CN103074350A - Identification and application of specifically expressed gene GhMADS14 of cotton fiber - Google Patents

Abstract

The invention discloses identification and application of a specifically expressed gene GhMADS14 of a cotton fiber. The total length of the cDNA of the gene GhMADS14 is 786bp, and an MADS-box type transcription factor is encoded. The specificity of the mRNA of the gene in an extension and development period of a cotton fiber cell is accumulated greatly, and fact indicates that the gene is the specifically expressed gene of the fiber. The GhMADS14 protein is located in a nucleus and exerts influence for the transcriptional activation of the factor in the cotton fiber development. Fibers obtained after 10 days since the blossom of cotton are treated by GA, and the GhMADS14 is in a down-regulated expression. The GhMADS14 is over expressed in arabidopsis thaliana, so that seedlings grow slowly; compared with a wild type, a transgenetic seedling has smaller leaves, lengths of a hypocotyledonary axis and the root becomes short obviously, the gene expression quantity of the GA20ox1 is reduced, but the gene expression quantity of the GA2ox8 is increased. The fact indicates that the GhMADS14 is regulated and controlled by the GA, and constructive metabolism related genes of the GA is regulated and controlled, so that the expansion and growth of the cotton fibers can be regulated.

Description

GhMADS14 gene identification and the application of cotton fibre specifically expressing

Technical field

The present invention relates to a cotton gene.The MADS transcription factor of a cotton fibre specifically expressing (MADS-box transcription factor) gene specifically GhMADS14Clone and Function Identification.

Background technology

Cotton is most important natural fiber crop in the world, and China is the important cotton state of product and country of consumption, and cotton occupies very consequence in Chinese national economy.Cotton fibre is the main products of Cotton Production, and its yield and quality has directly determined the output value and the benefit of Cotton Production.Therefore, improve production of cotton fibers and quality is the major objective of cotton breeding always.Yet, owing to have negative correlation between the yield and quality of cotton fibre, seriously hampering the synchronous improvement of cotton fiber yield and quality, the negative correlation that traditional breeding way is difficult to break yield and quality reaches both and improves synchronously.

Cotton fiber cell is the not branch unicellular hair shape projection that is formed by ovule exterior skin differentiation, and it is grown by four overlapping but differentiable etap and forms, and is respectively origin of fibers, elongation, secondary wall is synthetic and dehydration is ripe.The developmental state of cotton fiber in this four-stage determined its yield and quality, the fibrocellular initial quantity that determines final fiber, and fibrocellular elongation has determined the length of mature fibers, the synthetic intensity that has determined fiber of secondary wall.Therefore, clone identification cotton fiber development genes involved is also studied mutual regulation relationship, resolves the molecular mechanism of cotton fiber development, not only has important theory significance, and improvement has important using value to the cotton fiber yield and quality.

The growth course of cotton fiber is subject to the regulation and control of hormonal readiness.The hormones such as brassinolide, growth hormone, Plant hormones regulators,gibberellins (GA) all play vital effect in fibrocellular initial sum growth course, execute certain density above-mentioned hormone outward and can promote fibrocellular growth.Although in cotton, found some genes relevant with hormone signal at present, but the function of these genes in the cotton hormone signaling pathways and the mechanism of action of cotton fiber development impact be it be unclear that, some crucial modulins also remain clone identification and illustrate its function.Therefore, the pipeline of hormone signal in the research cotton, particularly on the impact of cotton fiber development, the important regulating and controlling factor gene that isolation identification is relevant has important theory significance and using value.

The title of MADS is from the initial of yeast saccharomyces cerevisiae transcription factor MCM1, thaliana flower homeotic gene AGAMOUS, Common Snapdragon floral homeotic genes DEFICIENS and 4 kinds of albumen of human serum response factors SRF, and these 4 kinds of protein are all by the MADS box (MADS-box) of a high conservative that is comprised of 56～58 amino acid.The gene that coding contains this conserved sequence protein factor is called the MADS gene.Cis-acting elements specific combination in the MADS genes encoding one class MADS transcription factor, itself and eukaryotic gene promoter region, thus make target gene with specific intensity at specific spatial-temporal expression.The MADS gene all has multi-form spatial and temporal expression pattern in floral meristem, floral organ and the various vegetative organ of plant, play different separately functions.

