CN105671076B - A kind of plant expression vector and its application in raising output of cotton - Google Patents
A kind of plant expression vector and its application in raising output of cotton Download PDFInfo
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- CN105671076B CN105671076B CN201610199869.2A CN201610199869A CN105671076B CN 105671076 B CN105671076 B CN 105671076B CN 201610199869 A CN201610199869 A CN 201610199869A CN 105671076 B CN105671076 B CN 105671076B
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Abstract
The present invention relates to genetic engineering fields, and in particular to Ustilago maydis sucrose transporter UmSRT1 gene, expression vector and its application in raising output of cotton.The present invention merges specific promoter with Ustilago maydis sucrose transporter UmSRT1 gene, construct the plant expression vector of specifically expressing sucrose transporter gene, the plant expression vector is converted into host, obtain transformant, the host is converted into plant again, the unginned cotton and lint yield of cotton can be improved in the transgene cotton of the specifically expressing Ustilago maydis sucrose transporter UmSRT1 gene of acquisition.
Description
Technical field
The present invention relates to genetic engineering fields, and in particular to a kind of specifically expressing Ustilago maydis sucrose transporter
The plant expression vector of UmSRT1 gene and the method that output of cotton is improved by technique for gene engineering.
Background technique
Plant passes through the fixed CO of photosynthesis2The carbohydrate of synthesis assimilation quotient is mainly carried out in the form of sucrose
Distribution.Sucrose provides carbon source not just for the growth and development of plant, and is other compositions in plant, such as plant hormone,
Trafficking signal (Turgeon R et al, 2008) is provided.It is passive between cell and cell that sucrose can be carried out by plasmodesmus
Transport, can also be discharged into apoplast by the albumen dependent on ATP and proton carry out active transport (Norbert Sauer,
2007).Sucrose is loaded in " source " organ sieve tube element, and by the long-distance transportation of bast, it is tough then to arrive " library " organ
Skin zone's unloading, the sucrose transporter (SUT or SUC) on plasma membrane play an important role wherein.
UmSRT1 comes from Ustilago maydis, is positioned at fungi plasma membrane, is a kind of specificity to intracellular transport sucrose
Transport protein, Km value are 26 ± 4.3 μM, and the affinity to sucrose is 20-200 times of sucrose transporter in higher plant.
And UmSRT1 and ZmSUT1 has competitive relation to the absorption of sucrose, ZmSUT1 affinity is well below UmSRT1 (Wahl R et
Al, 2010).BAN donor organism is arabidopsis, and long 335bp, controllable target gene is in cotton kind skin specifically expressing
(CN102296085A, Southwest University, 2011).
China is the consumption big country of cotton, self-supporting insufficient, belongs to cotton net importer, international market share is very low.
2010 years, Cotton in China market there are nearly 4,000,000 tons of insufficiency of supply-demand, make up cotton needed for notch mainly from the U.S., India,
The states such as Pakistan import (Zhang Yanna etc., 2013).China's cultivated land resource is limited, and cotton is improved on limited cultivated land resource
Yield is the main breeding objective of Cotton in China to realize the self-sufficient of Cotton in China.But between the yield and quality of cotton
To be hereditary negatively correlated, the yield and product for improving cotton can not be significantly synchronized in a relatively short period of time by traditional breeding mode
Matter, therefore utilize transgenic technology means, the heredity that can break cotton is negatively correlated, can enhance the disease-resistant of cotton, antiweed, with
And the yield and quality of insect resistance capacity and synchronous raising cotton.Since cotton fiber is attached on seed coat, the development of seed
The yield and quality of cotton is had a major impact.Therefore, the quantity of gene regulation seed can be passed through by improving cotton fiber yield
(Ruan Y L, 2013) is realized by force in how many and library.Meanwhile cotton seeds be also important vegetable protein with it is adipose-derived.
It but for a long time, is the SOYBEAN IN HIGH-YIELD BREEDING of selection target often to sacrifice seed weight as cost (Miller using fiber production
and Rawlings,1967；Zhang et al,2005).Therefore, synchronous raising seed and fiber production, which have become, improves cotton
The task of top priority of flower yield.
Summary of the invention
It is an object of the present invention to provide a kind of plant expression vector, contain the sugarcane in Ustilago maydis
Saccharide transporter UmSRT1 gene, can be used for improving output of cotton in transgene cotton.
It is another object of the present invention to provide the transformant for containing above-mentioned plant expression vector.
It is yet a further object of the present invention to provide purposes of the above-mentioned plant expression vector in prepare transgenosis plant.
It is yet a further object of the present invention to provide the sides of a kind of preparation method of transgene cotton and raising output of cotton
Method.
The present invention is according to the sucrose transporter UmSRT1 in Ustilago maydis to the superelevation affinity of sucrose
The characteristics of, proposition places it under the control of specific promoter, promotion photosynthate targeting transport and distribution, raising cotton boll,
" the library gesture " of seed and fiber is realized that cotton seeds are synchronous with fiber production and is improved.Strategy proposed by the present invention is to other works
The output increased of object also has significant application value.
According to an aspect of the present invention, it is an object of the present invention to provide a kind of specifically expressing Ustilago maydis sugarcanes
The plant expression vector of saccharide transporter UmSRT1 gene, using technique for gene engineering by Ustilago maydis sucrose transporter
UmSRT1 gene (SEQ ID NO.4) is operably connected with specific promoter, building plant expression vector, after transformed plant,
Ustilago maydis sucrose transporter UmSRT1 gene is expressed under the action of specific promoter.Wherein, specific promoter can
To improve the promoter that target gene is expressed in specific plant tissues and specific stage of development, preferably BAN promoter.It is preferred to plant
Object expression vector has carrier structure as shown in Figure 4.Wherein, Ustilago maydis sucrose transporter UmSRT1 gene is positive
After being connected to BAN promoter, the plant expression vector pLGN-BAN-UmSRT1 of the specifically expressing gene is formed.
According to another aspect of the present invention, preparation is provided and includes Ustilago maydis sucrose transporter UmSRT1 gene
The method of transgene cotton, by obtaining Ustilago maydis sucrose transporter UmSRT1 gene from Ustilago maydis
(SEQ ID NO.4), it is operably connected with specific promoter, and building specifically expressing Ustilago maydis sucrose transports egg
Carrier conversion host is obtained transformant, then the transformant is transferred to cotton and is planted by the plant expression vector of white UmSRT1 gene
Strain obtains transgene cotton.
In accordance with a further aspect of the present invention, a kind of method improving output of cotton is provided, specifically expressing in cotton is passed through
Ustilago maydis sucrose transporter UmSRT1 gene improves output of cotton, and this method includes that specifically expressing maize is black
The plant expression vector of powder bacterium sucrose transporter UmSRT1 gene is integrated into the step in cotton gene group.
Base is encoded the present invention provides Ustilago maydis sucrose transporter UmSRT1 or its encoding gene or containing it
Cotton unginned cotton and lint yield can be improved by the gene integration into cotton gene group in the recombinant vector of cause.
The present invention can provide the transformant containing above-mentioned plant expression vector.
The preparation method of genetically modified plants of the present invention offer based on plant expression vector of the present invention.
The present invention is achieved through the following technical solutions:
A kind of plant expression vector, includes at least Ustilago maydis sucrose transporter gene UmSRT1 and kind skin is special
Different promoter BAN, and the BAN promoter is located at the 5 ' upstreams of Ustilago maydis sucrose transporter gene UmSRT1.
Plant expression vector of the invention, with structure as shown in Figure 4.
Above-mentioned plant expression vector is improving the application in cotton seeds and fiber production.
A kind of nucleotide encoding Ustilago maydis sucrose transporter gene, has as shown in SEQ ID No.4
Nucleotide sequence.
A kind of transformant containing above-mentioned plant expression vector.
A kind of preparation method containing the above-mentioned genetically modified plants for stating plant expression vector, comprising the following steps:
1) acquisition of skin and fiber specific expression promoter BAN is planted；
2) acquisition of Ustilago maydis sucrose transporter gene；
3) the Ustilago maydis sucrose being cloned into specific promoter and step 2) isolated in step 1) is turned
Fortune protein gene is merged, and the plant expression vector of specifically expressing sucrose transporter gene is constructed；
4) plant expression vector obtained in step 3) is integrated into cotton gene group；
5) cotton that will be obtained by step 4) is further cultivated, cultivation, the final cotton plants for obtaining transgenosis.
Wherein, specific promoter described in step 3) and UmSRT1 Gene Fusion are to construct specifically expressing sucrose transhipment egg
White expression vector method is the conventional method of this field, and the carrier used is conventional carrier used in plant transgene field.
Wherein, expression vector is integrated into method used in cotton gene group described in step 4) is Agrobacterium tumefaciems
Mediated method is common plant transgenic method.
Signified " transgene cotton " refers to through biotechnological method in the present invention, and the gene of other biological is gone to cotton
Hua Zhong, thus the cotton that the cotton inhereditary material being modified is transformed.Gene for transformation can derive from plant, move
Object and microorganism or artificial synthesized transformation.
Signified seed refers to as the weight of hundred cotton seeds in the present invention, is indicated with g.
Signified " ginning outturn " refers to that fibre weight is expressed as a percentage to unginned cotton weight ratio in unginned cotton in the present invention.Also
It is the ratio that fibre weight accounts for entire seed and total weight of fiber.
Signified " output of cotton " refers to the unginned cotton and gined cotton weight harvested in Cotton Production, yield forming in the present invention
Factor includes single basal munure, bell weight and ginning outturn etc..
The experiment proves that Ustilago maydis sucrose transporter UmSRT1 is passed through transgenic approach by the present invention
It imports in wild type cotton and obtains transgene cotton, transgene cotton seed size, fiber number, Single boll weight, single plant unginned cotton
Yield, lint yield are above wild type cotton, to prove that cotton unginned cotton and lint yield can be improved in the albumen.
Detailed description of the invention
Fig. 1: pLGN carrier figure (NPT II, new enzyme element phosphoric acid transferase gene；GUS, β-gluconic acid glycoside enzyme gene；
CaMV35S-P, from the plant constitutive promoter of cauliflower mosaic virus；Pnos, Opines synthase gene promoter；
Nos, Opines synthase gene terminator；LB, T-DNA left margin；RB, T-DNA right margin.)
Fig. 2: pCambia2300 carrier figure (NPT II, new enzyme element phosphoric acid transferase gene；CaMV35S-P, from flower coconut palm
The plant constitutive promoter of cauliflower mosaic virus；Pnos, Opines synthase gene promoter；Nos, Opines synthase gene
Terminator；LB, T-DNA left margin；RB, T-DNA right margin.)
Ustilago maydis sucrose transporter gene expression vector pLGN- under Fig. 3: specific promoter BAN regulation
The building flow chart of BAN-UmSRT1.
Fig. 4 is the Ustilago maydis sucrose transporter gene expression vector pLGN- under specific promoter BAN regulation
The structure chart of BAN-UmSRT1.
Fig. 5 is that specifically expressed Ustilago maydis sucrose transporter UmSRT1 gene is turning BAN::UmSRT1 gene
RT-PCR analysis in cotton；BUR1, BUR2, BUR3 are that different BAN::UmSRT1 transformants is numbered.WT is wild type cotton
Plant is used as control.As a result BUR1, BUR2, BUR3 strain detect the obvious expression of UmSRT1 gene.
Fig. 6 is the specific data of Soluble adhesion molecule in 15 days seeds of transgene cotton and fiber.
Fig. 7 is Soluble adhesion molecule in transgene cotton mature seed.
Fig. 8 is that transgene cotton bell becomes larger, the phenotypic map that seed becomes larger.
Fig. 9 is the phenotypic map that transgene cotton fiber shortens.
Figure 10 is mature fibers quantity statistics on every seed of transgene cotton.
Specific embodiment
Below in conjunction with attached drawing, the present invention is further described in detail, but does not limit the invention below,
Any pair of deformation and change of the invention, as long as it does not depart from the spirit of the invention, should belong to appended claims institute of the present invention
The range of definition.Experimental method used in following embodiments is conventional method unless otherwise specified.
Material agents as used in the following examples etc., are commercially available unless otherwise specified.
The clone of [embodiment 1] Ustilago maydis sucrose transporter (UmSRT1) gene and its building of expression vector
1. the acquisition of specific promoter
According to arabidopsis BANYULS gene (GenBank accession number: AF092912) and its genome sequence, designs BAN and open
Mover primer (SEQ ID No.1, SEQ ID No.2).Capitalization is the restriction enzyme site of addition, from arabidopsis gene group
PCR amplification obtains the segment of 300bp or so.Sequencing analysis is shown to be the BAN spy of arabidopsis after amplification of DNA fragments is cloned
Specific Promoters, nucleotide sequence are shown in SEQ ID No.3.
The acquisition of Ustilago maydis sucrose transporter 2. (UmSRT1) gene
According to Ustilago maydis sucrose transporter (UmSRT1) gene order (GenBank accession number: XM_
011390372.1) design primer, with Ustilago maydis (Ustilago maydis) (purchased from China Microbiological resource
The heart, number 5.121) bacterium solution be template, with following primer
F1:5 '-ATGGCGTCGTCTTCTCCCATTCGT-3 ', R1:5 '-TCATTGTGGACTCGGCTGCAGAG-3 ' into
The PCR product of row PCR amplification acquisition 1641bp.UmSRT1 gene order is obtained after sequencing analysis (see SEQ ID No.4).
The building process of specifically expressing Ustilago maydis sucrose transporter (UmSRT1) genophore is shown in Fig. 3, specifically
The structure chart of Ustilago maydis sucrose transporter gene expression vector pLGN-BAN-UmSRT1 under promoter BAN regulation
See Fig. 4.All restriction enzymes are purchased from Roche company, operate according to operation instructions.PLGN (Fig. 1) be this laboratory by
One binary plant expression vector of pCambia2300 (Fig. 2) transformation.Its T-DNA section (region between RB and LB) replaces
Change the track fusion box of the reporter gene gus and marker gene nptII of constitutive promoter CaMV35S-P control into, and
LoxpFRT recombination enzyme recognition site is respectively added at this expression cassette both ends and another is controlled by CaMV35S-P
Expression cassette.Expression element contained by the T-DNA of pLGN derives from pBI121, extends the method that splicing is connected with digestion by PCR
Building.The site LoxpFRT derives from yeast.Recombinase can recognize that the DNA sequence dna between two sites is cut in the site LoxpFRT
It removes.PCambia2300 and pBI121 plant expression vector and related elements are widely applied in genetic manipulation of plants, do not send out
Existing its has pathogenic and may develop into the relevant report for having pathogenic carrier.NptII gene comes from bacterial transposon Tn5
On aph2, which encodes aminoglycoside -3'- phosphotransferase, which makes aminoglycoside antibiotics phosphorylation and inactivate,
Render transgenic plant obtains the resistance to this kind of resistance element, does not have other harmful influences, Amp, ammonia benzyl blueness to genetically modified plants
Mycin resistant gene；Km, kalamycin resistance gene；NPT II, new enzyme element phosphoric acid transferase gene；GUS, β-gluconic acid glycosides
Enzyme gene；35S, from the plant constitutive promoter of cauliflower mosaic virus；Pnos, Opines synthase gene promoter
Son；Nos, Opines synthase gene terminator；LB, T-DNA left margin；RB, T-DNA right margin.For constructing plant expression
The pLGN carrier that the skeleton carrier of carrier is transformed on the basis of being pCambia2300, with the GUS under the regulation of CaMV 35S promoter
Gene, convenient for carrying out the screening of GUS dyeing to transformant during Genetic Transformation in Higher Plants.
The preparation of [embodiment 2] transformant and genetically modified plants
1. the plant expression carrier plasmid of building is imported Agrobacterium LBA4404 with electrization.
With reference to Bio-RAD MicroPulser instruction manual book, above-mentioned carrier is imported into Agrobacterium by Electroporation conversion
LBA4404。
2. specifically expressing Ustilago maydis sucrose transporter (UmSRT1) genophore passes through Agrobacterium tumefaciens mediated
Method be integrated into upland cotton Ji cotton 14 (given in Agricultural University Of Hebei Ma Zhiying teach) genome.
The Agrobacterium tumefaciens mediated Cotton Transformation culture medium of table one
MS:Murashige&Skoog, 1962
B5:Gamborg, 1986
Gelrite:Sigma, article No.: G1910
SH:Schenk&Hildebrandt, 1972
Target gene, using Cotton Hypocotyl as receptor, is integrated into cultivation product by mediated by agriculture bacillus by above-mentioned expression vector
In kind of Ji cotton 14 (Luo et al., 2007).The specific method is as follows:
0.1% (w/v) HgCl is added after the sterilizing of 75% ethyl alcohol is added in the 14 cotton seeds decladding of Ji cotton2Sterilize 12min, nothing
About 40mL sterile water will be added after washing 6~8 times in bacterium.Sterile water twice is changed daily in 100rpm, 30 DEG C of culture 2d.It will show money or valuables one carries unintentionally
Cotton kernel insertion seed germination medium in, lost in 28 DEG C of dark culturing 2d, when until hypocotyl elongation is to 1~2cm
Pass conversion.Picking Agrobacterium single bacterium is fallen in liquid YSK culture medium, 200rpm, and 28 DEG C of shaken cultivations are overnight to bacterium solution OD600nmIt reaches
It is centrifuged 1min to 0.8~1.2,10000rpm, collects thallus.Base weight is co-cultured with the liquid containing 200 μm of ol/L acetosyringones
Outstanding thallus, 100rpm, 30 DEG C of culture 1h.
