CN104313032B

CN104313032B - The function of cotton GhTT8A genes and the application using animal nutrition in brown fibre cotton is improved

Info

Publication number: CN104313032B
Application number: CN201410380168.XA
Authority: CN
Inventors: 李付振; 祝水金; 邱新棉; 吴学龙; 王美兴
Original assignee: Zhejiang Academy of Agricultural Sciences; Zhejiang University ZJU
Current assignee: Zhejiang Academy of Agricultural Sciences; Zhejiang University ZJU
Priority date: 2014-08-04
Filing date: 2014-08-04
Publication date: 2017-06-06
Anticipated expiration: 2034-08-04
Also published as: CN104313032A

Abstract

The application in cotton brown fibre color and luster is improved is carried out using cotton transgenic technology the present invention relates to a kind of method that utilization animal nutrition improves cotton brown fibre color and luster, more particularly to a kind of plant binary vector using cotton gene and its structure.One kind is by cottonGhTT8AThe protein of gene code, the amino acid sequence such as SEQ ID of the protein：Shown in NO.2.CottonGhTT8AThe plant positive sense complementary binary expression vector of gene, with plant binary vector pFGC5941 as skeleton carrier, selection contains the expression vectorGhTT8aOne section of sequence of gene whole protein encoder block and is introduced respectively as the just expression structure of the geneBamHIWithXhoIRestriction enzyme site, so as to construct containingGhTT8aThe sense expression vector of gene.

Description

The function of cotton GhTT8A genes is simultaneously improveing brown fibre using animal nutrition Application in cotton

Technical field

The present invention relates to a kind of method that utilization animal nutrition improves cotton brown fibre color and luster, more particularly to one kind Carried out using cotton transgenic technology using the plant binary vector of cotton gene and its structure fine in improvement cotton brown Application in dimension color and luster.

Background technology

Natural color cotton is the specific type cotton that a kind of cotton fiber has natural colour, the Natural color because of it with uniqueness Coloured silk, need not bleaching and dyeing in weaving and process, to the mankind and environmental nonpollution, comfortable and easy to wear, be particularly suitable for making underlinen Thing, is real " ecology ", " environmental protection " cotton, so as to cause domestic and international cotton breeder and weaving, the extensive pass of clothing industry Note, is described as the most potential ecological textile in 21 century international market.Though the research and development starting of Chinese color cotton is relatively late, But have developed rapidly.From 1998 to 2010, Chinese natural color cotton cultivated area was expanded to more than annual 20 ten thousand from annual 10000 mu Mu, lint yield increases between annual more than 20,000 tons, 10 years from annual 800 tons, and color cotton cultivated area and lint yield increase respectively 20 times and 25 times.

Color cotton mainly has brown and the big series color of green two, in terms of color cotton breeding of new variety, by means of routine Some breeding units of breeding technique, countries in the world and China conduct a research and select a collection of color cotton new varieties one after another, one Determine to be improved in degree the quality and proterties of color cotton.The colored cotton variety that domestic each research unit selects has：New coloured silk cotton system Row kind (No. 1 to No. 20), CCRI 51, Zhejiang coloured silk cotton No. 2, the color kind such as miscellaneous No. 1 of Soviet Union.Compared with white cotton, colored cotton variety Still there are problems that following prominent：Fiber strength is not high, and fibre length is partially short, ginning outturn is relatively low, and pigment stability is poor, color It is single, so as to limit application of the brown cotton in textile industry.Therefore, innovate the fiber colour of color cotton and improve brown cotton Fibre strength, fibre length be related to two of China's textile industry and color cotton industry development success or failure it is crucial because Element, is also an important topic that vast cotton breeding researcher faces.

In nature, phytochrome can be divided into photosynthetic pigments (such as chlorophyll) and non-photosynthetic pigments (such as flavonoids and class Carrotene).Research shows, natural colorful cotton fibre pigment belongs to non-photosynthetic pigments, and with the specific period of cotton fiber development It is relevant.Domestic and foreign scholars by Cotton Fiber of Natural Brown Cotton pigment forming process and biochemical character this Quality Research, it was initially believed that in the presence of 2 kinds of suppositions below：(1) Ryser, Xiao and Zhan etc. speculate that thinking that natural liquor storeroom fiber pigment is initially originated is located in vacuole Catechin derivation formed proanthocyanidin (Proanthocyanidins), also referred to as condensed tannin (Condensed Tannins), and aoxidized by condensed tannin ingress of air and form the quinones with brown；(2) Zhao etc. thinks brown cotton Fiber pigment belongs to flavonoids.And, the experiment of Xiao and Zhan all confirms the precursor substance category of Cotton Fiber of Natural Brown Cotton pigment In proanthocyanidin (condensed tannin).Zhan etc. is analyzed the pigment content being distributed in brown cotton, white cotton fiber and kind skin, is ground Study carefully and think：The main cause for determining fiber color is allocation proportion of the pigment between fiber and kind skin.Cotton fiber is by kind of a skin Cell differentiation, the forming process of cotton fiber is in close relations with the development for planting chrotoplast.Arabidopsis, cotton belong to dicotyledonous Plant, understands the anabolism and regulatory pathway of arabidopsis seed coat pigment for disclosing cotton kind skin brown pigment, cotton fiber brown The co-deposition molecule mechanism of element development, fiber and pigment has most important theories meaning.

