CN102399792A

CN102399792A - Cloning and identification of cotton fiber cell secondary wall development-associated GhNAC1 gene

Info

Publication number: CN102399792A
Application number: CN201110375620XA
Authority: CN
Inventors: 黄耿青; 李学宝; 李鹏; 张�杰; 李雯
Original assignee: Huazhong Normal University
Current assignee: Huazhong Normal University
Priority date: 2011-11-23
Filing date: 2011-11-23
Publication date: 2012-04-04

Abstract

The invention discloses a novel cotton fiber secondary development specific expression gene GhNAC1 full-length sequence, and relates to an important cotton fiber development-associated regulator gene element. The GhNAC1 gene contains three exons and two introns, and is coded with an NAC (N Acetyl L Cysteine) transcription factor. A large quantity of mRNAs (messenger Ribonucleic Acids) of the gene are accumulated specifically at the secondary development stage of the cotton fiber cells, which indicates that the gene is a fiber secondary wall development specific gene. GhNAC1 protein is positioned in a cell nucleolus and has the capability of activating transcription independently, which indicates that the protein serving as a transcription activating factor plays a role in cotton fiber development. GhNAC1 is expressed excessively in arabidopsis, so that leaves crimp upwards, cell walls are thickened, ectopic sedimentation of lignin and the like is detected in foliar epidermic cells, so that the hemicellulose content of foliar cells is greatly increased. As proved by the results, the GhNAC1 can be used for regulating the biosynthesis of cell secondary walls, and plays a key role in the developing process of cotton fiber.

Description

The cotton fiber cell secondary wall is grown relevant GhNAC1 gene clone and is identified

Technical field:

The present invention relates to cotton gene.Specifically be clone and the Function Identification of NAC transcription factor (NAC transcription factor) the gene GhNAC1 of a cotton fiber cell secondary wall specifically expressing.

Background technology:

Cotton is the important cash crop of China, and is closely bound up with China 200,000,000 peasants' income and nearly 1,900 ten thousand textile workers' employment.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.It is the first that China's gined cotton gross output the first in the world, per unit area yield also occupy the cotton state of five big products, but the Cotton in China fibrous quality is 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 gined cottons that need of China cause homemade raw cotton to overstock.The fibrous 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 fiber.The yield and quality of cotton fibre not only receives Effect of Environmental, and receives 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.And this work carry out at first depend on to the identification of cotton fiber development related gene and controlling element thereof with separate.Through key function gene and core controlling elements such as clone fibre strength, fineness and length; Analyze the biological function of mutual work, expression of gene regulation and control and expression product between the fiber development related gene; Illustrate the molecular mechanism that fiber is grown; Disclose the hereditary basis that the cotton fibre quality proterties forms, for efficient fibres modified quality provides theoretical foundation and gene element.The clone of high specific functional gene is the important content of cotton fibre quality improvement and molecular breeding with evaluation.It is reported that country such as the U.S., Japan, Australia and Israel all has the patent of a large amount of cotton fiber development genes.

Plant NAC (gets NAM, ATAF1/2 with CThree of UC contain the initial of NAC domain protein and name) transcription factor is newfound plant specific transcriptional regulator over past ten years.Such transcription factor up to now, is found in species such as a lot of angiosperms, fern and liver moss as one of transcription factor gene family maximum in the Plant Genome in succession.Wherein, in Arabidopis thaliana, find 105 NAC members altogether, found more than 140 member in the paddy rice.

In the NAC transcription factor, topmost constructional feature is the NAC structural domain that each member's N end contains high conservative, and this zone is DNA and protein binding domain.In the NAC conserved domain, comprise 5 subdomains (A, B, C, D, E), wherein subdomain A, C, D high conservative explain that it has vital role to the NAC function.The C-terminal sequence of NAC transcription factor is the transcriptional activation function district, and the aminoacid sequence height variety in this district, total characteristics are that some amino acid such as Serine, Threonine, proline(Pro), the L-glutamic acid frequency that repeats to occur is high.

