CN101818148B - Identification of cotton fiber specificity promoter GhFLA1 - Google Patents

Abstract

The invention discloses a cotton fiber specificity promoter GhFLA1 and a gene complete sequence thereof, relating to an important regulatory element related to cotton fiber development. The FLA1 gene complete sequence has totally 1920bp and comprises a 5' end promoter and a gene coding sequence. The gene does not contain an intron and encodes an arabinogalactan protein (AGP). The cotton fiber contains a great amount of the AGP protein, and that AGP irreversible inhibitor can inhibit fiber elongation shows that the protein has an important role for cotton fiber development. That the FLA1 gene mRNA has a great amount of specificity accumulation in the process of cotton fiber cell development shows that the gene promoter plays an important role in the regulation of fiber specificity expression. Experiments also show that the promoter has strong expression activity in tobacco leaf epidermal hair, which further proves that the promoter is the specificity regulatory element of the cotton fiber. The promoter can be used as a gene cis-regulatory element to be applied to cotton molecular breeding research so as to improve the cotton fiber quality and to enhance the cotton yield.

Description

The evaluation of a cotton fiber specific promoter GhFLA1

Technical field:

The present invention relates to a cotton fiber specific promoter.The clone who specifically is the promoter sequence that is encoded into bundle protein appearance Arabinogalactan-Protein (fasciclin-like arabinogalactan protein) gene GhFLA1 of a fiber specifically expressing identifies and activation analysis.

Background technology:

Cotton fibre is the important raw and processed materials of China's textile industry and related industries thereof.Along with textile industry fast development and living standards of the people improve constantly, textile industry and related industries thereof are more and more high to the requirement of cotton fibre quality.Cotton in China output ranks first in the world, but cotton fibre quality is relatively poor, can not be as the raw materials for production of some high added value high quality textile products.Therefore, annual more than 100 ten thousand tons of the import high-quality cottons that need of China cause a large amount of of homemade raw cotton to overstock.The cotton fibre quality problem has become one of major obstacle of restriction Cotton in China industry Sustainable development.

The ultimate aim of cotton genetic improvement is to improve the yield and quality of cotton fibre.Production of cotton fibers and quality not only receive Effect of Environmental, and receive the particularly regulation and control of fiber specific expression gene of genes involved.At present, except utilizing the conventional breeding means, also progressively use genetic engineering that cotton fibre quality is carried out genetic improvement both at home and abroad.With excellent agronomic characters, the good character that particularly fibrous quality is relevant is introduced the cotton main breed, not only can improve output of cotton and fibrous quality, reduces production costs, and can alleviate the disadvantageous effect to environment.Therefore, critical function gene and specificity controlling element thereof that the separating clone cotton fiber development is relevant have extremely important using value.In order to make foreign gene in destination organization, be able to give full expression to, different traits needs different promotors.In the quality-improving of cotton fibre, cotton fiber specific promoter is an indispensable cis-regulating element in the genetic manipulation of cotton fibre quality improvement, fiber-specific expression regulation of its decision target gene and expression intensity regulation and control.The important goal gene that is used for improveing cotton fibre quality need be at the cotton fibre specifically expressing, thereby must use this expression of gene of fiber-specific promoters driven, reaches the target of fibres modified quality.And, the disadvantageous effect that the tissue specific expression of external source target gene also can avoid foreign gene that non-purpose tissue growth is grown.Use the cotton fibre specific expression promoter; Can be at the specified phase expression alien gene of cotton fiber development; Acceleration is to the improvement of the important indicator of fibrous quality such as intensity, fineness, length etc., can also increase that new quality trait is crease-resistant, nonshrink to fiber, color and luster etc.Existing research proves that clone's cotton fibre specific expression promoter is the prerequisite of genetic engineering approach improvement cotton fibre.Therefore, the clone of high specific, highly active promotor identifies the important content of the genetic engineering that is the cotton fibre quality improvement.

Arabinogalactan-Protein (arabinogalactan protein; AGP) be one of plant four big main cell wall structure albumen; Be the albumen of high glycosylation; They participate in the expansion and the elongation of regulation and control, cell proliferation and the cell of body embryonic development, participate in the interaction of cell walls and cytoskeleton.Fasciclin appearance Arabinogalactan-Protein ( fAsciclin- lIke aRabinogalactan protein; FLA) be a subclass among the AGP; Except structure with typical AGP; Also have a fasciclin spline structure territory (fasciclin-like domain), the albumen that contains fasciclin spline structure territory generally all has the function of molecule adhesion, and this structural domain has vital role to the performance of whole protein function.Up to the present, in various plants isolation identification some FLA genes.Johnson etc. have separated 21 FLA protein genes from Arabidopis thaliana, separation such as Faik obtain 22 paddy rice and 34 wheat FLA genes.In addition, scientist also is separated to some FLA genes from willow (Populus tremula L.), torch pine (pinus taeda L.) and Herba Zinnia elegansae (Zinnia elegans L.).The FLA gene of these isolation identification is expressed in different plant tissues, relates to expression regulation element-promotor separately.

Summary of the invention:

The object of the present invention is to provide a cotton fiber specific controlling element-GhFLA1 promotor.Analyze and disclose this promoter expression activity, explore its molecular mechanism, and then use this controlling element improvement cotton fibre quality, create the cotton improved seeds the fiber developmental regulation.

The applicant's screening from the cotton fiber cDNA library that this laboratory makes up obtains 4 GhFLA genes.In order to obtain more cotton FLA, the technological method of information biology and transcription group is used to screen the public est database of cotton, finally obtains 19 cotton FLA genes, called after GhFLA1-GhFLA19.Through designing these FLA sequence 3 ' end RT primers, the applicant has been real-time RT-PCR to these FLA expression of gene and has analyzed, and the result finds to have 7 GhFLA genes a large amount or specifically expressing in cotton fiber; And its expression receives the fiber developmental regulation (to see Huang GQ; Xu WL, GongSY, Li B; Wang XL; Li XB, 2008, Characterization of the 19novel cotton FLA genes and theirexpression profiling in fiber development and in response to phytohormones and salt stress.Physiol Plant 134:348-359).One of them cotton fiber specific gene, the applicant's called after GhFLA1, it comprises the open reading frame of 732bp, one the 243 amino acid whose albumen of encoding; Its molecular weight is 25.62KD, and iso-electric point (pI) is 9.27, is rich in Pro (7%); Thr (11.9%), Ser (9.5%), Gln (7.8%); Ara (9.5%), Leu (12.4%), Val (7%).GhFLA1 albumen contains typical A GP structure, i.e. the signal peptide of N-end, and the hydrophobic region that Pro district and C-hold is rich in the centre, and in addition this albumen also has a fasciclin spline structure territory.AtFLA11/12 homology in this albumen and the Arabidopis thaliana is the highest, and existing research shows that AtFLA11 participates in the growth of Arabidopis thaliana secondary wall, is hinting that GhFLA1 possibly grow relevant with cotton fibre cell walls.Utilize the quantitative RT-PCR technology that the express spectra of this gene in the cotton different tissues analyzed, the result shows GhFLA1 gene specifically expressing in cotton fiber cell, in other tissue of cotton, express very weak or almost detect less than.Further research GhFLA1 finds at the expression of cotton fiber development different steps; This gene begins to express at cotton fiber development early stage (blooming back 2 days); In the back 10 days cotton fibre of blooming, express the highest; Expression amount descends to some extent in the back 15 days cotton fibre of blooming, and it is expressed and begins again to rise in the fiber secondary wall growth period (blooming back 20 days).

Be the further tissue specific expression regulation and control of research GhFLA1 gene, the applicant has separated the promotor of this gene.Promotor is separated employing genomic walking test kit (Genome Walker Universal Kit; BD BiosciencesClontech); At first set up cotton gene group walker kit, then according to GhFLA1 gene order designs C P1 and CP2 primer, this cotton gene group walker kit of pcr amplification; Separate the 5 '-upstream sequence that has obtained the GhFLA1 gene, this sequence length is 925bp.Find when utilizing PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to analyze the cis-acting elements in the promotor; Remove the CAAT box that exists eukaryote typical case promotor to be had; Outside the TATA frame; This promotor also contains some other cis-acting elements, as the light response element ( AAAACGTTTA, ATTAATTTTACA, ATTAAT, TTTCAAA), the Plant hormones regulators,gibberellins response element ( TCTGTTG), the heat stress response element AAAAAATTTC, the low temperature response element ( CCGAAA), and the MeJA response element ( CGTCA, TGACG), the hypoxia inducible element ( TGGTTT) wait controlling element.Because tobacco leaf epidermal hair and cotton fibre (kind fur) all have homology in origin, structure and heredity, and some investigators have successfully utilized this allos system to detect the activity of cotton fibre specific promoter both at home and abroad.Therefore, the present invention also uses tobacco and studies the GhFLA1 promoter activity.The applicant has made up the fusion expression vector of GhFLA1 promotor and gus reporter gene, utilizes agriculture bacillus mediated DNA transfer techniques that it is imported Arabidopis thaliana and tobacco, obtains transfer-gen plant.Histochemical stain result to transgenic tobacco plant shows that GhFLA1 promoters driven gus gene is expressed, and has confirmed that this promotor is that cotton fibre is special in tobacco petiole epidermal hair, can be used as the gene regulating element of genetically engineered improvement cotton fibre quality.

In addition, the irreversible inhibitor β-Yariv that uses the AGP proteinoid handles the ovule that exsomatizes, and the result causes cotton fibre elongation speed to slow down, and the speed of inhibition elongate fiber is directly proportional with the concentration of β-Yariv.Ovule with the sugar component antibody JIM4/8/13 of AGP and bloom back 1 day, 2 days and 3 days carries out immunoreation; The result show bloom back 1 day, 2 days and 3 days fiber can with these several kinds of antibody mediated immunities reactions, this shows and has a large amount of AGP albumen (comprising FLA albumen) in the fiber.FLA (as the subclass of AGP) specifically expressing in cotton fibre explains that gene pairs cotton fiber developments such as FLA1 play an important role.

Advantage of the present invention:

1, the GhFLA1 promoter sequence of a new cotton fibre specifically expressing is provided, has analyzed its cis-acting elements that contains.Its gene of this promoter regulation is at cotton fibre high level expression elongating stage, and can drive gus gene specifically expressing in the tobacco leaf epidermal hair.The present invention provides special controlling element for the fibrous quality improvement.

2, a new cotton fibre specifically expressing GhFLA1 gene order is provided, the protein sequence of its translation and Arabidopis thaliana AtFLA11 homology show that up to 54% this gene maybe be relevant with the growth of cotton fiber cell secondary wall.If suppress such genetic expression, then cotton fiber development is obstructed, and length shortens, and explains that gene such as GhFLA1 plays an important role in the cotton fiber development process.

The present invention does further elaboration through following accompanying drawing and embodiment, but does not limit the scope of the invention.

Description of drawings:

The fluorescence quantitative RT-RCR analysis that Fig. 1 GhFLA1 gene is expressed in each tissue of cotton

Among the figure: 1) root; 2) hypocotyl; 3) cotyledon; 4) blade; 5) petal; 6) flower pesticide; 7) fiber; 8) ovule.

The fluorescence quantitative RT-RCR analysis that Fig. 2 GhFLA1 expresses in the cotton fibre different developmental phases

Among the figure: 1) 2 days (day post anthesis, the back fate of blooming) fibers; 2) 5 days fibers; 3) 10 days fibers; 4) 15 days fibers; 5) 20 days fibers.

Fig. 3 GhFLA1 promotor is analyzed in tobacco epidermal hair and the GUS histochemical stain of planting expression activity in the skin

Among the figure: tobacco seed A) tobacco petiole B).The result shows that petiole epidermal hair and seed epidermis all dyed blueness; And other histocyte is not colored; Be illustrated under the regulation and control of GhFLA1 promotor gus gene at epidermal hair or plant specifically expressing in the chrotoplast, thereby confirm that this promotor is a fiber-specific in cotton.

The proteic immunolocalization analysis of Fig. 4 AGP

Among the figure: with JIM4/8/13 antibody respectively with bloom back 1 day (1DPA), the ovule of 2 days (2DPA) and 3 days (3DPA) carries out immunohistochemical analysis; On behalf of antibody, green fluorescence act on mutually with antigen A GP sugar component in the fiber among the figure.The result shows that AGP (comprising FLA) works in cotton fibre initial sum elongation process.(annotate: 1-3DPA is the cotton fiber development initial period, and 3-15DPA is the elongate fiber stage; JIM8 antibody and AGP sugar component be specific combination mutually, and JIM4 and JIM13 combine with part sugar side chain among the AGP.DPA: the back fate of blooming.)

Fig. 5 different concns β-Yariv agent treated cotton isolated culture ovule

Among the figure: 1) the back 1 day ovule of blooming was cultivated 10 days on the BT minimum medium; 2) back 1 day ovule additional 10 μ M β-Yariv (for the irreversible inhibitor of AGP) cultivation 10 days on the BT minimum medium of blooming; 3) additional 25 μ M β-Yariv; 4) additional 100 μ M β-Yariv additional 50 μ M β-Yariv 5); 6) additional 100 μ M α-Yariv (be the analogue of β-Yariv, but do not act on AGP), this result show the regulation and control of AGP albumen (comprising FLA albumen) participation cotton fiber cell growth course.

Embodiment:

The FLA1 promotor that cotton is new and separation, evaluation and the expression activity analysis of gene order thereof.

1, the isolation identification of cotton FLA1 gene order

From cotton fibre cDNA library, separated 10; More than 000 cotton cDNA clone; Through bioinformatic analysis, 4 GhFLA cDNA sequences have been identified, one of them called after GhFLA1; With full length sequence design primer GhFLA1Fup and the GhFLA1Fdn of GhFLA1cDNA, obtain full length gene again through pcr amplification.

2, real-time fluorescence quantitative RT-PCR is analyzed the GhFLA1 expression of gene

(1) extraction of total tissue RNA (is pressed Li XB; Cai L; Cheng NH; Liu JW, Molecular characterizationof the cotton GhTUB1 gene that preferentially expressed in fiber.Plant Physiol.2002, the method for 130:666-674 is carried out).

(2) real-time fluorescence quantitative RT-PCR research expression of gene is (according to Li XB; Fan XP; Wang XL; Cai L, YangWC, 2005.The Cotton ACTIN1 gene is functionally expressed in fibers and participates in fiberelongation.Plant Cell 17:859-875 carries out).At first, the total RNA (2 μ g/ appearance) with cotton different tissues (root, hypocotyl, cotyledon, leaf, petal, flower pesticide, bloom back 2 days fibers, 5 days fibers, 10 days fibers and 10 days ovules, 15 days fibers, 18 days fibers, 20 days fibers etc.) uses SuperScript ^TMII RNase H ^-Reverse Transcriptase (Invitrogen Life Technologies) reverse transcription becomes cDNA; Then, be template with cDNA, (TOYOBO Japan) carries out the quantitative PCR reaction with the primer (GhFLA1RTP1 and GhFLA1RTP2) of gene specific and Real-time PCR Master Mix.Cotton polyubiquitin gene (GhUBI1) is as the interior mark of RT-PCR reaction, and each round-robin amplification of target gene is all by the SYBR-Green fluoroscopic examination.The horizontal relative value of each expression of gene is Y=10 by formula ^{Δ Ct/3.57}* 100% calculate (Δ Ct=CtGhUBI1-CtGhFLA1 wherein, the 3.57th, utilize the inverse of slope among the typical curve y=-0.28x+9.87 of GhUBI1 preparation, expression genetic expression differs 10 times PCR cycle number).Repeat the statistical study experimental result 3 times.

3. the separation of promotor

(1) with genomic walking test kit (Genome Walker Universal Kit; BD Biosciences Clontech; Cat.No.638904) make up cotton gene group walker kit; By the method that is provided, match respectively at AP1 in the test kit and AP2 primer again, from cotton gene group walker kit, separated GhFLA1 gene 5 '-upstream 935bp fragment (comprising the promotor and 5 ' the untranslated district) through two-wheeled PCR with GSP1 and GSP2 primer.

This project the primer sees the following form 1:

Table 1

4. the expression analysis of promotor

The cis-acting elements of promotor according to plantCARE ( Http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/) carry out, the result sees table 2.Make up the GhFLA1 promotor: gus gene fusion expression vector (primer sequence is respectively pFLA1P1 and pFLA1P2), electric shocking method transforms Agrobacterium LBA4404, transformation of tobacco then.The active histochemical stain analysis of GUS is according to Li XB; Cai L; ChengNH; Liu JW (Molecular characterization of the cotton GhTUB1 gene that preferentiallyexpressed in fiber.Plant Physiol.2002, carry out by the method for 130:666-674) introducing.

The cis-acting elements of table 2PlantCARE prediction pGhFLA1

The functional element motif of supposing of taking advantage of a situation	The position	The motif sequence	Suppose function
				The AAAC-motif	+459，+845	CAACAAAAACCT	The light response element
The AAGAA-motif	-692	GAAAGAA	Unknown
				ACE-motif r	-331	AAAACGTTTA	The light response element
The ARE-motif	-11，+276	TGGTTT	Hypoxia inducible
				The ATCT-motif	+642	AATCTAATCT	The light response element
Box?I	+279，+700	TTTCAAA	The light response element
				The CGTCA-motif	+901	CGTCA	The methyl jasmonic is replied
The GA-motif	+184	ATAGATAA	The light response element
				The GAG-motif	-541	AGAGAGT	The light response element
The GARE-motif	-649，-716	AAACAGA， TCTGTTG	The Plant hormones regulators,gibberellins response element
				The GATA-motif	-354，-413	AAGATAAGATT	The light response element
HSE	-19，+61，+283	AAAAAATTTC	Heat stress is replied
				The I-motif	-413	GATAAGATT	The light response element
LTR	+43	CCGAAA	The low temperature response element
				Sp1	+600	CC(G/A)CCC	The light response element
TGACG	-901	TGACG	The methyl jasmonic is replied
				circadian	+412，-491	CAANNNNATC	The clock rhythm and pace of moving things is relevant

5.GhFLA1 gene function analysis

The ovule that exsomatizes is cultivated according to Bealsey CA and Ting IP (Effects of Plant Growth Substances on inVitro Fiber Development from Unfertilized Cotton Ovules.Amer.J.Bot.1974; The method of 61:188-194) introducing is carried out; Handle the ovule that exsomatizes with different concns β-Yariv again, observe and measure cotton length after 10 days.The result shows, β-Yariv handles back cotton fibre elongation and receives remarkable inhibition, and the inhibition degree is directly proportional with β-Yariv, shows that AGP albumen (comprising FLA albumen) plays an important role to cotton fiber development.Simultaneously; With the proteic different antibodies of AGP with bloomed back 1 day, the ovule of 2 days and 3 days carries out immunohistochemical analysis and (presses Tang XC, He YQ; Wang Y; Sun MX, The role ofarabinogalactan proteins binding to Yariv reagents in the initiation, cell developmental fate; Andmaintenance of microspore embryogenesis in Brassica napus L.cv.Topas.J Exp Bot.2006; The method of 57:2639-2650 is carried out), the result finds JIM8 antibody capable that combines with AGP protein sugar component and the fiber immunoreation of blooming back 1/2/3 day, and is particularly very strong with the fiber immunoreation of blooming back 3 days; JIM4 that combines with AGP protein part sugar side chain and JIM13 antibody then with the immunoreation of the back 1/2/3 day fiber of blooming a little less than.Explain that AGP (comprising FLA) albumen plays an important role in the cotton fiber development process, particularly in cotton fibre elongating stage.

The GhFLA1 upstream region of gene promoter sequence (comprising that 5 ' holds untranslated district) of cotton fiber specific regulation and control is as follows:

1 ATCTTATTTA?AAAACCATCG?AAATTTTTAT?TACTCAATCA?TTACCGAAAT?AGAATCGGGC

61 TAAAATATTT?CGAAAACTAA?AAGTTTCACT?TTTTATATTG?AAAAACGAGG?CTTTGTGATT

121?CTTATAAATT?TAATTCATTG?AAATTTCATC?AAGTAAAATG?GAAGAATTAT?AAATCTCTAA

181?AATGATAGAT?AAAAATGTCG?CAAATAAAGC?CATTGTGACA?CCCATTAAAG?GAGTCTTTCC

241?CCATCCAGGG?GCTACCTTAC?CATATTCCGA?ATTCAATGGT?TTCAAAAAAT?CTCCTACAAC

301?AATTCGTCTT?GGACTAGATC?TAGAACTACT?ATTAACGTTT?TGTGTAGCCA?TAAATCTTAT

361?TTTGTATTCA?TTGAGATTTG?TTAACTTTGT?ATATCATTTC?ACTATAAATA?AACAATCTTA

421?TCATAGACCC?ATTGTGATCA?TGAAATTATA?TAATAATGGA?AACAAAAACC?TTAGTTGCAA

481?TCTCTATATC?TGATTTACTT?GTAAGTTTTA?CTAGGTACAC?CTTATATACT?GCTTTTGGGT

541?AACTCTCTCA?ACAATTAAGA?GATCCATTCG?AGGAATACGG?GAAATAATTG?AAGAAACGAG

601?CCTCCCCATA?TGTATGTCTA?TTTGGTCACA?TATCCACTCC?AAAATCTACT?CTGTTTTCCC

661?TGCTTTTCCA?AGCAATGTAG?TTGCTCATCA?ATTTCTTTCC?TTT

AA?AAATCCCAAC

721?AGAGTTTGCT?AAAGTTGATA?AGTATGTCTT?CTAGTTAACT?AAAGTAGCAT?ATTTTACCTA

781?ACTTCACCCT?CCAATATCC

AAG?CTCCCATTTC?TTATCCAATC?A

AACATCCA

841?TAACATTTTT?GTTCAAGGAC?CACTTCTTCC?CTTCCATTTT?ACTTTGTTTT?AGTTGCCATA

901?ACGTCACCTT?CCAATACAAC?CCACA

FLA1 promoter sequence length is 925bp.Utilize NNPP prediction transcription initiation site, with the possible controlling element of PlaceCARE prediction.The result is following: square frame

is the transcription initiation site of supposition; Grey shading letter is the CAAAT frame of supposition, and the letter of black shading is the TATA frame of supposition.

GhFLA1 genes encoding region sequence (comprises that ORFs and 3 ' holds untranslated district; Open reading frame and3 '-untranslated region, be called for short ORF and 3 '-UTR) as follows:

1 ATGAGGCAAC?AATATGTCTT?CACTACTCTC?ACGTTGCTCA?TCCTGTTTTC?CCTCAGTTGT

61 TCAACAACAT?TAGCCCAATC?TCCGGCACTG?GCCCCGGCAC?CTTCTGGCCC?GACAAACGTC

121?ACCAAGATCC?TCGAAAAAGC?CGGTCAATTC?ACCCTCTTCA?TTCGTCTTCT?AAAGTCCACT

181?CAAGTGGCCA?ACCAGCTGCT?CGGTCAGCTC?AACAATTCCA?ACAATGGTAT?GACCGTTTTT

241?GCACCAACGG?ACAATGCTTT?CTCCAGCCTT?AAATCGGGCA?CATTGAATTC?ACTCACCGAT

301?GAACAAAAAG?TGCAGCTGGT?GCAATTCCAC?ATCGTCCCAA?CATACCTCAC?CTCGTCTCAG

361?TTCCAAACCA?TTAGCAACCC?TTTGAGAACC?CAAGCTGGTG?ATAGTGGCGA?TGGCAAGTTC

421?CCTCTCAATG?TCACCACTTC?GGGGAACTCT?GTGAATATAA?CAACAGGGTT?GACAAACACC

481?AGTGTTTCCG?GCACTATTTA?CACTGATGGT?CAGCTTGCTG?TTTATCAAAT?CGATCAAGTT

541?CTTCAACCAT?TGCAAATATT?TGCACCTAGG?CCTCCAGCTC?CCGCACCGGC?ACCGGCAAAG

601?TCGAAGAATA?AGAAGGCTAC?CACTGTTGCT?GATAGCCCCG?ATGTTACCCC?AGCTGATAAC

661?TCCAAAGCGG?CCACCTTGCA?AAATGTTGGT?TTGTTTGGAG?TTGCTGCTCT?AGTTATTGCA

721?CTTTCTTTGT?GACCATGAAA?ATGGAGAAAA?GAAGAAGACA?GTGATTTTGA?TGGTGGTGAT

781?CAAGATGGCG?AGTGTTTTTT?AATTTTTCAA?TAATTATCAT?TTAAAAAATT?TATGTTCTGT

841?ATGAAGATTG?AATTTTGAGT?TCGTGTTGTT?GATTTCATTT?ATTTTTGTTT?TGAAATTTTC

901?TTTGTTATCT?CTTACTTCTC?AATTGTAATT?GTGAATTTTG?TGTCACTTTC?TTCATCCTTT

961?CTCTTTTTAA?GATTTTGCAT?CATTGCTTGT?TTTTG

The coding region of gene (ORF) to terminator codon TGA (1-732bp), does not contain intron (promptly this gene order is identical with its cDNA sequence) from initiator codon ATG.

The protein sequence of GhFLA1 gene coding region translation is following:

1 MRQQYVFTTL?TLLILFSLSC?STTLAQSPAL?APAPSGPTNV?TKILEKAGQF?TLFIRLLKST

61 QVANQLLGQL?NNSNNGMTVF?APTDNAFSSL?KSGTLNSLTD?EQKVQLVQFH?IVPTYLTSSQ

121?FQTISNPLRT?QAGDSGDGKF?PLNVTTSGNS?VNITTGLTNT?SVSGTIYTDG?QLAVYQIDQV

181?LQPLQIFAPR?PPAPAPAPAK?SKNKKATTVA?DSPDVTPADN?SKAATLQNVG?LFGVAALVIA

241?LSL

Claims (1)

1. cotton fiber specific controlling element GhFLA1 upstream region of gene promotor, its sequence is following:

1 ATCTTATTTA?AAAACCATCG?AAATTTTTAT?TACTCAATCA?TTACCGAAAT?AGAATCGGGC

61 TAAAATATTT?CGAAAACTAA?AAGTTTCACT?TTTTATATTG?AAAAACGAGG?CTTTGTGATT

121?CTTATAAATT?TAATTCATTG?AAATTTCATC?AAGTAAAATG?GAAGAATTAT?AAATCTCTAA

181?AATGATAGAT?AAAAATGTCG?CAAATAAAGC?CATTGTGACA?CCCATTAAAG?GAGTCTTTCC

241?CCATCCAGGG?GCTACCTTAC?CATATTCCGA?ATTCAATGGT?TTCAAAAAAT?CTCCTACAAC

301?AATTCGTCTT?GGACTAGATC?TAGAACTACT?ATTAACGTTT?TGTGTAGCCA?TAAATCTTAT

361?TTTGTATTCA?TTGAGATTTG?TTAACTTTGT?ATATCATTTC?ACTATAAATA?AACAATCTTA

421?TCATAGACCC?ATTGTGATCA?TGAAATTATA?TAATAATGGA?AACAAAAACC?TTAGTTGCAA

481?TCTCTATATC?TGATTTACTT?GTAAGTTTTA?CTAGGTACAC?CTTATATACT?GCTTTTGGGT

541?AACTCTCTCA?ACAATTAAGA?GATCCATTCG?AGGAATACGG?GAAATAATTG?AAGAAACGAG

601?CCTCCCCATA?TGTATGTCTA?TTTGGTCACA?TATCCACTCC?AAAATCTACT?CTGTTTTCCC

661?TGCTTTTCCA?AGCAATGTAG?TTGCTCATCA?ATTTCTTTCC?TTTCAAATAA?AAATCCCAAC

721?AGAGTTTGCT?AAAGTTGATA?AGTATGTCTT?CTAGTTAACT?AAAGTAGCAT?ATTTTACCTA

781?ACTTCACCCT?CCAATATCCT?AAATAAAAAG?CTCCCATTTC?TTATCCAATC?AAaAACATCCA

841?TAACATTTTT?GTTCAAGGAC?CACTTCTTCC?CTTCCATTTT?ACTTTGTTTT?AGTTGCCATA

901?ACGTCACCTT?CCAATACAAC?CCACA。

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