The MADS-box transcription factor of MADS genes encoding mainly is comprised of MADS box, K box, I district, C end and 5 parts of N-terminal.Wherein, the structural domain of the high conservative that the MADS box is comprised of about 56～58 amino acid, in all MADS transcription factors of finding so far, this zone has 9 amino acid just the same.Studies show that, the MADS box be a sequence-specific DNA in conjunction with primitive, it is interacted with the DNA ditch by the formed specific dimeric structure of antiparallel alpha-helix primitive.The MADS box mainly is combined with DNA, sometimes also forms dimer or is combined with cofactor.The K box is comprised of about 70 amino acid, and moderate is conservative, and is with Keratin sulfate (keratin) portion homologous, also undiscovered in animal and fungi so far.Secondary structure analysis shows that the K structural domain contains 3 alpha-helixs (K1, K2 and K3), with hydrophobic residue conservative regular spacing is arranged, and can form two affinity spirane structures in the albumen dimer, regulates the interaction between the protein.The K structural domain is the characteristic sequence of plant MADS transcription factor, also is Dimerized structural motif occurs.The I district is positioned between MADS structural domain and the K structural domain, and by the non-conservative zone of containing more hydrophilic residue that about 35 amino acid form, its effect is to help dimeric transcription factor to be combined with DNA to form complex body.The I district is that selectivity forms DNA in conjunction with dimeric molecule determinative.C-terminal is positioned at K box downstream, by the non-conservative zone of being rich in hydrophobic residue that about 30 amino acid form, may be regional as the MADS gene transcription, and relevant with MADS polymer higher structure.Utilize yeast two-hybrid verified, rice Os MADS16 albumen contains a transcriptional activation domain in the C-terminal zone.Some vegetable-proteins contain additional N-terminal residue in the MADS upstream, be the hydrophilic-structure territory of being rich in basic aminoacids, form NMIKC type albumen.MADS box, K box, I district, C-terminal and N-terminal play different separately functions in the Dimerized and DNA of MADS albumen and dimeric combination.

The MADS gene is the very important transcriptional regulator gene of a class, and in plant growth and development process, the MADS gene may affect growing of plant at different steps, different sites, different tissues organ.Most study be the MADS gene relevant with flower development, comprise the control flowering time, the determining of floral meristem, the foundation of floral organ identity, fruit development, inner seed coat grows and the control of nutritional development etc.For example, Tzeng etc. from long cylinder lily cloning and identification a MADS gene LMADS1, it is taken turns in the floral organ at 4 of flower and all expresses.Drive LMADS1 gene and arabidopsis thaliana transformation with 35S promoter, short petal and the stamen of colored tool of transfer-gen plant do not observed the phenomenon that floral organ reverses.Intercepting LMADS1Gene M ADS-box regional sequence arabidopsis thaliana transformation, transfer-gen plant shows similar AP3The like that obvious recessive ectopic expression sudden change of gene, i.e. petal tool sepal spline structure, stamen has been transformed into the carpel spline structure, shows LMADS1Corresponding in the lily AP3The corresponding function gene of gene.The MADS gene function is various, the adjusting of except the characteristic that determines meristematic tissue and floral organ, also participate in nourishing and growing, ovary development, the growth of kind skin, the formation of root, embryo morphogenesis, symbiosis being induced etc.Take the AG gene as probe, from Arabidopis thaliana, be separated to more than 20 AGL gene, they are expressed at the different sites such as different steps, root tissue and embryo of flower development respectively, thereby growth and development of plants is produced different impacts, finish different biological functions.In addition, research finds that SaMADSD gene pairs inflorescence development and flower organ morphology have built up double influence.