NAC expresses the regulation and control that receive developmental stage and multiple environmental factors, like plant different developmental phases, pathogenic agent, fungi, arid, low temperature, hypoxemia, physical abuse and hormone etc.; Simultaneously, the expression of some NAC has tissue specificity, as SNAC1 in the paddy rice mainly in the stomata guard cell of pore by abduction delivering (Hu et al., 2006).Research shows, some NAC form at apical meristem and floral organ differentiation, lateral root, bringing into play important effect in the specific growth course of plants such as aging of the branch of branch and blade.Some NAC transcription factor involved in plant are to stress response such as pathogenic agent, fungi, virus, arid, salt, low temperature, hypoxemia, physical abuse and hormones.In recent years, the investigator finds that membrane-mediated regulation and control have played vital role in the process of the quick transcription response external stimulus of plant, some NAC (like the NTL8 in the Arabidopis thaliana) in this process, work (Kim et al., 2006).About the regulating effect of NAC albumen in the Plant Secondary Materials wall, then just obtain some progress in recent years.2005; Kubo etc. find 7 relevant NAC gene VND1-7 (vascular related NAC domain1-7) of dimension pipe in Arabidopis thaliana; Their expression is relevant with the differentiation of root vascular tissue xylem, discovers that further VND6 and VND7 are respectively the pass key switches that activates metaxylem (metaxylem) and protoxylem (protoxylem) generation in the Arabidopis thaliana primary root.Overexpression VND6 or VND7 can both cause the anormogenesis of metaxylem like cell in the root or protoxylem like cell; And suppress VND6 or VND7 expression through the dominance inhibition test; Then suppress the growth of metaxylem or protoxylem respectively; This is to have reported that NAC is in the developmental regulating effect of xylem (Kubo et al., 2005) for the first time.The same year, the tactful gene NST1 that has disclosed two coding NAC transcription factors of employing dominance such as Mitsuda inhibition ( NAC sEcondary wall tHickening promoting factor1) and NST2, thickening is very important to flower pesticide endodermis cell secondary wall, and overexpression NST1 or NST2 have also activated the expression of secondary wall synthesis related gene and caused the dystopy deposition of secondary wall.The T-DNA knock-out experiment shows; There is functional redundancy in these two genes of NST1 and NST2; And in the double-mutant of two genes, causing the endothelium secondary wall not form fully, this is a function (Mitsuda et al., 2005) of having reported NACs performance regulon in the thickening of endothelium secondary wall for the first time.Discovery such as Zhong SND1 ( SEcondary wall-associated NAC dOmain protein also is NST3 or ANAC012) between the Arabidopis thaliana vascular bundle, work to transcribe valve in the fiber secondary wall thickening program.Dominance suppresses SND1 and expresses; Cause the rapid attenuation of fiber secondary wall thickness; And overexpression SND1; Activated the expression of secondary wall synthesis related gene and caused the dystopy deposition of secondary wall, further test showed also that expression SND1 made several fiber associated transcription factor up-regulateds (Zhong et al., 2006).Shortly after that, two Testing Team such as Zhong and Mitsuda have studied NST1 and the effect of NST3 in the fiber secondary wall respectively, and the result shows that NST1 and NST3 exist functional redundancy (Mitsuda et al., 2007 in the fiber secondary wall is grown; Zhong et al., 2007).Zhong etc. suppress this two NAC expression of gene simultaneously with RNAi, cause not having in the Arabidopis thaliana stem the synthetic of fiber secondary wall, and in the fiber finer cell wall, to lack three types of secondary wall components be Mierocrystalline cellulose, xylan and xylogen (Zhong et al., 2007).The phenotypes that Mitsuda etc. also utilize can the complementary NST1/NST2 two sudden changes of the expression of NST3 promoters driven VND6 or VND7 to cause.See that from evolutionary relationship VND1-7, NST1-3 belong to a differentiation together and prop up in NAC family, so they possibly exercise similar function in different cell types.Discover that further NST1 and NST2 promote Arabidopis thaliana pod cracking through accelerating the sophisticated mode of secondary wall, and have funtion part redundant (Mitsuda and Ohme-Takagi, 2008).Recently, Zhao etc. find one with VND1-7 and NST1-3 not at a NAC albumin X ND1 on evolving, it can the negative regulation lignocellulose synthesizes and the programmed death of tracheid.Zhong etc. propose in nearest research, and SND1 is the MS that secondary wall is grown in the fiber, and the author finds 11 transcription factor genes relevant with SND1 (SND2, SND3; MYB103, MYB85, MYB52; MYB54, MYB69, MYB42; MYB43, MYB20 and KNAT7), these expression of gene are relevant with the thickening of secondary wall.In these 11 genes, dominance suppresses SND2, SND3, and MYB103, MYB85, MYB52, MYB54 and KNAT7 expression of gene, result make the thickening of fibrocyte secondary wall significantly reduce.Adopt direct target analysis, the result shows MYB46, and SND3, MYB103 and KNAT7 are the direct action targets of SND1 and homologous protein NST1, NST2, VND6 and VND7.Analyze through these, Zhong etc. think that SND1 and homology NACs albumen and their downstream targets albumen thereof form a tight transcriptional control network, regulate and control the secondary wall biosynthesizing (Zhong et al., 2008) in the dissimilar cells jointly.Above-mentioned research has also shown well-bedded transcription factor network regulation Plant Secondary Materials wall synthetic, and influence the content of some and cell walls development associated protein, thus influence the assembling of wall composition.Therefore, further these functions of participating in regulating cell wall transcription factors and cell walls synthesis associated protein thereof of research and inquirement cell (particularly cotton fiber cell) that will make us better understand the different sorts type is content and assembling how to control wall fraction.