The metabolic pathway of synthesizing of arabidopsis seed coat pigment is regulated and controled by several genes, belongs to Secondary Metabolism of Plant.Pass through Research to arabidopsis kind skin Transparent testa series mutants shows：Phenylalanine (Phenylpropanoid) is The initial amino acid of condensed tannin is formed, it ultimately forms colourless condensed tannin, then pass through under a series of catalytic action of enzymes Air oxidation and show the brown feature of kind of skin.Wherein, TT4 encodes chalcone synthase (CHS, chalcone synthase), TT5 encodes enzyme, namely chalcone isomerase (CHI, chalcone isomerase) etc. as in structural gene coding flavonoids metabolic pathway Enzyme, the transcription as transcription factor to structural gene such as TT1, TT2, TTG1, TTG2, TT8 regulates and controls, and TT8 and TT19 are then Coding pigment transport protein participates in the transhipment and accumulation of flavonoid substances.In the correlative study of Cotton Fiber of Natural Brown Cotton colour development, Domestic and foreign scholars use different molecular biology methods, and multiple and arabidopsis seed coat pigment anabolism has been cloned from cotton The related homologous gene of approach, such as GhCHS, GhCHI, GhF3H, GhDFR, GhANS, GhANR etc., these genes participate in pigment and close Into protein function speculate be based on homologous sequence analysis and bioinformatics possible function, whether simultaneously participate in cotton actually Seeds of flowering plants skin pigment, the pigment synthesis metabolism of fiber need to be further characterized by.For above-mentioned heavy present in the research of cotton brown pigment Theoretical problem is wanted, as reference, is cloned in cotton related in the urgent need to arabidopsis kind skin brown pigment route of synthesis related gene Homologous gene, and functional verification is subject to by molecular biology and transgenic technology, with clear and definite these related genes whether simultaneously Participate in the synthesis of cotton fiber brown pigment.The research confirmation of above mentioned problem, will further carry out Cotton Fiber of Natural Brown Cotton pigment for us The research of metabolic pathway of synthesizing provides theories integration, and improves Cotton Fiber of Natural Brown Cotton quality by molecular biology method, breaks through cotton Fiber colour Study on Diversity has important theory and practice meaning.

In plant gene function research, transgenic function complementation and gene silencing are the effective technologies for disclosing gene function Means.Have complementary functions and convert the deletion mutant body by building the sense expression vector of genes of interest, so as to verify the base Because of the function of being performed in plant.For the checking of homologous gene function, it would however also be possible to employ the mutant of the approximate species of conversion, So as to be rapidly completed the functional verification of homologous gene., single traits mutant library more long for the cotton transgenic cycle lacks, and adopts The functional verification that homologous gene or similar genes domain are carried out with arabidopsis thaliana transformation mutant is a kind of fast method. Gene silencing is a kind of ancient mechanism of organism, and it by degradation of rna, suppression translation or is modified during growth and development The modes such as chromosome play a role.In recent years, the technology as an important genetic manipulation instrument in molecular biology of plants Research and genetic improvement aspect are used widely, and various plants gene silent technology is set up in succession, including antisense RNA, DsRNAi (double-stranded RNA interference) and artificial microRNA, particularly dsRNAi and microRNA gene silencing accuracy and The aspects such as high efficiency have new breakthrough.DsRNAi and microRNA is in the efficiency of degraded target gene mRNA than antisense RNA skill Art is higher, expresses under less than the concentration of the several orders of magnitude of antisense RNA target gene and drop to extremely low level even function Completely lose；And, in transgenic protocol, in the absence of the problem of insertion point randomness, any gene all can quilt in theory Targetedly dsRNAi or m icroRNA specificity silence.

The content of the invention

In order to clone and understand cotton fiber brown pigment synthesis related gene and and then Cotton Fiber of Natural Brown Cotton quality is changed It is good, the variation of cotton fiber color and luster is realized using means such as biotechnologys, first purpose of the invention is to provide cotton GhTT8A genes and the protein by the gene code, and be experimentally confirmed the gene and both participated in kind of a formation for skin brown pigment, The formation of fiber brown pigment is participated in again, is expected to be applied in brown cotton breeding.Second object of the present invention is to provide and contains There are the expression vector and host cell of above-mentioned gene.Third object of the present invention is to provide above-mentioned gene in improvement cotton Application in brown fibre color and luster.