MADS genetic expression is subjected to the regulation and control of hormon.Rony etc. (2000) study discovery, Arabidopis thaliana MADS gene AGL20Regulated and control by GA.Patriick etc. (2007) report, plant hormone ethylene increase the DELLA protein accumulation, and DELLA albumen further suppresses to bloom the expression of positive regulatory factor LFY and SOC1, thereby makes the delay of blooming.Proved that simultaneously Ethylene Signal path and GA3-DELLA-MADS signal path that the CTR1/EIN3 gene relies on are interconnected to form regulated and control network, the coordinate plant growth cycle is so that the changeable external environment of plant antagonism.There is research to find, behind GA3 processing Arabidopis thaliana, can removes DELLA to the MADS gene AP3, PI, AGRestraining effect, play the effect of Accelerate bloom.AGL15 is at the embryonic development period high expression level, Agl15In the mutant, AtGA2ox6Expression amount descends, AGL15Overexpression plant lotus throne leaf GA content decrease, plant have the phenotype of GA disappearance, and plant is short and small, the delay of blooming.AGL15 can by in conjunction with special dna sequence dna, regulate and control the target gene in downstream, thereby affect growing of plant.In sum, the MADS transcription factor of research and inquirement cotton fibre specifically expressing will make us understand better the function of MADS transcription factor in cotton fiber development.

Summary of the invention

The object of the present invention is to provide a new cotton fibre specifically expressing the MADS gene ( GhMADS14), analyze and disclose this gene function, explore it to the molecular mechanism of Fibre Development regulation and control, and then use this improvement of genes cotton fibre, create the cotton improved seeds.

In nearest research, the contriver is more than 10000 cotton cDNA of random choose cloning and sequencing from the cotton fiber cDNA library.The contriver finds that STK (agamous-like MADS-box protein AGL11) homology is very high in the MADS albumen of one of them genes encoding and the Arabidopis thaliana, with its called after GhMADS14 GhMADS14CDNA comprises the open reading frame of 672 bp, the 223 amino acid whose MADS albumen of encoding, and molecular weight is 25.7KD.GhMADS14 albumen contains MADS-box (M), I district, K box and C-end.Utilize Northern blot hybridization technique that this gene is analyzed at the express spectra of cotton different tissues and cotton fibre different developmental phases, the result shows GhMADS14Gene specifically expressing in 10 days cotton fibre after blooming.External source applies GA, the GhMADS14 down-regulated expression in the cotton ovule of isolated culture and fiber.On the contrary, add GA synthetic inhibitor PAC, the GhMADS14 up-regulated.Studies show that further the GhMADS14 Subcellar location shows that GhMADS14 may play a role as transcription factor in nucleus.

In order to study GhMADS14The function of gene, the contriver will GhMADS14Overexpression in Arabidopis thaliana.17 strains have been obtained GhMADS14The arabidopsis thaliana of gene overexpression, the phenotype of these strains is consistent, and the overexpression growth of seedling that its most obvious feature is normal growth is slow, and blade is less than wild-type, and hypocotyl and root are shorter than wild-type.Add external source GA in the MS substratum after, significantly elongation appears in wild-type and overexpression seedling hypocotyl and root.Overexpression seedling phenotype coexists and compares on the normal substratum, and phenotype is recovered to some extent, but on the substratum that adds GA, hypocotyl and the root of the transgenosis seedling of overexpression still are shorter than wild-type.Add GA synthetic inhibitor PAC, wild-type and overexpression seedling grow and all are suppressed, and compare with the seedling of growing on the MS substratum, and hypocotyl and root all shorten, but after PAC processed, overexpression seedling hypocotyl and root length and wild-type did not have significant difference.Extracted the total RNA of blade, done semi-quantitative RT-PCR analysis, the result shows that some relate to the obviously change of expression generation of two key enzymes (GA 20-oxydase and the GA 2-oxydase) gene in the Plant hormones regulators,gibberellins metabolic pathway of synthesizing in transfer-gen plant.Promote the GA 20-oxidase gene that active GA is synthetic GA20ox-1Express significantly downward modulation, and the GA of activity is converted into the GA 2-oxidase gene of nonactive GA GA2ox8Express significantly and raise.This has shown that GhMADS14 may be by the key enzyme in the regulation and control Plant hormones regulators,gibberellins metabolic pathway of synthesizing, thereby affects the conversion of active GA and nonactive GA, so that the content of endogenous GA changes, thereby affects growth and development of plants.Therefore, in cotton fibre Fiber elongation process, this gene may by the key enzyme of the intracellular Plant hormones regulators,gibberellins metabolic pathway of synthesizing of regulating cotton, so that endogenesis GA changes, affect the Fiber elongation of cotton fiber cell.