Summary of the invention

The object of the present invention is to provide a new cotton fibre secondary wall to grow the NAC gene GhNAC1 of specifically expressing; Analyze and disclose this gene function and activity; Explore its molecular mechanism, and then use this improvement of genes cotton fibre quality, create the cotton improved seeds the fiber developmental regulation.

In nearest research, the applicant is more than 10000 cotton cDNA of random choose cloning and sequencing from cotton fiber cDNA library.Wherein, 10 sequences are the GhNAC full-length gene.Through BLASTP, the applicant finds that NST1-3 and VND1-7 homology are very high in one of them GhNAC encoded protein sequence and the Arabidopis thaliana.The applicant is with its called after GhNAC1.GhNAC1 cDNA comprises the open reading frame of 1152bp, one the 383 amino acid whose albumen of encoding, and its molecular weight is 43.62KD, iso-electric point (pI) is 6.47.The GhNAC1 gene contains 3 exons and 2 introns, and is consistent with most of NAC gene structures.The long 66bp of its introne 1, inserting the site is the 90th amino acid whose codon (GAG) inside; Intron 2 long 128bp are inserted between the 179th and 180 amino acid whose codons.The proteic N-end of GhNAC1 contains the NAC structural domain; The C-end has a transcriptional activation domain; AtNST1 homology in this albumen and the Arabidopis thaliana the highest (reaching 59.7%); Existing research shows that AtNST1 is a regulating switch of intending southern stem and the growth of flower pesticide secondary wall parietal cell secondary wall, is hinting that GhNAC1 possibly play keying action in the cotton fibre secondary wall is grown.Utilize the quantitative RT-PCR technology that this gene is analyzed at the express spectra of cotton different tissues and cotton fibre different developmental phases; The result show the GhNAC1 gene in the cotton fibre secondary wall etap special and a large amount express, and a little less than in other tissue of cotton, expressing very or almost detect less than.

Further research shows that GhNAC1 albumen has the self activation of transcribing ability, and its self activation ability mainly is positioned at this proteic C-end; The inferior locating and displaying of cell, GhNAC1 is positioned in the nucleus, shows that GhNAC1 is acting as activating transcription factor.

