and the reaction cumulative volume is 25 μ l, comprises 10 * LA Taq buffer, 2.5 μ l, every kind of dNTP100 μ mol/L, 1.5mmol/L MgCl ₂, template DNA 10ng, each 400nmol/L of upstream and downstream primer, the 1 LA TaqDNA of unit polysaccharase (TaKaRa company).

Amplification condition: 94 ℃ of sex change 4min, continue with 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ of extension 1min, totally 35 circulations, last 72 ℃ are extended 10min.

Amplified production is containing on 1% sepharose of ethidium bromide behind the voltage electrophoresis with 5V/cm, and ultraviolet lamp is observed down and taken pictures.

Histochemical stain and the observation of [embodiment 11] GUS

Get fresh transgene tobacco, upland cotton material, in the rearmounted 1.5ml centrifuge tube that is cut into small pieces, add GUS staining fluid (10mmol/LEDTA, 100mmol/L sodium phosphate buffer pH7.0,0.1mol/L

K ₃[Fe(CN) ₆]，0.1mol/L?K ₄[Fe(CN) ₆]，0.1％(V/V)Triton?X-100，5mg/ml?X-Glue)。The centrifuge tube that will contain vegetable material and GUS dye liquor is put into 37.0 ℃ of constant temperature incubators, dyeing 3h.Staining fluid is removed in hypsokinesis, behind 70% ethanol decolorization, observes and takes a picture.If GUS dyeing back tobacco seed and cotton ovule are carried out the paraffin section observation, behind the staining fluid that then inclines, fix 3 to 24h with the FAA stationary liquid.The flush away stationary liquid, 6 grades of dehydrations of conventional ethanol---trimethyl carbinol method, waxdip, embedding.The plant tissue that paraffin embedding is good is cut into slices on slicing machine, and slice thickness is controlled at 10-15 μ m.

The microscopic examination of [embodiment 12] transgenic line GFP

The flesh tissue that cuts dropped into immediately contain 4% Paraformaldehyde 96,2.5% glutaraldehyde, 20mmol/L phosphoric acid buffer (pBS in stationary liquid pH7.0), after the 1h vacuum-treat, continues down fixedly to spend the night in 4 ℃.The fixed sample directly is wrapped on the sample with Tissue-Tek O.C.T. embedding medium after phosphoric acid buffer embathes 2～3 times, and is freezing rapidly with liquid nitrogen again.As early as possible sample is moved to subsequently in-80 ℃ of Ultralow Temperature Freezers preserve standby.Before section, about 1h, sample is taken out from-80 ℃ of Ultralow Temperature Freezers, put in the freezing microtome chamber, after rising again, be bonded at embedding medium again and carry out frozen section on the sample head.Slice thickness is 10 μ m, and the slicing machine cavity temperature is-20 ℃.After the section nature dries, under fluorescent microscope, observe and photograph.

[embodiment 13] transgenic plant are organized the GUS enzyme assay

The gus gene encoding beta-glucosidase, this enzyme is a kind of lytic enzyme, does not need coenzyme during show activity, the hydrolysis of the many beta-glucoside Esters of energy catalysis.The optimal pH of katalysis is 5.2～8.0, can adapt to the ionic strength scope of broad.With 4-methyl umbelliferone acyl-β-D glucuronide (4-MUG) is substrate, and it is hydrolyzed to 4-methyl umbelliferone (4-MU) and β-D glucuronic acid GUS catalysis.Hydroxyl in the 4-MU molecule dissociates the back by the optical excitation of 365nm, produce the fluorescence of 455nm, available spectrophotofluorometer is quantitative, because the pKa of 4-MU hydroxyl is between 8～9, desire to make a large amount of ionizations of hydroxyl, the pH of solution should generally use the yellow soda ash termination reaction and create alkaline condition greater than its pKa.

1, the extraction of GUS crude enzyme liquid

The transgene tobacco that selection has been bloomed, the mark flowering time of listing the same day of blooming.Take away the fruit (wherein 3～5 fruits of each individual plant) of spending back 5d, strip seed, ovary wall under the room temperature respectively.In addition, ordinary method is to blade, sepal, young stem and the sampling of the corolla on the same day of blooming.Vegetable material to be measured put in the liquid nitrogen pulverize, the GUS enzyme extraction damping fluid (100mmol/L sodium phosphate buffer pH8.0,1%PVP W/V, 10mmol/L beta-mercaptoethanol) that adds 2 times of volumes is made homogenate, places 1h on ice.In the centrifugal 10min of 13000rpm/min, collect supernatant, measure the GUS enzyme immediately and live, preserve ℃ preservation of adding sterile glycerol (50%v/v)-20 as need.

2, Bradford method (Wang Jiazheng, 2000 protein technical manuals 42～46, Beijing: the content of total protein in mensuration GUS crude enzyme liquid Science Press).

3, the making of 4-MU typical curve

When carrying out promoter Analysis,, in the different tissues of transgenic plant, can obtain different GUS enzymes and live based on the space-time specificity of promoter expression.Therefore, for guaranteeing the accuracy of experiment, made the typical curve of high and low two kinds of 4-MU concentration gradients.At first, use minor N, dinethylformamide dissolves the 4-MU solid, is diluted to the mother liquor of 1mmol/L then with distilled water.Secondly, with reaction terminating liquid the 4-MU mother liquor is diluted to the high density gradient sample of 100 μ mol/L, 80 μ mol/L, 60 μ mol/L, 40 μ mol/L and 20 μ mol/L and the lower concentration gradient sample of 10 μ mol/L, 8 μ mol/L, 6 μ mol/L, 4 μ mol/L and 2 μ mol/L.At exciting light 360nm, launching light 460nm on microplate reader, measure the fluorescence intensity of each sample under the condition of slit 25nm, is blank with the reaction terminating liquid, and every kind of concentration sample is provided with 3 repetitions, by calculating mean value drawing standard curve.

4, GUS enzyme-to-substrate reaction

Preheating reaction buffer in 37 ℃ of water-baths (in the 100mlGUS enzyme extraction damping fluid add 35.23mg 4-MUG) in 2 96 micropore elisa plates, adds reaction terminating liquid (200mmol/L yellow soda ash) with 180 μ l/ holes simultaneously.The reaction buffer and the 5 μ lGUS crude enzyme liquids that in the sample centrifuge tube, add 195 μ l preheatings, mixing takes out 20 μ l immediately and joins in No. 1 plate, and this is 0 o'clock sample of reaction (during fluorometric assay as blank), and strictness is clocked.Sptting plate is put into 37 ℃ of water-baths carry out enzyme reaction, when 20min, respectively get 20 μ l reaction solutions and join in No. 2 plates mixing, the sample during for enzyme reaction 20min.

5, GUS enzyme fluorometric assay alive

It is blank that fluorescent is measured with No. 1 plate, with exciting light 360nm, launches light 460nm on microplate reader, measures the fluorescence intensity of No. 2 each sample of plate under the condition of slit 25nm.Select corresponding standard curve according to the fluorescence intensity level that obtains, go out 4-MU content in No. 2 plates by regression equation calculation.Enzyme activity unit is defined as: the enzyme amount that per minute hydrolysis 4-MUG generates 1nmol4-MU is a unit.The expression activity of gus gene represents with the enzyme activity of every milligram of total protein, i.e. 4-MU nmol/mg/min.

The functional analysis of [embodiment 14] GhSCFP promotor

Through the GUS that changes GhSCFP1.0 ∷ gus gene tobacco different tissues is dyeed, found that and in blade, stem, corolla, stamen, column cap, all do not detect blue the appearance, and arrive blueness at tobacco tender seed of children and part placenta surface observation, there is very dark blueness to occur at seed-coat especially.Simultaneously blooming preceding and the back 12 days seed-coat of blooming are observed blueness, the blue disappearance after 12 days.The result shows: the GhSCFP1.0 promotor has activity in early days in seed development, and only expresses in seed.Further the painted seed sections observation of GUS is found that in five confluent monolayer cells of kind of skin, have only the outermost layer cell to be colored, promptly this promotor has kind of a skin expression specificity.

Carry out fluoroscopic examination to changeing GhSCFP1.0 ∷ GFP genetic tobacco, do not observe the green fluorescence of GFP equally at blade, stem, corolla, stamen, column cap, and on the tender seed of children, can observe stronger green fluorescence, not genetically modified Nicotiana gossei seed only is inspired red fluorescence.The above results shows GhSCFP ∷ GUS and GhSCFP ∷ GFP fusion gene, can express in the kind skin of the tender tobacco of children specifically.Therefore, in tobacco, this promotor has strict kind skin specificity and development time specificity.

Further detect the GUS enzymic activity of changeing GhSCFP1.0 ∷ gus gene tobacco different tissues, in the corolla of the visible transgene tobacco of result, blade, stem, sepal and the ovary wall, the GUS enzymic activity is the highest has only 88.5nmol/mg/min, but the GUS enzymic activity is more than 500nm0l/mg/min in the back 5 days seed of blooming.The result illustrates that equally this promotor has seed predominant expression specificity.

Above-mentioned is the detected result of transgene tobacco, through the GUS that changes GhSCFP1.0 ∷ gus gene cotton different tissues is dyeed, found that in blade, young stem, corolla, stamen, column cap and preceding 1-2 days ovule of blooming and all do not observe blue signal, and on the fiber of back ovule protrusion of surface of blooming, observe very dark blueness, special ovule surface on the same day of blooming, observe blue point, and the point of these bluenesss is exactly the fiber of projection, up to blooming back 8 days, still show blue.To blooming back 3 days and carrying out rip cutting through the painted ovule of GUS, observe profile and find, dyed blue part by GUS, be exactly the pars fibrosa of cotton.Further observe by paraffin section, on the ovule surface on the same day of blooming, the fibrocyte of projection is dyed blueness, and the epidermic cell of projection is painted very not shallow.The above results shows: the GhSCFP1.0 promotor has the upland cotton fiber development-specific.

[embodiment 15] cotton GhSCFP gene promoter nucleus is identified

According to 5 ' end disappearance principle, the GhSCFP1.0 promotor is deleted.Deleted-634 to-543 ,-634 to-336 ,-634 to-127 ,-634 to-61 and-634 to 146 behind totally 5 zones of this promotor, constructed plant expression vector with gus gene respectively and change in the tobacco.The transgene tobacco seed of blooming back 5 days is carried out the GUS enzyme activity assay, detected result shows behind-634 to-543 zones (SEQ ID NO.3) of this promotor of deletion, GUS enzymic activity in the transgene tobacco seed has reduced about 30 times, shows that this zone contains an enhancer sequence.After deleted respectively in all the other 4 zones, the GUS enzymic activity changed little in the seed.The GUS enzyme assay of transgene tobacco different tissues is found: after deletion GhSCFP1.0 promotor-635 arrives-543 zones, GUS enzyme work in stem and the blade is higher than seed, corolla, shows the element of controlling promotor specifically expressing in seed in this zone in addition.

Sequence table .ST25

SEQUENCE?LISTING

＜110〉Southwestern University

＜120〉cotton fiber specific promoter and application thereof

<130>06P103129

<160>3

<170>PatentIn?version?3.1

<210>1

<211>2436

<212>DNA

＜213〉upland cotton

<220>

<221>misc_feature

<222>(1)..(2436)

＜223〉cotton kind hide fiber specific gene

<400>1

<210>2

<211>1037

<212>DNA

＜213〉artificial sequence

<220>

<221>promoter

<222>(1)..(1037)

<223>

<220>

<221>enhancer

<222>(1)..(91)

<223>

<400>2

<210>3

<211>91

<212>DNA

＜213〉artificial sequence

<220>

<221>enhancer

<222>(1)..(91)

<223>

<400>3

Claims (10)

1. an enhanser is characterized in that it is the nucleotide sequence shown in SEQ ID NO.3.

2. a cotton fiber specific promoter is characterized in that it is the nucleotide sequence shown in SEQ ID NO.2.

3. plant expression vector that contains the described cotton fiber specific promoter of claim 2.

4. plant expression vector according to claim 3 is characterized in that it has structure as shown in Figure 4.

5. plant expression vector according to claim 3 is characterized in that it has structure as shown in Figure 5.

6. a transformant comprises described cotton fiber specific promoter of claim 2 and host.

7. transformant according to claim 6 is characterized in that, described host is an agrobacterium tumefaciens.

8. the application of the described cotton fiber specific promoter of claim 2 in the preparation transgenic plant.

9. application according to claim 8 is characterized in that described transgenic plant are tobacco or upland cotton.

10. a preparation method who contains the transgenic plant of the described cotton fiber specific promoter of claim 2 comprises the steps:

(1) promoter sequence with cotton fiber specific operationally inserts in the expression vector, makes up plant expression vector;

(2) transform the host with described plant expression vector, obtain transformant;

(3) described transformant is transformed plant, obtain transgenic plant.

Images