Advantage of the present invention

1, provides new cotton fibre specifically expressing GhMADS14Gene (cDNA) sequence.This gene specifically expressing in 10 days the cotton fiber cell after blooming shows that this gene may play an important role in control cotton fibre Fiber elongation process.

2, in cotton fiber, GhMADS14Expression is subjected to Plant hormones regulators,gibberellins (GA) regulation and control.Applying external source GA can suppress GhMADS14Express.On the contrary, add GA synthetic inhibitor PAC, suppress the GA biosynthesizing of cotton fiber cell, then GhMADS14Express significantly and rise.

3, the GhMADS14 protein localization shows that this albumen plays a role as transcription factor in nucleus.

4, overexpression in Arabidopis thaliana GhMADS14The gene inhibition transfer-gen plant grows.Under the normal growth condition, the transgenosis seedling of overexpression GhMADS14 is shorter and smaller than wild-type, and seedling hypocotyl and root length shorten.Statistical study shows that transfer-gen plant hypocotyl length significantly is shorter than wild-type.RT-PCR the analysis showed that, two the key enzyme GA20-oxydase and the oxidasic expression activity of GA2-that relate to Plant hormones regulators,gibberellins (GA) metabolic pathway of synthesizing in the transfer-gen plant occur obviously to change.Promote what active GA synthesized GA20ox-1Down-regulated expression, and the GA of activity is converted into nonactive GA's GA2ox8Up-regulated.This shows GhMADS14Gene is brought into play its function in the cotton fiber development process by regulation and control GA signal path.

The present invention is further elaborated by the following drawings and embodiment, but does not limit the scope of the invention.

Description of drawings

Fig. 1-1 is Northern blot analyzes GhMADS14Expression in cotton tissue;

Among the figure: (1) root; (2) hypocotyl; (3) cotyledon; (4) blade; (5) petal; (6) flower pesticide; (7) the rear 5 days fiber of blooming; (8) the rear 10 days fiber of blooming; (9) the rear 15 days fiber of blooming; (10) the rear 10 days ovules of blooming; (11) the rear 15 days ovules of blooming.

Fig. 1-2 is Fluorescence quantitative RT-RCR is analyzed GhMADS14Expression in the cotton fiber development stage;

Among the figure:The ovule that 0d-is bloomed the same day; The 3d-rear 3 days fiber of blooming; The 6d-rear 6 days fiber of blooming; The 9d-rear 9 days fiber of blooming; The 12d-rear 12 days fiber of blooming; The 15d-rear 15 days fiber of blooming; The 18d-rear 18 days fiber of blooming; The 21d-rear 21 days fiber of blooming.