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

A kind of protein by cotton GhTT8A gene codes, the amino acid sequence such as SEQ ID of the protein：NO.2 institutes Show.

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

The plant positive sense complementary binary expression vector of cotton GhTT8A genes, the expression vector is with plant binary vector PFGC5941 is skeleton carrier, choose one section of sequence containing GhTT8a gene whole protein encoder blocks as the gene just Adopted expression structure, and BamHI and XhoI restriction enzyme sites are introduced respectively, carried so as to construct the justice expression containing GhTT8a genes Body.

A kind of host cell, the host cell is expressed using the plant positive sense complementary double base of described cotton GhTT8A genes Carrier is converted.Preferably, the bacterial strain of described conversion uses Agrobacterium EHA105.Further, described host cell is adopted With osmosis arabidopsis thaliana transformation mutant tt8.

The double-stranded RNA interference expression vector of cotton GhTT8a genes, the expression vector is with plant binary vector PCAMBia2301 is skeleton carrier, chooses the expression nuclear structure of plant binary vector pBI121, and digestion, connection are built into Carrier pCAMBIA2301-121, then with SEQ ID：One section of sequence is interfered as double base is built in cotton gene group shown in NO.1 The middle Loop cyclic structures of carrier, the sequence SEQ ID of Loop cyclic structures：Shown in NO.3, primer is sense primer： AGAAGCGAGATGAGGGATGT, anti-sense primer：CATTTTCATGCGAAGGATTC.

A kind of host cell, the double-stranded RNA interference expression vector of cotton GhTT8a gene of the host cell described in Conversion.

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

Nucleotide sequence such as SEQ ID：The application of gene shown in NO.1 in cotton brown fibre color and luster is improved.

The present invention has been cloned one and new has been contained bHLH-MYC_N using the method for bioinformatic analysis and electronic cloning With the gene of HLH conserved domains, the gene is with arabidopsis kind skin brown pigment synthesis TT8 gene amino acid sequences in conserved structure Region (there is about 81% sequence homology in 10--195 amino acid positions, 480--540 amino acid positions, but in remaining region Sequence homology is not high, and only 60% or so.The gene has been cloned followed by the method for RT-PCR, GhTT8A has been named as, 684 amino acid residues of the gene code, molecular weight about 76.5KD belongs to a member in bHLH-MYC supergene families. GhTT8a genes are analyzed in development in different stages fiber, the expression characteristic of kind skin using quantitative fluorescent PCR, are as a result shown： GhTT8a genes equal predominant expression in the brown fibre of tetraploid cotton, white cotton seed skin and brown cotton seed skin, it is fine in white cotton Hardly expressed in dimension, illustrate that the gene had both participated in kind of a formation for skin brown pigment, the formation of fiber brown pigment is participated in again.For cotton The functional analysis of flower GhTT8a genes, builds plant justice binary expression vector promoter 35S：：GhTT8A：：Nos terminates sublist Up to nuclear structure complement Arabidopsis mutant tt8, Molecular Identification and the analysis of transgenosis physiology show that the gene can make arabidopsis Mutant tt8 recovers wild type phenotype, so as to prove that the gene participates in the synthesis of kind of skin brown pigment；Molecule knot according to the gene Structure and conserved domains characteristic, build 2 kinds of different types of double-stranded RNA interference carriers (dsRNAi, carrier sequence sees appendix), lead to The pollen tube passage method conversion brown cotton damaged is crossed, to 40 transgenic line T of brown cotton₀Generation and T₁The Molecular Identification in generation and The analysis of transgene cotton physiologic index shows that the gene can in various degree reduce the color and luster of cotton brown fibre, so as to demonstrate,prove The bright gene participates in the metabolism of cotton fiber brown pigment.In research process, using GhTT8A genes and by biotechnology side Method obtains 8 parts of the brown fibre cotton germ plasm resource of different depth degrees, and these germplasm materials of innovation are expected in Cotton Fiber of Natural Brown Cotton Applied on quality-improving.

Brief description of the drawings

Fig. 1 compares figure for cotton GhTT8A genes with the amino acid sequence of other species TT8 homologous genes.

Fig. 2 is the amino acid sequence phylogenetic analysis of cotton GhTT8A genes.

Fig. 3 is GhTT8A genes in brown, the expression figure of white cotton (upland cotton) fiber different development stage.

Fig. 4 is the structure figure containing GhTT8A gene plant sense expression vectors.

Fig. 5 is to contain 2 structure flow charts of different structure double base interference vector of GhTT8A genes.

Fig. 6 expands qualification figure by the PCR that cotton GhTT8A genes turn the positive strain that arabidopsis TT8 mutant is obtained；M： DNA marker DL2000,1：Positive plasmid pFGC5941-GhTT8a containing cotton GhTT8A genes；2、3、4、5、6：Spray Spill the resistant arabidopsis individual plants of Basta；7：Wild-type Arabidopsis plants.

