CN104313032A - Functions of cotton GhMTT8a gene and application of the cotton GhMTT8a gene in brown cellucotton modification by utilization of biological technical means - Google Patents

Abstract

The invention relates to a method of modifying color and luster of cotton brown fiber by utilization of biological technical means, and particularly relates to an application in modification of color and luster of the cotton brown fiber by utilization of a cotton transgenic technology and by utilization of a cotton gene and a plant binary expression vector constructed by the cotton gene. Protein coded by a cotton GhMTT8a gene is provided. The amino acid sequence of the protein is shown as SEQID:NO.2. A plant positive-sense complementary binary expression vector of the cotton GhMTT8a gene is provided. The expression vector adopts the plant expression binary vector pFGC5941 as a skeleton vector, adopts a sequence segment containing a complete protein open reading frame of the cotton GhMTT8a gene as a positive-sense expression structure of the gene, and separately introduces BamHI and XhoI enzyme digestion sites, thus constructing the positive-sense expression vector containing the cotton GhMTT8a gene.

Description

The function of cotton GhMTT8a gene also utilizes the application of animal nutrition in improvement brown fibre cotton

Technical field

The present invention relates to a kind of method utilizing animal nutrition to improve cotton brown fibre color and luster, particularly relate to a kind of plant binary vector of cotton gene and structure thereof that utilizes and adopt cotton transgenic technology to carry out the application in improvement cotton brown fibre color and luster.

Background technology

Natural color cotton is the specific type cotton that a kind of cotton fibre has natural colour, because it has unique natural colour, without the need to bleachinging and dyeing in weaving and the course of processing, to the mankind and environmental nonpollution, comfortable and easy to wear, be particularly suitable for making underclothes, be that real " ecology ", " environmental protection " are cotton, thus cause the extensive concern of domestic and international cotton breeding man and weaving, Clothing industry circle, be described as the ecological textile of 21 century world market most potentiality.Though the research and development starting of China's color cotton is comparatively slow, develop very fast.From 1998 to 2010, Chinese natural color cotton cultivated area expanded annual more than 20 ten thousand mu to from annual 10000 mu, and lint yield is increased to annual more than 20,000 tons from annual 800 tons, and between 10 years, color cotton cultivated area and lint yield increase 20 times and 25 times respectively.

Color cotton mainly contains brown and green two large serial colors, in color cotton breeding of new variety, by means of traditional breeding method, countries in the world and some breeding units of China conduct a research one after another and select a collection of color cotton new variety, improve quality and the proterties of color cotton to a certain extent.The color cotton kind that domestic each research unit selects has: the kind such as new color cotton series of products (No. 1 to No. 20), CCRI 51, color cotton No. 2 of Zhejiang, color assorted No. 1 of Soviet Union.Compared with white cotton, color cotton kind still also exists following outstanding problem: fiber strength is not high, and staple length is partially short, ginning outturn is on the low side, and pigment stability is poor, and color is single, thus limits the application of brown cotton in textile industry.Therefore, the fiber colour of innovation color cotton and improve the fibre strength of brown cotton, staple length is two key factors being related to China's textile industry and color cotton industry development success or failure, is also the important topic that vast cotton breeding researcher faces.

At occurring in nature, plant pigments can be divided into photosynthetic pigments (as chlorophyll) and non-photosynthetic pigments (as flavonoid and carotenoid).Research shows, natural colorful cotton fibre pigment belongs to non-photosynthetic pigments, and relevant with the specific period of cotton fiber development.Chinese scholars is passed through Cotton Fiber of Natural Brown Cotton chromogenesis process and this Quality Research of biochemical character, tentatively think to there are following 2 kinds of suppositions: (1) Ryser, Xiao and Zhan etc. infers that thinking that natural liquor storeroom fiber pigment is originated at first is the proanthocyanidin (Proanthocyanidins) that the catechin derivation be positioned in vacuole is formed, also referred to as condensed tannin (Condensed Tannins), and be oxidized by condensed tannin ingress of air and formed there is brown quinones; (2) Zhao etc. think that Cotton Fiber of Natural Brown Cotton pigment belongs to flavonoids.And the experiment of Xiao and Zhan all confirms that the precursor substance of Cotton Fiber of Natural Brown Cotton pigment belongs to proanthocyanidin (condensed tannin).Zhan etc. analyze the pigment content distributed in brown cotton, white cotton fiber and seed coat, and research is thought: the major cause of decision fiber color is the allocation proportion of pigment between fiber and seed coat.Cotton fibre is by kind of an epithelial cell differentiation, and the forming process of cotton fibre is in close relations with the growth of planting chrotoplast.Arabidopis thaliana, cotton belong to dicotyledons, understand the anabolism of Arabidopis thaliana seed coat pigment and regulatory pathway for disclosing cotton seed coat brown pigments, cotton fibre brown pigments is grown, the codeposition molecule mechanism of fiber and pigment has most important theories meaning.

The metabolic pathway of synthesizing of Arabidopis thaliana seed coat pigment is subject to the regulation and control of several genes, belongs to Secondary Metabolism of Plant.By showing the research of Arabidopis thaliana seed coat Transparent testa series mutants: phenylalanine (Phenylpropanoid) is the initial amino acid forming condensed tannin, it is under the katalysis of a series of enzyme, the condensed tannin that final formation is colourless, the browner feature presenting seed coat through atmospheric oxidation.Wherein, tT4coding chalkane synthetase ( cHS, chalcone synthase), tT5coding enzyme, namely chalcone isomerase ( cHI, chalcone isomerase) etc. as the enzyme in structural gene coding flavonoid pathways metabolism, tT1, TT2, TTG1, TTG2, TT8transcribe to structure gene Deng as transcription factor, tT8with tT19pigment translocator of then encoding participates in transhipment and the accumulation of flavonoid substances.In the correlative study of Cotton Fiber of Natural Brown Cotton colour development, Chinese scholars adopts different molecular biology methods, has cloned multiple homologous gene relevant to Arabidopis thaliana seed coat pigment metabolic pathway of synthesizing from cotton, as ghCHS, GhCHI, GhF3H, GhDFR, GhANS, GhANRdeng, the protein function of these genes participation pigment synthesis infers the possible function all based on homologous sequence analysis and information biology, whether participates in cotton seed coat pigment actually simultaneously, the pigment synthesis metabolism of fiber needs confirmation further.For the above-mentioned most important theories problem existed in the research of cotton brown pigments, in the urgent need to Arabidopis thaliana seed coat brown pigments route of synthesis genes involved for reference, associated homologous gene is cloned in cotton, and by the in addition functional verification of molecular biology and transgenic technology, whether participate in the synthesis of cotton fiber brown pigments with these genes involveds clear and definite simultaneously.The research of the problems referred to above confirms, there is provided theories integration by the research carrying out Cotton Fiber of Natural Brown Cotton pigment synthesis pathways metabolism for us further, and by molecular biology method improvement Cotton Fiber of Natural Brown Cotton quality, breakthrough cotton fibre color and luster Study on Diversity, there is important theory and practice meaning.