Because GhNAC1 and Arabidopis thaliana NST1 have very high homology, in order to study the function of GhNAC1 gene, the applicant is with GhNAC1 overexpression in Arabidopis thaliana.Obtained 12 strains of arabidopsis thaliana system of GhNAC1 gene overexpression, the phenotype of this several strains system is consistent, and its obvious characteristics is to roll up on the blade.The applicant has extracted the total RNA of blade, has done the RT-PCR analysis, and the result is illustrated in the transfer-gen plant some expression that relate to the marker gene of the synthetic regulation and control of secondary wall and strengthens.The phenotype that produces for further distinct GhNAC1 genetic expression is because it influence due to the cell walls growth; The applicant carries out free-hand section to the stem of the co-located of transgenic and wild-type plant; And with Phloroglucinol-HCl xylogen that dyes; The result shows xylogen dystopy accumulation in the stem epidermic cell of transgenic plant, and can see that through dyeing stem epidermic cell wall has had obvious thickening.The monosaccharide component analysis revealed, the content of synthetic required wood sugar, pectinose, semi-lactosi and the glucose of semicellulose improves greatly in the overexpression transgenic plant blade.These have shown that GhNAC1 can synthesize by the regulating cell secondary wall, infers that it plays a crucial role in cotton fibre secondary wall growth course.

Advantage of the present invention:

1. provide a new cotton fibre secondary wall to grow the GhNAC1 gene order and the cDNA sequence thereof of specifically expressing.This gene shows that at cotton fibre secondary wall developmental stage specifically expressing this gene possibly play an important role in control cotton fibre secondary wall growth course.

2.GhNAC1 albumen is positioned in the nucleus, has the self-excitation of transcribing activity, shows that this albumen is acting as activating transcription factor.

3.GhNAC1 gene overexpression in Arabidopis thaliana promotes xylogen dystopy accumulation in the stem epidermic cell, causes epidermic cell to thicken, and influences the increase of hemicellulose level in the leaf tissue.Result of study shows that also the transcription factor of some control cell secondary walls generations and the genetic expression of the synthetic relevant enzymes of secondary wall polysaccharide strengthen.These results show that GhNAC1 has the function that regulating cotton cell secondary wall is grown.

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

Description of drawings:

Fig. 1. fluorescence quantitative RT-RCR is analyzed GhNAC1 in each tissue of cotton and the expression of fiber etap

Among the figure: 1--root, 2--hypocotyl; The 3--cotyledon; The 4--blade; The 5--petal; 6--flower pesticide; 7--10dpa (day post anthesis, the back fate of blooming) ovule; The 8--0dpa ovule; 9--3dpa fiber and ovule; The 10--6dpa fiber; The 11--9dpa fiber; The 12--12dpa fiber; The 13--15dpa fiber; The 14--18dpa fiber; The 15--20dpa fiber.

The gene structure mode chart of Fig. 2 .GhNAC1

Fig. 3 .GhNAC1 transcribes the self activation experiment.

Wherein: 1 is the growth of Trp-Ade+ substratum, and the yeast position of demonstration is consistent with its synoptic diagram;

2 is the two scarce substratum growths of Trp-Ade-, and wherein the yeast of growth is NAC1-AH109 and NAC1-Y187, and the bacterium that does not grow is BD-AH109 and BD-Y187;

3 is the active test experience of X-gal.

Fig. 4. the inferior positioning analysis of cell.The GFP green fluorescence shows that GhNAC1 albumen is positioned in the nucleus.

Fig. 5. be the expression analysis of the transgenic arabidopsis plant of overexpression GhNAC1.

M is molecular weight marker DL2000 ,-negative contrast, and+positive contrast, 1-19 is transgenic plant.

Fig. 6. be the transgenic plant phenotype analytical of overexpression GhNAC1 (NAC structural domain) and GhNAC1 total length.