Fig. 7 is in turning cotton GhTT8A genes in complement Arabidopsis TT8 mutant positive strains root, stem, leaf, silique and spending Expression figure.

Fig. 8 is to turn cotton GhTT8A genes depth PAs and insoluble in complement Arabidopsis TT8 mutant positive strains PAs containing spirogram.

Fig. 9 is PCR amplification identification of the Partial Fragment in nigger-brown fibre cotton is turned in cotton GhTT8A gene interference vectors Figure A；M：DNA marker DL2000,1：Positive plasmid pFGC5941- containing cotton GhTT8A gene interference vectors GhTT8aRA；2、3、4、5、6、7：The resistant transgenic cotton individual plant of 4/1000ths card sodium mycins of sprinkling；8：Wild type is dark brown Color cotton P158 individual plants；

Fig. 9 B；M：DNA marker DL2000,1：Positive plasmid containing cotton GhTT8A gene interference vectors pFGC5941-GhTT8ARIB；2、3、4、5、6、7：The resistant transgenic cotton individual plant of 4/1000ths card sodium mycins of sprinkling；8： The dark brown color cotton P158 individual plants of wild type..

Figure 10 is the application drawing that cotton GhTT8A interferes dark-brown cotton fiber；ZM11：Non- transgenosis white cotton Zhejiang cotton 11； P158：Dark brown color cotton P158 strains (transgenosis parent plant)；T0A：Dark brown color cotton interferes strain 1；T0B：Dark brown color cotton interferes strain 2； T0C：Dark brown color cotton interferes strain 3；T0D：Dark brown color cotton interferes strain 4；T0E：Dark brown color cotton interferes strain 5；T0F：Dark brown color cotton interference Strain 6.

Specific embodiment

1. materials and methods

1.1 vegetable materials and growth conditions

The color cotton No. 2 in upland cotton brown fibre Zhejiang, white fiber cotton Zhejiang cotton 11, nigger-brown fibre cotton P158 are by Zhejiang Province Academy of Agricultural Sciences crop and industrial crops research department of nuclear technology research on utilization institute provide and live at the beginning of 5 months 2011 are seeded in this Hangzhou academy proper of institute experimental field.9d (Post flowering, DPA, Day Post Anthesis), 12d, 15d in cotton Post flowering, 18d, 21d and 24d, choose the cotton fiber tissue and seed coat tissue development correlative study developed respectively.

1.2 experimental techniques

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

Cotton genomic dna is extracted and uses CTAB methods.The side provided using polyphenol, polysaccharide plant RNA extraction kit Method extracts cotton fiber tissue, seed coat tissue total serum IgE, and kit is purchased from the Tyke Bioteck Bioisystech Co., Ltd of Beijing hundred.

1.2.2 cotton GhTT8A genes are screened and cloned using bioinformatic analysis

According to the cotton brown fibre pigment GhTT8A genes and arabidopsis kind skin brown pigment AtTT8 genes cloned Protein conserved domain sequence, search cotton ESTs databases (http://blast.ncbi.nlm.nih.gov/), choosing altogether Go out 3 sequences (GenBank accession number is ES825879.1, DT551422.1, ES796809.1).Searched with this 3 est sequences again Rope ESTs databases simultaneously splice, and finally obtain a sequence, and the sequence contains entire open reading frame (ORF, an Opening Reading Frame), the protein predicted according to sequence ORF exists about with arabidopsis TT8 albumen in conserved domain part 82% homology.According to the sequence signature with ORF, 1 couple of special primer (P1 of PCR amplifications of design: TCTCCAAAGGGAAAGTTGTG and P2:TTATGGTATTATTTGATTTATCGCCCTCTTTACC).

The chains of cDNA first with cotton fiber tissue's total serum IgE reverse transcription acquisition of No. 2 development 15DPA of Zhejiang coloured silk cotton are as mould respectively Plate, with reference to round pcr, amplification condition is:94 DEG C of predegeneration 4min；94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 68 DEG C of extensions 2min；30 PCR cycles, last 68 DEG C of extensions 10min, 15 DEG C of insulations.Amplification system is 50ul：2X KOD amplification reaction solutions, 10ul dNTP, 2ul P1 primers, 2ul P2 primers, product, 5ul ultra-pure waters after 1ul KOD FX, 5ul transcriptions；PCR is produced again Thing adds A, plus A reaction systems are 10ul：5ul connects buffer solution, and 1ul pTA2 carriers, 1ul ligases, 1ul adds A to connect liquid, 2ul KOD amplified productions.Conversion E. coli competent TG1, screening positive clone send Nanjing Genscript Biotechnology Co., Ltd. to survey Sequence.Reverse transcription reagent box using MBI companies synthesizes the chains of cDNA first, and the GhTT8a bases for speculating are obtained using KOD FX enzymatic amplifications Because of (bio tech ltd is spun by TOYOBO, Shanghai, Japan).The involved primer of this experiment synthesizes from Shanghai gives birth to work Biotechnology Engineering Service Co., Ltd.