In plant gene function research, transgenic function complementation and gene silencing are the effective technology means disclosing gene function.Have complementary functions and transform this deletion mutant body by building the sense expression vector of goal gene, thus verify the function that this gene performs in plant materials.For the checking of homologous gene function, also can adopt the mutant transforming approximate species, thus complete homogenic functional verification fast., single traits mutant library longer for the cotton transgenic cycle lacks, and the functional verification adopting arabidopsis thaliana transformation mutant to carry out homologous gene or similar genes structural domain is a kind of fast method.Gene silencing is the ancient mechanism of one of organism, and it is played a role by modes such as degradation of rna, suppression translation or modification karyomit(e)s in g and D process.In recent years, this technology is used widely in molecular biology of plants research and genetic improvement as an important genetic manipulation instrument, various plants gene silent technology is set up in succession, comprise sense-rna, dsRNAi (double-stranded RNA interference) and artificial microRNA, particularly dsRNAi and microRNA and had new breakthrough in the accuracy and high efficiency etc. of gene silencing.DsRNAi and microRNA is higher than Antisense RNA Technique in the efficiency of degraded target gene mRNA, target gene can be made under the concentration lower than the several order of magnitude of sense-rna to express drop to extremely low level even function completely lose; And in transgenic protocol, there is not the problem of insertion point randomness, any gene all can by dsRNAi or microRNA specificity is reticent targetedly in theory.

Summary of the invention

In order to clone and understand cotton fiber brown pigments synthesis related gene and and then Cotton Fiber of Natural Brown Cotton quality is improved, utilize the means such as biotechnology realize cotton fiber color and luster variation, first object of the present invention is to provide cotton ghTT8Agene and the protein by this genes encoding, and prove that this gene had both participated in the formation of seed coat brown pigments by experiment, participate in again the formation of fiber brown pigments, be expected to be applied in brown cotton breeding.Second object of the present invention is to provide expression vector containing above-mentioned gene and host cell.3rd object of the present invention is to provide the application of above-mentioned gene in improvement cotton brown fibre color and luster.

In order to realize first above-mentioned object, present invention employs following technical scheme:

A kind of by cotton ghTT8Athe protein of genes encoding, the aminoacid sequence of this protein is as shown in SEQ ID:NO.2.

In order to realize second above-mentioned object, present invention employs following technical scheme:

Cotton ghTT8Athe plant positive sense complementary binary expression vector of gene, this expression vector for skeleton carrier with plant binary vector pFGC5941, is chosen and is contained ghTT8aone section of sequence of gene whole protein encoder block as the just expression structure of this gene, and is introduced respectively bamHIwith xhoIrestriction enzyme site, thus construct and contain ghTT8athe sense expression vector of gene.

A kind of host cell, the cotton described in this host cell adopts ghTT8Athe plant positive sense complementary binary expression vector of gene transforms.As preferably, the bacterial strain of described conversion adopts Agrobacterium EHA105.Further, described host cell adopts osmose process arabidopsis thaliana transformation mutant tt8.

Cotton ghTT8athe double-stranded RNA interference expression vector of gene, this expression vector with plant binary vector pCAMBia2301 for skeleton carrier, choose the expression nuclear structure of plant binary vector pBI121, enzyme is cut, connect and be built into carrier pCAMBIA2301-121, again in the cotton gene group shown in SEQ ID:NO.1 one section of sequence as the middle Loop ring texture building double base interference vector, shown in the sequence SEQ ID:NO.3 of Loop ring texture, primer is upstream primer: AGAAGCGAGATGAGGGATGT, downstream primer: CATTTTCATGCGAAGGATTC.

A kind of host cell, the cotton described in this host cell adopts ghTT8athe double-stranded RNA interference expression vector of gene transforms.

In order to realize the 3rd above-mentioned object, present invention employs following technical scheme:

The application of the gene of nucleotide sequence as shown in SEQ ID:NO.1 in improvement cotton brown fibre color and luster.

The present invention utilizes the method for bioinformatic analysis and electronic cloning, clone a new gene containing bHLH-MYC_N and HLH conserved domain, this gene and Arabidopis thaliana seed coat brown pigments synthesize TT8 gene amino acid sequence at conserved structure region (10--195 amino acid position, there is the sequence homology of about 81% in 480--540 amino acid position, but not high in all the other regional sequence homologys, only have about 60%.The method of RT-PCR is utilized to clone this gene, called after subsequently ghTT8A, this genes encoding 684 amino-acid residues, molecular weight is about 76.5KD, belongs to a member in bHLH-MYC supergene family.Quantitative fluorescent PCR is utilized to analyze ghTT8agene is at the expression characteristic of development in different stages fiber, seed coat, and result shows: ghTT8agene equal predominant expression in the brown fibre of tetraploid cotton, white cotton seed coat and brown cotton seed coat, expresses hardly, illustrates that this gene had both participated in the formation of seed coat brown pigments, participate in again the formation of fiber brown pigments in white cotton fiber.For cotton ghTT8athe functional analysis of gene, builds plant justice binary expression vector promotor 35S::GhTT8A::Nosterminator expresses nuclear structure complement Arabidopsis mutant tt8, Molecular Identification and transgenosis physiologic analyses all show that this gene can make Arabidopsis Mutants tt8recover wild type phenotype, thus prove that this gene participates in the synthesis of seed coat brown pigments; According to molecular structure and the conserved domains characteristic of this gene, build 2 kinds of dissimilar double-stranded RNA interference carriers (dsRNAi, carrier sequence sees appendix), transform brown cotton, to brown cotton 40 transgenic line T by the pollen tube passage method of damage ₀generation and T ₁the Molecular Identification in generation and the analysis of transgene cotton physiologic index all show that this gene all can reduce the color and luster of cotton brown fibre in various degree, thus prove that this gene participates in the metabolism of cotton fiber brown pigments.In research process, utilize ghTT8Agene also obtains the cotton germ plasm resource 8 parts of brown fibre of different depth degree by biotechnological means, these germplasm materials of innovation are expected to be applied on Cotton Fiber of Natural Brown Cotton quality-improving.

Accompanying drawing explanation

Fig. 1 is cotton ghTT8Agene and other species tT8homogenic aminoacid sequence comparison diagram.

Fig. 2 is cotton ghTT8Athe aminoacid sequence phylogenetic analysis figure of gene.

Fig. 3 is ghTT8Agene is at the expression level figure of brown, white cotton (upland cotton) fiber different development stage.

Fig. 4 is for containing ghTT8Athe design of graphics of gene plant sense expression vector.

Fig. 5 is for containing ghTT8Athe structure schema of gene 2 different structure double base interference vectors.

Fig. 6 is cotton ghTT8Agene turns Arabidopis thaliana tT8the pcr amplification qualification figure of the positive strain that mutant obtains; M:DNA marker DL2000,1: containing cotton ghTT8Athe positive plasmid pFGC5941-of gene ghTT8a; 2,3,4,5,6: spray the Arabidopis thaliana individual plant that Basta has resistance; 7: wild-type Arabidopsis plants.