Among the figure: 1. wild-type plant; 2. overexpression GhNAC1 (NAC structural domain) strain is; 3-6. the different transgenic lines of overexpression GhNAC1 full length sequence.2 strains that screening is obtained are that T3 plants 30 (totally 60) separately for seedling; 95% little seedling leaf upsweep (5 and 6); And roll up phenomenon on wild-type and overexpression GhNAC1 (NAC structural domain) the transfer-gen plant on-bladed, explain that the functional domain of GhNAC1 is positioned at its C end.

The content of lignin of GhNAC1 gene arabidopsis thaliana stem xylem is changeed in Fig. 7 Phloroglucinol-HCl staining analysis

Arrow is depicted as epidermic cell among the figure.GhNAC1 gene overexpression in Arabidopis thaliana promotes xylogen dystopy accumulation in the stem epidermic cell (the red xylogen composition of representing), causes epidermic cell to thicken.

In the transgenic arabidopsis of Fig. 8 .GhNAC1 overexpression, some are positioned at the expression analysis of VNDs and NSTs downstream gene

Form the monosaccharide component analysis of semicellulose in Fig. 9 .GhNAC1 transgenic arabidopsis blade

Among the figure: the 1MKOH analytical results shows: with respect to wild-type (WT), wood sugar (Xyl), glucose (Glu) and semi-lactosi (Gal) total amount all significantly raise in the overexpression GhNAC1 transgenic plant blade.4M KOH analytical results shows: relative wild-type (WT), wood sugar (Xyl), seminose (Man), glucose (Glu) and semi-lactosi (Gal) total amount all significantly raise in the overexpression GhNAC1 transgenic plant blade.

Embodiment:

The clone of the new gene GhANC1 of a cotton NAC family full length sequence identifies and functional analysis:

1, the isolation identification of GhNAC1 full length sequence and cDNA sequence thereof

From cotton fibre cDNA library, separate more than 10,000 cotton cDNA clone, carried out dna sequencing.Through bioinformatic analysis, obtain 10 GhNAC full length gene cDNA sequences, one of them called after GhNAC1.According to GhNAC1cDNA sequences Design primer GhNAC1 OEup and GhNAC1 OEdn, be template with the cotton genomic dna, adopt the round pcr amplification to obtain the DNA full length sequence of this gene.

2. quantitative RT-PCR is analyzed the GhNAC1 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 GhTUB1 gene that preferentially expressed in fiber.Plant Physiol.130:666-674 carries out).

(2) real-time fluorescence quantitative RT-PCR research expression of gene is (according to Li XB; Fan XP; Wang XL; Cai L, Yang WC, 2005.The Cotton ACTIN1 gene 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/ appearance) of cotton different tissues (root, hypocotyl, cotyledon, leaf, petal, flower pesticide, the

bloom

0 and 10 day ovule in back, bloom back 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, be template with cDNA, (TOYOBO Japan) carries out the quantitative PCR reaction with the primer (GhNAC1 RTP1 and GhNAC1 RTP2) of gene specific and Real-time PCR Master Mix.Cotton GhUBI1 gene 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-CtGhNAC1 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.GhNAC1 transcribe the self activation analysis

Make up pGBKT7-GhNAC1 and pGBKT7-GhNAC1 (NAC) and pGBKT7-GhNAC1 (TAF) carrier, be transformed into respectively among yeast strain AH109 and the Y187.The AH109 positive transformant that on the SD/-Trp flat board, grow lines the dull and stereotyped and SD/-Trp/-Ade flat board of SD/-Trp/-His respectively, if on above two kinds of flat boards, all can grow, explains that then bait protein has the self activation effect; If on above two kinds of flat boards, all can not grow, explain that then bait protein does not have the self activation effect.Simultaneously; The coupling reaction of carrying out the Y187 positive transformant that will on the SD/-Trp flat board, grow detects the expression that whether is activated of LacZ reporter gene; Concrete operation steps is following: in petridish, soak an aseptic filter paper with 5mL Z buffer/X-gal solution, the yeast list bacterium colony of picking fresh culture was placed on room temperature in freezing 15 seconds and melts to another aseptic filter paper and in liquid nitrogen.Carefully will be with zymic filter paper to be put on the wetted filter paper, cover lid places 30 ℃ of incubators, and the positive of blueness appears in bacterium colony in 8 hours, explains that bait protein has the self activation effect.And, explain that bait protein does not have the self activation effect greater than occurring blue in 8 hours or still not showing blue negative.