1.2.3 design of primers and bioinformatic analysis

The design of primers being related in research using Primer Premier 5.0 (http:// www.premierbiosoft.com/index.html) complete, using the softwares of DNAman 6.0 (http:// www.lynnon.com) tetraploid rice and nucleic acid Phylogenetic analysis, protein conserved structure are carried out to amino acid sequence The supposition in domain using in Genbank databases Blast on-line analysis systems (http:// blast.ncbi.nlm.nih.gov), specific domain position analysis using SMART online softwares analysis (http:// smart.embl-heidelberg.de)。

1.2.4 the expression characteristic of GhTT8a genes in dliploid cotton and four times of cottons is analyzed using quantitative fluorescent PCR

The cDNA of fibr tissue, seed coat tissue according to cotton different development stage, uses real-time fluorescence quantitative RT-PCR skill Art detects relative expression levels of the GhTT8a genes in development in different stages fiber, seed coat tissue.Cotton GBQ7 genes are used as The reference gene (amplimer) of quantitative fluorescent PCR, the specificity amplification primer of GhTT8a genes is shown in Table 2.Experimental implementation is joined Kit specification (bio tech ltd is spun by TOYOBO SYBR Green Supermixture, Shanghai, Japan) is examined, it is glimmering The temperature cycles of Fluorescent Quantitative PCR：50 DEG C, 1min；94 DEG C of predegeneration 3min；94 DEG C of denaturation 30s；56 DEG C of annealing 30s；72 DEG C of extensions 45s；30 PCR cycles altogether.

Primer used and sequence in the quantitative fluorescent PCR of table 1

1.2.5 the complementation test of the structure of cotton GhTT8a genes sense expression vector and Arabidopsis Mutants TT8

The GhTT8A gene cDNA sequences obtained according to sequencing and the entire protein coding sequence for determining the gene.Due to Arabidopsis TT8 mutant is inserted by T-DNA and produced, therefore, we select plant to express double carrier-free pFGC5941 as this The positive sense complementary carrier of gene, restriction enzyme site XhoI is introduced in initiating terminal, and restriction enzyme site BamHI is introduced in clearing end (GhTT8aP1：

CCGCTCGAGTCTCCAAAGGGAAAGTTGTG；GhTT8aP2：

CGCGGATCCTTATGGTATTATTTGATTTATCGCCCTCTTTACC), so as to build complete just gene table Up to structure.

Using osmosis arabidopsis thaliana transformation mutant TT8.Recombinant plasmid Agrobacterium will be contained bacterium will be shaken at 28 DEG C overnight to OD 600 values are between 1.0-2.0,8 000rpm centrifugation 8min abandon supernatant collects thalline.Precipitation is 50g/ L with containing mass concentration Sucrose, pH value 5.7 MS culture mediums it is resuspended to the values of OD 600 about 0.5, and 0.02%Silwet70 is added, after fully mixing Arabidopsis floral is immersed, bacterium solution is stirred every 2min, taken out after 10min and kept flat and moisturizing 12--24 hours, 4 Zhou Houshou The seed of arabidopsis TT8 after collection conversion.

1.2.6 the structure of cotton GhTT8A genes double-stranded RNA interference carrier

The hairpin ring structure that one section of sequence in cotton gene group is designed as interference vector, this section of sequence sees appendix 4, amplimer INA, INB introduce restriction enzyme site BamHI, SacI and KpnI in hair clip Loop ring design.Cotton GhTT8a bases Just end primer the restriction enzyme site XbaI, BamHI of the different interference structures of 2 kinds of cause；Antisense end introduce restriction enzyme site SacI and KpnI, so as to build complete hairpin ring structure, the primer sequence information being related to is shown in Table 2.The amplification of Loop ring structures is with cotton Flower genomic DNA is template, the GhTT8a gene PCR products that the amplification of genetic fragment is obtained with reverse transcription in interference vector structure It is template, purpose fragment (bio tech ltd is spun by TOYOBO, Shanghai, Japan) is obtained using KOD FX enzymatic amplifications.

Primer used and sequence in the quantitative fluorescent PCR of table 2

1.2.7 improved method pollen tube passage method converting cotton

Treatment containing the purposeful fresh bacterium solution of interference vector Agrobacterium LBA4404：Recombinant plasmid Agrobacterium will be contained at 28 DEG C Bacterium is shaken overnight to the values of OD 600 between 1.0-2.0,8 000rpm centrifugation 8min abandon supernatant collects thalline.Precipitation is with containing quality Concentration is the sucrose of 50g/L, the MS culture mediums of pH value 5.7 are resuspended to the values of OD 600 about 0.5, and adds 0.02% Silwet70, is used immediately after fully mixing, and bacterium solution is stirred every 2min.

The operation of improved method pollen tube passage method converting cotton：I.e. between cotton blooms pollination that afternoon 3--6 point, Cotton column cap is rived with blade, is dipped with very small cotton mass and soaked by processing and the agriculture bar containing purposeful carrier The fresh bacterium solution of bacterium LBA4404, then grips the cotton mass containing bacterium solution and is clipped in the column cap rived with tweezers, then uses straw (plastic grip) clamps the cotton column cap and moisturizing rived.The line of conversion treated cotton bell different colours pricks cotton boll base portion To show difference, it is easy to be distinguished during sowing.

1.2.8 transgenic arabidopsis, cotton plants field test and Molecular Identification

After the arabidopsis seed that transgenosis is collected is dried, with 75% ethanol (contain 0.2% polysorbas20) sterilization 10min after, Supernatant is abandoned, after then rinsing 10s with absolute ethyl alcohol, is placed in and 30min is dried up on aseptic filter paper, be seeded in MS culture mediums, Yu Guang After 8h, the illumination box culture 14d of dark 16h, the Basta solution for spraying 3/1000ths concentration carries out the preliminary of positive strain Identification.

The identification of transgene cotton positive strain uses cotyledon period card sodium mycin Rapid identification method.

Select T0, T1 for being respectively provided with resistance to Basta identifications and the identification of card sodium mycin is carried out for arabidopsis and cotton plants PCR is detected.Genes of interest is expanded with interference vector fragment amplification and identification sequence referring to table 2, PCR cycle reaction condition following 94 DEG C, 30s；64 DEG C, 30s；68 DEG C, 90s.

1.2.9 the measure of transgenic arabidopsis, cotton kind skin brown pigment and fiber brown cellulose content

P-Dimethylaminocinnamaldehyde is first adopted in transgenic positive strain and the extraction of offspring's brown pigment and detection (DMACA) analyze, seed, fiber are immersed in 4 days in 2%DMACA and 3M hydrochloric acid (volume ratio) solution respectively, then with 70% Ethanol is cleaned up to be observed under optical microphotograph；Second, the Flavonoid Content of material is determined with high performance liquid chromatography (HPLC), Concrete operation step is referring to document^[11,16]。

2. result and analysis

2.1 cotton GhTT8A gene clonings, sequence information feature

According to the speculated sequence information that est sequence splicing is obtained, the encoder block region that primer covers the gene is separately designed, Aim sequence is obtained in conjunction with RT-PCR method, the result that sequencing is obtained is consistent with the data that EST splicings are obtained, it was demonstrated that the sequence Row exactly GhTT8A genes.From 46bp to the 1516bp of GhTT8A gene open reading frames, 497 amino acid, molecule are encoded altogether Amount size is 53.6KD, with reference to SMART On-line analysis programs (http://smart.embl-heidelberg.de/), show 15th (see the table below middle line to 195 amino acids, 485 to there is continuous 2 domains between 561 amino acids Amino acid sequence), belong to bHLH-MYC_N domains and bHLH domains.

Underscore amino acid moiety is 2 conserved domains of the gene, is respectively bHLH-MYC_N domains and bHLH Domain ,=the initiation codon position of the gene is represented, * represents the position of terminator codon.

The phyletic evolution and homology analysis of 2.2 cotton GhTT8A genes

Amino acid sequence and other species TT8 protein to cotton GhTT8A gene codes carry out BlastP and many repeated orders Row compare (mutiple alignment) analysis, as a result show, cotton GhTT8A genes divide as other species TT8 genes Han You not bHLH-MYC_N domains and bHLH domains, and TcTT8 with cocoa, the AtTT8 of arabidopsis, soybean Albumen coded by GmTT8, the MtTT8 genes of clover and the FvTT8 of strawberry is respectively provided with certain amino acid sequence similarity, especially It contains the bHLH domain portions between bHLH-MYC_N and 485--560 amino acid between amino acid sequence 10--195 Sequence is more consistent, shows that TT8 genes are more guarded in evolution, as shown in Figure 1.

The TT8 homologous geneses for choosing other 6 kinds of species carry out Multiple sequence alignments together with cotton GhTT8a genes, together When build 7 Phylogenetic trees of species TT8 homologous geneses.As shown in Fig. 2 cotton GhTT8a genes and cocoa chocolate tree TcTT8 is closest on evolutionary relationship, and amino acid sequence homology reaches 83%, in soybean GmTT8 genes and crowtoe LjTT8 genes between exist about 78% homology.

Arabidopsis (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), crowtoe (Lotus japonicus) LjTT8 (BAH28881.1), clover (Medicago truncatula) MtTT8 (XP_003590656.1) and cocoa (Theobroma cacao) TcTT8 (XP_ 007050639) TT8 homologous genes.

Expression analysis of the 2.3 cotton GhTT8a genes in cotton different developmental phases fiber, seed coat tissue

In order to study expression pattern of the GhTT8a genes in cotton fiber different phase, we use real time fluorescent quantitative The method of RT-PCR detects the expression of the gene.Boll Development to Post flowering 9d, 12d, 15d, 18d, 21d, 24d is selected respectively Fibr tissue, extracts total serum IgE, by the use of reverse transcription synthesis the chains of cDNA first as fluorescence quantitative PCR detection template, fluorescence is fixed Amount testing result is as shown in Figure 3.The expression of GhTT8A is able to detect that in all samples.In brown fibre cotton Zhejiang coloured silk cotton 2 In number, with the growth of Fibre Development time, the expression quantity of GhTT8A have it is different degrees of increase, especially after 15DPA, table There is rapidly raising up to amount；But in cotton fiber in white cotton Zhejiang cotton 11, with the growth of development time, the expression of the gene The following presentation of amount increases substantially, and maintains your the low level that compares always.In 18DPA and 21DPA periods, GhTT8A Expression quantity in brown fibre is respectively 5 times and 6 times of expression quantity in white fiber.Result above tentatively shows：GhTT8A Gene is presented predominant expression in brown fibre.

The structure of 2.4 cotton GhTT8a gene plant positive sense complementary binary expression vectors

In order to verify whether cotton GhTT8a genes participate in the anabolism of cotton kind skin brown pigment, experiment is expressed with plant Binary vector pFGC5941 is skeleton carrier, chooses one section of sequence containing GhTT8A gene whole protein encoder blocks as this The just expression structure of gene, and BamHI and XhoI restriction enzyme sites are introduced respectively, building process is shown in Fig. 4.Most afterwards through double digestion, PCR is identified and is sequenced, in showing that purpose fragment has been connected to binary expression vector, so as to construct containing GhTT8A genes just Adopted expression vector, names pFGC-GhTT8A.

2.5 cotton GhTT8a gene plants interfere the structure of expression vector

With plant binary vector pCAMBia2301 as skeleton carrier, choose plant binary vector pBI121's Expression nuclear structure, digestion, connection are built into carrier pCAMBIA2301-121, then using one section of sequence in cotton gene group as structure The middle Loop cyclic structures of double base interference vector are built, the sequence and primer location of Loop cyclic structures see the table below.

Underlined sequences are the primer sequence of hairpin loop structures.

According to bHLH-MYC_N the and bHLH domain regions of cotton GhTT8a gene protein sequences, 2 sections are selected respectively , as the positive-sense strand of the interference expression structure of the gene, its corresponding antisense strand is as the reverse complemental for building for different sequences Structure, the just end of the chain introduces XbaI and BamHI restriction enzyme sites, and anti-sense strand complementary introduces KpnI and SacI restriction enzyme sites.Built Journey is shown in Fig. 5.Most identify and be sequenced through incomplete double digestion, PCR afterwards, in showing that purpose fragment has been connected to binary expression vector, So as to construct containing 2 interference expression vectors of different interference structures, pGhTT8ARI-A and pGhTT8ARI- is respectively designated as B。

The identification of 2.6 cotton GhTT8a gene complementation Arabidopsis Mutants TT8

The sense expression vector pFGC-GhTT8a containing cotton GhTT8A genes for building is transferred to Agrobacterium EHA105, By agriculture bacillus mediated titbit dip method arabidopsis thaliana transformation TT8 mutant, and obtain seed.In order to detect cotton GhTT8A Gene whether successful conversion TT8 mutant, obtain arabidopsis thaliana transformation T0 individual plants grow to 3----4 piece leaves when, with thousand points Three the Arabidopsis plant that is growing of Basta spray solutions, after 2 weeks, choose 5 plants of arabidopsis resistant to Basta Individual plant, with Arabidopsis Mutants TT8 (N5740, http://www.arabidopsis.org/) strain genomic DNA conduct Template, is reacted by PCR and is detected.Result shows that 5 transgenic arabidopsis strains all amplify purpose band (Fig. 6).Table Bright cotton GhTT8A genes are successfully transferred to Arabidopsis Mutants, and Bar gene normal expressions.

In order to verify the cotton GhTT8A genes being transferred to whether normal expression, we using the method for quantitative fluorescent PCR come Detect the expression of the gene.Control is expressed as with wildtype Arabidopsis thaliana TT8 genes, more than selection 5 T1 of strain are for Basta Positive strain, is detected respectively by strain.The expression intensity of cotton GhTT8A genes is shown in Fig. 7 in arabidopsis TT8 mutant.Cotton In 5 plants of T1 generations positive strain of GhTT8a genes success complement Arabidopsis mutant TT8, root, stem, leaf and the expression quantity in spending are poor Different is not that clearly, but expression quantity between strain has notable difference, it may be possible to composition type expression promoter CaMV35S's Expression is relevant.

For further determine that GhTT8a whether perform with arabidopsis TT8 identical functions, we are from complementary TT8 mutant Flavone compound PAs polymer is extracted in the seed that positive individual plant is harvested, with reference to acid-catalyzed hydrolysis and liquid chromatography mass (LC-MS) analysis determines its content.In highly acid and oxicracking condition, the monomer yield of PAS pink anthocyanidin can be very Easily quantitative analysis.Insoluble PAs is 5 times of content in wild type seeds in arabidopsis TT8 mutant seeds, and soluble PAs is more or less the same with insoluble PAs contents.Positive individual plant measurement result after complementation shows that soluble PAs is compared with mutant TT8 In content greatly reduced, reduction ratio is respectively 53%, 73%, 71%, 70% and 83%, insoluble PAs in positive strain It is 3.81,3.17,4.79,4.27,4.45 times of soluble PAs, result above is as shown in Figure 8 (data summary).Experiment above is tied Fruit shows：Cotton GhTT8A genes are formed in cotton kind skin brown pigment and perform critical function, similar with AtTT8 gene functions.

2.7 cotton GhTT8A gene dsRNAi carriers reduce dark-brown cotton fiber during expression, in various degree Color and luster

According to the gene structure of cotton GhTT8A, we have separately designed 2 kinds of different double-stranded RNA interference carrier structures, Nigger-brown fibre cotton P158 is converted using improved method pollen tube passage method, and Molecular Identification has been carried out for positive strain to T0, confirmed Genes of interest has rotated into dark-brown color cotton (Fig. 9), and transgenosis T0 is presented the brown that depth degree differs for some individual plants Cotton (Figure 10).Above test result indicate that cotton GhTT8A genes also assist in the anabolism of cotton fiber brown pigment simultaneously, Critical function is played in the development of fiber brown pigment.

3. discuss

The present invention is using bioinformatic analysis in conjunction with homologous clone and possible function.According to cotton in ncbi database Huge EST data, the est sequence higher to similitude splices, integrates so as to obtain the aim sequence of initial guess, in conjunction with Molecular biology correlation technique obtains the real sequence of genes of interest, so as to be cloned into genes of interest faster.This method is outstanding It is applied to and compare further investigation and establish the species in EST storehouses having studied.Up to the present, in ncbi database, only cotton Spend more than the 310000 and new cotton est sequence of the est sequence of different tissues still in being continuously increased, make full use of These data messages, are a kind of effective ways for quickly obtaining cotton new gene.

Contain typical membrane spaning domain in cotton GhTT8A protein sequences.Sequence alignment analysis show, GhTT8a genes Certain uniformity is there is in sequence with the gene on TcTT8a genes and other species, but whole code area protein Homology contains bHLH-MYC_N and bHLH conserved domains up to 83%.Cotton is the important industrial crops of China, fiber Crop.The height of brown cellulose content has influence on the depth of cotton fiber color and luster again in natural liquor storeroom, and the precursor substance of brown pigment is Proanthocyanidin.This research is started with from cotton GhTT8A genes, using different cultivars color and luster fibrous material to kind of a skin, fiber brown pigment Biosynthesis has carried out Primary Study, it is thus understood that important function of the GhTT8A genes in the synthesis of cotton proanthocyanidin is In the Cotton Fiber of Natural Brown Cotton pigment synthesis metabolism related gene of clone, such as GhCHS1, GhCHI, GhF3H, GhDFR, GhANS, GhANR Have with arabidopsis kind skin brown pigment metabolic pathway of synthesizing related gene and compare homology high Deng, these genes, but participation The research of cotton kind skin brown pigment function has no report always.

Initial analysis of the cotton GhTT8A genes in tetraploid cotton fiber is shown：Gene table in Cotton Fiber of Natural Brown Cotton It is and minimum in white cotton fiber up to amount highest.This research demonstrates cotton gene GhTT8A and had both participated in cotton kind skin brown The anabolism of element participates in the anabolism of fiber brown pigment again, so as to prove conclusions from molecular biology level, For the anabolism for studying cotton fiber brown pigment provides a kind of Research Thinking.Cotton kind skin brown pigment and fibre are also implied simultaneously The synthesis for tieing up brown pigment may share similar metabolic pathway.According to above result of study, the fiber color depth can be filtered out not One brown cotton individual plant, is 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 functions and the application using biotechnology in cotton brown fibre color and luster is improved

<160>3

<210> 1

<211> 2280

<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> 685

<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

1. cottonGhTT8AGene, it is characterised in that：The nucleotide sequence of the gene such as SEQ ID：Shown in NO.1.

2. a kind of protein, it is characterised in that the amino acid sequence of the protein such as SEQ ID：Shown in NO.2.

3. application of the gene described in claim 1 in cotton brown fibre color and luster is improved.