Fig. 7 is for turning cotton ghTT8Agene is at complement Arabidopsis tT8the positive strain root of mutant, stem, leaf, angle fruit and the expression level figure in spending.

Fig. 8 is for turning cotton ghTT8Agene is at complement Arabidopsis tT8in the positive strain of mutant can deeply property PAs and insoluble PAs contain spirogram.

Fig. 9 is cotton ghTT8Ain gene interference vector, Partial Fragment is turning the pcr amplification qualification figure A in nigger-brown fibre cotton; M:DNA marker DL2000,1: containing cotton ghTT8Athe positive plasmid pFGC5941-GhTT8aRA of gene interference vector; 2,3,4,5,6,7: spray the transgenic cotton individual plant that 4/1000ths card sodium mycins have resistance; 8: the cotton P158 individual plant of wild-type dark-brown; Fig. 9 B; M:DNA marker DL2000,1: containing cotton ghTT8Athe positive plasmid pFGC5941-GhTT8ARIB of gene interference vector; 2,3,4,5,6,7: spray the transgenic cotton individual plant that 4/1000ths card sodium mycins have resistance; 8: the cotton P158 individual plant of wild-type dark-brown.。

Figure 10 is cotton ghTT8Ainterfere the application drawing of dark-brown cotton fibre; ZM11: non-transgenosis white cotton Zhejiang cotton 11; P158: cotton P158 strain (transgenosis parent plant) of dark-brown; T0A: dark-brown cotton interferes strain 1; T0B: dark-brown cotton interferes strain 2; T0C: dark-brown cotton interferes strain 3; T0D: dark-brown cotton interferes strain 4; T0E: dark-brown cotton interferes strain 5; T0F: dark-brown cotton interferes strain 6.

Embodiment

. materials and methods

1.1 vegetable materials and growth conditions

Color cotton No. 2 of upland cotton brown fibre Zhejiang, white fiber cotton Zhejiang cotton 11, the cotton P158 of nigger-brown fibre provides by Zhejiang Academy of Agricultural Science crop and cash crop research department of nuclear technique research on utilization institute and field is tested by live Hangzhou, the court academy proper that is seeded at the beginning of 2011 5 months.At the 9d(Post flowering of cotton Post flowering, DPA, Day Post Anthesis), 12d, 15d, 18d, 21d and 24d, choose the cotton fiber tissue of growing respectively and seed coat tissue carries out correlative study.

.2 experimental technique

1.2.1 the extraction of cotton genomic dna and cotton fibre, seed coat tissue total serum IgE

Cotton genomic dna extracts and adopts CTAB method.The method that employing polyphenol, polyose plant RNA extraction test kit provide extracts cotton fiber tissue, seed coat tissue total serum IgE, and test kit is purchased from Tyke, Beijing hundred Bioteck Bioisystech Co., Ltd.

.2.2 bioinformatic analysis is utilized to screen and clone cotton ghTT8Agene

According to the cotton brown fibre pigment of having cloned ghTT8Agene and Arabidopis thaliana seed coat brown pigments atTT8the protein conserved domain sequence of gene, search cotton ESTs database (http://blast.ncbi.nlm.nih.gov/), selects 3 sequences (GenBank accession number is ES825879.1, DT551422.1, ES796809.1) altogether.Splice with these 3 EST sequence search ESTs databases again, final acquisition sequence, this sequence contains an entire open reading frame (ORF, Opening Reading Frame), there is the homology of about 82% in conserved domain part in the protein predicted according to this sequence ORF and Arabidopis thaliana TT8 albumen.According to the sequence signature with ORF, the special primer (P1:TCTCCAAAGGGAAAGTTGTG and P2:TTATGGTATTATTTGATTTATCGCCCTCTTTACC) of design 1 pair of pcr amplification.

CDNA first chains that cotton fiber tissue's total serum IgE reverse transcriptions of growing 15DPA with the color cotton No. 2 in Zhejiang respectively obtain are for template, and in conjunction with round pcr, amplification condition is: 94 DEG C of denaturation 4 min; 94 DEG C of sex change 30 s, 56 DEG C of annealing 30 s, 68 DEG C extend 2 min; 30 PCR circulations, last 68 DEG C extend 10 min, 15 DEG C of insulations.Amplification system is 50ul:2 X KOD amplification reaction solution, 10ul dNTP, 2 ul P1 primers, 2ul P2 primer, 1ul KOD FX, and 5ul transcribes after product, 5ul ultrapure water; Again PCR primer is added A, adding A reaction system is that 10ul:5ul connects damping fluid, and 1ul pTA2 carrier, 1ul ligase enzyme, 1ul adds A connecting fluid, 2ul KOD amplified production.Transformation of E. coli competence TG1, screening positive clone send Nanjing Genscript Biotechnology Co., Ltd. to check order.Adopt Reverse Transcription box synthesis cDNA first chain of MBI company, utilize the acquisition of KOD FX enzymatic amplification to infer ghTT8agene (bio tech ltd is spun by Japan for TOYOBO, Shanghai).Primer involved by this experiment all synthesizes the raw work biotechnology Engineering Service company limited from Shanghai.

.2.3 design of primers and bioinformatic analysis

The design of primers related in research utilizes Primer Premier 5.0 (http://www.premierbiosoft.com/index.html) to complete, DNAman 6.0 software (http://www.lynnon.com) is utilized to carry out tetraploid rice and nucleic acid Phylogenetic analysis to aminoacid sequence, the supposition of protein conserved domain adopts the Blast on-line analysis system (http://blast.ncbi.nlm.nih.gov) in Genbank database, concrete structural domain position analysis utilizes the online software analysis of SMART (http://smart.embl-heidelberg.de).

. 2. 4 utilize quantitative fluorescent PCR to analyze in diploid cotton and four times of cottons ghTT8athe expression characteristic of gene

According to the fibrous tissue of cotton different development stage, the cDNA of seed coat tissue, detect with real-time fluorescence quantitative RT-PCR ghTT8athe relative expression levels of gene in development in different stages fiber, seed coat tissue.Cotton GBQ7 gene is used as the reference gene (amplimer) of quantitative fluorescent PCR, ghTT8athe specificity amplification primer of gene is in table 2.Experimental implementation equal reference reagent box specification sheets (bio tech ltd is spun by Japan for TOYOBO SYBR Green Supermixture, Shanghai), the temperature cycle of quantitative fluorescent PCR: 50 DEG C, 1 min; 94 DEG C of denaturation 3 min; 94 DEG C of sex change 30 s; 56 DEG C of annealing 30 s; 72 DEG C extend 45 s; Amount to 30 PCR circulations.

Primer used in table 1 quantitative fluorescent PCR and sequence

1. 2. 5 cottons ghTT8athe structure of gene sense expression vector and Arabidopsis Mutants tT8complementation test

Obtain according to order-checking ghTT8Agene cDNA sequence also determines the entire protein coding sequence of this gene.Due to Arabidopis thaliana tT8mutant is inserted by T-DNA and produces, and therefore, we select the two carrier free pFGC5941 of expression of plants as the positive sense complementary carrier of this gene, introduce restriction enzyme site at initiating terminal xhoI,restriction enzyme site is introduced at clearing end bamHI(GhTT8aP1:CCG cTCGAGtCTCCAAAGGGAAAGTTGTG; GhTT8aP2:CGC gGATCCthus build complete just genetic expression structure TTATGGTATTATTTGATTTATCGCCCTCTTTACC).

Adopt osmose process arabidopsis thaliana transformation mutant tT8.Will containing recombinant plasmid Agrobacterium 28 DEG C shake bacterium spend the night to OD 600 value be between 1. 0-2. 0, the centrifugal 8m in of 8 000 rpm, abandon supernatant collect thalline.Precipitation be the sucrose of 50 g/ L containing mass concentration, the MS substratum of pH value 5. 7 is resuspended is about 0. 5 to OD 600 value, and add 0. 02% Silwet70, after abundant mixing, Arabidopis thaliana inflorescence is immersed, bacterium liquid is stirred every 2 m in, take out after 10 m in and keep flat and moisturizing 12--24 hour, after 4 weeks, collect Arabidopis thaliana after transforming tT8seed.

cotton ghTT8Athe structure of gene double-stranded RNA interference vector

According to the hairpin ring structure that section sequence of in cotton gene group designs as interference vector, this section of sequence sees appendix 4, amplimer INA, INB, in hair clip Loop ring design, introduce restriction enzyme site bamHI, SacIwith kpnI.Cotton ghTT8athe justice end primer restriction enzyme site of 2 kinds of different interference structures of gene xbaI, BamHI; Antisense end introduces restriction enzyme site sacIwith kpnI, thus building complete hairpin ring structure, the primer sequence information related to is in table 2.The amplification of Loop ring structure take cotton genomic dna as template, and in interference vector structure, the amplification of gene fragment obtains with reverse transcription ghTT8agene PCR product is template, all adopts KOD FX enzymatic amplification to obtain object fragment (bio tech ltd is spun by Japan for TOYOBO, Shanghai).

Primer used in table 2 quantitative fluorescent PCR and sequence

1.2. 7 improved method pollen tube passage method converting cottons

Process containing the fresh bacterium liquid of object interference vector Agrobacterium LBA4404: will containing recombinant plasmid Agrobacterium 28 DEG C shake bacterium spend the night to OD 600 value be between 1. 0-2. 0, the centrifugal 8m in of 8 000 rpm, abandon supernatant collect thalline.Precipitation be the sucrose of 50 g/ L containing mass concentration, the MS substratum of pH value 5. 7 is resuspended is about 0. 5 to OD 600 value, and adds 0. 02% Silwet70, use immediately after fully mixing, every 2 m in stirring bacterium liquid.

The operation of improved method pollen tube passage method converting cotton: namely bloom between pollination 3--6 point that afternoon in cotton, with blade, cotton column cap is rived, dip with very little cotton mass and soak through processing and containing the fresh bacterium liquid of the Agrobacterium LBA4404 of object carrier, then be clipped in the cotton mass that tweezers gripping contains bacterium liquid in the column cap of riving, the cotton column cap moisturizing using straw (plastic grip) to clamp subsequently to rive.The line of conversion treated cotton bell different colours pricks cotton boll base portion to show difference, distinguishes and come when being convenient to sowing.

transgenic arabidopsis, cotton plants field test and Molecular Identification

After the Arabidopis thaliana seed that transgenosis is collected is dried, to sterilize 10 m in 75% ethanol (containing 0. 2% polysorbas20)

After, abandon supernatant, after then using dehydrated alcohol rinsing 10 s, be placed on aseptic filter paper and dry up 30 m in, be seeded in MS substratum, after the illumination box of illumination 8 h, dark 16 h cultivates 14 d, the Basta solution spraying 3/1000ths concentration carries out the preliminary evaluation of positive strain.

The qualification of the positive strain of transgene cotton adopts cotyledon period card sodium mycin Rapid identification method.

T0, T1 Basta qualification and the qualification of card sodium mycin all to resistance is selected to carry out PCR detection for Arabidopis thaliana and cotton plants.Goal gene amplification and interference vector fragment amplification and identify sequence see table 2 and table 4, following 94 DEG C of PCR circulating reaction condition, 30s; 64 DEG C, 30s; 68 DEG C, 90s.

the mensuration of transgenic arabidopsis, cotton seed coat brown pigments and fiber brown pigments content

the isolation and determination of transgenic positive strain and offspring's brown pigments is first adopted p-Dimethylaminocinnamaldehyde (DMACA) and is analyzed, respectively seed, fiber to be immersed in 2%DMACA and 3M hydrochloric acid (volume ratio) solution 4 days, more totally to observe under optical microphotograph with the ethanol purge of 70%; The second, measure the Flavonoid Content of material by high performance liquid chromatography (HPLC), concrete operation step is see document ^[11,16].

results and analysis

2. 1 cotton ghTT8Agene clone, sequence information feature

Splice the speculated sequence information obtained according to est sequence, design the encoder block region that primer covers this gene respectively, then obtain aim sequence in conjunction with RT-PCR method, it is consistent that the result that order-checking obtains and EST splice the data obtained, and proves this sequence just ghTT8Agene.From ghTT8A46 bp of gene open reading frame are to 1516 bp, coding 497 amino acid altogether, molecular size range is 53.6 KD, in conjunction with SMART On-line analysis program (http://smart.embl-heidelberg.de/), show to there are continuous 2 structural domains (aminoacid sequence of the middle line that sees the following form) at the 15th to 195 amino acids, between 485 to 561 amino acids, belong to bHLH-MYC_N structural domain and bHLH structural domain.

1 TAGTTGGCTTGGAACCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCCAA

61 TACTGTATCTCCAAAGGGAAAGTTGTGAATTTTGTTTGTGGTTTTTTTTTTAACGTAAAA

121 ATCCAAGCTTTAAATCTGAAAGTTA ATGGCGGCGCCGCCGAGTAGTAGGCTACAAAAAAT

1 M A A P P S S R L Q K M

181 GTTACAGGCAGCGGTGCAATCGGTTCAATGGACGTATAGTCTTTTCTGGCAAATTTGTCC

13 L Q A A V Q S V Q W T Y S L F W Q I C P

241 TCAACAAGGTATCTTAATTTGGTCCGATGGATACTACAATGGAGCAATCAAAACAAGAAA

33 Q Q G I L I W S D G Y Y N G A I K T R K

301 AACAGTGCAACCAATGGAAGTTAGTACAGAAGAAGCATCTTTACAAAGAAGCCAACAACT

53 T V Q P M E V S T E E A S L Q R S Q Q L

361 CCGAGAACTTTACGACTCGTTGTCTTCCGGCGACACCAACCAACCGGCTCGACGACCGTC

73 R E L Y D S L S S G D T N Q P A R R P S

421 TGCCGCGTTGTCGCCGGAAGATTTAACGGAATCCGAATGGTTCTATCTAATGTGTGTCTC

93 A A L S P E D L T E S E W F Y L M C V S

481 CTTCTCTTTTCCCCCTGGTGTAGGGTTACCTGGAGAGGCATATGCAAGGAGGCAACATGT

113 F S F P P G V G L P G E A Y A R R Q H V

541 ATGGCTTACAGGTGCAAACGAAGTTGATAGCAAAACATTTTCAAGAGCTATTCTTGCCAA

133 W L T G A N E V D S K T F S R A I L A K

601 GAGTGCTTGTATACAGACTGTGCTTTGCATTCCTATTCTTGATGGTGTTCTTGAACTTGG

153 S A C I Q T V L C I P I L D G V L E L G

661 ATATACCGAAAAGGTGCAAGAAGATTTAGGGTTAGTCCAGCACGTTAAAACTTTCTTCAA

173 Y T E K V Q E D L G L V Q H V K T F F N

721 CGATGGAAAAGCACCAAACCCTCCGCCACCAAAACCGGCTCTCTCCGAACATTCAACGTC

193 D G K A P N P P P P K P A L S E H S T S

781 GAACCCGGCCGCTTCATCGGACTATACTCGATTTCATTCCCCTTCCGTTCCTCTCGTTTA

213 N P A A S S D Y T R F H S P S V P L V Y

841 CGCAGCAGCGGACCCGCCGGTGAACGCGGACCAAGGAGAGTATAACAACGTTGATGAAGA

233 A A A D P P V N A D Q G E Y N N V D E E

901 GGAGGAGGAAGAAGAAGAAGATGACGACGATGAAGAGGGAGACGAAGAACCCGAATCGTA

253 E E E E E E D D D D E E G D E E P E S Y

961 CTCGGCCGAAACGGGTCGAACCACGCAACAAGTCCCGACGGAAAACACTCTGGCAGTAGT

273 S A E T G R T T Q Q V P T E N T L A V V

1021 GGCGGCGGAGGAGCCGAGCGAGTTAATGCAATTGGATATGTCGGAAGATATCGAGCTCGG

293 A A E E P S E L M Q L D M S E D I E L G

1081 TTCACCTGACGATGCATCGAATAACTTGGACTCGGATTTCAATATGTTGGCGGTGAGTCA

313 S P D D A S N N L D S D F N M L A V S Q

1141 AAGCGGGAACCCAACCGACAATCAACGGCGAGCTGAGTTGTTTAGAGATGAGTCGAATCG

333 S G N P T D N Q R R A E L F R D E S N R

1201 GAGGTGGCAAATGCTACAAGAACCCTCGTGCGGTAGTCTTCAACCACTTTCAACAGGGCC

353 R W Q M L Q E P S C G S L Q P L S T G P

1261 GCAGCCAGTGGACGAAATGTCACAAGAAGACACGCACTATTCACAGACCGTCTCCACTAT

373 Q P V D E M S Q E D T H Y S Q T V S T I

1321 CCTCCAAGCTCAACTGGTCCGGTGGACCGAGTCATCGTCAACCGTCAACGCCGCCACCTA

393 L Q A Q L V R W T E S S S T V N A A T Y

1381 CTCCACCCAATCAGCATTTGCCAAGTGGACAACCCATTCAGACCACCACCTCCACTCCGC

413 S T Q S A F A K W T T H S D H H L H S A

1441 CGTCGTCGAAGGCACGTCACAATGGCTCCTCAAATACATCCTATTCACCGTACCATTCCT

433 V V E G T S Q W L L K Y I L F T V P F L

1501 CCACAACAAATACCACGACGAGAACTCCCCCAAATTACGCGACGCCGCCGTCGCCGCCGC

453 H N K Y H D E N S P K L R D A A V A A A

1561 ACGGTTTCGGAAAGGAACTCCCAACGAAGACCTCAGCGCCAACCACGTCCTGGCGGAGAG

473 R F R K G T P N E D L S A N H V L A E R

1621 ACGGAGGCGCGAAAAGCTCAACGAGCGGTTCATAATATTAAGATCCTTGGTCCCCTTCGT

493 R R R E K L N E R F I I L R S L V P F V

1681 CACTAAAATGGACAAAGCTTCCATCCTCGGCGACACCATCGAGTACGTCAAACAGCTCCG

513 T K M D K A S I L G D T I E Y V K Q L R

1741 TAAAAAGATTCAAGACCTCGAAACCCGAAACAAGCAAATGGAGGCCGATAACGACCGGCC

533 K K I Q D L E T R N K Q M E A D N D R P

1801 GAGATCGGCCGATTCGATGCAGAGAAATAGCAGCTCTAAGGATCAAAGAAGTGGGTTAAC

553 R S A D S M Q R N S S S K D Q R S G L T

1861 GACACGATCATCGGGTCCTGATAAAAAGAAGATGAGGATAGCGGAAGCAACAAAGCAGAA

573 T R S S G P D K K K M R I A E A T K Q K

1921 GACGGTGGAACCTCCACCGCAAACGGCGGAAACGTCGGTCGAAGTGTCGATAATCGAAAG

593 T V E P P P Q T A E T S V E V S I I E S

1981 CGACGCGTTGCTGGAGTTGCAGTGCGGGTACAGAGAAGGGTTGTTGCTTGATATAATGCA

613 D A L L E L Q C G Y R E G L L L D I M Q

2041 GATGTTAAGGGAAAAGCTACGGATCGAGATAACGGCGGTTCAGTCTTCATTGAACAATGG

633 M L R E K L R I E I T A V Q S S L N N G

2101 GTTGTTTACAGCTGAATTAAGAGCCAAGGTGAAGGATAATATGAACGGGAAGAAAGTAAG

653 L F T A E L R A K V K D N M N G K K V S

2161 CATAATGGAGGTAAAGAGGGCGATAAATCAAATAATACCATAATTGTAATATTTGACTAT

673 I M E V K R A I N Q I I P *

Underscore amino acid moiety is 2 conserved domains of this gene, is bHLH-MYC_N structural domain and bHLH structural domain respectively ,=representing the initiator codon position of this gene, * represents the position of terminator codon.

cotton ghTT8Athe phyletic evolution of gene and homology analysis

To cotton ghTT8Athe aminoacid sequence of genes encoding and other species TT8 protein carry out BlastP and Multiple sequence alignments (mutiple alignment) is analyzed, and result shows, cotton ghTT8Agene and other species tT8gene is the same, respectively containing bHLH-MYC_N structural domain and bHLH structural domain, and with cocoa tcTT8, Arabidopis thaliana atTT8,soybean gmTT8, clover mtTT8gene and strawberry fvTT8coded albumen all has certain amino acid sequence similarity, and the sequence especially containing the bHLH domain portion between bHLH-MYC_N and 485--560 amino acid between aminoacid sequence 10--195 is more consistent, shows tT8gene is comparatively conservative in evolution, as shown in Figure 1.

Choose other 6 kinds of species tT8homologous genes and cotton ghTT8agene carries out Multiple sequence alignments together, builds 7 species simultaneously tT8the Phylogenetic tree of homologous genes.As shown in Figure 2, cotton ghTT8agene and cocoa tree tcTT8the most close on evolutionary relationship, amino acid sequence homology reaches 83%, with soybean gmTT8the homology of about 78% is there is between LjTT8 gene in gene and Root or stem of Littleleaf Indianmulberry.

Arabidopis thaliana ( arabidopsis thaliana) atTT8(AJ277509.1), strawberry ( fragaria vesca) tvTT8(XP_004290663), oranges and tangerines ( citrus sinensis) csTT8(XP_006479737.1), soybean ( glycine max) gmTT8(XP_006575074.1), Root or stem of Littleleaf Indianmulberry ( lotus japonicus) ljTT8(BAH28881.1), clover ( medicago truncatula) mtTT8(XP_003590656.1) and cocoa ( theobroma cacao) tcTT8(XP_007050639) tT8homologous gene.

cotton ghTT8a the expression analysis of gene in cotton different developmental phases fiber, seed coat tissue

In order to study ghTT8athe expression pattern of gene in cotton fibre different steps, we adopt the method for real-time fluorescence quantitative RT-PCR to detect the expression of this gene.Select Boll Development to the fibrous tissue of Post flowering 9d, 12d, 15d, 18d, 21d, 24d respectively, extract total serum IgE, cDNA first chain utilizing reverse transcription to synthesize is as the template of fluorescence quantitative PCR detection, and fluorescent quantitation detected result as shown in Figure 3.All can detect in all samples ghTT8Aexpression.In color cotton No. 2 of the cotton Zhejiang of brown fibre, along with the growth of Fibre Development time, ghTT8Aexpression amount all have increasing in various degree, especially after 15DPA, expression amount has and improves rapidly; But in white cotton Zhejiang cotton 11 in cotton fibre, along with the growth of development time, this gene expression amount to present future and increase substantially, and maintain your the low level that compares always.In 18 DPA and 21 DPA periods, ghTT8Aexpression amount in brown fibre is 5 times and 6 times of expression amount in white fiber respectively.Above result tentatively shows: ghTT8Agene presents predominant expression in brown fibre.

2. 4 cottons ghTT8a the structure of gene plant positive sense complementary binary expression vector

In order to verify whether cotton GhTT8a gene participates in the anabolism of cotton seed coat brown pigments, to test with plant binary vector pFGC5941 as skeleton carrier, choosing and contain ghTT8Aone section of sequence of gene whole protein encoder block as the just expression structure of this gene, and is introduced respectively bamHIwith xhoIrestriction enzyme site, building process See Figure 4.Finally by double digestion, PCR qualification and order-checking, show that object fragment is connected in binary expression vector, thus construct and contain ghTT8Athe sense expression vector of gene, name pFGC-GhTT8A.

cotton ghTT8agene plant interferes the structure of expression vector

With plant binary vector pCAMBia2301 for skeleton carrier, choose the expression nuclear structure of plant binary vector pBI121, enzyme is cut, connect and be built into carrier pCAMBIA2301-121, in cotton gene group, one section of sequence is as the middle Loop ring texture building double base interference vector again, and sequence and the primer location of Loop ring texture see the following form.

1 TGAAAAGT AG AAGCGAGATG AGGGATGTGC ATCGCTAAAA TTTCATCACA CAAATTTAAA

61 CAATGTTAAA TAAATATACT GAACATTATA TGGATTATTA TTAGTTATAG TGGTAGTTGA

121 CAGTTGGTTT TTGAAAGCTA TCATGTGCTT TTAATTAGTT TTTTTATATA TTTAAAAAAA

181 ATAATATAAG AATACTTGAT AATTTCTGAA ATTTGCTTGT GCAGTTTTTT TAATTTCATT

241 CTTAATATGC AACTTAATTA TCCTATGTTT AAATACAATT AAATATATTT AATGTATGTT

301 TTGAATGTTG ACATATTTAA TTATATTTAT GAAATTGATA TGAAAATAAT ACGAGACATA

361 GAACACAGTT ACAAATCGTC GAATCTGGGT AATAT CATTT TCATGCGAAG GATTCAAACA

Underlined sequences is the primer sequence of hairpin loop structures.

According to cotton ghTT8abHLH-MYC_N and the bHLH domain region of gene protein sequence, the sequence that selection 2 sections is different is respectively as the positive-sense strand of the interference expression structure of this gene, and the antisense strand of its correspondence is as the reverse complemental structure built, and the just end of the chain is introduced xbaIwith bamHIrestriction enzyme site, anti-sense strand complementary is introduced kpnIwith sacIrestriction enzyme site.Building process is shown in Fig. 5.Finally by incomplete double digestion, PCR qualification and order-checking, show that object fragment is connected in binary expression vector, thus construct the interference expression vector containing 2 different interference structures, respectively called after pGhTT8ARI-A and pGhTT8ARI-B.

cotton ghTT8a gene complementation Arabidopsis Mutants tT8qualification

The sense expression vector pFGC-GhTT8a containing cotton GhTT8A gene built is proceeded to Agrobacterium EHA105, by agriculture bacillus mediated titbit dip method arabidopsis thaliana transformation tT8mutant, and obtain seed.In order to detect cotton ghTT8Agene whether successful conversion tT8mutant, when the arabidopsis thaliana transformation T0 individual plant obtained grows to 3----4 sheet leaf, the Arabidopsis plant that the Basta spray solution with 3/1000ths is growing, after 2 weeks, chooses 5 strains have resistance Arabidopis thaliana individual plant to Basta, with Arabidopsis Mutants tT8the genomic dna of (N5740, http://www.arabidopsis.org/) strain, as template, is detected by PCR reaction.Result shows, 5 transgenic arabidopsis strains all amplify object band (Fig. 6).Show cotton ghTT8Agene successfully proceeds to Arabidopsis Mutants, and Bar gene normal expression.

In order to verify the cotton proceeded to ghTT8Agene whether normal expression, we adopt the method for quantitative fluorescent PCR to detect the expression of this gene.Be expressed as contrast with wildtype Arabidopsis thaliana TT8 gene, select the T1 of above 5 strains for the positive strain of Basta, detect respectively by strain.Arabidopis thaliana tT8cotton in mutant ghTT8Athe expression intensity of gene is shown in Fig. 7.Cotton ghTT8agene success complement Arabidopsis mutant tT85 strain T1 in positive strain, root, stem, leaf and the expression amount difference in spending are not clearly, but the expression amount between strain has notable difference, may be relevant with the expression of composition type expression promoter CaMV35S.