4.GhNAC1 the inferior positioning analysis of proteic cell

The N-terminal sequence and the eGFP of coding NAC structural domain in the GhNAC1 gene order are merged; And structure pBI121-GhNAC1 (NAC domain): eGFP plant expression vector; This carrier is changed in the GV3101 Agrobacterium through electrotransformation, and again through the flower-dipping method arabidopsis thaliana transformation, screening obtains the transgenic seedling; And be transplanted in the basin, T1 is for seed for results.T1 for seed germination, is got the root of 7 days seedlings of growth, place and observe GFP fluorescence under the Laser Scanning Confocal Microscope.

5.RT-PCR analyzing GhNAC1 crosses and expresses that some synthesize relevant genetic expression with cell wall polysaccharides in the plant

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

6. change the monosaccharide component analysis of cell wall polysaccharides in the GhNAC1 gene Arabidopsis leaf

The insoluble cell-wall component of alcohol is after CDTA and Na2CO3 (pectin fraction in the extracting cell-wall component) extracting; Carry out 1M KOH and 4M KOH extracting continuously, obtain respectively to combine with Mierocrystalline cellulose untight semicellulose and with the compact semicellulose component of Mierocrystalline cellulose.We adopt inositol (Ino) as interior mark; Contents of monosaccharides through different components polysaccharide in the gc analysis cell walls; The result shows that contents of monosaccharides changes not obvious (data does not provide) in the pectin fraction, and the monosaccharide component in the semicellulose changes obviously (result is as shown in Figure 9).

The primer of the present invention is seen table 1:

Table 1 gene-specific primer

1. cotton GhNAC1 gene order is following:

ATGCAAAGTT CATTTGGTAT ATATCATCAC TTATTATTTC TCTCTCAATC TTCCTCTACT 60

TATATAGTTT CCATCTTTGT TAAAATGCCG GAAAACATGA ACATATCTGT AAACGGACAA 120

TCCCAAGTCC CTCCCGGATT CCGATTTCAT CCCACCGAAG AGGAGCTCTT ACAATACTAT 180

CCCCGAAAGA AGGTTTCTTA TGAGAAGATC GATTTAGATG TGATTCGAGA GGTTGATCTT 240

AACAAGCTCG AGCCATGGGA TATTCAAG GT ACATATCTGT GTGTGTGTGT GTGTGTGTGT 300

AGTACCATGA TTTATGTTCC ACTGTTATTT ACAGAGAGGT GCAAGATTGG AACTACGCCG 360

CAGAATGATT GGTATTTTTT TAGCCACAAA GACAAGAAGT ACCCGACCGG GACTCGGACT 420

AACCGTGCCA CGGCGGCGGG TTTTTGGAAA GCCACCGGCC GTGACAAGGT CATATATAGC 480

AACTGCCGGC GGATTGGAAT GAGGAAGACG TTGGTGTTTT ACAAAGGGAG AGCCCCTCAT 540

GGTCAAAAAT CTTATTGGAT CATGCATGAA TATAGACTAG ACGATAACAT CGTTGAAACC 600

AAC GTAAGCC TTTTAAAGCT CAAATTTACG TCATAATATT AAAAAAAATT GTTATAGGGT 660

CCTAAAATAT ATTTTGATGC TATTTTTTGT TCGTTAAAAT AATAATAAAT AGTACATTTT 720

TTTTAATGAA GGTGTCAAAT GGTATGATGG AGGGGACACA AGAAGAAGGA TGGGTGGTAT 780

GTCGTATTTT CAAGAAGAAA AATCATCATA AAACCCTAGA AAACCCTATT AGCACATCCT 840

TGTCTGAAAA AACCAGGAAC CTGCATATGT TCAACACATG TAATGAAGGA GCCTTGGAAC 900

ATATACTTGA ATACATGGGA AGGAGTTGTA AGGAAGACAA CGAAGCAAAT AATAACAACA 960

CGAGATTTGT CATGACCATG GAGCTTGCTA TCAACAACAA TGGCTACTAT GACTCCTTCA 1020

CGAAGCTGCC AAGTCTAGAC AGTCCAAACT CAGCCAGCAG TTACCAACCC ATTATGACAG 1080

AAAACGAAGG CTCAATCATC AACTCGGTGA GTGGTGGTGA TTTCAACTCG GTTTATAACA 1140

ATGACTCGGG GCTAACCAAC TGGGCAGCAC TCGACCGACT CGTAGCTTCA CATCTCAATG 1200

GACAAACGGA GACATCGAGA CAACTGGCTT GTTTCAATGA CCAACACATG GCCTACTGCA 1260

ATAACACCAA CAACACTGAT CATCAAGATC TTGAATTACC AGCCGTTCGA TCAACATCAC 1320

CATCATCATC ATCATCAATA GACATCTGGA CTAGTTTCAC TACCAGATCA TCATCTTTGT 1380

CATCATCCGT TATCGACCCA CTATGCCACG TGGTTAATGC TAGTGTATAA 1430

Cotton GhNAC1 gene contains two introns, and underscore partly is an intron sequences.

2. the cDNA sequence of cotton GhNAC1 gene is following:

ATGCCGGAAA ACATGAACAT ATCTGTAAAC GGACAATCCC AAGTCCCTCC CGGATTCCGA 60

TTTCATCCCA CCGAAGAGGA GCTCTTACAA TACTATCTCC GAAAGAAGGT TTCTTATGAG 120

AAGATCGATT TAGATGTGAT TCGAGAGGTT GATCTTAACA AGCTCGAGCC ATGGGATATT 180

CAAGAGAGGT GCAAGATTGG AACTACGCCG CAGAATGATT GGTATTTTTT TAGCCACAAA 240

GACAAGAAGT ACCCGACCGG GACTCGGACT AACCGTGCCA CGGCGGCGGG TTTTTGGAAA 300

GCCACCGGCC GTGACAAGGT CATATATAGC AACTGCCGGC GGATTGGAAT GAGGAAGACG 360

TTGGTGTTTT ACAAAGGGAG AGCCCCTCAT GGTCAAAAAT CTTATTGGAT CATGCATGAA 420

TATAGACTAG ACGATAACAT CGTTGAAACC AACGTGTCAA ATGGTATGAT GGAGGGGACA 480

CAAGAAGAAG GATGGGTGGT ATGTCGTATT TTCAAGAAGA AAAATCATCA TAAAACCCTA 540

GAAAACCCTA TTAGCACATC CTTGTCTGAA AAAACCAGGA ACCTGCATAT GTTCAACACA 600

TGTAATGAAG GAGCCTTGGA ACATATACTT GAATACATGG GAAGGAGTTG TAAGGAAGAC 660

AACGAAGCAA ATAATAACAA CACGAGATTT GTCATGACCA TGGAGCTTGC TATCAACAAC 720

AATGGCTACT ATGACTCCTT CACGAAGCTG CCAAGTCTAG ACAGTCCAAA CTCAGCCAGC 780

AGTTACCAAC CCATTATGAC AGAAAACGAA GGCTCAATCA TCAACTCGGT GAGTGGTGGT 840

GATTTCAACT CGGTTTATAA CAATGACTCG GGGCTAACCA ACTGGGCAGC ACTCGACCGA 900

CTCGTAGCTT CACATCTCAA TGGACAAACG GAGACATCGA GACAACTGGC TTGTTTCAAT 960

GACCAACACA TGGCCTACTG CAATAACACC AACAACACTG ATCATCAAGA TCTTGAATTA 1020

CCAGCCGTTC GATCAACATC ACCATCATCA TCATCATCAA TAGACATCTG GACTAGTTTC 1080

ACTACCAGAT CATCATCTTT GTCATCATCC GTTATCGACC CACTATGCCA CGTGGTTAAT 1140

GCTAGTGTAT AACAAAATCT ATAATCATAC CTTAGTACCT AAAGTCACAA AGTTATTATT 1200

ATTAGAGTAT TACATCACAG TTATGTTGAT GAAATATATA TAAATAAAAA TTAAAAAAAA 1260

AAAAAAAAAA 1270

Gene coding region (ORF) from initiator codon ATG to terminator codon TAA (1-1152bp).

3. the protein sequence of cotton GhNAC1 genes encoding is following:

Met Pro Glu Asn Met Asn Ile Ser Val Asn Gly Gln Ser Gln Val Pro Pro Gly Phe Arg Phe

1 5 10 15 20

His Pro Thr Glu Glu Glu Leu Leu Gln Tyr Tyr Leu Arg Lys Lys Val Ser Tyr Glu Lys Ile

25 30 35 40

Asp Leu Asp Val Ile Arg Glu Val Asp Leu Asn Lys Leu Glu Pro Trp Asp Ile Gln Glu Arg

45 50 55 60

Cys Lys Ile Gly Thr Thr Pro Gln Asn Asp Trp Tyr Phe Phe Ser His Lys Asp Lys Lys Tyr

65 70 75 80

Pro Thr Gly Thr Arg Thr Asn Arg Ala Thr Ala Ala Gly Phe Trp Lys Ala Thr Gly Arg Asp

85 90 95 100 105

Lys Val Ile Tyr Ser Asn Cys Arg Arg Ile Gly Met Arg Lys Thr Leu Val Phe Tyr Lys Gly

110 115 120 125

Arg Ala Pro His Gly Gln Lys Ser Tyr Trp Ile Met His Glu Tyr Arg Leu Asp Asp Asn Ile

130 135 140 145

Val Glu Thr Asn Val Ser Asn Gly Met Met Glu Gly Thr Gln Glu Glu Gly Trp Val Val Cys

150 155 160 165

Arg Ile Phe Lys Lys Lys Asn His His Lys Thr Leu Glu Asn Pro Ile Ser Thr Ser Leu Ser

170 175 180 185

Glu Lys Thr Arg Asn Leu His Met Phe Asn Thr Cys Asn Glu Gly Ala Leu Glu His Ile Leu

190 195 200 205 210

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

215 220 225 230

Met Thr Met Glu Leu Ala Ile Asn Asn Asn Gly Tyr Tyr Asp Ser Phe Thr Lys Leu Pro Ser

235 240 245 250

Leu Asp Ser Pro Asn Ser Ala Ser Ser Tyr Gln Pro Ile Met Thr Glu Asn Glu Gly Ser Ile

255 260 265 270

Ile Asn Ser Val Ser Gly Gly Asp Phe Asn Ser Val Tyr Asn Asn Asp Ser Gly Leu Thr Asn

275 280 285 290

Trp Ala Ala Leu Asp Arg Leu Val Ala Ser His Leu Asn Gly Gln Thr Glu Thr Ser Arg Gln

295 300 305 310 315

Leu Ala Cys Phe Asn Asp Gln His Met Ala Tyr Cys Asn Asn Thr Asn Asn Thr Asp His Gln

320 325 330 335

Asp Leu Glu Leu Pro Ala Val Arg Ser Thr Ser Pro Ser Ser Ser Ser Ser Ile Asp Ile Trp

340 345 350 355

Thr Ser Phe Thr Thr Arg Ser Ser Ser Leu Ser Ser Ser Val Ile Asp Pro Leu Cys His Val

360 365 370 375

Val Asn Ala Ser Val *

380 383

Claims

1. cotton GhNAC1 gene, its sequence is following: