CN104560906B - Specifically expressed protein C YP734A1 like 1 and its application in fibrocyte - Google Patents
Specifically expressed protein C YP734A1 like 1 and its application in fibrocyte Download PDFInfo
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Abstract
The invention belongs to plant genetic engineering field, and in particular to specifically expressed protein C YP734A1 like 1 and its application in fibrocyte.The technical problem to be solved in the present invention is for improve that current fiber is special or the gene of predominant expression still very little, be not met by the needs of improvement cotton fiber yield and quality MOLECULE DESIGN.Protein C YP734A1like 1 disclosed by the invention, has the amino acid sequence as shown in SEQ ID NO.3.Present invention also offers the plant expression vector for expressing the albumen.The present invention provides new selection to improve cotton fiber quality and improvement cotton variety.
Description
Technical field
The invention belongs to plant genetic engineering field, and in particular to specifically expressed protein C YP734A1 in fibrocyte
Like-1 and its application.
Background technology
Cotton is one of fibre crops main in the world.Cotton fiber cell is cotton ovule external integument epidermal cell warp
Differentiation projection and polarity elongation form unicellular.Its growth growth course be divided into four not only substantially distinguished but also it is overlapping when
Phase：Fibrocyte starting phase, fibrocyte elongation phase, secondary wall thickening phase and dehydration maturity period.Each period has respective
Feature, but adjacent developmental stage is again overlapping.There is shadow in each period of Fibre Development to the yield and/or quality of fiber
Ring, the differentiation starting of fiber determines the quantity of fiber in simple grain ovule, and fibrocyte extension speed and duration mainly determine
The length of fiber is determined, the mode of secondary wall deposition and duration essentially dictate the intensity of mature fibers and secondary wall thickness
Degree.The elongate fiber phase be (primary wall synthesize phase) since the same day of blooming, can continue to Post flowering 25 days (25DPA), mainly
Cell polarity elongation and the synthesis of primary cell wall are carried out, the elongate fiber speed of this period and duration determine fiber most
Whole length, upland cotton fiber draw ratio is up to 1000-3000.But so far, we are to regulating and controlling the molecule machine of its growth course
System is still known little about it.
Since cotton fiber cell is a special cell, must there are special gene expression and phase in its growth course
Close regulatory mechanism.Therefore the gene of screen fibre cell specific expression becomes the hot spot of cotton fiber molecular biology research.It is logical
CDNA library differential screening *, cDNA reprensentative difference analysis, Subtractive hybridization and mRNA differential display techniques are crossed, and in recent years
Come develop biochip technology, high throughput sequencing technologies etc., clone and analyze special or predominant expression base in fiber
Cause.John and Crow cloned Fibre Development early stage specifically expressed E6 in first passage cDNA library differential screening in 1992
Gene [John M E, Crow L J.1992.Gene expression in cotton (Gossypium hirsutum L.)
fiber：Cloning of the mRNAs.Proc Natl Acad Sci USA, 89 (13)：5769-5773] utilizations are equally
Method, Rinehart etc. (1996) be isolated to Fibre Development middle and later periods specific expression gene FbL2A [Rinehart J A,
Petersen M W, John M E.1996.Tissue specific and developmental regulation of
cotton gene FbL2A.Plant Physiology,112(3):1331-1334].In recent years, fiber is special or advantage table
The microtubule protein gene that reaches, extensin gene, lipid transfer protein gene etc. cloned and analyzed in succession [Lv Shaopu, Wang Xujing,
Tang Qiaoling, waits the progress Biotechnological Advances of .2014. cotton fibers specific gene and its specific promoter, 4 (1):1-6].
These results of study not only confirm that the development that fiber is special or predominant expressed gene is to fibrocyte plays an important roll, but also
Experimental data is provided to have parsed the regulatory mechanism of fibroblast growth development.But up to the present, the fiber of acquisition is special
The gene of exclusive or predominant expression still very little, is not met by the needs of improvement cotton fiber yield and quality MOLECULE DESIGN.
The content of the invention
The technical problem to be solved in the present invention is a kind of new selection is provided to improve cotton fiber quality, to improve current obtain
Fiber is special or the gene of predominant expression still very little, be not met by improvement cotton fiber yield and quality MOLECULE DESIGN
Need.
The technical scheme is that the specifically expressed protein C YP734A1 like-1 in fibrocyte, have (a)
Amino acid sequence as shown in SEQ ID NO.3.
Specifically, the coding GhCYP734A1L-1 genes of the albumen have：
(a) cDNA as shown in SEQ ID NO.1 (cDNA, that is, complementary DNA (cDNA), for with mRNA (couriers
RNA) chain is in the single stranded DNA of complementary base sequence)；
Or the genomic DNA (genomic DNA, that is, gDNA) of (b) as shown in SEQ ID NO.2.
Present invention also offers the plant expression vector for expressing the protein.
Specifically, the plant expression vector is overexpression GhCYP734A1L-1 genes or Antisense Suppression
The carrier of GhCYP734A1L-1 genes.
Present invention also offers the host cell containing the carrier.
Specifically, the host cell is Agrobacterium.
Present invention also offers the CYP734A1 like-1 or plant expression vector answering in cotton variety is improved
With.
Present invention also offers the CYP734A1 like-1 or plant expression vector in improving cotton fiber quality
Using.
In the present invention, protein G hCYP734A1L-1 includes, and has the amino acid sequence shown in SEQ ID NO.3；Or will
The amino acid residue sequence of SEQ ID NO.3 pass through one or several amino acid residues substitution, missing or addition, and with
The derivative protein of the identical bioactivity of 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 genes, SEQ
Nucleotides sequence shown in ID NO.2 is classified as gDNA (genomic DNA) sequence of gGhCYP734A1L-1 genes.Wherein described cDNA
Sequence includes 5'- non-translational regions sequence, open reading frame (ORF) sequence and 3'- non-translational region sequences, wherein open reading frame sequence
Coded sequence is classified as, gDNA sequences contain extron and intron sequences.
The present invention plant expression vector, can using at least one of enhanced, composing type or inducible promoter come
Regulate and control the expression of GhCYP734A1L-1 genes.Promoter for building plant expression vector of the present invention preferably constitutes type startup
Son or tissue-specific promoter, more preferably the plant constitutive promoter CaMV35S from cauliflower mosaic virus.It is logical
Often, it is GhCYP734A1L-1 is gene constructed in the downstream of CaMV35S.For building the load of setting out of plant expression vector of the present invention
Body can be any one double base agrobacterium vector or the plant expression vector available for via Particle Bombardment Transformation.In order to screen and table
The needs reached, in expression vector comprising screening-gene sequence, reporter sequences and others are alternatively also gene work
The needs of journey operation and the various restriction enzyme sites that are inserted into, screening-gene and reporter gene can be from gene sequences commonly used in the art
Selected in row, it is preferable that plant expression of the invention has structure as shown in Figure 5.For example, can be in the expression vector
The enzyme of color change or the gene of luminophor can occur for the coding that adding can express in plant, such as GUS (beta-glucosidases
Sour enzyme) gene, GFP (green fluorescent protein) gene, luciferase etc.；Resistant antibiotic marker, such as anti-celebrating are big mould
Plain label, anti-kalamycin label etc.；Anti- chemical reagent marker gene, such as anti-herbicide gene.
The present invention uses excess or suppresses the method for GhCYP734A1L-1 expression vector prepare transgenosis plants, including with
Lower step：
1) GhCYP734A1L-1 genes are operably connected with promoter；
2) plant expression vector containing GhCYP734A1L-1 genes and promoter is built；
3) host is converted with the plant expression vector, obtains transformant；
4) plant is converted with the transformant, obtains genetically modified plants.
, can be by using Ti-plasmids, Ri plasmids, plant or microbiosis poisonous carrier, directly delivered DNA, micro- in the present invention
Injection, conductance or the conventional biology methods such as agriculture bacillus mediated, by the expression vector containing GhCYP734A1L-1 genes of the present invention
Transfect cotton and obtain transformant.
Beneficial effects of the present invention：
The coding protein of presently disclosed GhCYP734A1L-1 genes can promote cotton fiber extension, improve cotton
Fibre length, increases fiber quality and yield；Therefore can be using the gene come improving cotton fiber quality, while can also basis
Need to select different types of promoter, carry out improving cotton fiber quality or improve cotton plant type, to be conducive to output of cotton
The raising and special cultivation operation (such as mechanical harvesting operation and high-density planting) of quality.
Brief description of the drawings
Fig. 1：The tetraploid rice of GhCYP734A1L-1 protein and other species related proteins；
gi144905179：Pea (Pisum sativum) Cytochrome P450 family protein；gi15225777：Arabidopsis
(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：Middle cotton is false to intend
Albumen.It is higher same that this figure shows that the cytochrome p450 protein (CYP734A1) of GhCYP734A1L-1 and other species has
Source property, GhCYP734A1L-1 genes are the homologous genes of CYP734A1.
Fig. 2：The phylogenetic analysis of GhCYP734A1L-1；
This figure shows that GhCYP734A1L-1 genes and the homologous gene affiliation in middle cotton and cocoa are nearest.
Fig. 3：The expression analysis of GhCYP734A1L-1 genes；
Wherein A：Expression pattern of the GhCYP734A1L-1 genes in different tissues, organ and cell；
Wherein B：GhCYP734A1L-1 genes are in fibrocyte and the expression of ovule different development stage；OF-0DPA
~OF-4DPA：Ovule (fibre-bearing cell) on the day of blooming arrives 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 blooming
The ovule of 16 days afterwards.This figure shows GhCYP734A1L-1 genes specifically expressing in fibrocyte, in root, stem, leaf, flower, son
Leaf, hypocotyl and ovule are hardly expressed.Faint expression in figure in ovule be probably fibrocyte from ovule sur-face peeling when
Caused by remaining fibrocyte.
Fig. 4：GhCYP734A1L-1 genes upland cotton wild type TM-1 and its superbhort fiber mutant li-1 fiber and
Expression in ovule；
TM-1：Upland cotton wild type；li-1：Superbhort fiber mutant；FO-0DPA:Ovule fiber on the 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 embryo of 10 days
Pearl.This figure shows GhCYP734A1L-1 genes in the superbhort fiber mutant Post flowering ovule fiber of 6 days and Post flowering 10 days
Fibrocyte in expression developmental stage identical well below its wild type correspond to expression in fiber and ovule.Explanation
GhCYP734A1L-1 genes play an important roll during elongate fiber.
Fig. 5：The structure chart of the plant expression vector of the gene containing GhCYP734A1L-1；
Wherein CaMV 35S represent CaMV 35S promoters；GhCYP734A1L-1 represents GhCYP734A1L-1 genes
cDNA；Ter represents terminator；LB represents T-DNA left margins；RB represents T-DNA right margins.
Fig. 6：Currently preferred plant expression vector pC-CaMV 35S-GhCYP734A1L-1 structure charts；
Wherein GusPlus-His6 represents GUSPlus reporter genes, which has merged His6 sequence labels in C-terminal；
NPTII represents neomycin phosphotransferase gene, has kalamycin resistance；Ter：Nos terminators；CaMV35S：From flower
The plant composition promoter of cauliflower mosaic virus；LB：T-DNA left margins；RB：T-DNA right margins.Plant expression vector is to change
The pCambia carriers (detailed in Example three) made.
Fig. 7：Currently preferred Antisense Suppression GhCYP734A1L-1 gene plants expression vector pC-CaMV 35S- antisenses
GhCYP734A1L-1 structure charts.Wherein GusPlus-His6 represents GUSPlus reporter genes, which has merged His6 in C-terminal
Sequence label；NPTII represents neomycin phosphotransferase gene, has kalamycin resistance；Ter：Nos terminators；
CaMV35S：From the plant composition promoter of cauliflower mosaic virus；LB：T-DNA left margins；RB：T-DNA right margins.
Plant expression vector is the pCambia carriers (detailed in Example three) of transformation.
Fig. 8：The identification of transgene cotton；
Wherein A：Histochemistry's identification of transgene cotton, WT：Non-transgenic cotton (wild type), compares；pC-
CaMV35S- antisenses GhCYP734A1L-1 is the transgene cotton blade of Antisense Suppression GhCYP734A1L-1 genes；pC-
CaMV35S-GhCYP734A1L-1 represents the transgene cotton blade of overexpression GhCYP734A1L-1 genes；
Wherein B：The amplification verification of overexpression GhCYP734A1L-1 transgene cottons, M：DNA marker2000；H2O：
Using water as PCR amplification template, make blank control；+：Using pC-CaMV 35S-GhCYP734A1L-1 plasmids as PCR amplification template,
Make positive control；1 and 2：Overexpression GhCYP734A1L-1 transgene cottons 1# and 2#.
Wherein C：The amplification verification of Antisense Suppression GhCYP734A1L-1 transgene cottons, M：DNA marker15；-：Non- turn
Gene cotton (wild type), makees negative control；H2O：Using water as PCR amplification template, make blank control；+：With pC-CaMV35S-
Antisense GhCYP734A1L-1 plasmids are PCR amplification template, make positive control；1~3：Antisense Suppression GhCYP734A1L-1 turns base
Because of cotton 1#~3#.
Fig. 9：The expression analysis of GhCYP734A1L-1 genes in transgene cotton；
Wherein A：The expression of GhCYP734A1L-1 genes point in Antisense Suppression GhCYP734A1L-1 transgene cotton fibers
Analysis, WT:Non-transgenic cotton (wild type)；S1~S13:Antisense Suppression GhCYP734A1L-1 transgene cottons 1#~13#.
Wherein B：The expression analysis of GhCYP734A1L-1 in overexpression GhCYP734A1L-1 transgene cotton blades,
WT：Non-transgenic cotton (wild type)；O1~O14:Overexpression GhCYP734A1L-1 transgene cottons 1#~14#.
This chart is bright, by Cotton Transformation, obtains the increase of GhCYP734A1L-1 gene expression doses and turn reduced
Gene cotton.
Figure 10：The influence of overexpression GhCYP734A1L-1 gene pairs cotton plants growth and development；
Wherein A：Non-transgenic cotton (wild type) and the growth of overexpression GhCYP734A1L-1 transgenic cotton plants
Situation；A left side is wild type cotton plant, and the right side is overexpression GhCYP734A1L-1 transgenic cotton plants, scale=10cm.
Wherein B：The comparison of wild type cotton blade and transgene cotton blade；A left side is wild type cotton mature leaf, right
Upper 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 blades.
Wherein D：The stomatal movement situation of wild type cotton blade and transgene cotton blade；Figure below is non-transgenic cotton
The opening and closing situation of stomata on (wild type) blade, upper figure are the opening and closing situation of stomata on transgene cotton blade.
Figure 11：The influence of Antisense Suppression GhCYP734A1L-1 gene pairs cotton fiber lengths；
Wherein A：The length of cotton mature fibers compares；WT：Non-transgenic cotton (wild type)；S1:Antisense Suppression
GhCYP734A1L-1 transgene cotton 1# strains；
Wherein B：In cotton ovule culture in vitro system, non-transgenic cotton (wild type) and Antisense Suppression
The growing state of GhCYP734A1L-1 transgene cotton fibers；WT：Non-transgenic cotton (wild type)；S1:Antisense Suppression
GhCYP734A1L-1 transgene cotton 1# strains；
Wherein C：The fibre length measurement statistics of mature fibers, wild type and transgenic fibre respectively detection 20, by fibre
Dimension measures its length after combing.WT：Non-transgenic cotton (wild type)；S1:Antisense Suppression GhCYP734A1L-1 transgene cottons
1# strains.
Embodiment
Reagent chemicals in present example do not do illustrate be it is common commercially available, MATERIALS METHODS does not illustrate
Refer to《Molecular Cloning:A Laboratory guide》(Sambrook and Russell, 2001).
In following examples of the present invention, cotton experiment material used is (the Gossypium hirsutum of Ji cotton 14
Cv.Jimian14), it is the upper cultivar of production；TM-1 (Gossypium hirsutum cv.TM-1) and its superbhort fiber are dashed forward
Variation (ligon lintless-1, li-1).Wild type TM-1 separates that (li-1 dashes forward from the li-1 mutant sowed every time
Variation is a dominant mutant, and homozygote is lethal.Therefore every season only needs to harvest mutant seeds, which after planting grows
Cotton seedling in some be the wild type separated, i.e. TM-1), li-1 mutant is ground from the Chinese Academy of Agricultural Sciences cotton
Study carefully institute's Germplasm Bank, it is open to provide.
The Cloned culturing of 1 GhCYP734A1L-1 genes of embodiment
1st, the extraction of cotton RNA
The fresh cotton materials of about 3g are chosen, wear into fine powder rapidly in liquid nitrogen, load 50mL centrifuge tubes, are added
15ml65 DEG C preheating RNA extracting solutions (2%CTAB (W/V), 2%PVP (W/V), 100mmol/L Tris-HCl (pH8.0),
0.5g/L spermidines, 2.0mol/L NaCl, 2% mercaptoethanol (V/V), uses preceding addition)), overturn and mix.65 DEG C of water-baths 3~
10min, during which mixes 2~3 times.Lv Fang ︰ isoamyl alcohol (24 ︰ 1) extracts 2 times (10,000r/min, room temperature, 5min).Take supernatant,
Add 1/4 volume 10mol/L LiCl solution, 4 DEG C of placement 6h, with Lv Fang ︰ isoamyl alcohol (24 ︰ 1) respectively extracting 1 time (10,000r/
Min, room temperature, 5min).Add the absolute ethyl alcohol of 2 times of volumes, more than 30min is precipitated in -70 DEG C of refrigerators.12,000r/min, 4 DEG C from
Heart 20min, abandons supernatant.The precipitation DEPC processing water of 200 μ L dissolves.Phenol (pH4.5) ︰ Lv Fang ︰ isoamyl alcohol (25 ︰, 24 ︰ 1), chlorine
Each extracting 1 time (10,000r/min, room temperature, 5min) of Fang ︰ isoamyl alcohol (24 ︰ 1).Add 1/10 volume 3mol/L NaAc solution and
The absolute ethyl alcohol of 2.5 times of volumes, more than 30min is precipitated in -70 DEG C of refrigerators.12,000r/min, 4 DEG C of centrifugation 20min, abandon supernatant.
Precipitation is rinsed once with 70% alcohol, is air-dried.The DEPC of 200 μ L is added to handle water dissolving.With non denatured agarose gel electrophoresis
With the quality of ultraviolet specrophotometer Scanning Detction RNA.
2nd, cDNA is synthesized
With the various sample total serum IgEs of cotton, mono- chains of cDNA are synthesized with kit (Fermentas).Specific method is：Take about 10
In the amplification pipe that μ g total serum IgEs are handled to DEPC, 1 μ L, 2.5 μm of ol/L Oligo-dT are added, add the water that DEPC is handled to final volume
After 12 μ L, 70 DEG C of water-bath 5min are denatured RNA, ice bath 3min immediately.Then 45 × bufferings of μ L are sequentially added into amplification pipe
Liquid, 2 μ L 10mmol/L dNTPs, 1 μ L RNase inhibitor (20U), 37 DEG C of processing 5min.Add 1 μ L reverse transcriptases AMV
After Rtase (5U), insulation program is 42 DEG C, 60min；70 DEG C, 5min；5 DEG C, 5min.After EP (end of program), mono- chain products of cDNA
Frozen in -20 DEG C.
3rd, the extraction of cotton genomic dna
Cotton genomic dna is extracted using the CTAB methods of improvement.0.5g cotton spires are taken, are ground into rapidly in liquid nitrogen
Powder, adds the CTAB extracting solutions of 65 DEG C of preheatings of 3mL, and quick oscillation mixes.65 DEG C of water-bath 30min, then add 1mL 5mol/L
After KAc, ice bath 20min, 1 time (12,000r/min, 4 DEG C of centrifugation 5min) is extracted with isometric Lv Fang ︰ isoamyl alcohol (24 ︰ 1),
Supernatant is taken, adds -20 DEG C of pre- cold isopropanols of 2/3 times of volume, is mixed, about 30min is stood, chooses flocculent deposit, 75% second
Alcohol rinses 3 times repeatedly, then is rinsed 1 time with absolute ethyl alcohol, and drying, is dissolved in 500 μ L TE again.Add 1 μ L RNaseA (10mg/
ML), 37 DEG C of processing 1h.Again Yong Fen ︰ Lv Fang ︰ isoamyl alcohol (25 ︰, 24 ︰ 1) and Lv Fang ︰ isoamyl alcohol (24 ︰ 1) respectively extracting 1 time (12,
000r/min, 4 DEG C of centrifugation 5min), supernatant is taken, adds the absolute ethyl alcohol precipitation DNA of 2 times of volumes.- 20 DEG C of placement about 30min, from
The heart (12,000r/min, 4 DEG C of centrifugation 5min), abandons supernatant, and precipitation is rinsed with 75% ethanol, air-dries, be dissolved in 200 μ L TE ,-
20 DEG C save backup.
4th, high homology cotton est sequence is screened
With the cotton in the protein sequence search ncbi database of arabidopsis Brassinosteroids metabolic gene CYP734A1
Flower EST (Expressed Sequence Tag, EST) sequence, to the high homology cotton est sequence searched
Confluence analysis is carried out, obtains an integration sequence, then integration sequence is dealt into ncbi database and is compared, determines to integrate sequence
Whether row encode the homologous gene of CYP734A1, if it is, in ORF contained by integration sequence (open reading frame, open
Reading frame) both sides design amplimer, carry out gene cloning.
5th, the amplification of GhCYP734A1L-1 gene cDNA sequences and genome sequence
Based on integration sequence, amplimer is designed in the both sides of its ORF, 5' ends primer is GhCYP734A1L-1-3
(SEQ ID NO.4) and 3' ends primer are GhCYP734A1L-1-4 (SEQ ID NO.5), using 12DPA fibers cDNA as template into
Row PCR.The cDNA amplification systems of 25 μ L contain 2.5 μ L 10 × Ex PCR buffer solutions (no Mg2+), 2 μ L 2.5mmol/L dNTPs, 2
μL 25mmol/L MgCl2, 1 μ L special primers 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 polymerases, the production of 1 μ L cDNA, mono- chains
Thing.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 of extensions
10min。
Expanded at the same time by template and identical primer of cotton genomic dna, the genomic DNA amplification system of 20 μ L
Containing 10 μ 2 × PrimerStar of L mix, 1 μ L special primers 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 of extension 10min.
6th, amplified fragments recycle, and connection, converts escherichia coli DH5a
(1) electrophoresis
Amplified production is separated by electrophoresis in 1.0% (W/V) Ago-Gel.
(2) recycle
Use QIAquick Gel Extraction Kit：Recycling step is carried out according to kit specification, and recycling fragment is powered in Ago-Gel
Swimming is quantitative.
(3) clone and be sequenced
The fragment of recycling is quantified through agarose gel electrophoresis.By kit specification, by recycling fragment and cloning vector
Connection, Escherichia coli conversion, the culture of positive bacterium colony and the plasmid enzyme restriction verification of connection product, recycling fragment is cloned into
On pGEm-T (Shanghai Sangon) carrier.Sequencing is completed by Ying Jun companies.
The fragment of recycling establishes following linked system with pGEm-T (Shanghai life work) carrier：10 × T4 DNA connection buffer solutions
1 μ L, 1 μ L of vector DNA fragment, 1 μ L, T4 DNA ligase of external source connection product DNA fragmentation, 1 μ L, volume is supplied extremely with distilled water
10μL。
Vector DNA fragment is 1 ︰, 3,16 DEG C of connection 12h with external source connection product DNA fragmentation molar ratio.Connection is produced afterwards
Thing converts escherichia coli DH5a.
7th, GhCYP734A1L-1 gene sequencings
Using 14 12DPA fibers cDNA of upland cotton Ji cotton as template, the special band of an about 1700bp is amplified.Sequencing
After find amplifying specific piece segment length 1730bp (SEQ ID NO.1), include a complete open reading frame (ORF), it is long
1575bp.The protein (SEQ ID NO.3) of 524 amino acid residue is encoded, the molecular weight of the protein prediction is
59.8kD, isoelectric point 9.498.
The homologous protein of GhCYP734A1L-1 is searched on NCBI, the results showed that GhCYP734A1L-1 belongs to cytochromes
The CYP734A subfamilies of P450 families.The albumen and pea (Pisum sativum) CYP734A1 albumen (gi144905179)；
Soybean (Glycine max) cytochromoid P450 albumen (CYP734A1-like) (gi356563055)；Arabidopsis
(Arabidopsis thaliana) cytochrome p450 protein (CYP734A1) (gi15225777)；Grape (Vitis
Vinifera) cytochromoid P450 albumen (CYP734A1-like) (gi225445412)；Castor-oil plant (Ricinus
Communis) cytochromoid P450 albumen (gi255566913)；Cocoa (Theobroma cacao) Cytochrome P450 egg
The albumen such as (gi590577824) has higher homology, the same amino acid residue of GhCYP734A1L-1 and these albumen in vain
79%, 63%, 66%, 72% and 65% is respectively reached, similar amino acid residue respectively reaches 88%, 85%, 84%, 83% and
78% (Fig. 1).It is the homologous gene of CYP734A1 genes to illustrate GhCYP734A1L-1.
Phylogenetic analysis is the result shows that GhCYP734A1L-1 has with cocoa, grape, willow and the CYP734A1 of castor-oil plant
Nearer affiliation, and with the affiliation of the CYP734A subfamily albumen of the species such as pea and arabidopsis farther out (Fig. 2).
The genomic dna sequence of GhCYP734A1L-1 genes, the long 2978bp of the sequence have been cloned from upland cotton at the same time
(SEQ ID NO.2), by being compared with the cDNA sequence of GhCYP734A1L-1 genes and being identified according to GT-AG intrones
Law-analysing, it turns out that, GhCYP734A1L-1 genome sequences contain 5 extrons and 4 intrones.
Expression analysis of the 2 GhCYP734A1L-1 genes of embodiment in cotton plants and Fibre Development
Using mono- chains of cDNA of synthesis as template, PCR is carried out using real-time quantitative PCR kit (Bio-Rad).Expression is drawn
Thing is designed according to the cDNA sequence of GhCYP734A1L-1 genes, and 5 ' end primers are GhCYP734A1L-1-1 (SEQ ID NO.6),
3 ' end primers are GhCYP734A1L-1-2 (SEQ ID NO.7).Include 10 μ L MIX buffer solutions in the reaction system of 20 μ L
(including PCR buffer solutions, archaeal dna polymerase, dNTPs and MgCl2), 5'- ends and each 1 μ L of 3'- ends primer (5 μm of ol/L).Circulation ginseng
Number is 94 DEG C of pre-degeneration 3min；94 DEG C, 30sec, 55 DEG C, 30sec, 72 DEG C, 30sec, preset cycle number 40.Use cotton
GhHISTONE3 genes (GenBank accession number：AF024716 internal standard) is made, the 5'- primers of GhHISTONE3 genes are GhHIS-1
(SEQ ID NO.8), 3'- primers are GhHIS-2 (SEQ ID NO.9).
1st, the expression pattern of the different times of different tissues organ and fiber Ovule Development
Upland cotton (Gossypium hirsutum L.) the respectively total serum IgE of tissue and organ is extracted, and synthesizes mono- chains of cDNA.
Expression of the GhCYP734A1L-1 genes in different tissues and organ is have detected with real-time quantitative RT-PCR.Testing result
Show, GhCYP734A1L-1 genes specifically expressing in fibrocyte is several in root, stem, leaf, flower, cotyledon, hypocotyl and ovule
Do not express.Faint expression in figure in ovule be probably fibrocyte from ovule sur-face peeling when remaining fibrocyte caused by
(Fig. 3 A).It is a fibrocyte specific expression gene to illustrate GhCYP734A1L-1 genes.
Further detect expressions of the GhCYP734A1L-1 in cotton ovule fiber different development stage.The result shows that
(Fig. 3 B), which hardly expresses in the ovule fiber of (0DPA) on the day of blooming and Post flowering 2 days (2DPA), in Post flowering
There is faint expression in 4 days (4DPA) ovule fibers；In the fibrocyte of Post flowering 6 days (6DPA) to Post flowering 10 days (10DPA)
Middle great expression, and as the growth of fibrocyte, expression gradually rise, expression top is reached in 10DPA；
After 10DPA, with the continued growth of fibrocyte, the expression of GhCYP734A1L-1 genes gradually reduces, but is blooming
Still there is higher expression in (16DPA) fibrocyte within 16 days afterwards.Expression in ovule is all well below identical development
Expression in period fiber, is nearly no detectable the expression of GhCYP734A1L-1 genes in 16DPA ovules.In ovule
Faint expression be probably fiber from ovule sur-face peeling when caused by remaining few fibers (Fig. 3 B).This expression pattern is shown
GhCYP734A1L-1 genes are in fibrocyte rapid elongation phase expression highest, its expression and fibrocyte elongation speed
Rate is directly proportional, illustrates that the gene plays an important roll in fibrocyte elongation.
2nd, the differential expression in superbhort fiber mutant fiber
Ligonless-1 (li-1) is a superbhort fiber mutant, its fiber is in mesoderm growing early stage and its wild type TM-1
Fibre Development there is no obvious difference, during Post flowering 5 days, both Fibre Developments start difference occur, during to fiber maturation,
The fibre length of li-1 mutant only has 6 millimeters, and the fibre length of wild type TM-1 is 29 millimeters.The fibre of li-1
Dimension cell elongation receives serious suppression, therefore the mutant is the good material for studying fibrocyte elongation.In order to further bright
Effect of the true GhCYP734A1L-1 genes in fibrocyte elongation, TM-1 and li-1 are have detected using real-time quantitative RT-PCR
Mutant bloom the same day ovule fiber (FO-0DPA), the Post flowering ovule fiber (FO-6DPA) of 6 days, the Post flowering fibre of 10 days
Tie up the differential expression of GhCYP734A1L-1 genes in cell (F-10DPA) and the Post flowering ovule of 10 days (O-10DPA).As a result
Show that the expression of the gene significant difference occurs in 6DPA ovules fiber and 10DPA fibers, particularly in 10DPA fibers
In, which hardly expresses in li-1 fibers, and the gene has high expression in wild fiber type.
In 0DPA ovules fiber and 10DPA ovules, which does not express (Fig. 4) in wild type and li-1 mutant.Thus speculate
Differential expression of the GhCYP734A1L-1 genes in 6DPA ovule fibers mainly also derives from the difference table in 6DPA fibrocytes
Reach.This result illustrates that GhCYP734A1L-1 genes play an important roll in fibrocyte elongation.
The structure and cotton of 3 overexpression of embodiment and Antisense Suppression GhCYP734A1L-1 gene plant expression vectors
Genetic transformation
1st, the structure of excess and antisense expression vector
PGEm-GhCYP734A1L-1 carriers are built when cloning GhCYP734A1L-1 genes, thereon
GhCYP734A1L-1 fragments have been sequenced.(the lucky match biotechnology in Guangzhou is limited for the pCambia carriers of transformation for plant expression vector
Company), (design primer is from pBI121 carriers with NPT II genes are replaced with after XhoI single endonuclease digestions for the HPT II genes of the carrier
Amplification obtains, design of primers both ends band XhoI sites), restricted digestion and sequencing result demonstrate the direction of NPT II genes.
Amplification obtains CaMV35S promoters and NOS terminator in pBI121 carriers, while the two element both ends have also been introduced accordingly
Restriction enzyme site, the two elements are finally directed respectively into the multiple cloning sites of pCambia, form CaMV35S：：MCS：：NOS is mono-
Member.Plant expression elements of the plant expression vector (Fig. 5) containing a set of 2 × CaMV35S promoters control NPTII genes, one
Cover the plant expression elements of CaMV35S promoter control report genes GRP-GusPlus-His6 and a set of CaMV35S promoters
The plant expression elements of control targe gene are, it can be achieved that the double labelling of Kan and GUS activity is screened.In multiple cloning sites
(Multiple cloning site, MCS) is inserted into foreign gene, it is possible to achieve the overexpression of foreign gene.
In order to which overexpression GhCYP734A1L-1 genes in transgenic plants, it is necessary to by GhCYP734A1L-1 genes just
Into insertion plant expression vector, and started with CaMV35S promoters promoter and expressed, construct overexpression
GhCYP734A1L-1 gene plant expression vector pCambia-35S-GhCYP734A1L-1-NOS (abbreviation pC-
GhCYP734A1L-1) (Fig. 6).GhCYP734A1L-1 genes are reversely inserted into plant expression vector pCambia, are used
CaMV35S promoters start expression, construct the plant expression vector pC- containing GhCYP734A1L-1 gene antisense sequences
CaMV 35S- antisense GhCYP734A1L-1, (abbreviation pC- antisense GhCYP734A1L-1) (Fig. 7).
2. the genetic transformation of cotton
The plant expression carrier plasmid of structure is imported Agrobacterium LBA4404 bacterial strain and carries out cotton heredity with electrization and is turned
Change.With reference to Bio-RAD MicroPulser instruction manual books, above-mentioned carrier is imported into Agrobacterium by Electroporation conversion
LBA4404 bacterial strains.
Above-mentioned plant expression vector imports cotton by agriculture bacillus mediated Cotton Hypocotyl method for transformation.Specific method is such as
Under：
14 seed of Ji cotton peels off shell, with 0.1% mercuric chloride (HgCl2) sterilizing 10min, with a large amount of aseptic water washings 8 times.
The concussion of about 35mL sterile waters is added in 125mL triangular flasks overnight, next day changes a sterile water.When seed grows hypocotyl root
Afterwards, it is seeded on seed germination medium, 2-3d is sprouted under 28 DEG C, dark condition, seed hypocotyl initially enters soon at this time
In the period of speed elongation, suitably carry out genetic transformation.
The antisense GhCYP734A1L-1 plants containing pC-CaMV35S-GhCYP734A1L-1 and pC-CaMV35S- of conversion
The agrobacterium strains of expression vector activate on the YEB solid mediums of Km containing 50mg/L and 125mg/L Sm.Its single bacterium of picking
Fall, be inoculated in YEB fluid nutrient mediums of the 5ml containing identical antibiotic, 28 DEG C, 200r/min shake cultures stay overnight.After culture
Agrobacterium bacterium solution presses 1：20 ratio is transferred in YEB fluid nutrient mediums of the 25ml containing identical antibiotic, continues culture to OD600
Value is about 0.6-0.8,10,000r/min, 1min be collected by centrifugation thalline, thalline with isometric liquid co-culture base weight hang it is standby
With.
During conversion, Cotton Hypocotyl is cut into the segment of 1.5-2.0cm, is put into triangular flask with the Agrobacterium bacterium prepared
Liquid infects, and condition infects 30min for 28 DEG C of shaking table 120r/min.Then bacterium solution is blotted, hypocotyl section is gone to and is co-cultured on base,
28 DEG C of light cultures 2-3 days.
After co-cultivation, hypocotyl section is transferred to lower embryo section screening and culturing medium, 28 DEG C of illumination cultivations, about 20 days subcultures once,
Until there are a large amount of callus.Callus is transferred to embryo callus subculture inducing culture, about 15 days subcultures one together with lower embryo section
It is secondary, until there is substantial amounts of light yellow embryo callus subculture.Embryo callus subculture is chosen in embryo callus subculture suspension medium, 28 DEG C, 120r/
Min shaking table cultures one week or so.The body embryo that fine sand shape is drawn with the 1.0mL pipette tips for subtracting tip is laid in body embryo elongation culture
, there is the substantial amounts of small body embryo of green in base after 20-30 days.The good body embryo squamous subculture of picking growth conditions, treats that body embryo is elongated to
During 1-2cm, it is transferred into seedling culture medium and takes root emergence.When growth of seedling is to 3-5cm high, pass through the side of grafting or transplanting
Formula, which is transferred in greenhouse flowerpot, to be grown.Wherein, culture medium used in this experimental example is as shown in table 1.
The Agrobacterium tumefaciens mediated Cotton Hypocotyl genetic transformation used medium of table 1
MS:Murashige&Skoog,1962；B5:Gamborg,1986.
SH culture mediums：phytoTechnologyProduction number：S816, lot：11L081602613.
The verification of 4 transgene cotton of embodiment
1st, histochemistry identifies
Due to having a set of CaMV35S promoters control report base GusPlus-His6's in the plant expression vector of structure
Plant expression elements, therefore can utilize whether histochemical method identification is transformed plant.Plant to be transformed is in root media
On take root and when growing to 5~8cm, seedling is taken out from blake bottle and is cleaned, is transferred to 2~3d of water planting hardening on triangular flask.Meanwhile
Take a fritter blade and a bit of progress GUS dyeing.As a result as shown in Figure 8 A, wild-type leaves GUS dyeing is negative, resistance
Plant leaf GUS dyeing is the positive, and positive plant is directly transplanted in nutritive cube.
2nd, amplification verification
Treat cotton plants transplant survival and grow into a certain size, take 0.5g blades to extract cotton genomic dna, with GUS
The upstream and downstream primer GUS-up (SEQ ID NO.10) and GUS-down (SEQ ID NO.11) of gene are expanded.The expansion of 25 μ L
Increasing system contains 2.5 μ 10 × PCR of L buffer, 2 μ L 2.5mmol/L dNTPs, 1.5 μ L 25mmol/L MgCl2, each 1 μ L draw
Thing GUS-up and GUS-down (5 μm of ol/L), 1U Taq archaeal dna polymerases, 1 μ L genomic DNAs (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 of extension 10min.Expressed with positive and negative plant
Vector plasmid makees positive control, makees negative control with water and wild type cotton genomic DNA.The results show GUS stained positives
Transgenic cotton plant can amplify a band consistent with positive control, illustrate the T-DNA sections of plant expression vector
Through being incorporated into transgene cotton genome (Fig. 8 B and 8C).
Embodiment 5 detects expression change of the GhCYP734A1L-1 genes in transgene cotton
According to the method that cotton RNA is extracted in embodiment 1, the total serum IgE of extraction transgene cotton and wild type cotton blade,
And synthesize mono- chains of cDNA.The expression of target gene in transgene cotton is analyzed with Real-Time PCR methods, PCR is fixed in real time
Amount PCR instrument on carry out, the reaction system of 25 μ L include 12.5 μ L MIX buffer (including PCR buffer solutions, DNA polymerize
Enzyme, dNTPs and MgCl2, the offer of real-time quantitative RT-PCR kit, Bio-Rad).GhCYP734A1L-1 gene primers
GhCYP734A1L-1-1 (SEQ ID NO.6) and GhCYP734A1L-1-2 (SEQ ID NO.7) amplifications, internal standard use cotton
GhHISTONE3 (GenBank accession number：AF024716), primer is GhHIS-1 (SEQ ID NO.8) and GhHIS-2 (SEQ ID
NO.9).Amplification program is：94 DEG C of pre-degeneration 3min；94 DEG C, 30sec；56 DEG C, 30sec；72 DEG C, 30sec；Preset cycle number is
35.Before real-time quantitative PCR is run, expanded once in identical temperature change program with same primers and template, pass through expansion
Increase production the electrophoresis of thing, it is ensured that amplified production is single tape.Real-time quantitative RT-PCR analysis the result shows that GhCYP734A1L-1 genes
Expression in overexpression GhCYP734A1L-1 transgene cotton blades is than compareing high (Fig. 9 B), due to excess table
Growth up to the gene render transgenic cotton plants is suppressed, it is impossible to is yielded positive results, therefore cannot be examined in transgenic fibre
The expression change of the gene is surveyed, therefore have detected the expression of the gene in blade, the results showed that the gene is in transgenosis
Cotton leaf expression is higher.Conversely in the transgene cotton fiber of all suppression GhCYP734A1L-1 genes, the table of the gene
All (Fig. 9 A) is reduced up to horizontal in various degree.This result illustrates to have obtained overexpression and Antisense Suppression GhCYP734A1L-1's
Transgenic cotton plant.
6 transgene cotton of embodiment is compared with the phenotype of wild type cotton
1st, overexpression GhCYP734A1L-1 suppresses the growth of cotton plants
PC-GhCYP734A1L-1 transgenosis and wild type cotton are planted at the same time in experimental plot, carries out normal production pipe
Reason.The increased transgene cotton of GhCYP734A1L-1 expressions is observed, finds overexpression GhCYP734A1L-1
Transgenic cotton flower growth receives suppression, and plant short and small (Figure 10 A), stem internode shortens, side shoot is less or without side shoot, and side shoot
Internode shorten, blade diminishes (Figure 10 B), the dim light of night is dark green, chlorophyll content increase (Figure 10 C), blade face stomata is in desiccation
Under do not close (Figure 10 D).This result illustrates that overexpression GhCYP734A1L-1 genes can suppress the growth of cotton plants, turns
The plant type of gene plant has significant change.If use the table of the specific promoter of stem or branch control GhCYP734A1L-1 genes
Reach, will effectively change the plant type of transfer-gen plant, there is larger application value on Plant-type Breeding.
Embodiment 7 suppresses GhCYP734A1L-1 gene expressions and promotes cotton fiber extension
The transgene cotton of Antisense Suppression GhCYP734A1L-1 and control are planted to experimental plot at the same time, carried out normal
Production management, observes the growth and development of cotton fiber and the length of comparative maturity fiber.The results show that suppress GhCYP734A1L-
The transgenic cotton plant of 1 gene does not have notable difference in growth with wild type cotton plant, further illustrates the gene not
Do not expressed in the organs such as root, stem, leaf and flower.Suppress the transgene cotton mature fibers of GhCYP734A1L-1 genes than control
Long, its average length is 34.6 ± 0.25 millimeters, and the average length of control fiber is 31.9 ± 0.54 millimeters, and fibre length increases
8.46% (Figure 11 A and C) is added.Meanwhile the growth of transgene cotton fiber and control fiber is observed by ovules culture in vitro.
The result shows that (Figure 11 B), the fiber for suppressing to grow on the transgene cotton ovule of GhCYP734A1L-1 expression is than wild type ovule
The fiber length of upper growth.It is important that this result further illustrates that GhCYP734A1L-1 genes have in fibrocyte elongation process
Effect, its expression have substantial connection with fibre length.
Claims (10)
1. plant expression vector, it is characterised in that：Specifically expressed protein C YP734A1 in the carrier expression fibrocyte
like-1；The amino acid sequence of the protein C YP734A1 like-1 is as shown in SEQ ID No.3；It is described
The nucleotide sequence of the cDNA of the encoding gene GhCYP734A1L-1 of CYP734A1like-1 as shown in SEQ ID NO.1 or
Its genomic DNA is as shown in SEQ ID NO. 2.
2. carrier as claimed in claim 1, it is characterised in that：The plant expression vector is overexpression
The carrier of GhCYP734A1L-1 genes.
3. the host cell containing the carrier of claim 1 or 2.
4. host cell as claimed in claim 3, it is characterised in that：The host cell is Agrobacterium.
5. application of the 1 or 2 any one of them plant expression vector of claim in cotton variety is improved.
6. plant expression vector, it is characterised in that：The encoding gene of the carrier marking protein CYP734A1 like-1
The antisense sequences of GhCYP734A1L-1, the amino acid sequence such as SEQ ID NO.3 of the protein C YP734A1 like-1
It is shown；The nucleotide sequence of the cDNA of the encoding gene GhYP734A1L-1 of the CYP734A1like-1 such as SEQ ID
Shown in NO.1.
7. carrier as claimed in claim 6, it is characterised in that：The plant expression vector is Antisense Suppression
The carrier of GhCYP734A1L-1 genes.
8. the host cell containing the carrier of claim 6 or 7.
9. the host cell as described in claim 8, it is characterised in that：The host cell is Agrobacterium.
10. application of the plant expression vector described in claim 6 or 7 in improving cotton fiber quality.
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| KHG02010;Mudge J.等;《Genbank》;20141204 * |
| PAG1, a cotton brassinosteroid catabolism gene, modulates fiber elongation;Zuoren Yang等;《New Phytologist》;20140502;第203卷(第2期);437-448 * |
| XP_007013339.1;Mockaitis K.等;《Genbank》;20140306 * |
| 棉花T-DNA激活标签突变体pag1分子机制的研究和应用;杨作仁;《中国博士学位论文全文数据库》;20141015;摘要，图3.6和3.7 * |
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