For determining further ghTT8awhether perform the function identical with Arabidopis thaliana TT8, we extract flavonoid compound PAs polymkeric substance from the seed of the positive individual plant results of complementary TT8 mutant, and combined acid catalytic hydrolysis and liquid chromatography mass (LC-MS) analysis measure its content.In strongly-acid and oxicracking condition, the monomer output of PAS pink cyanidin(e) can quantitative analysis easily.Arabidopis thaliana tT8in mutant seeds, insoluble PAs is 5 times of content in wild type seeds, and solubility PAs and insoluble PAs content are more or less the same.Positive individual plant measurement result after complementation shows, solubility PAs is mutant comparatively tT8in content greatly reduce, minimizing ratio is respectively 53%, 73%, 71%, 70% and 83%, and in positive strain, insoluble PAs is 3.81,3.17,4.79,4.27,4.45 times of solubility PAs, more than the results are shown in Figure (data are slightly) shown in 8.Above experimental result shows: cotton ghTT8Agene is formed at cotton seed coat brown pigments and performs critical function, with atTT8gene function is similar.

Cotton ghTT8Agene dsRNAi carrier, in expression process, reduces the color and luster of dark-brown cotton fibre in various degree

According to cotton ghTT8Agene structure, we devise 2 kinds of different double-stranded RNA interference carrier structures respectively, improved method pollen tube passage method is utilized to transform the cotton P158 of nigger-brown fibre, and for positive strain, Molecular Identification has been carried out to T0, confirm that goal gene has turned the brown color cotton of depth (Fig. 9), and transgenosis T0 presents for some individual plants the brown cotton (Figure 10) that depth degree differs.Above experimental result shows cotton ghTT8Agene also participates in the anabolism of cotton fiber brown pigments simultaneously, in fiber brown pigments is grown, play critical function.

. discuss

The present invention utilizes bioinformatic analysis again in conjunction with homologous clone and possible function.According to the EST data that cotton in ncbi database is huge, to the higher est sequence splicing of similarity, integrate thus obtain the aim sequence of initial guess, binding molecule biology correlation technique obtains the real sequence of goal gene again, thus is cloned into goal gene faster.This method is particularly useful for studying the species comparing and further investigate and establish EST storehouse.Up to the present, in ncbi database, only the est sequence of cotton different tissues is more than 310,000, and new cotton est sequence is still in constantly increasing, and making full use of these data messages, is a kind of effective ways obtaining cotton new gene fast.

Containing typical membrane spaning domain in cotton GhTT8A protein sequence.Sequence alignment analysis shows, ghTT8agene with tcTT8agene on gene and other species also exists certain consistence in sequence, but the homology of whole coding region protein reaches 83%, and containing bHLH-MYC_N and bHLH conserved domain.Cotton is the important cash crop of China, fibre crops.In natural liquor storeroom, the height of brown pigments content has influence on again the depth of cotton fibre color and luster, and the precursor substance of brown pigments is proanthocyanidin.This research is from cotton ghTT8Agene is started with, and utilizes different varieties color and luster filamentary material to carry out preliminary study to seed coat, the biosynthesizing of fiber brown pigments, recognizes ghTT8Athe vital role of gene in the synthesis of cotton proanthocyanidin. in the Cotton Fiber of Natural Brown Cotton pigment synthesis metabolism related gene of having cloned, as ghCHS1, ghCHI, ghF3H, ghDFR, ghANS, ghANRdeng, these genes all have higher homology with Arabidopis thaliana seed coat brown pigments metabolic pathway of synthesizing genes involved, but the research participating in cotton seed coat brown pigments function has no report always.

To cotton ghTT8Athe initial analysis of gene in tetraploid cotton fibre shows: this gene expression amount in Cotton Fiber of Natural Brown Cotton is the highest, and minimum in white cotton fiber.This research demonstrates cotton gene ghTT8Anot only participate in the anabolism of cotton seed coat brown pigments but also participate in the anabolism of fiber brown pigments, thus proved conclusions from molecular biology level, also for the anabolism of research cotton fiber brown pigments provides a kind of Research Thinking.Also imply that cotton seed coat brown pigments may share similar pathways metabolism with the synthesis of fiber brown pigments simultaneously.According to above result of study, the brown cotton individual plant that fiber color is deep mixed can be filtered out, be expected to be applied in breeding.

Sequence table

<110> Zhejiang Academy of Agricultural Science; Zhejiang University's agricultural and Biotechnology Institute

<120> cotton GhTT8A gene function and utilize the application of biotechnology in improvement cotton brown fibre color and luster

<160>3

<210> 1

<211> 1654

<212> DNA

<213> cotton

<400> 1

1 TAGTTGGCTT GGAACCCCTC GAGGTCGACG GTATCGATAA GCTTGATATC GAATTCCCAA

61 TACTGTATCT CCAAAGGGAA AGTTGTGAAT TTTGTTTGTG GTTTTTTTTT TAACGTAAAA

121 ATCCAAGCTT TAAATCTGAA AGTTAATGGC GGCGCCGCCG AGTAGTAGGC TACAAAAAAT

181 GTTACAGGCA GCGGTGCAAT CGGTTCAATG GACGTATAGT CTTTTCTGGC AAATTTGTCC

241 TCAACAAGGT ATCTTAATTT GGTCCGATGG ATACTACAAT GGAGCAATCA AAACAAGAAA

301 AACAGTGCAA CCAATGGAAG TTAGTACAGA AGAAGCATCT TTACAAAGAA GCCAACAACT

361 CCGAGAACTT TACGACTCGT TGTCTTCCGG CGACACCAAC CAACCGGCTC GACGACCGTC

421 TGCCGCGTTG TCGCCGGAAG ATTTAACGGA ATCCGAATGG TTCTATCTAA TGTGTGTCTC

481 CTTCTCTTTT CCCCCTGGTG TAGGGTTACC TGGAGAGGCA TATGCAAGGA GGCAACATGT

541 ATGGCTTACA GGTGCAAACG AAGTTGATAG CAAAACATTT TCAAGAGCTA TTCTTGCCAA

601 GAGTGCTTGT ATACAGACTG TGCTTTGCAT TCCTATTCTT GATGGTGTTC TTGAACTTGG

661 ATATACCGAA AAGGTGCAAG AAGATTTAGG GTTAGTCCAG CACGTTAAAA CTTTCTTCAA

721 CGATGGAAAA GCACCAAACC CTCCGCCACC AAAACCGGCT CTCTCCGAAC ATTCAACGTC

781 GAACCCGGCC GCTTCATCGG ACTATACTCG ATTTCATTCC CCTTCCGTTC CTCTCGTTTA

841 CGCAGCAGCG GACCCGCCGG TGAACGCGGA CCAAGGAGAG TATAACAACG TTGATGAAGA

901 GGAGGAGGAA GAAGAAGAAG ATGACGACGA TGAAGAGGGA GACGAAGAAC CCGAATCGTA

961 CTCGGCCGAA ACGGGTCGAA CCACGCAACA AGTCCCGACG GAAAACACTC TGGCAGTAGT

1021 GGCGGCGGAG GAGCCGAGCG AGTTAATGCA ATTGGATATG TCGGAAGATA TCGAGCTCGG

1081 TTCACCTGAC GATGCATCGA ATAACTTGGA CTCGGATTTC AATATGTTGG CGGTGAGTCA

1141 AAGCGGGAAC CCAACCGACA ATCAACGGCG AGCTGAGTTG TTTAGAGATG AGTCGAATCG

1201 GAGGTGGCAA ATGCTACAAG AACCCTCGTG CGGTAGTCTT CAACCACTTT CAACAGGGCC

1261 GCAGCCAGTG GACGAAATGT CACAAGAAGA CACGCACTAT TCACAGACCG TCTCCACTAT

1321 CCTCCAAGCT CAACTGGTCC GGTGGACCGA GTCATCGTCA ACCGTCAACG CCGCCACCTA

1381 CTCCACCCAA TCAGCATTTG CCAAGTGGAC AACCCATTCA GACCACCACC TCCACTCCGC

1441 CGTCGTCGAA GGCACGTCAC AATGGCTCCT CAAATACATC CTATTCACCG TACCATTCCT

1501 CCACAACAAA TACCACGACG AGAACTCCCC CAAATTACGC GACGCCGCCG TCGCCGCCGC

1561 ACGGTTTCGG AAAGGAACTC CCAACGAAGA CCTCAGCGCC AACCACGTCC TGGCGGAGAG

1621 ACGGAGGCGC GAAAAGCTCA ACGAGCGGTT CATAATATTA AGATCCTTGG TCCCCTTCGT

1681 CACTAAAATG GACAAAGCTT CCATCCTCGG CGACACCATC GAGTACGTCA AACAGCTCCG

1741 TAAAAAGATT CAAGACCTCG AAACCCGAAA CAAGCAAATG GAGGCCGATA ACGACCGGCC

1801 GAGATCGGCC GATTCGATGC AGAGAAATAG CAGCTCTAAG GATCAAAGAA GTGGGTTAAC

1861 GACACGATCA TCGGGTCCTG ATAAAAAGAA GATGAGGATA GCGGAAGCAA CAAAGCAGAA

1921 GACGGTGGAA CCTCCACCGC AAACGGCGGA AACGTCGGTC GAAGTGTCGA TAATCGAAAG

1981 CGACGCGTTG CTGGAGTTGC AGTGCGGGTA CAGAGAAGGG TTGTTGCTTG ATATAATGCA

2041 GATGTTAAGG GAAAAGCTAC GGATCGAGAT AACGGCGGTT CAGTCTTCAT TGAACAATGG

2101 GTTGTTTACA GCTGAATTAA GAGCCAAGGT GAAGGATAAT ATGAACGGGA AGAAAGTAAG

2161 CATAATGGAG GTAAAGAGGG CGATAAATCA AATAATACCA TAATTGTAAT ATTTGACTAT

2221 TAATTAATTG ATAATTATTA TATAAGAATA ATTATATAAA TGTTAGACTA GACCCCACCT

<210> 2

<211> 499

<212> amino acid

<213> cotton

<400> 2

1 MAAPPSSRLQ KMLQAAVQSV QWTYSLFWQI CPQQGILIWS DGYYNGAIKT RKTVQPMEVS

61 TEEASLQRSQ QLRELYDSLS SGDTNQPARR PSAALSPEDL TESEWFYLMC VSFSFPPGVG

121 LPGEAYARRQ HVWLTGANEV DSKTFSRAIL AKSACIQTVL CIPILDGVLE LGYTEKVQED

181 LGLVQHVKTF FNDGKAPNPP PPKPALSEHS TSNPAASSDY TRFHSPSVPL VYAAADPPVN

241 ADQGEYNNVD EEEEEEEEDD DDEEGDEEPE SYSAETGRTT QQVPTENTLA VVAAEEPSEL

301 MQLDMSEDIE LGSPDDASNN LDSDFNMLAV SQSGNPTDNQ RRAELFRDES NRRWQMLQEP

361 SCGSLQPLST GPQPVDEMSQ EDTHYSQTVS TILQAQLVRW TESSSTVNAA TYSTQSAFAK

421 WTTHSDHHLH SAVVEGTSQW LLKYILFTVP FLHNKYHDEN SPKLRDAAVA AARFRKGTPN

481 EDLSANHVLA ERRRREKLNE RFIILRSLVP FVTKMDKASI LGDTIEYVKQ LRKKIQDLET

541 RNKQMEADND RPRSADSMQR NSSSKDQRSG LTTRSSGPDK KKMRIAEATK QKTVEPPPQT

601 AETSVEVSII ESDALLELQC GYREGLLLDI MQMLREKLRI EITAVQSSLN NGLFTAELRA

661 KVKDNMNGKK VSIMEVKRAI NQIIP

<210> 3

<211> 420

<212> DNA

<213> cotton

<400> 3

1 TGAAAAGTAG AAGCGAGATG AGGGATGTGC ATCGCTAAAA TTTCATCACA CAAATTTAAA

61 CAATGTTAAA TAAATATACT GAACATTATA TGGATTATTA TTAGTTATAG TGGTAGTTGA

121 CAGTTGGTTT TTGAAAGCTA TCATGTGCTT TTAATTAGTT TTTTTATATA TTTAAAAAAA

181 ATAATATAAG AATACTTGAT AATTTCTGAA ATTTGCTTGT GCAGTTTTTT TAATTTCATT

241 CTTAATATGC AACTTAATTA TCCTATGTTT AAATACAATT AAATATATTT AATGTATGTT

301 TTGAATGTTG ACATATTTAA TTATATTTAT GAAATTGATA TGAAAATAAT ACGAGACATA

361 GAACACAGTT ACAAATCGTC GAATCTGGGT AATATCATTT TCATGCGAAG GATTCAAACA

Claims (8)

1. cotton ghTT8Agene, is characterized in that: the nucleotide sequence of this gene is as shown in SEQ ID:NO.1.

2. the plant positive sense complementary binary expression vector of gene according to claim 1, is characterized in that: this expression vector for skeleton carrier with plant binary vector pFGC5941, is chosen and contained ghTT8Aone section of sequence of gene whole protein encoder block as the just expression structure of this gene, and is introduced respectively bamHIwith xhoIrestriction enzyme site, thus construct and contain ghTT8Athe sense expression vector of gene.

3. a host cell, is characterized in that: this host cell adopts expression vector according to claim 2 to transform.

4. a kind of host cell according to claim 2, is characterized in that: the bacterial strain transformed adopts Agrobacterium EHA105.

5. the double-stranded RNA interference expression vector of gene according to claim 1, it is characterized in that: this expression vector with plant binary vector pCAMBia2301 for skeleton carrier, choose the expression nuclear structure of plant binary vector pBI121, enzyme is cut, connect and be built into carrier pCAMBIA2301-121, again in the cotton gene group shown in SEQ ID:NO.1 one section of sequence as the middle Loop ring texture building double base interference vector, shown in the sequence SEQ ID:NO.3 of Loop ring texture, primer is upstream primer: AGAAGCGAGATGAGGGATGT, downstream primer: CATTT TCATGCGAAGGATTC.

6. a host cell, is characterized in that: this host cell adopts expression vector according to claim 5 to transform.

7. a protein, is characterized in that the aminoacid sequence of this protein is as shown in SEQ ID:NO.2.

8. the application of gene according to claim 1 in improvement cotton brown fibre color and luster.