Fig. 2 is the quantitative RT-PCR analysis GhMADS14Regulated and control by GA;

Among the figure: CK is that fiber isolated culture after blooming 10 days is in the BT liquid nutrient medium; 0.5 μ M GA is that fiber isolated culture after blooming 10 days is in the BT liquid nutrient medium that has added 0.5 μ M GA; 5 μ M GA are that fiber isolated culture after blooming 10 days is in the BT liquid nutrient medium that has added 5 μ M GA; 5 μ M PAC are that fiber isolated culture after blooming 10 days is in the BT liquid nutrient medium that has added 5 μ M PAC; 10 μ M PAC are that fiber isolated culture after blooming 10 days is in the BT liquid nutrient medium that has added 10 μ M PAC.

Fig. 3 is the Subcellar location analysis of GhMADS14 albumen;

Among the figure: the GFP fluorescence micrograph under the A-dark-field; 5 minutes fluorescence micrograph is processed in DAPI dyeing under the B-dark-field; Cell Photomicrograph under the C-bright field; Overlaping of D-GFP and DAPI fluorescence photo and bright field photo; The GFP green fluorescence shows that the GhMADS14 protein localization is in nucleus.

Fig. 4-1 is overexpression under the light culture condition GhMADS14The expression analysis of transgenic arabidopsis plant;

Among the figure: 1, wild-type; 2-17, the transfer-gen plant strain.

Fig. 4-2 is overexpression under the light culture condition GhMADS14The transgenic plant phenotype analytical;

Among the figure: B is under the normal illumination condition, and wild-type (WT) and transgenic plant (L1, L3, L9, L11) are grown on the MS substratum; C is under the normal illumination condition, and wild-type (WT) and transgenic plant (L1, L3, L9, L11) are grown in MS and add 5 μ M GA ₃On the substratum; D is under the normal illumination condition, and wild-type (WT) and transgenic plant (L1, L3, L9, L11) are grown in MS and add on the 0.2 μ M PAC substratum; E is under the normal illumination condition, and wild-type (WT) and transgenic plant (L1, L3, L9, L11) are grown in MS and add 0.2 μ M PAC+5 μ M GA ₃On the substratum.

Fig. 4-3 is under the normal illumination condition, and wild-type (WT) and transgenic plant (L1, L3, L9, L11) are grown in MS, 5 μ M GA ₃, 0.2 μ M PAC, 0.2 μ M PAC+5 μ M GA ₃Seedling hypocotyl length statistical study on the substratum.

Fig. 5 GhMADS14The expression analysis of Plant hormones regulators,gibberellins synthesis related gene in the overexpression Arabidopis thaliana;

Among the figure: WT, wild-type; (L1, L3, L9, L11), the transfer-gen plant strain.

Embodiment

The new gene of cotton MADS family GhMADS14Clone identification and functional analysis

1. GhMADS14(cDNA) isolation identification of sequence

(1) structure of cotton fibre cDNA library (is pressed Li XB etc., method among 2002, the Molecular characterization of the cotton GhTUB1 gene that is preferentially expressed in fiber. Plant Physiol 130:666-674 is carried out).

(2) from the cotton fibre cDNA library, separate more than 10,000 cotton cDNA clone, carried out dna sequencing.By bioinformatic analysis, obtain a plurality of GhMADSThe full length cDNA sequence of gene, one of them called after GhMADS14

Quantitative RT-PCR is analyzed GhMADS14The expression of gene

(1) total RNA of cotton tissue extracts and (presses Li XB, Cai L, Cheng NH, Liu JW, 2002. Molecular characterization of the cotton GhTUB1Gene that is preferentially expressed in fiber. Plant Physiol. 130:666-674 carries out).

(2) GA processes cotton fiber and extracts total RNA.Cotton ovule after will blooming 10 days and fiber isolated culture are added respectively 0.5 and 5 μ M GA in the BT liquid nutrient medium ₃, 5 and 10 μ M PAC (GA synthetic inhibitor), then dark the cultivation 6 hours extract respectively total RNA of cotton fibre as stated above under 30 degree conditions Celsius.

(3) expression of real-time fluorescence quantitative RT-PCR research gene (according to Li XB, Fan XP, Wang XL, Cai L, Yang WC, 2005. The Cotton ACTIN1Gene is functionally expressed in fibers and participates in fiber elongation. Plant Cell 17:859 – 875 carries out).At first, with total RNA (2 μ g/ sample) of cotton different tissues (root, hypocotyl, cotyledon, leaf, petal, flower pesticide, the rear 10 days ovules of blooming, bloom rear 3 days fibers, 6 days fibers, 9 days fibers, 12 days fibers, 15 days fibers, 18 days fibers, 20 days fibers etc.) with M-MLV RNase H ^-Reverse Transcriptase (Promega) reverse transcription becomes cDNA.Then, take cDNA as template, use the primer (GhMADS14 RTP1 and GhMADS14 RTP2) of gene specific and Real-time PCR Master Mix (TOYOBO, Japan) to carry out the quantitative PCR reaction.Cotton GhUBI1Gene is as the interior mark of RT-PCR reaction, and the amplification of each circulation of target gene is by the SYBR-Green fluoroscopic examination.The expression level relative value of each gene is Y=10 by formula ^△Ct/3.57 * 100% calculate (△ Ct=CtGhUBI1 – CtGhMADS14 wherein, the 3.57th, utilize GhUBI1The inverse of slope in the typical curve y of preparation=– 0.28x+9.87, expression genetic expression differs 10 times PCR cycle number).Repeat the statistical study experimental result 3 times.

The used RT-PCR primer of this experiment is as follows:

GhMADS14Gene primer p1:5 '-AGAGTTTGTCTCCGAACCAAG-3 ', p2 5 '-CAAGCACACGACAGAAGTAGT-3 ';

GhUBI1Gene primer p1:5 '-CTGAATCTTCGCTTTCACGTTATC-3 ', p2 5 '-GGGATGCAAATCTTCGTGAAAAC-3 '.

The Subcellular Localization of albumen

Make up the pBI121-GhMADS14:eGFP plant expression vector, this carrier is changed in the LBA4404 Agrobacterium by electrotransformation, by Agrobacterium-mediated Transformation Cotton Hypocotyl explant, screening obtains positive callus cell again, places and observes GFP fluorescence under the Laser Scanning Confocal Microscope.

The expression analysis of transgenic arabidopsis plant phenotype and GA synthesis related gene thereof

Make up the GhMADS14 Overexpression vector, utilize agriculture bacillus mediated flower-dipping method arabidopsis thaliana transformation, obtain transfer-gen plant.Treat that the Arabidopis thaliana vine growth and development is extremely ripe, collect seed.Wild-type and transgenic arabidopsis seed germination and growth seedling on the MS substratum is observed transgenic arabidopsis seedling phenotype under illumination cultivation, measure hypocotyl length and carry out statistical study.

Utilize Trizol test kit (Invitrogen) to extract the total RNA of Arabidopsis leaf, with M-MLV RNase H ^-Reverse Transcriptase (Promega) reverse transcription becomes cDNA; Then, take cDNA as template, with Arabidopis thaliana AtACT2Gene is as the confidential reference items of RT-PCR reaction, and it is quantitative to carry out RT-PCR with the AtACT2 primer first, and then the primer with gene specific carries out semi-quantitative RT-PCR analysis.Repeat the comparative analysis experimental result 3 times.

Cotton GhMADS14The cDNA sequence of gene is as follows:

ATGGGAAGAG GAAAAATAGA GATAAAGAGG ATCGAAAACA CAACAAATCG TCAGGTTACC TTTTGCAAAC 60 GCAGGAATGG CCTGCTGAAG AAAGCTTACG AACTGTCAGT CCTCTGTGAT GCTGAAGTTG CTCTCATTGT 120 CTTCTCCAGT CGAGGCCGTC TGTATGAGTA CTCCAACAAC AACATAAGAT CAACAATAGA CAGGTACAAG 180 AAGGCTTGCT CAGATACTTC TAACACAAAC ACTGTTACTG AAATCAATGC TCAGTATTAT CAACAAGAAT 240 CAGCCAAGTT GAGACAGCAG ATTCAAATGT TACAGAATTC TAACAGGCAC CTAATGGGAG ATTCCTTGAG 300 TTCCTTAACT GTGAAAGAGT TAAAGCAGGT AGAAAACAGG CTTGAAAGAG GAATTACTAG GATCAGGTCC 360 AAGAAGCACG AAATGCTACT AGCTGAAATA GAGTTTTTGC AGAAAAGGGA AATCGAATTG GAAAATGAAA 420 GTGTTTGTCT CCGAACCAAG ATTGCAGAAA TTGAGAGGCT TCAGCAGGCA AACATGGTGA CTGGACCTGA 480 GCTTAATGCT ATTCAAGCTT TAGCTTCTCG CAATTTCTTT AGCCCCAATG TCATTGAGCA TCCATCTGCT 540 TACTCCCATC TCTCTGACAA GAAGATTCTC CATCTTGGG T AGAGGAGTTG GAGAAAACAA ATATGGAAAA 600 TGGTGTTTTC CATAATATAA TATGTTATAT CATATTATAT TCTATATTGA AAGTTAAATA AAATAAGGAA 660 ATGACAACTC TTGCGAAGTT CGTATGATGT AAAACTGTGA CTACTTCTGT CGTGTGCTTG TTTTTGGAGT 720 ATCACTTATT ACAAAGAAGC ATAAATTTGG ATATATTTAT TTTTGGATTT TGCTTTTGAA AAAAAAAAAA 780 AAAAAA 786

Gene coding region (ORF) is from initiator codon ATGTo terminator codon TAG1 –, 672 bp.

2. cotton GhMADS14The protein sequence of genes encoding is as follows:

Met Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Thr Thr Asn Arg Gln Val Thr

1 5 10 15 20

Phe Cys Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala Tyr Glu Leu Ser Val Leu Cys Asp

25 30 35 40

Ala Glu Val Ala Leu Ile Val Phe Ser Ser Arg Gly Arg Leu Tyr Glu Tyr Ser Asn Asn

45 50 55 60

Asn Ile Arg Ser Thr Ile Asp Arg Tyr Lys Lys Ala Cys Ser Asp Thr Ser Asn Thr Asn

65 70 75 80

Thr Val Thr Glu Ile Asn Ala Gln Tyr Tyr Gln Gln Glu Ser Ala Lys Leu Arg Gln Gln

85 90 95 100

Ile Gln Met Leu Gln Asn Ser Asn Arg His Leu Met Gly Asp Ser Leu Ser Ser Leu Thr

105 110 115 120

Val Lys Glu Leu Lys Gln Val Glu Asn Arg Leu Glu Arg Gly Ile Thr Arg Ile Arg Ser

125 130 135 140

Lys Lys His Glu Met Leu Leu Ala Glu Ile Glu Phe Leu Gln Lys Arg Glu Ile Glu Leu

145 150 155 160

Glu Asn Glu Ser Val Cys Leu Arg Thr Lys Ile Ala Glu Ile Glu Arg Leu Gln Gln Ala

165 170 175 180

Asn Met Val Thr Gly Pro Glu Leu Asn Ala Ile Gln Ala Leu Ala Ser Arg Asn Phe Phe

185 190 195 200

Ser Pro Asn Val Ile Glu His Pro Ser Ala Tyr Ser His Leu Ser Asp Lys Lys Ile Leu

205 210 215 220

His Leu Gly

223

223 amino acid of GhMADS14 genes encoding.

Claims (2)

1. cotton GhMADS14The cDNA sequence of gene is as follows:

ATGGGAAGAG GAAAAATAGA GATAAAGAGG ATCGAAAACA CAACAAATCG TCAGGTTACC TTTTGCAAAC 60 GCAGGAATGG CCTGCTGAAG AAAGCTTACG AACTGTCAGT CCTCTGTGAT GCTGAAGTTG CTCTCATTGT 120 CTTCTCCAGT CGAGGCCGTC TGTATGAGTA CTCCAACAAC AACATAAGAT CAACAA TAGA CAGGTACAAG 180 AAGGCTTGCT CAGATACTTC TAACACAAAC ACTGTTACTG AAATCAATGC TCAGTATTAT CAACAAGAAT 240 CAGCCAAGTT GAGACAGCAG ATTCAAATGT TACAGAATTC TAACAGGCAC CTAATGGGAG ATTCCTTGAG 300 TTCCTTAACT GTGAAAGAGT TAAAGCAGGT AGAAAACAGG CTTGAAAGAG GAATTAC TAG GATCAGGTCC 360 AAGAAGCACG AAATGCTACT AGCTGAAATA GAGTTTTTGC AGAAAAGGGA AATCGAATTG GAAAATGAAA 420 GTGTTTGTCT CCGAACCAAG ATTGCAGAAA TTGAGAGGCT TCAGCAGGCA AACATGGTGA CTGGACCTGA 480 GCTTAATGCT ATTCAAGCTT TAGCTTCTCG CAATTTCTTT AGCCCCAATG TCATTGAGCA TCCATCTGCT 540 TACTCCCATC TCTCTGACAA GAAGATTCTC CATCTTGGGT AGAGGAGTTG GAGAAAACAA ATATGGAAAA 600 TGGTGTTTTC CATAATATAA TATGTTATAT CATATTATAT TCTATATTGA AAGTTAAATA AAATAAGGAA 660 ATGACAACTC TTGCGAAGTT CGTATGATGT AAAACTGTGA CTACTTCTGT CGTGTGCTTG TTTTTGGAGT 720 ATCACTTATT ACAAAGAAGC ATAAATTTGG ATATATTTAT TTTTGGATTT TGCTTTTGAA AAAAAAAAAA 780 AAAAAA 786

Gene coding region is 1 – 672bp from initiator codon ATG to terminator codon TAG.

2. cotton GhMADS14The protein sequence of genes encoding is as follows:

Met Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Thr Thr Asn Arg Gln Val Thr

1 5 10 15 20

Phe Cys Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala Tyr Glu Leu Ser Val Leu Cys Asp

25 30 35 40

Ala Glu Val Ala Leu Ile Val Phe Ser Ser Arg Gly Arg Leu Tyr Glu Tyr Ser Asn Asn

45 50 55 60

Asn Ile Arg Ser Thr Ile Asp Arg Tyr Lys Lys Ala Cys Ser Asp Thr Ser Asn Thr Asn

65 70 75 80

Thr Val Thr Glu Ile Asn Ala Gln Tyr Tyr Gln Gln Glu Ser Ala Lys Leu Arg Gln Gln

85 90 95 100

Ile Gln Met Leu Gln Asn Ser Asn Arg His Leu Met Gly Asp Ser Leu Ser Ser Leu Thr

105 110 115 120

Val Lys Glu Leu Lys Gln Val Glu Asn Arg Leu Glu Arg Gly Ile Thr Arg Ile Arg Ser

125 130 135 140

Lys Lys His Glu Met Leu Leu Ala Glu Ile Glu Phe Leu Gln Lys Arg Glu Ile Glu Leu

145 150 155 160

Glu Asn Glu Ser Val Cys Leu Arg Thr Lys Ile Ala Glu Ile Glu Arg Leu Gln Gln Ala

165 170 175 180

Asn Met Val Thr Gly Pro Glu Leu Asn Ala Ile Gln Ala Leu Ala Ser Arg Asn Phe Phe

185 190 195 200

Ser Pro Asn Val Ile Glu His Pro Ser Ala Tyr Ser His Leu Ser Asp Lys Lys Ile Leu

205 210 215 220

His Leu Gly

223

223 amino acid of GhMADS14 genes encoding.

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