CN1478791A - Beta tubulin of cotton fiber and its coding gene and application - Google Patents

Abstract

A para-beta-microtubulin Gh-BtubL of cotton fibre, its coding gene and its application are disclosed. Said Gh-BtubL is the protein with the amino acid residue sequence of SEQ ID No.2. Its coding gene is one of the DNA sequence of SEQ ID No.1 and the DNA sequence, which has 85% or more homology to the DNA sequence limited by SEQ ID No.1 and can code the protein with same functions.

Description

The beta tubulin of cotton fiber and encoding gene thereof and application

Technical field

The present invention relates to vegetable-protein and encoding gene thereof and application, particularly relate to beta tubulin and the encoding gene and the application of cotton fiber.

Background technology

Cotton is one of most important cash crop in the world, is primary fibre crops and important oil crops.The growth of cotton fiber occurs with unicellular form, and metamorphosis is big, and its length/diameter can reach 1000-3000.Aspect RESEARCH ON CELL-BIOLOGY, sophisticated cotton fiber secondary cell wall almost is made up of pure Mierocrystalline cellulose, can get rid of the cell fission of complexity and the interference that many cells are grown, and is that research carbon participates in the synthetic ideal material that reaches cell elongation of Mierocrystalline cellulose.Except the meaning of studying basic cytobiology aspect, illustrate cell and molecule mechanism that fiber is grown, can find target gene----the cotton fiber output of genetic engineering improvement cotton fibre and the basic factor of determination of quality, have huge potential economic worth.

1992, John and Crow clone also analyzed first fiber specific expression gene E6, had started the upsurge of clone's fiber specific gene.The E6 transcripton can both detect in the whole process that fiber is grown.The mRNA of E6 and protein concentration are synthetic the highest in early days in synthetic late period of primary wall and secondary wall, do not find known protokaryon or eukaryotic gene with the E6 dna homolog.Yet, change inverted defined gene in the cotton [John, 1996], do not find that the growth of fiber and quality have obvious phenotypes to change, and show that the E6 gene is not vital for the normal development and the structural integrity of fiber.

Nineteen ninety-five, Delmer etc. are cloned into the special gtp binding protein gene Rac13 of cotton fibre, efficiently express in the fiber of growing, and the highest to the synthetic transition period expression amount of secondary cell wall by elongating stage, this moment, cytoskeleton just reorganized.Therefore Rac13 may play a role in the signal transmission of cytoskeletal organization.

Nineteen ninety-five, John[1995] be cloned into a fiber cance high-expression gene B6, long 1.2kb, it all has expression at primary wall and secondary wall synthesis phase, and albumen has a hydrophobic N-terminal and two zones of being rich in Pro.And obtained upstream 2.3kb promoter region, can guide the transient expression of GUS.But the function of this gene is not quite clear.

Nineteen ninety-five, John and Keller[1995] be cloned into the gene H6 of fiber specifically expressing, be rich in Pro in the albumen, there is 17 pentaamino primitives (motif): Ala (Ser)-Thr (Ser)-Pro-Pro-Pro.Its mRNA forms early stage gathering at primary wall, is to assemble when beginning the secondary wall synthesis phase but its albumen forms late period at primary wall, shows that may there be the adjusting of translation skill in H6 and plays a role in the secondary wall assembling.Infer that from its amino acid composition and solubility it may belong to arabinogalactan-proteins (AGP), embeds growth and the framework that composition participates in the cotton fiber secondary wall as cytolemma.

Nineteen ninety-five, Ma etc. are cloned into GH3, and its fat transfer protein (LTP) of encoding may be positioned the fibrocyte adventitia.The GH3 gene is subjected to developmental regulation, elongating stage expression amount the highest, may participate in transmembrane transport chitin monomer, promote chitin synthetic.

1996, Reinhart etc. were cloned into the gene FbL2A of cotton fibre specifically expressing.Its expression is subjected to developmental regulation, in synthetic late period of primary wall with secondary wall is synthetic is activated in early days.The albumen of a 43.4KD of this genes encoding, except that the hydrophobic highly-hydrophilic of N end, 62% is made up of repeating group unit, one 55 amino acid whose regional repetition is arranged 4 times more than.The function of this albumen in fibrocyte is not quite clear.

1997, Ma etc. were cloned into fat transfer protein Ltp6 gene, with the amino acid identity of GH3 be 64%.Also expression amount is the highest elongating stage in fibrocyte for Ltp6, and just expression level is low.1999, Hsu etc. were separated to the promotor of Ltp6, and the Ltp6 promotor has tissue expression specificity.

