CN104560906A

CN104560906A - Protein CYP634A1 like-1 specifically expressed in fiber cells and application of protein

Info

Publication number: CN104560906A
Application number: CN201510063409.2A
Authority: CN
Inventors: 罗明; 李芳�; 隗廷; 翟云兰; 曾志锋; 裴炎; 肖月华; 侯磊; 李先碧
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2015-02-06
Filing date: 2015-02-06
Publication date: 2015-04-29
Anticipated expiration: 2035-02-06
Also published as: CN104560906B

Abstract

The invention belongs to the field of plant genetic engineering, and in particular relates to protein CYP634A1 like-1 specifically expressed in fiber cells and application of the protein. The invention aims at solving a technical problem that the requirements on the fiber output and quality molecular design of improved cotton cannot be satisfied due to the lack of genes for improving specific or preferential expression of fiber at present. The amino acid sequence of the protein CYP634A1 like-1 disclosed by the invention is as shown in SEQ ID NO.3. Furthermore, the invention also provides a plant expression vector for expressing the protein. The invention offers a new choice for improving cotton fiber quality and cotton variety.

Description

The protein C YP734A1 like-1 of specifically expressing and application thereof in fibrocyte

Technical field

The invention belongs to plant genetic engineering field, be specifically related to protein C YP734A1 like-1 and the application thereof of specifically expressing in fibrocyte.

Background technology

Cotton is one of main in the world fibre crops.Cotton fiber cell be cotton ovule outer integument epidermic cell through differentiation projection and polarity extend unicellular.Its growth and development process is divided into four not only obviously to distinguish but also period overlapping to some extent: fibrocyte initial phase, fibrocyte elongating stage, secondary wall thicken phase and dehydration ripening stage.There is respective feature in each period, but adjacent developmental stage is overlapping to some extent again.There is impact in each period of Fibre Development on the output of fiber and/or quality, the initial quantity determining fiber in simple grain ovule of the differentiation of fiber, fibrocyte extension speed and time length essentially dictate the length of fiber, and the mode of secondary wall deposition and time length essentially dictate intensity and the secondary wall thickness of mature fibers.The elongate fiber phase is that (primary wall synthesis phase) was from the same day of blooming, Post flowering 25 days (25DPA) can be lasted till, mainly carry out the synthesis of cell polarity elongation and primary cell wall, the final lengths of the elongate fiber speed of this period and time length decision fiber, upland cotton fiber length-to-diameter ratio can reach 1000-3000.But up to now, we still know little about it to the molecular mechanism of its growth course of regulation and control.

Because cotton fiber cell is a special cell, special genetic expression and associated regulatory mechanism in its growth course, must be had.Therefore the gene of screen fibre cell specific expression becomes the focus of cotton fiber molecular biology research.By cDNA library differential screening *, cDNA reprensentative difference analysis, Subtractive hybridization and mRNA differential display technique, and the biochip technology of development in recent years, high throughput sequencing technologies etc., clone and analyze special in fiber or predominant expressed gene.John and Crow was in first passage cDNA library differential screening in 1992, clone E6 gene [the John M E of the early stage specifically expressing of Fibre Development, Crow L is expression in cotton (Gossypium hirsutum L.) fiber:Cloningof the mRNAs.Proc Natl Acad Sci USA J.1992.Gene, 89 (13): 5769-5773]. profit uses the same method, Rinehart etc. (1996) have been separated to Fibre Development middle and later periods specific expression gene FbL2A [Rinehart J A, Petersen M W, John ME.1996.Tissue specific and developmental regulation of cotton gene FbL2A.Plant Physiology, 112 (3): 1331-1334].In recent years, [Lv Shaopu is cloned and analyzed to fiber special or predominant expression microtubule protein gene, extensin gene, lipid transfer protein gene etc. in succession, Wang Xujing, Tang Qiaoling, Deng the progress of .2014. cotton fibre specific gene and specific promoter thereof. Biotechnological Advances, 4 (1): 1-6].These results of study not only confirm that the special or predominant expressed gene of fiber has vital role to fibrocellular growth, and the regulatory mechanism of growing for having resolved fibroblast growth provides experimental data.But up to the present, the gene of the special or predominant expression of the fiber of acquisition still very little, can't meet the needs of improvement cotton fiber yield and quality molecular designing.

Summary of the invention

The technical problem to be solved in the present invention provides a kind of new select for improving cotton fiber quality, to improve the gene of the special or predominant expression of current fiber still very little, can't meet the needs of improvement cotton fiber yield and quality molecular designing.

Technical scheme of the present invention is the protein C YP734A1 like-1 of specifically expressing in fibrocyte, has (a) aminoacid sequence as shown in SEQID NO.3.

Concrete, the coding GhCYP734A1L-1 gene of described albumen has:

(a) cDNA as shown in SEQ ID NO.1 (cDNA and complementary DNA (cDNA), for having and the single stranded DNA of mRNA (messenger RNA(mRNA)) chain in complementary base sequence);

Or (b) genomic dna as shown in SEQ ID NO.2 (genomic dna and gDNA).

Present invention also offers the plant expression vector of expressing described protein.

Concrete, described plant expression vector is the carrier of overexpression GhCYP734A1L-1 gene or Antisense Suppression GhCYP734A1L-1 gene.

Present invention also offers the host cell containing described carrier.

Concrete, described host cell is Agrobacterium.

Present invention also offers described CYP734A1 like-1 or the application of plant expression vector in improvement cotton variety.

Present invention also offers described CYP734A1 like-1 or the application of plant expression vector in improving cotton fiber quality.

In the present invention, protein G hCYP734A1L-1 comprises, and has the aminoacid sequence shown in SEQ ID NO.3; Or by the replacement of one or several amino-acid residue of the amino acid residue sequence of SEQ ID NO.3 process, disappearance or interpolation, and there is bioactive derivative protein identical with the amino acid residue sequence of SEQ ID NO.3.

Nucleotides sequence shown in SEQ ID NO.1 is classified as the cDNA sequence of coding cotton GhCYP734A1L-1 gene, and the nucleotides sequence shown in SEQID NO.2 is classified as gDNA (genomic dna) sequence of gGhCYP734A1L-1 gene.Wherein said cDNA sequence comprises 5'-non-translational region sequence, open reading frame (ORF) sequence and 3'-non-translational region sequence, and wherein open reading frame sequence is encoding sequence, and gDNA sequence contains exon and intron sequences.

Plant expression vector of the present invention, can adopt at least one in enhancement type, composing type or inducible promoter to regulate and control the expression of GhCYP734A1L-1 gene.The preferred constitutive promoter of promotor for building plant expression vector of the present invention or tissue-specific promoter, more preferably derive from the plant constitutive promoter CaMV35S of cauliflower mosaic virus.Usually, by gene constructed for the GhCYP734A1L-1 downstream at CaMV35S.Can be any one double base agrobacterium vector or the plant expression vector that can be used for via Particle Bombardment Transformation for building the carrier that sets out of plant expression vector of the present invention.In order to the needs screened and express, in expression vector, also comprise screening-gene sequence, reporter sequences and other the needs for genetically engineered operation and the various restriction enzyme sites that insert alternatively, select the gene order that screening-gene and reporter gene can be commonly used from this area, preferably, expression of plants of the present invention has structure as shown in Figure 5.Such as, the coding can expressed in plant can be added in described expression vector the enzyme of colour-change or the gene of luminophor can occur, as GUS (β-glucuronidase) gene, GFP (green fluorescent protein) gene, luciferase etc.; There is the antibiotic marker thing of resistance, as anti-gentamicin marker, anti-kalamycin marker etc.; Chemical resistance reagent marker gene, as anti-herbicide gene etc.

The present invention adopts excess or suppresses GhCYP734A1L-1 expression vector to prepare the method for transgenic plant, comprises the following steps:

1) GhCYP734A1L-1 gene is operably connected with promotor;

2) plant expression vector containing GhCYP734A1L-1 gene and promotor is built;

3) transform host with described plant expression vector, obtain transformant;

4) with described transformant conversion of plant, transgenic plant are obtained.

In the present invention, by using Ti-plasmids, Ri plasmid, plant or microbiosis poisonous carrier, directly delivered DNA, microinjection, conductance or the conventional biology methods such as agriculture bacillus mediated, the expression vector transfection cotton containing GhCYP734A1L-1 gene of the present invention is obtained transformant.

Beneficial effect of the present invention:

Disclosed in the present invention, the coded protein of GhCYP734A1L-1 gene can promote cotton fiber extension, improves cotton fiber length, increases fibrous quality and output; Therefore this gene can be utilized to carry out improving cotton fiber quality, dissimilar promotor can also be selected as required simultaneously, carry out improving cotton fiber quality or improve cotton plant type, to be conducive to raising and the special cultivation operation (as mechanical harvesting operation and high-density planting etc.) of output of cotton quality.

Accompanying drawing explanation

The tetraploid rice of Fig. 1: GhCYP734A1L-1 protein and other species related proteins;

Gi144905179: pea (Pisum sativum) Cytochrome P450 family protein; Gi15225777: Arabidopis thaliana (Arabidopsis thaliana) cytochrome p450 protein (CYP734A1); Gi225445412: grape (Vitis vinifera) cytochromoid P450 albumen (CYP734A1-like); Gi255566913: castor-oil plant (Ricinus communis) cytochromoid P450 albumen; Gi356563055: soybean (Glycine max) cytochromoid P450 albumen (CYP734A1-like); Gi590577824: cocoa (Theobroma cacao) cytochrome p450 protein; Gi728846626: the false albuminoid of middle cotton.The cytochrome p450 protein (CYP734A1) that this figure shows GhCYP734A1L-1 and other species has higher homology, and GhCYP734A1L-1 gene is the homologous gene of CYP734A1.

The phylogenetic analysis of Fig. 2: GhCYP734A1L-1;

The homologous gene sibship that this figure shows in GhCYP734A1L-1 gene and middle cotton and cocoa is nearest.

The expression analysis of Fig. 3: GhCYP734A1L-1 gene;

The wherein expression pattern of A:GhCYP734A1L-1 gene in different tissues, organ and cell;

Wherein B:GhCYP734A1L-1 gene is at the expression level of fibrocyte and ovule different development stage; OF-0DPA ~ OF-4DPA: the ovule (fibre-bearing cell) on the same day of blooming is to the Post flowering ovule of 4 days (fibre-bearing cell); F-6DPA ~ F-16DPA: the Post flowering fiber of 6 days is to the Post flowering fiber of 16 days; O-6DPA ~ O-16DPA: the Post flowering ovule of 6 days is to the Post flowering ovule of 16 days.This figure shows GhCYP734A1L-1 gene specifically expressing in fibrocyte, expresses hardly at root, stem, leaf, flower, cotyledon, hypocotyl and ovule.Faint expression in figure in ovule may be fibrocyte from caused by fibrocyte residual during ovule sur-face peeling.

The expression of Fig. 4: GhCYP734A1L-1 gene in the fiber and ovule of upland cotton wild-type TM-1 and its superbhort fiber mutant li-1;

TM-1: upland cotton wild-type; Li-1: superbhort fiber mutant; FO-0DPA: the ovule fiber on the same day of blooming; FO-6DPA: the Post flowering ovule fiber of 6 days; F-10DPA: the Post flowering fibrocyte of 10 days; O-10DPA: the Post flowering ovule of 10 days.This figure show GhCYP734A1L-1 gene in the superbhort fiber mutant Post flowering ovule fiber of 6 days with the expression in the Post flowering fibrocyte of 10 days well below the expression in the corresponding fiber of the identical developmental stage of its wild-type and ovule.Illustrate that GhCYP734A1L-1 gene has vital role in elongate fiber process.

Fig. 5: containing the structure iron of the plant expression vector of GhCYP734A1L-1 gene;

Wherein CaMV 35S represents CaMV 35S promoter; GhCYP734A1L-1 represents GhCYP734A1L-1 gene cDNA; Ter represents terminator; LB represents T-DNA left margin; RB represents T-DNA right margin.

Fig. 6: the present invention preferred plant expression vector pC-CaMV 35S-GhCYP734A1L-1 structure iron;

Wherein GusPlus-His6 represents GUSPlus reporter gene, and this gene has merged His6 sequence label at C end; NPTII represents neomycin phosphotransferase gene, has kalamycin resistance; Ter:Nos terminator; CaMV35S: the plant composition promotor deriving from cauliflower mosaic virus; LB:T-DNA left margin; RB:T-DNA right margin.Plant expression vector is the pCambia carrier (detailed in Example three) of transformation.

Fig. 7: the present invention's preferred Antisense Suppression GhCYP734A1L-1 gene plant expression vector pC-CaMV 35S-antisense GhCYP734A1L-1 structure iron.Wherein GusPlus-His6 represents GUSPlus reporter gene, and this gene has merged His6 sequence label at C end; NPTII represents neomycin phosphotransferase gene, has kalamycin resistance; Ter:Nos terminator; CaMV35S: the plant composition promotor deriving from cauliflower mosaic virus; LB:T-DNA left margin; RB:T-DNA right margin.Plant expression vector is the pCambia carrier (detailed in Example three) of transformation.

Fig. 8: the qualification of transgene cotton;

Wherein A: histological chemistry's qualification of transgene cotton, WT: non-transgenic cotton (wild-type), compares; PC-CaMV35S-antisense GhCYP734A1L-1 is the transgene cotton blade of Antisense Suppression GhCYP734A1L-1 gene; PC-CaMV35S-GhCYP734A1L-1 represents the transgene cotton blade of overexpression GhCYP734A1L-1 gene;

The amplification checking of wherein B: overexpression GhCYP734A1L-1 transgene cotton, M:DNA marker2000; H ₂o: be pcr amplification template with water, makes blank; +: with pC-CaMV 35S-GhCYP734A1L-1 plasmid for pcr amplification template, make positive control; 1 and 2: overexpression GhCYP734A1L-1 transgene cotton 1# and 2#.

The amplification checking of wherein C: Antisense Suppression GhCYP734A1L-1 transgene cotton, M:DNA marker15;-: non-transgenic cotton (wild-type), makes negative control; H ₂o: be pcr amplification template with water, makes blank; +: with pC-CaMV35S-antisense GhCYP734A1L-1 plasmid for pcr amplification template, make positive control; 1 ~ 3: Antisense Suppression GhCYP734A1L-1 transgene cotton 1# ~ 3#.

Fig. 9: the expression analysis of GhCYP734A1L-1 gene in transgene cotton;

The expression analysis of GhCYP734A1L-1 gene in wherein A: Antisense Suppression GhCYP734A1L-1 transgene cotton fiber, WT: non-transgenic cotton (wild-type); S1 ~ S13: Antisense Suppression GhCYP734A1L-1 transgene cotton 1# ~ 13#.

The expression analysis of GhCYP734A1L-1 in wherein B: overexpression GhCYP734A1L-1 transgene cotton blade, WT: non-transgenic cotton (wild-type); O1 ~ O14: overexpression GhCYP734A1L-1 transgene cotton 1# ~ 14#.

This figure shows, by Cotton Transformation, obtains the transgene cotton that GhCYP734A1L-1 gene expression dose increases and reduces.

The impact that Figure 10: overexpression GhCYP734A1L-1 gene pairs cotton plants grows;

Wherein A: the upgrowth situation of non-transgenic cotton (wild-type) and overexpression GhCYP734A1L-1 transgenic cotton plant; A left side is wild type cotton plant, and the right side is overexpression GhCYP734A1L-1 transgenic cotton plant, scale=10cm.

Wherein B: the comparison of wild type cotton blade and transgene cotton blade; A left side is wild type cotton mature leaf, and upper right is transgene cotton spire, and bottom right is transgene cotton mature leaf, scale=10cm.

Wherein C: the chlorophyll content of wild type cotton blade and transgene cotton blade compares; WT: non-transgenic cotton (wild-type) blade; O1: overexpression GhCYP734A1L-1 transgene cotton blade.

Wherein D: the stomatal movement situation of wild type cotton blade and transgene cotton blade; Figure below is the opening and closing situation of pore on non-transgenic cotton (wild-type) blade, and upper figure is the opening and closing situation of pore on transgene cotton blade.

The impact of Figure 11: Antisense Suppression GhCYP734A1L-1 gene pairs cotton fiber length;

Wherein A: the length of cotton mature fibers compares; WT: non-transgenic cotton (wild-type); S1: Antisense Suppression GhCYP734A1L-1 transgene cotton 1# strain;

Wherein B: in cotton ovule culture in vitro system, the growing state of non-transgenic cotton (wild-type) and Antisense Suppression GhCYP734A1L-1 transgene cotton fiber; WT: non-transgenic cotton (wild-type); S1: Antisense Suppression GhCYP734A1L-1 transgene cotton 1# strain;

Wherein C: the staple length of mature fibers measures statistics, and wild-type and transgenic fibre respectively detect 20, measure its length after fiber combing.WT: non-transgenic cotton (wild-type); S1: Antisense Suppression GhCYP734A1L-1 transgene cotton 1# strain.

Embodiment

It is common commercially available that reagent chemicals in example of the present invention does not do being of illustrating, and MATERIALS METHODS does not do equal reference " Molecular Cloning: A Laboratory guide " (Sambrook and Russell, 2001) that illustrate.

In following example of the present invention, cotton experiment material used is Ji cotton 14 (Gossypium hirsutumcv.Jimian14), for producing upper Cultivar; TM-1 (Gossypium hirsutum cv.TM-1) and superbhort fiber mutant (ligon lintless-1, li-1) thereof.Wild-type TM-1 separates from the li-1 mutant of each sowing that (li-1 mutant is a dominant mutant, and homozygote is lethal.Therefore per only need in season to gather in the crops mutant seeds, in the cotton seedling grown after this planting seed, some is the wild-type separated, i.e. TM-1), li-1 mutant, from Cotton Inst., Chinese Agricultural Academy's Germplasm Bank, is openly provided.

The Cloned culturing of embodiment 1 GhCYP734A1L-1 gene

1, the extraction of cotton RNA

Choose about 3g fresh cotton floral material, fine powder is worn into rapidly in liquid nitrogen, load 50mL centrifuge tube, add RNA extracting solution (2%CTAB (W/V), the 2%PVP (W/V) of 15ml65 DEG C of preheating, 100mmol/L Tris-HCl (pH8.0), 0.5g/L spermidine, 2.0mol/L NaCl, 2% mercaptoethanol (V/V), add before using)), put upside down mixing.65 DEG C of water-bath 3 ~ 10min, period mixing 2 ~ 3 times.Lv Fang ︰ primary isoamyl alcohol (24 ︰ 1) extracting 2 times (10,000r/min, room temperature, 5min).Get supernatant, add 1/4 volume 10mol/L LiCl solution, place 6h, with Lv Fang ︰ primary isoamyl alcohol (24 ︰ 1) each extracting 1 time (10,000r/min, room temperature, 5min) for 4 DEG C.Add the dehydrated alcohol of 2 times of volumes, at-70 DEG C of refrigerator precipitation more than 30min.12,000r/min, 4 DEG C of centrifugal 20min, abandon supernatant.Precipitate the DEPC process water dissolution with 200 μ L.Phenol (pH4.5) ︰ Lv Fang ︰ primary isoamyl alcohol (25 ︰ 24 ︰ 1), Lv Fang ︰ primary isoamyl alcohol (24 ︰ 1) each extracting 1 time (10,000r/min, room temperature, 5min).Add the dehydrated alcohol of 1/10 volume 3mol/L NaAc solution and 2.5 times of volumes, at-70 DEG C of refrigerator precipitation more than 30min.12,000r/min, 4 DEG C of centrifugal 20min, abandon supernatant.Precipitate with 70% alcohol rinsing once, air-dry.Add the DEPC process water dissolution of 200 μ L.By the quality of non denatured agarose gel electrophoresis and ultraviolet spectrophotometer Scanning Detction RNA.

2, cDNA synthesis

With the various sample total serum IgE of cotton, synthesize cDNA mono-chain with test kit (Fermentas).Concrete grammar is: get about 10 μ g total serum IgE in the amplification pipe of DEPC process, add 1 μ L, 2.5 μm of ol/L Oligo-dT, adds the water of DEPC process to final volume 12 μ L, after 70 DEG C of water-bath 5min make RNA sex change, and ice bath 3min immediately.Then in amplification pipe, 4 μ L 5 × damping fluids are added successively, 2 μ L 10mmol/L dNTPs, 1 μ L RNase inhibitor (20U), 37 DEG C of process 5min.After adding 1 μ L reversed transcriptive enzyme AMV Rtase (5U), insulation program is 42 DEG C, 60min; 70 DEG C, 5min; 5 DEG C, 5min.After EP (end of program), cDNA mono-chain product is frozen in-20 DEG C.

3, the extraction of cotton genomic dna

Improved method of CTAB is adopted to extract cotton genomic dna.Get 0.5g cotton spire, pulverize rapidly in liquid nitrogen, add the CTAB extracting solution of 3mL 65 DEG C of preheatings, quick oscillation mixes.65 DEG C of water-bath 30min, then add 1mL 5mol/LKAc, after ice bath 20min, with isopyknic Lv Fang ︰ primary isoamyl alcohol (24 ︰ 1) extracting 1 time (12,000r/min, 4 DEG C of centrifugal 5min), get supernatant, add-20 DEG C of pre-cold isopropanols of 2/3 times of volume, mixing, leave standstill about 30min, choose flocks, the ethanol of 75% rinsing 3 times repeatedly, then use dehydrated alcohol rinsing 1 time, dry up, be heavily dissolved in 500 μ L TE.Add 1 μ L RNaseA (10mg/mL), 37 DEG C of process 1h.Yong Fen ︰ Lv Fang ︰ primary isoamyl alcohol (25 ︰ 24 ︰ 1) and Lv Fang ︰ primary isoamyl alcohol (24 ︰ 1) each extracting 1 time (12,000r/min, 4 DEG C of centrifugal 5min), get supernatant again, adds the dehydrated alcohol precipitation DNA of 2 times of volumes.Place about 30min for-20 DEG C, centrifugal (12,000r/min, 4 DEG C of centrifugal 5min), abandon supernatant, and precipitate the ethanol rinse with 75%, air-dry, be dissolved in 200 μ L TE ,-20 DEG C save backup.

4, high homology cotton est sequence is screened

With cotton EST (the Expressed Sequence Tag in the protein sequence of Arabidopis thaliana Brassinosteroids metabolic gene CYP734A1 search ncbi database, expressed sequence tag) sequence, confluence analysis is carried out to the high homology cotton est sequence searched, obtain an integration sequence, again integration sequence is dealt in ncbi database and compares, determine whether integration sequence encodes the homologous gene of CYP734A1, if, then ORF (open reading frame contained by integration sequence, openreading frame) both sides design of amplification primers, carry out gene clone.

5, the amplification of GhCYP734A1L-1 gene cDNA sequence and genome sequence

Based on integration sequence, in the both sides design of amplification primers of its ORF, 5' holds primer to be that GhCYP734A1L-1-3 (SEQID NO.4) and 3' hold primer to be GhCYP734A1L-1-4 (SEQ ID NO.5), with 12DPA fiber cDNA for template carries out PCR.The cDNA amplification system of 25 μ L contains 2.5 μ L 10 × Ex PCR damping fluids (without Mg ²⁺), 2 μ L 2.5mmol/LdNTPs, 2 μ L 25mmol/L MgCl ₂1 μ L special primer GhCYP734A1L-1-3 (SEQ ID NO.4) (5 μm of ol/L), 1 μ L GhCYP734A1L-1-4 (SEQ ID NO.5) (5 μm of ol/L), 0.2 μ L Ex Taq archaeal dna polymerase, 1 μ L cDNA mono-chain product.Amplification program is: 94 DEG C, 5min; 94 DEG C, 30sec, 56 DEG C, 30sec, 72 DEG C, 1.5min, 30 circulations; 72 DEG C extend 10min.

Be simultaneously that template increases with identical primer with cotton genomic dna, the genomic DNA amplification system of 20 μ L is containing 10 μ L 2 × PrimerStar mix, 1 μ L special primer GhCYP734A1L-1-3 (SEQ ID NO.4) (5 μm of ol/L), 1 μ L GhCYP734A1L-1-4 (SEQ ID NO.5) (5 μm of ol/L), 1 μ L gDNA and 7 μ L water.Amplification program is: 94 DEG C, 5min; 94 DEG C, 30sec, 56 DEG C, 30sec, 72 DEG C, 20sec, 30 circulations; 72 DEG C extend 10min.

6, amplified fragments reclaims, and connects, transformation of E. coli DH5a

(1) electrophoresis

Amplified production is carried out electrophoretic separation in 1.0% (W/V) sepharose.

(2) reclaim

Use and reclaim test kit: recycling step carries out according to test kit specification sheets, reclaim fragment electrophoresis on sepharose quantitative.

(3) Cloning and sequencing

The fragment reclaimed is quantitative through agarose gel electrophoresis.By test kit specification sheets, transformed by the connection of reclaiming fragment and cloning vector, the intestinal bacteria that connect product, the cultivation of positive bacterium colony and plasmid enzyme restriction verify, recovery fragment is cloned on pGEm-T (Shanghai Sangon) carrier.Sequencing is completed by Ying Jun company.

The fragment reclaimed sets up following linked system with pGEm-T (the raw work in Shanghai) carrier: 10 × T4 DNA is connected damping fluid 1 μ L, vector DNA fragment 1 μ L, external source connects product D NA fragment 1 μ L, and T4 DNA ligase 1 μ L, supplies volume to 10 μ L with distilled water.

Vector DNA fragment is connected product D NA fragment mol ratio with external source be that 1 ︰ 3,16 DEG C connects 12h.Product conversion escherichia coli DH5a will be connected afterwards.

7, GhCYP734A1L-1 gene sequencing

With the cotton 14 12DPA fiber cDNA in upland cotton Ji for template, amplify the special band of an about 1700bp.Find this amplifying specific sheet segment length 1730bp (SEQ ID NO.1) after order-checking, comprise a complete open reading frame (ORF), long 1575bp.The protein (SEQ ID NO.3) of coding 524 amino-acid residues, the molecular weight of this protein prediction is 59.8kD, and iso-electric point is 9.498.

NCBI searches for the homologous protein of GhCYP734A1L-1, and result shows that GhCYP734A1L-1 belongs to the CYP734A subfamily of Cytochrome P450 family.This albumen and pea (Pisum sativum) CYP734A1 albumen (gi144905179); Soybean (Glycine max) cytochromoid P450 albumen (CYP734A1-like) (gi356563055); Arabidopis thaliana (Arabidopsis thaliana) cytochrome p450 protein (CYP734A1) (gi15225777); Grape (Vitisvinifera) cytochromoid P450 albumen (CYP734A1-like) (gi225445412); Castor-oil plant (Ricinus communis) cytochromoid P450 albumen (gi255566913); The albumen such as cocoa (Theobroma cacao) cytochrome p450 protein (gi590577824) have higher homology, the same amino acid residue of GhCYP734A1L-1 and these albumen reaches 79%, 63%, 66%, 72% and 65% respectively, and similar amino acid residue reaches 88%, 85%, 84%, 83% and 78% (Fig. 1) respectively.Illustrate that GhCYP734A1L-1 is the homologous gene of CYP734A1 gene.

Phylogenetic analysis result show GhCYP734A1L-1 and cocoa, grape, willow and castor-oil plant CYP734A1 there is nearer sibship, and with the sibship (Fig. 2) comparatively far away of the CYP734A subfamily albumen of the species such as pea and Arabidopis thaliana.

From upland cotton, cloned the genomic dna sequence of GhCYP734A1L-1 gene simultaneously, the long 2978bp of this sequence (SEQID NO.2), by to compare with the cDNA sequence of GhCYP734A1L-1 gene and according to GT-AG intron identification law-analysing, found that, GhCYP734A1L-1 genome sequence contains 5 exons and 4 introns.

The expression analysis of embodiment 2 GhCYP734A1L-1 gene in cotton plants and Fibre Development

Using cDNA mono-chain of synthesis as template, real-time quantitative PCR test kit (Bio-Rad) is adopted to carry out PCR.Express the cDNA sequence design of primer according to GhCYP734A1L-1 gene, 5 ' end primer is GhCYP734A1L-1-1 (SEQ IDNO.6), and 3 ' end primer is GhCYP734A1L-1-2 (SEQ ID NO.7).Comprise 10 μ LMIX damping fluids in the reaction system of 20 μ L and (comprise PCR damping fluid, archaeal dna polymerase, dNTPs and MgCl ₂), 5'-end and 3'-hold each 1 μ L of primer (5 μm of ol/L).Loop parameter is 94 DEG C of denaturation 3min; 94 DEG C, 30sec, 55 DEG C, 30sec, 72 DEG C, 30sec, preset cycle number is 40.Mark in doing with cotton GhHISTONE3 gene (GenBank accession number: AF024716), the 5'-primer of GhHISTONE3 gene is GhHIS-1 (SEQ ID NO.8), and 3'-primer is GhHIS-2 (SEQ ID NO.9).

1, the expression pattern of the different times of different tissues organ and fiber Ovule Development

Extract the total serum IgE of each tissue of upland cotton (Gossypium hirsutum L.) and organ, and synthesize cDNA mono-chain.The expression of GhCYP734A1L-1 gene in different tissues and organ is have detected with real-time quantitative RT-PCR.Detected result shows, GhCYP734A1L-1 gene specifically expressing in fibrocyte, expresses hardly at root, stem, leaf, flower, cotyledon, hypocotyl and ovule.Faint expression in figure in ovule may be fibrocyte from (Fig. 3 A) caused by fibrocyte residual during ovule sur-face peeling.Illustrate that GhCYP734A1L-1 gene is a fibrocyte specific expression gene.

Further detection GhCYP734A1L-1 is at the expression of cotton ovule fiber different development stage.Result shows (Fig. 3 B), this gene bloomed the same day (0DPA) and Post flowering 2 days (2DPA) ovule fiber in express hardly, within 4 days, in (4DPA) ovule fiber, have faint expression at Post flowering; Great expression in Post flowering 6 days (6DPA) to the fibrocyte of Post flowering 10 days (10DPA), and along with fibrocellular growth, expression level raises gradually, reaches express climax when 10DPA; After 10DPA, along with fibrocellular continued growth, the expression level of GhCYP734A1L-1 gene reduces gradually, but within 16 days, still has higher expression level in (16DPA) fibrocyte at Post flowering.Expression level in ovule all well below the expression level in identical developmental stage fiber, almost can't detect the expression of GhCYP734A1L-1 gene in 16DPA ovule.Faint expression in ovule may be fiber from (Fig. 3 B) caused by few fibers residual during ovule sur-face peeling.This expression pattern display GhCYP734A1L-1 gene is the highest at fibrocyte rapid elongation phase expression level, and its expression level is directly proportional to fibrocyte extension speed, illustrates that this gene has vital role in fibrocyte extends.

2, the differential expression in superbhort fiber mutant fiber

Ligonless-1 (li-1) is a superbhort fiber mutant, the Fibre Development that its fiber is growing early stage and its wild-type TM-1 does not have obvious difference, during Post flowering 5 days, both Fibre Developments start to occur difference, during to fiber maturation, the staple length of li-1 mutant only has 6 millimeter, and the staple length of wild-type TM-1 is 29 millimeter.The fibrocyte of li-1 extends and receives serious suppression, and therefore this mutant is the good material that research fibrocyte extends.In order to the effect of further clear and definite GhCYP734A1L-1 gene in fibrocyte extends, the differential expression of the middle GhCYP734A1L-1 gene of the ovule fiber (FO-0DPA), the Post flowering ovule fiber (FO-6DPA) of 6 days, the Post flowering fibrocyte of 10 days (F-10DPA) and the Post flowering ovule of 10 days (O-10DPA) that utilize real-time quantitative RT-PCR to have detected TM-1 and li-1 mutant to bloom the same day.Result shows that the expression level of this gene occurs significant difference in 6DPA ovule fiber and 10DPA fiber, and particularly in 10DPA fiber, this gene is expressed hardly in li-1 fiber, and this gene has high expression level in wild fiber type.In 0DPA ovule fiber and 10DPA ovule, this gene is not expressed (Fig. 4) in wild-type and li-1 mutant.Infer that the differential expression of GhCYP734A1L-1 gene in 6DPA ovule fiber mainly also derives from the differential expression in 6DPA fibrocyte thus.This result illustrates that GhCYP734A1L-1 gene has vital role in fibrocyte extends.

The structure of embodiment 3 overexpression and Antisense Suppression GhCYP734A1L-1 gene plant expression vector and the genetic transformation of cotton

1, the structure of excess and antisense expression vector

PGEm-GhCYP734A1L-1 carrier builds when cloning GhCYP734A1L-1 gene, and the GhCYP734A1L-1 fragment on it checks order.Plant expression vector is the pCambia carrier (Ji Sai bio tech ltd, Guangzhou) of transformation, (design primer increases and obtains from pBI121 carrier to replace with NPT II gene after the HPT II gene XhoI single endonuclease digestion of this carrier, design of primers two end band XhoI site), restriction enzyme digestion and sequencing result demonstrate the direction of NPT II gene.In pBI121 carrier, amplification obtains CaMV35S promotor and NOS terminator, these two element two ends have also been introduced corresponding restriction enzyme site simultaneously, these two elements finally import the multiple clone site of pCambia respectively, form CaMV35S::MCS::NOS unit.The plant expression elements of this plant expression vector (Fig. 5) containing a set of 2 × CaMV35S promotor control NPTII gene, the plant expression elements of a set of CaMV35S promotor control report gene GRP-GusPlus-His6 and the plant expression elements of a set of CaMV35S promotor control objectives gene, can realize the double-tagging screening of Kan and GUS activity.Insert foreign gene in multiple clone site (Multiple cloning site, MCS), the overexpression of foreign gene can be realized.

In order to overexpression GhCYP734A1L-1 gene in transgenic plant, GhCYP734A1L-1 gene forward is needed to insert in plant expression vector, and start expression by CaMV35S promotor promotor, construct overexpression GhCYP734A1L-1 gene plant expression vector pCambia-35S-GhCYP734A1L-1-NOS (being called for short pC-GhCYP734A1L-1) (Fig. 6).GhCYP734A1L-1 gene is oppositely inserted in plant expression vector pCambia, start by CaMV35S promotor and express, construct the plant expression vector pC-CaMV 35S-antisense GhCYP734A1L-1 containing GhCYP734A1L-1 gene antisense sequence, (being called for short pC-antisense GhCYP734A1L-1) (Fig. 7).

2. the genetic transformation of cotton

With electrization, the plant expression carrier plasmid of structure is imported Agrobacterium LBA4404 bacterial strain carry out Cotton Transformation.With reference to Bio-RAD MicroPulser instruction manual book, above-mentioned carrier is imported Agrobacterium LBA4404 bacterial strain by Electroporation conversion.

Above-mentioned plant expression vector imports cotton by agriculture bacillus mediated Cotton Hypocotyl method for transformation.Concrete grammar is as follows:

Cotton 14 seeds in Ji peel off shell, with the mercuric chloride (HgCl of 0.1% ₂) sterilizing 10min, with a large amount of aseptic water washing 8 times.In 125mL triangular flask, add the concussion of about 35mL sterilized water spend the night, change a sterilized water next day.After seed grows hypocotyl root, be seeded on seed germination medium, at 28 DEG C, sprout 2-3d under dark condition, now seed hypocotyl starts the period entering rapid elongation, is suitable for carrying out genetic transformation.

The agrobacterium strains containing pC-CaMV35S-GhCYP734A1L-1 and pC-CaMV35S-antisense GhCYP734A1L-1 plant expression vector transformed is activating containing on the YEB solid medium of 50mg/L Km and 125mg/L Sm.Its single bacterium colony of picking, is inoculated in 5ml containing in identical antibiotic YEB liquid nutrient medium, 28 DEG C, 200r/min shakes overnight incubation.Agrobacterium bacterium liquid after cultivation is transferred to 25ml containing in identical antibiotic YEB liquid nutrient medium in the ratio of 1:20, continue to be cultured to OD600 value and be about 0.6-0.8,10,000r/min, 1min collected by centrifugation thalline, the isopyknic liquid Dual culture basic weight of thalline hangs for subsequent use.

During conversion, Cotton Hypocotyl is cut into the segment of 1.5-2.0cm, put into the triangular flask Agrobacterium bacterium liquid prepared and infect, condition is that 28 DEG C of shaking table 120r/min infect 30min.Then blot bacterium liquid, hypocotyl section is forwarded on Dual culture base, 28 DEG C of light culture 2-3 days.

After Dual culture, hypocotyl section is transferred to lower embryo section screening culture medium, 28 DEG C of illumination cultivation, about 20 days subcultures once, until there is a large amount of callus.Callus transfers to embryo callus subculture inducing culture together with lower embryo section, about 15 days subcultures once, until there is a large amount of light yellow embryo callus subcultures.Embryo callus subculture is chosen in embryo callus subculture suspension medium, 28 DEG C, 120r/min shaking table cultivation about a week.The body embryo drawing fine sand shape with the 1.0mL rifle head deducting tip is laid in body embryo elongation medium, within 20-30 days, occurs a large amount of green corpusculum embryos afterwards.The good body embryo subculture of picking growth conditions is cultivated, and when body embryo is elongated to 1-2cm, is transferred to seedling substratum and takes root and emerge.When growth of seedling is to 3-5cm height, is transferred in greenhouse flowerpot by the mode of grafting or transplanting and grow.Wherein, substratum used in this experimental example is as shown in table 1.

The Cotton Hypocotyl genetic transformation used medium that table 1 is Agrobacterium tumefaciens mediated

MS:Murashige&Skoog,1962；B5:Gamborg,1986。

SH substratum: phytoTechnology production number: S816, lot:11L081602613.

The checking of embodiment 4 transgene cotton

1, histological chemistry's qualification

Whether have the plant expression elements of a set of CaMV35S promotor control report base GusPlus-His6 in plant expression vector due to structure, it is transformed plant that histochemical method therefore can be utilized to identify.When plant to be transformed takes root and grows to 5 ~ 8cm on root media, seedling is taken out from culturing bottle and cleans, proceed to water planting hardening 2 ~ 3d on triangular flask.Meanwhile, get a fritter blade and a bit of carry out GUS dyeing.As shown in Figure 8 A, wild-type leaves GUS dyes as negative result, and resistant plant blade GUS dyes as positive, and positive plant is directly transplanted in nutrition pot.

2, amplification checking

Treat cotton plants transplant survival and grow into a certain size, getting 0.5g blade and extract cotton genomic dna, increase with the upstream and downstream primer GUS-up of gus gene (SEQ ID NO.10) and GUS-down (SEQ ID NO.11).The amplification system of 25 μ L is containing 2.5 μ L 10 × PCR buffer, 2 μ L 2.5mmol/L dNTPs, 1.5 μ L 25mmol/L MgCl ₂, each 1 μ L primer GUS-up and GUS-down (5 μm of ol/L), 1U Taq archaeal dna polymerase, 1 μ L genomic dna (50ng).Amplification program is: 94 DEG C, 5min; 94 DEG C, 30sec, 56 DEG C, 30sec, 72 DEG C, 1min, 35 circulations; 72 DEG C extend 10min.Make positive control with positive and negative plant expression carrier plasmid, make negative control with water and wild type cotton genomic dna.The transgenic cotton plant of result display GUS stained positive can amplify a band consistent with positive control, illustrates that the T-DNA section of plant expression vector has been incorporated into (Fig. 8 B and 8C) in transgene cotton genome.

Embodiment 5 detects the expression change of GhCYP734A1L-1 gene in transgene cotton

According to the method extracting cotton RNA in embodiment 1, extract the total serum IgE of transgene cotton and wild type cotton blade, and synthesize cDNA mono-chain.Analyze the expression of goal gene in transgene cotton by Real-Time PCR method, PCR carries out on real-time PCR, comprises 12.5 μ L MIX buffer (comprise PCR damping fluid, archaeal dna polymerase, dNTPs and MgCl in the reaction system of 25 μ L ₂, real-time quantitative RT-PCR test kit provides, Bio-Rad).Primer GhCYP734A1L-1-1 (SEQ ID NO.6) and GhCYP734A1L-1-2 (the SEQ ID NO.7) amplification of GhCYP734A1L-1 gene, interior mark adopts cotton GhHISTONE3 (GenBank accession number: AF024716), and primer is GhHIS-1 (SEQ ID NO.8) and GhHIS-2 (SEQ ID NO.9).Amplification program is: 94 DEG C of denaturation 3min; 94 DEG C, 30sec; 56 DEG C, 30sec; 72 DEG C, 30sec; Preset cycle number is 35.Before operation real-time quantitative PCR, increase once with template by same primers in identical temperature variation program, by the electrophoresis of amplified production, guarantee that amplified production is single tape.The result of real-time quantitative RT-PCR analysis shows the expression level of GhCYP734A1L-1 gene in overexpression GhCYP734A1L-1 transgene cotton blade all than contrast high (Fig. 9 B), because the growth of this gene render transgenic cotton plants of overexpression is suppressed, can not yield positive results, therefore the expression change of this gene can not be detected in transgenic fibre, therefore in blade, have detected the expression of this gene, result shows that this gene is higher at transgene cotton leaf expression.On the contrary in the transgene cotton fiber of all suppression GhCYP734A1L-1 genes, the expression level of this gene reduces (Fig. 9 A) all in various degree.This result illustrates the transgenic cotton plant having obtained overexpression and Antisense Suppression GhCYP734A1L-1.

Embodiment 6 transgene cotton compares with the phenotype of wild type cotton

1, overexpression GhCYP734A1L-1 suppresses the growth of cotton plants

In experimental plot, plant pC-GhCYP734A1L-1 transgenosis and wild type cotton simultaneously, carry out normal production management.The transgene cotton that GhCYP734A1L-1 expression level increases is observed, find that overexpression GhCYP734A1L-1 transgenic cotton flower growth receives suppression, plant short and small (Figure 10 A), stem internode shortens, side shoot is less or do not have side shoot, and the internode of side shoot shortens, blade diminishes (Figure 10 B), the dim light of night is dark green, chlorophyll content increases (Figure 10 C), blade face pore is not closed (Figure 10 D) under desiccation.This result illustrates that overexpression GhCYP734A1L-1 gene can suppress the growth of cotton plants, and the plant type of transfer-gen plant has considerable change.If use the expression of the specific promoter control GhCYP734A1L-1 gene of stem or branch, will effectively change the plant type of transfer-gen plant, Plant-type Breeding has larger using value.

Embodiment 7 suppresses GhCYP734A1L-1 genetic expression to promote cotton fiber extension

The transgene cotton of Antisense Suppression GhCYP734A1L-1 and contrast plantation simultaneously to experimental plot, are carried out normal production management, growing and the length of comparative maturity fiber of observation cotton fiber.Result shows, and suppresses the transgenic cotton plant of GhCYP734A1L-1 gene not have notable difference with wild type cotton plant in growth, further illustrates this gene and does not express in the organs such as root, stem, leaf and flower.Suppress the transgene cotton mature fibers of GhCYP734A1L-1 gene longer than contrast, its mean length is 34.6 ± 0.25 millimeters, and the mean length of control fiber is 31.9 ± 0.54 millimeters, and staple length adds 8.46% (Figure 11 A and C).Meanwhile, the growth of transgene cotton fiber and control fiber is observed by ovules culture in vitro.Result shows (Figure 11 B), and the fiber that the transgene cotton ovule suppressing GhCYP734A1L-1 to express grows is longer than the fiber that wild-type ovule grows.This result further illustrates GhCYP734A1L-1 gene and have vital role in fibrocyte elongation process, and its expression level and staple length have substantial connection.

Claims

1. the protein C YP734A1like-1 of specifically expressing in fibrocyte, has the aminoacid sequence as shown in SEQ ID NO.3.

2. protein as claimed in claim 1, is characterized in that: the encoding gene GhCYP734A1L-1 of described CYP734A1like-1 has:

(a) cDNA as shown in SEQ ID NO.1;

Or (b) genomic dna as shown in SEQ ID NO.2.

3. express the plant expression vector of protein described in claim 1 or 2.

4. carrier as claimed in claim 3, is characterized in that: described plant expression vector is the carrier of overexpression GhCYP734A1L-1 gene or Antisense Suppression GhCYP734A1L-1 gene.

5. the host cell containing carrier described in claim 3 or 4.

6. host cell as claimed in claim 5, is characterized in that: described host cell is Agrobacterium.

7. the application of the CYP734A1like-1 protein described in claim 1 or 2 in improvement cotton variety.

8. the application of the plant expression vector described in claim 3 or 4 in improvement cotton variety.

9. the application of CYP734A1like-1 protein in improving cotton fiber quality described in claim 1 or 2.

10. the application of the plant expression vector described in claim 3 or 4 in improving cotton fiber quality.