1997, Oxford and Timmis were cloned into fat transfer protein FS6 (LTP) and and are rich in Pro albumen (PRP) FS17, and the both grows early stage high expression level at fiber.The signal peptide sequence that exists shows that LTP and PRP are positioned in the extracellular matrix of fiber.FS6 and the cotton LTP GH3[Ma et al. that found in the past, 1995] amino acid identity is 88%, but both hydrophobicity difference are very big.FS17 has the function of strengthening cell walls, it is connected with the structure of cell walls complexity, be rich in FS17 in the fibrocyte of elongation fast, may be relevant with the lasting supply of this growth needs wall material, exist typical PRP to repeat primitive (motif) PPVYK, with former report be rich in Pro albumen H6[John and Keller, 1995] do not have a remarkable similarity.The protein B 6[John of the special unknown function of another fiber, 1995], two zones of being rich in Pro are arranged, but these two zones all with FS17 in the tumor-necrosis factor glycoproteins dissmilarity.

1997, Song and Allen were cloned into the gene acyl carrier protein (ACP) of cotton fibre specifically expressing.Northern blot shows that this gene efficiently expressed in the elongate fiber phase, Southern blot analyzes the ACP that finds single copy and may exist in cotton A and D genome, during evolution, a member specifically expressing of ACP gene family becomes fiber, may be the synthetic film fat of the fiber of elongation rapidly.

1998, Kawai etc. were cloned into GhCAP (adenyl cyclase-associated) gene.The reading frame of GhCAP coding 471aa.Northern blot shows that GhCAP mainly expresses in the elongate fiber phase.GhCAP works in the microfilament tissue, may play the signal transfer function in the cytoskeleton reorganization.

1998, Oxford and Timmis were cloned into pGhEX1.Its expansin that encodes.This gene is only expressed in cotton fibre, is subjected to developmental regulation in elongating stage, and is promptly the highest during 16DPA, descends afterwards.Studies show that it may be relevant with the growth that the vegetable cell turgescence drives, find that also cotton fiber cell genetic expression is regulated and control simultaneously on transcriptional level.

1999, Loguercio etc. were cloned into and severally may and be grown relevant myb genoid with cotton fiber differentiation.6 expression characterizations of cotton myb genoid (GhMYB) in allotrtraploid upland cotton that belong to R2R3-MYB family have been studied.Active several structural domains of GhMYB and primitive in trans regulatory region, have been found to regulate.One is 40 the amino acid whose alkalescence zones (TRR1) that are positioned at DNA binding domains (DBD) downstream just that are present among 5 GhMYB.And, be present in the same position that conservative primitive GIDxxH among the class plant MYB also is present in GhMYB1 and GhMYB6TRR1 structural domain.(5 '-uORFs) has not qualitative reading frame at least two GhMYBs (GhMYB4 and GhMYB5), and it is proteic synthetic to regulate these GhMYB in translation skill at 5 ' leader sequence.The existence of a plurality of DNA binding domainss (DBD) shows that the synthetic relevant plant R2R3-MYB of GhMYB and phenylpropionic acid has structural similarity.GhMYB5 and cotton R2R3-MYB relation is in an isolated gene cluster together with drought-induced AtMYBG11 farthest.Except conservative primitive, sequential analysis does not find that GhMYB, MIXTA (AmMYBMx) and G11 (AtMYBG11) have other similaritys at DBD and TRR.Yet evolutionary tree analysis finds that GhMYB2, GhMYB3 and GhMYB4 belong to the gene cluster that comprises GLABROUS1, and GhMYB1 and GhMYB6 belong to close gene cluster.Sxemiquantitative RT-PCR analyzes demonstration, and GhMYB genetic expression has two different types: a type GhMYB (GhMYB-1 ,-2 and-3) transcripton has in a organized way in institute, and than two type abundance height; Two type GhMYB (GhMYB-4 ,-5 and-6) high expression level in fiber.The developmental regulation that GhMYB expresses is consistent with the function of these dna binding factors in fiber differentiation and elongation.

1999, Oxford etc. were cloned into a full length cDNA clone FS18, and it is 71 the amino acid whose albumen of only encoding, and with LTP some similaritys is arranged, and function is not quite clear.This gene high expression level in fiber, especially outstanding is it the synthetic beginning of secondary wall and after (18-24DPA) expression amount the highest.

Calendar year 2001, Cui etc. are cloned into new gene C FL1, this gene and FKS1 yeast β-1,3-dextran (callose) synthase catalytic subunit homology.CFL1 is at fiber primary wall synthesis phase high expression level, and its expression amount in young root is also higher.Sequential analysis and CaM-gel overlay assay infer has a CaM binding site at its hydrophilic N-terminal.Product-entrapment assay shows that this albumen can combine with external synthetic callose precipitation.CFL1 is yeast β-1, and the plant homologue of 3-glucan synthase catalytic subunit FKS1 may work in callose is synthetic.

Calendar year 2001, Zhao etc. are cloned into 10 special cDNA of fiber.Wherein 5 encode respectively bisphosphate nucleic acid enzyme (bisphosphate nucleotidase), α-tubulin, the sweet enzyme of β semi-lactosi, symphysis albumen (annexin) and reversible glycosylated polypeptide; 5 functions are uncertain in addition.These 10 cDNA efficiently express at fibrocyte, most early stage accumulations of growing at fiber, and except F14, it is the highest at fiber growth expression amount in late period.

Calendar year 2001, Tan etc. have cloned the gene of a specifically expressing in cotton fiber and organization of root tips: ghprp1 (proline-rich proteins), and the protein of 299 amino residue of this genes encoding has a hydrophobic signal peptide at this proteic N end.This gene is present in the A1 of cotton gene group, and A2 is on D1 and the D5 karyomit(e).

2002, Zhao etc. are cloned into GhRGP1 (reversibly glycosylated polypeptide) gene from cotton fiber, the protein of 359 amino-acid residues of this genes encoding, only in cotton fiber, express through this gene of experiment confirm, and in elongating stage and thicken the phase great expression, this albumen may participate in the biosynthesizing of the non-fiber saccharan of cell walls.

Though cotton fiber development and elongation aspect have been done a large amount of intensive work, because the complicacy of cotton fiber development and elongation adjusting aspect still has the field of a large amount of the unknowns to require study.

Summary of the invention

The purpose of this invention is to provide with cotton fiber extension and have closely-related beta tubulin and encoding gene thereof.

The name of cotton fiber beta tubulin provided by the present invention is called Gh-BtubL, be protein, or the amino acid residue sequence of sequence 2 is passed through replacement, disappearance or the interpolation of one or several amino-acid residue and has identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2 with sequence 2 amino acid residue sequences in the sequence table.

The protein of sequence 2 is made up of 444 amino-acid residues in the sequence table.

The encoding gene of cotton fiber beta tubulin Gh-BtubL is one of following nucleotide sequences:

1) dna sequence dna of sequence 1 in the sequence table;

2) with sequence table in the dna sequence dna that limits of sequence 1 have 85% above homology, and the identical function protein DNA sequence of encoding.

The gene of sequence 1 is by 1580 based compositions, and the reading frame of this gene is to hold the 1st to the 1335th totally 1335 bases from 3 '.The class β microtubule sample Protein G h-BtubL that encodes, closely related with cotton fiber extension, have the function of the cell elongation of making and the radial extent of cell walls and have substantial connection, cotton fiber development open phase beginning transcriptional start, and transcribe in a large number in elongating stage.

The growth of cotton fiber and elongation are the metabolic regulation processes of a complexity, are participated in by up to a hundred genes, only are cloned into more than 30 associated gene at present, and the gene of wherein understanding function has tens.Gh-BtubL gene provided by the present invention, specifically expressing in cotton fiber has the function that promotes that eukaryotic cell increases.

The present invention confirms that the fission yeast (Schizosaccharomyces pombe) that has forward Gh-BtubL gene shows fairly obvious physiological characteristic, has changed the morphological specificity of yeast cell.Under the normal circumstances, the length of fission yeast cell is about 10 μ m in stationary phase, begin binary fission then and breed, and the yeast that forward is expressed the Gh-BtubL gene is at abduction delivering after 20 hours, its thalline length can reach about 18 μ m, has increased about 1.7 times than the length of wild-type yeast.

Normal function according to β-tubulin gene is inferred, Gh-BtubL is a kind of constitutive protein of microtubule, in many vital movements of cell, bring into play critical function, for example in vegetable cell, microtubule participates in cell fission, the bioprocesss such as formation of intracellular organic matter transportation and cell walls, if so Gh-BtubL full-length gene of the present invention or its functional fusion gene are connected to as fiber-specific expression promotor by forward mode, the downstream of the promotor that the nmt1 promotor of yeast/colibacillary shuttle plasmid pREP5N or 35S promoter can efficiently express in various plant tissues and organ like that, be built into the Plant Transformation intermediate carrier, use agrobacterium co-cultivation then, particle bombardment or pollen tube passage method import this gene in the leaf class cash crop genome, and the transfer-gen plant that efficiently expresses in the whole growth phase of screening, lasting and vigorous cell elongation that the Gh-BtubL gene is mediated and division may increase substantially the output of the unit surface green nourishing tissue of transgenic plant, significantly improve the economic benefit of leaf vegetables cash crop.If in the herbaceous plant genomes such as Plant Transformation intermediate carrier agrobacterium co-cultivation, particle bombardment or pollen tube passage method importing bamboo that make up, and the transfer-gen plant that efficiently expresses in the whole growth phase of screening, lasting and vigorous cell elongation that the Gh-BtubL gene is mediated and division may increase substantially the speed of growth and the plant height of transgenic plant, significantly improve economic worth and the benefit of draft class plants such as bamboo.If in the textile plant genomes such as Plant Transformation intermediate carrier agrobacterium co-cultivation, particle bombardment or pollen tube passage method importing fiber crops that make up, and the transfer-gen plant that efficiently expresses in the whole growth phase of screening, lasting and vigorous cell elongation that the Gh-BtubL gene is mediated and division may increase substantially fiber growth speed and the length of transgenic plant, significantly improve economic worth and the benefit of textile plants such as fiber crops.Gh-BtubL gene of the present invention has important science and economic worth.Established solid basis for realizing artificial controlling plant cell elongation.

Description of drawings

Fig. 1 is the elongation situation of fission yeast cell under the different treatment condition.

Fig. 2 be Gh-BtubL mRNA expression level and with the analysis of elongate fiber relation.

Fig. 3 is the in situ hybridization result of Gh-BtubL on upland cotton and fls mutant ovule square section.

Fig. 4 is the collection of illustrative plates of fission yeast expression vector.

Embodiment

The clone of embodiment 1, Gh-BtubL gene and sequential analysis

No cellucotton flower seed mutant (fls) is carried out the analysis of cDNA difference formula, and (cDNA RepresentationalDifference Analysis, RDA), the clone obtains the gene Gh-BtubL of specifically expressing in the cotton fiber.

The basic step of carrying out the cotton fiber specific gene clone with cDNA difference formula analytical method is:

1, extract wild strain and the intracellular total RNA of fls mutant strain, reverse transcription becomes cDNA to digest with four base nickases later on, to reduce the complicacy of whole cDNA colony;

2, respectively above-mentioned two groups of cNDA are added behind the different joint sequences by 1: 100 (wild strain: mixed mutant strain), it is strand that 95 ℃ of pyroprocessing make the DNA sex change, slowly cool to 68 ℃ and kept 12 hours again, make the DNA in the system become two strands again;

3, pcr amplification.Because mutant strain is identical with most mRNA in the wild strain cell, according to the reaction kinetics principle, most cDNA will form the heterozygosis two strands with the mutant strain sample in the wild strain sample, the gene of specifically expressing in cotton fiber only, because can't in the sample of mutant strain, find complementary cDNA chain, can't form the sample of heterozygosis two strands, overwhelming majority cDNA can only increase by linear forms and (have the joint sequence that is complementary with primer because have only on the chain, another chain can not be complementary mutually with used primer from the sample of mutant strain).Owing on two chains of cotton fiber specifically-expressed gene the joint sequence that is complementary with primer is arranged all, they can be increased by exponential form, through 20-15 circulation, the cotton specific expression gene is hundreds thousand of to up to a million times by enrichment, but not specific expressed only increase 20-25 doubly.Through repeatability hybridization and pcr amplification, all be the gene fragment of specifically expressing in the cotton fiber absolutely almost in the reaction system;

4, clone after these fragments and to verify its expression characterization with the Northern hybrid method and be probe that screening cotton fiber cDNA subtraction library obtains cotton fiber specific expressing gene Gh-BtubL with one of them fragment.

5, by RT-PCR and the hybridization of mechanical manipulator point sample.According to operational manual S.N.A.P. ^TM(Invitrogen USA) from fiber, root, stem, the leaf of wild cotton with do not have the ovule of cellucotton flower seed mutant (fls) and extract total RNA respectively, is that template is according to operational manual SUPERSCRIPT with the total RNA of 5 μ g to Total RNAisolation Kit ^TMArticle one, the synthetic cDNA article one chain of chain synthesis system carries out pcr amplification as template then, and (Ubiquitin) makes positive control with the cotton ubiquitin.Amplified production passes through Biomek ^2000 LaboratoryAutomation workstation (Beckman Coulter Inc., USA) with PCR product point on nylon membrane, use ³³The P labeled DNA probe, after the hybridization, (data are used ArrayVision 6.0 (Imaging Research Inc. for Molecular Dyanmics, USA) exposure with the phosphorus screen, USA) software analysis, the result as shown in Figure 2, wherein upland cotton fiber (UF) is weighed with 0-20DPA (bloom back fate), as can be seen from the figure the variation tendency of genetic expression of the present invention, promptly this gene cotton fiber development open phase beginning transcriptional start, and transcribe in a large number in elongating stage.

6, in situ hybridization.According to the program of Ruan and Chourey sample specimens paraffin embedding slices with in situ hybridization.Use FAA (Formalin-Acetic Acid) stationary liquid to fix behind the cotton ovule sample collecting immediately, with serial trimethyl carbinol dehydration, use paraffin embedding at last, section then.With the dna probe and the section hybridization of DIG mark, the result as shown in Figure 3.Wherein, A is and the upland cotton ovule square section that utilizes the hybridization of Gh-BtubL cDNA synthetic antisense RNA probes that blue signal is represented the existence of Gh-BtubL mRNA; B is the square section with the ovule of the fls mutant of above-mentioned probe hybridization; C is and the upland cotton ovule square section that utilizes the hybridization of Gh-BtubL cDNA synthetic justice rna probe.D is and the upland cotton ovule square section that utilizes the hybridization of cotton ubiquitin (ubiquitin) cDNA synthetic antisense RNA probes that F is a fibrocyte among the figure; Oi is outer integument (outer integument); Ii is inner integument (innerintegumen).As can be seen from the figure, become the very strong hybridization signal of appearance in the fiber bulliform cell at cotton wild strain ovule outer integument, and in other outer integument epidermic cell and fls mutant cells, detect less than hybridization signal, show expression of gene tool fiber-specific of the present invention.

Embodiment 2, the expression of gene of the present invention in fission yeast

To insert yeast and bacillus coli shuttle plasmid pREP5N by positive dirction with the Gh-BtubL gene that embodiment 1 method is cloned, carrier construction pREP-Btub1 (+) (its collection of illustrative plates as shown in Figure 4), transform fission yeast (Schizosaccharomyces pombe), with restricted substratum screening positive clone.

Substratum and reagent used among this embodiment comprise:

1, substratum (, adding VITAMIN B4 and uridylic) to S.P.Q-01

Yeast extractive substance (Yeast Extract) 5g

Glucose (Glucose) 30g

VITAMIN B4 (Adenine 50 *) 20ml

Uridylic (Uracil 50 *) 20ml

Add water and be settled to 1L, it is 20g/L that solid medium adds agar to final concentration.

2, restricted (MM) substratum (, adding VITAMIN B4 and uridylic) to S.P.Q-01

Component addition ultimate density

O-2 potassium acid hydrogen potassium (KH Phthlate) 3g 14.7mM

Na ₂HPO4 2.2g 15.5mM

Sodium Glutamate (Glutamate monosodium) 5g 5g/L

Glucose (Glucose) 20g 111mM

Salt mother liquor (Salts Stock 50 *) 20ml 1 *

Mineral mother liquor (Mineral Stock 10,000 *) 0.1ml 1 *

VITAMIN mother liquor (Vitamins Stock 1,000 *) 1ml 1 *

VITAMIN B4 (Adenine 50 *) 20ml 1 *

Uridylic (Uracil 50 *) 20ml 1 *

Add water and be settled to 1 liter, it is 20g/L that solid medium adds agar to final concentration, the 10Psi/15-20min sterilization.

3, salt mother liquor (Salts Stock 50 *)

Component addition (g) ultimate density (mM)

MgCl ₂-6H ₂O 53.3 5.2

CaCl ₂-2H ₂O 0.735 0.1

KCl 50 13.4

Na ₂SO ₄ 2 0.28

Dissolve each component respectively with 50ml water, be settled to 1L after the mixing, frozen or be distributed into 4 ℃ of preservations of small volume.

4, mineral substance mother liquor (Mineral Stock 10,000 *)

Component addition (g) ultimate density (μ M)

H ₃BO ₃ 0.5 8.1

MnSO ₄ 0.4 2.37

ZnS0 ₄-7H ₂O 0.4 1.39

FeCl ₃-6H ₂O 0.2 0.74

MoO ₄-2H ₂O 0.16 0.25

KI 0.1 0.6

CuS0 ₄-5H ₂O 0.04 0.16

Citric acid (Citric Acid) 1 4.76

Dissolve each component respectively with 5ml water, be settled to 100ml after the mixing, filtration sterilization, frozen or 4 ℃ of preservations.

5, VITAMIN mother liquor (Vitamins Stock 1,000 *)

Component addition (g) ultimate density (μ M)

Nicotinic acid (Nicotinic acid) 1 81.2

Inositol (Inositol) 1 55.5

Vitamin H (Biotin) 1mg 40.8

Pantothenic acid (Pantothenic acid) 100mg 4.2

Each dissolves each component respectively with 10ml water, is settled to 100ml after the mixing, filtration sterilization, frozen or 4 ℃ of preservations.

6, other composition (Supplements)

Component addition (g/100ml) ultimate density (*)

Arginine-HCl (Arginine-HCl) 0.75 100

Leucine ¹(Leucine ¹) 0.75 100

Methionin-HCl (Lysine-HCl) 0.75 100

Sodium Glutamate ²(Glutamic monosodium ²) 0.75 100

Histidine-HCl (Histidine-HCl) 0.75 100

VITAMIN B4 (Adenine) 0.375 50

Uridylic (Uracil) 0.375 50

In basal culture medium, when 1 being during to Leu 1-32, concentration adds to 250mg/L.When 2 were the L-glutamic acid defective type, concentration was 75mg/L; Glu2.5g/L replaces NH ₄Cl.

The concrete operations step of this experiment is:

1, fission yeast 29 ℃, is cultured to 1 * 10 in the 300rpm shaking table in the YE liquid nutrient medium ⁷Cell/ml, the centrifugal collection of 3000rpm;

2, after ice-cold 1.2M sorbyl alcohol (Sorbitol) is given a baby a bath on the third day after its birth time, with 1 * 10 ⁹Cell/ml concentration is resuspended among the 1.2M Sorbitol;

3, add 1ng-1 μ g DNA in the 0.2ml cell, change over to rapidly in the ice-cold electric shock cup;

4, set electric shock index: 2.25kv, 200 Ω, 25 μ F, electric shock imports fission yeast (Schizosaccharomyces pombe) S.P.Q-01 (Leul-32h by electric shocking method with carrier pREP-Btub1 (+) ^-);

5, after the electric shock, add the ice-cold 1.2M Sorbitol of 0.5ml rapidly;

6, with ice-cold 1.2M Sorbitol dilution back (not above 5 times), get 0.5ml and be coated onto on the very dried selectivity MM culture medium flat plate, by the selective action of MM substratum, the proteic yeast cell of Gh-β-tubulin is expressed in screening;

7, bacterium colony appearred in 4-6 days in 30 ℃ of cultivations.The picking mono-clonal extracts plasmid and further identifies whether be transformant;

8, inoculate single bacterium colony (pREP+GhFas and pREP) in 5ml contain 2 μ M VB1 the MM liquid nutrient medium (+VB1) in, 29 ℃, cultivate 20 hours in the 300rpm shaking table to O.D ₆₀₀＜1.0 (0.8 is best);

9, be divided into two parts, under the room temperature, centrifugal 5 minutes of 3000rpm;

10, with the aseptic washing of 20ml 3 times, another part is constant;

That part of 11, washing changes the 50mlMM liquid nutrient medium over to; That that do not wash part switching go into equivalent the MM liquid nutrient medium (+VB1);

12,29 ℃, to cultivate in the 300rpm shaking table 20 hours, the culture of getting for 20 μ l logarithm late periods is put on the slide glass, covered, the microscope high power lens is observed down.

The result as shown in Figure 1, A, B, E, D are the photo that utilizes common bright field microscope to take among the figure, and F, G, I, J are the cell photo that utilizes after the DAPI dyeing that fluorescent microscope takes, wherein, A and F: contain the yeast cell of pREP-BtubL (+), non-induction type; B and G: contain the yeast cell of pREP-BtubL (+), non-induction type; E and I: contain the yeast cell of pREP5N carrier, induction type; D and I: unconverted yeast cell, induction type, as can be seen from the figure, Gh-β-tubulin gene that forward is expressed can increase the radical length of yeast cell significantly, shows that Gh-BtubL has the effect that stimulates yeast cell elongation, can improve zymic output.

＜160〉2＜210〉1＜211〉1580＜2 12〉DNA＜213〉 ( Gossypium hirsutum L. )＜400〉1atgagagaaa tcctccatgt tcaagccggt cagtgtggta atcaaattgg tggcaagttt 60tgggaagtag tatgtgatga acatgggata gatgccactg gtaactatgt cggcacttcg 120cctgttcagc ttgaaaggct taatgtttac tataatgaag ccagtggtgg cagatatgtg 180cctagagctg tgttaatgga tcttgagcca ggaactatgg acagtttacg aacaggtcct 240tatggaaaat tgtttagacc agataacttt gtgttcggcc aaaatggagc tggcaataac 300tgggctaagg gacattatac tgaaggagct gaattgattg attcagttct tgatgttgtt 360cgtaaagagg ctgagaattg tgattgttta caaggttttc aggtttgcca ttcactggga 420ggtggaacag ggtcagggat ggggacattg ttgatatcaa agatcaggga agaataccct 480gaccggatga tgctaacgtt ctcggtgttt ccatcaccta aagtatcgga tactgtggtt 540gaaccctata atgcgaccct gtcagtgcac cagcttgtgg aaaatgctga tgaatgcatg 600gtccttgaca atgaagctct ttatgatatc tgcttcagaa ctcttaagct cacaaatccc 660agctttggtg acttgaacca tttgatttca acaaccatga gtggagtcac atgctgcctt 720cgcttccctg gccaactcaa ttccgatctt cgaaaactag cagtaaactt gatcccattc 780ccacgcctcc attttttcat ggttggtttt gcacctttaa catcccgggg ttcacaacaa 840taccgagctt taacgatccc cgagctaact caacaaatgt gggattccaa aaacatgatg 900tgcgcggctg atcctcgtca tggaaggtac ttaacagcct cggcaatgtt tcgaggcaaa 960atgagcacca aggaagttga tgaacaaatg atcaatgtcc aaaacaagaa ctcttcgtac 1020tttgtggagt ggattccgaa cgatgttaaa tcaagtgttt gcgacatccc accaactggg 1080ttgacgatgt catcgacgtt tatggggaac tcgacatcga tacaagagat gtttcgacgt 1140gtttcggaac agttcacagt gatgtttagg aggaaagcat ttttgcattg gtatacaggg 1200gaagggatgg atgaaatgga gtttactgag gctgaaagta atatgaatga tttggtttct 1260gaatatcaac aatatcaaga tgctgtggtt gatgaagatg gtgaagggta tgaagatgaa 1320gctgaggaaa attgacggaa ttcttttcaa ctttctatag taatggccta ggaaaaaaaa 1380atcaaatatg gtgctcattt tgatggttct aaataaggag gccatgtttt ttcatttgtg 1440ctgggatttt tagtaatgtg aattgtgaat ttgtatattt atatcaaatg tgaattgtga 1500tttaatgaat ttggagtgta aatttatgta atgaatttgg aatcacaagt ttcaaaattt 1560caagacaaaa aaaaaaaaaa 1580＜210〉2＜211〉444＜212〉PRT＜213〉 ( Gossypium hirsutum L. )＜400〉2Met Arg Glu Ile Leu His Val Gln Ala Gly Gln Cys Gly Asn Gln1 5 10 15Ile Gly Gly Lys Phe Trp Glu Val Val Cys Asp Glu His Gly Ile

20 25 30Asp?Ala?Thr?Gly?Asn?Tyr?Val?Gly?Thr?Ser?Pro?Val?Gln?Leu?Glu

35 40 45Arg?Leu?Asn?Val?Tyr?Tyr?Asn?Glu?Ala?Ser?Gly?Gly?Arg?Tyr?Val

50 55 60Pro?Arg?Ala?Val?Leu?Met?Asp?Leu?Glu?Pro?Gly?Thr?Met?Asp?Ser

65 70 75Leu?Arg?Thr?Gly?Pro?Tyr?Gly?Lys?Leu?Phe?Arg?Pro?Asp?Asn?Phe

80 85 90Val?Phe?Gly?Gln?Asn?Gly?Ala?Gly?Asn?Asn?Trp?Ala?Lys?Gly?His

95 100 105Tyr?Thr?Glu?Gly?Ala?Glu?Leu?lle?Asp?Ser?Val?Leu?Asp?Val?Val

110 115 120Arg?Lys?Glu?Ala?Glu?Asn?Cys?Asp?Cys?Leu?Gln?Gly?Phe?Gln?Val

125 130 135Cys?His?Ser?Leu?Gly?Gly?Gly?Thr?Gly?Ser?Gly?Met?Gly?Thr?Leu

140 145 150Leu?Ile?Ser?Lys?Ile?Arg?Glu?Glu?Tyr?Pro?Asp?Arg?Met?Met?Leu

155 160 165Thr?Phe?Ser?Val?Phe?Pro?Ser?Pro?Lys?Val?Ser?Asp?Thr?Val?Val

170 175 180Glu?Pro?Tyr?Asn?Ala?Thr?Leu?Ser?Val?His?Gln?Leu?Val?Glu?Asn

185 190 195Ala?Asp?Glu?Cys?Met?Val?Leu?Asp?Asn?Glu?Ala?Leu?Tyr?Asp?Ile

200 205 210Cys?Phe?Arg?Thr?Leu?Lys?Leu?Thr?Asn?Pro?Ser?Phe?Gly?Asp?Leu

215 220 225Asn?His?Leu?Ile?Ser?Thr?Thr?Met?Ser?Gly?Val?Thr?Cys?Cys?Leu

230 235 240Arg?Phe?Pro?Gly?Gln?Leu?Asn?Ser?Asp?Leu?Arg?Lys?Leu?Ala?Val

245 250 255Asn?Leu?Ile?Pro?Phe?Pro?Arg?Leu?His?Phe?Phe?Met?Val?Gly?Phe

260 265 270Ala?Pro?Leu?Thr?Ser?Arg?Gly?Ser?Gln?Gln?Tyr?Arg?Ala?Leu?Thr

275 280 285Ile?Pro?Glu?Leu?Thr?Gln?Gln?Met?Trp?Asp?Ser?Lys?Asn?Met?Met

290 295 300Cys?Ala?Ala?Asp?Pro?Arg?His?Gly?Arg?Tyr?Leu?Thr?Ala?Ser?Ala

305 310 315Met?Phe?Arg?Gly?Lys?Met?Ser?Thr?Lys?Glu?Val?Asp?Glu?Gln?Met

320 325 330Ile?Asn?Val?Gln?Asn?Lys?Asn?Ser?Ser?Tyr?Phe?Val?Glu?Trp?Ile

335 340 345Pro?Asn?Asp?Val?Lys?Ser?Ser?Val?Cys?Asp?Ile?Pro?Pro?Thr?Gly

350 355 360Leu?Thr?Met?Ser?Ser?Thr?Phe?Met?Gly?Asn?Ser?Thr?Ser?Ile?Gln

365 370 375Glu?Met?Phe?Arg?Arg?Val?Ser?Glu?Gln?Phe?Thr?Val?Met?Phe?Arg

380 385 390Arg?Lys?Ala?Phe?Leu?His?Trp?Tyr?Thr?Gly?Glu?Gly?Met?Asp?Glu

395 400 405Met?Glu?Phe?Thr?Glu?Ala?Glu?Ser?Asn?Met?Asn?Asp?Leu?Val?Ser

410 415 420Glu?Tyr?Gln?Gln?Tyr?Gln?Asp?Ala?Val?Val?Asp?Glu?Asp?Gly?Glu

426 430 435Gly?Tyr?Glu?Asp?Glu?Ala?Glu?Glu?Asn

440 444

Claims (10)

1, the beta tubulin Gh-BtubL of cotton fiber, be protein, or the amino acid residue sequence of sequence 2 is passed through replacement, disappearance or the interpolation of one or several amino-acid residue and has identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2 with sequence 2 amino acid residue sequences in the sequence table.

2, albumen according to claim 1 is characterized in that: it is the protein with sequence 2 amino acid residue sequences in the sequence table.

3, the encoding gene of cotton fiber beta tubulin Gh-BtubL is one of following nucleotide sequences:

1) dna sequence dna of sequence 1 in the sequence table;

2) with sequence table in the dna sequence dna that limits of sequence 1 have 85% above homology, and the identical function protein DNA sequence of encoding.

4, gene according to claim 3 is characterized in that: the encoding gene of described cotton fiber beta tubulin Gh-BtubL is the dna sequence dna of sequence 1 in the sequence table.

5, gene according to claim 4 is characterized in that: the reading frame of this gene is for holding the 1st to the 1335th bit base from 3 '.

6, contain claim 3 or 4 described expression carrier.

7, carrier according to claim 6 is characterized in that: also contain promotor in the described expression vector.

8, carrier according to claim 7 is characterized in that: described promotor is nmt1 promotor or the 35S promoter that fiber-specific is expressed promotor, yeast/colibacillary shuttle plasmid pREP5N.

9, the clone that contains claim 3 or 4 described genes.

10, claim 3 or the 4 described genes application in the plant variety of cultivating the staple length increase.

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