Seedling hypocotyl 0.3cm~0.5cm is cut as explant, is put into dip dyeing liquid for shell, 100rpm, 30 DEG C of dip dyeings
45min~60min.Lower embryo section is transferred on solidified co-cultivation medium, 28 DEG C of dark culturing 2d.Lower embryo section is transferred to screening and culturing
In base, 28 DEG C of illumination screening and culturing 20d.Lower embryo section is transferred to subculture medium, calli induction media, embryo callus subculture in succession again
In liquid suspension culture base, body embryo growth medium and seedling culture medium, 28 DEG C of illumination cultivations.To growth of seedling to 5~10cm
Gao Shi is transplanted to greenhouse-grown.
In greenhouse-grown transgenic plant obtained, carry out Routine Management in greenhouse, in phenotype and growth and development with
The no significant difference of the control of wild type.After T1 is obtained after maturation for seed, and plants and be compared test in greenhouse.
Expression of the UmSRT1 gene that [embodiment 3] is imported with the method detection of real-time PCR in cotton ovule
Referring to EASYspin plant RNA rapidly extracting kit (Beijing Ai Delai Biotechnology Co., Ltd) specification into
Row extracts the total serum IgE of ovule on the day of rapidly extracting cotton blooms.1 μ L RNA agargel electrophoresis is taken after the completion of extracting, and is estimated dense
- 80 DEG C of refrigerators are stored in after degree.
It usesRT reagent Kit with gDNA Eraser kit (TaKaRa) is strictly pressed
The synthesis of mono- chain of cDNA is carried out according to reagent specification, according to the RNA concentration that agarose gel electrophoresis is estimated, is finally protected reaction product
It is stored in -20 DEG C of refrigerators.
CDNA template is quantitative: 80 μ L deionized waters are added in cDNA, 100 μ L are diluted to, in real-time PCR
Quantitative pcr amplification is carried out on (Bio-Rad, CFX96), checks cotton GhHIS (internal standard gene) expression quantity, GhHIS-F, GhHIS-
R is SEQ ID No.5 and SEQ ID No.6.Reaction system is (20 μ L): 10 2 × iTaq of μ LTM Green
Supermix(Bio-Rad)+7μL H2O+1μL cDNA+1μL GhHIS-F+1μL GhHIS-R.Amplification condition are as follows: first carry out
95 DEG C, after 3min；95 DEG C are carried out, 20sec；56 DEG C, 20sec；72 DEG C, 30sec；40 circulations.According to the Ct value of amplification,
Whether the amount of estimation template can be used for gene expression analysis, and Ct value is advisable between 16~22.
Primer amplification Efficiency testing: choosing concentration highest and the cDNA sample containing target gene, makes standard curve.With
10 times are gradient, are diluted to 6 concentration gradient points as template, using above-mentioned cDNA template quantify in reaction system and reaction
Condition amplification, the amplification effect of measurement UmSR1 gene primer (upstream and downstream primer is respectively SEQ ID No.7 and SEQ ID No.8)
Rate.
The detection of destination gene expression amount: using GhHIS3 gene as internal standard, each sample, which is repeated 3 times, to be expanded, simultaneously
It is arranged negative control (template is deionized water).Reaction system is (20 μ L): 10 2 × iTaq of μ LTM Universal
+ 1 μ L cDNA+7 μ L deionized water of Green Supermix+2 μ L target gene primer (upstream and downstream).Reaction condition are as follows: advanced
95 DEG C of row, after 3min；95 DEG C are carried out, 20sec；58 DEG C, 20sec；72 DEG C, 30sec；40 circulations；Finally carry out solubility curve
Detection shows that amplified production is uniform if it is in simple spike.It is counted using Bio-Rad CFX Manager and Excel software
It is analyzed according to processing, as a result as shown in figure 5, BUR1, BUR2, BUR3 strain detect the obvious expression of UmSRT1 gene.
Sucrose transporter UmSRT1 gene is in 15 days cottons of Post flowering in [embodiment 4] specifically expressing Ustilago maydis
Seed and the detection of Fiber Soluble sugar content
The content of total Soluble Sugar in seed and fiber is measured using anthrone-sulphuric acid method.By 15 days wild types of Post flowering and
Transgenic line respectively takes 3 cotton bolls, takes 105 DEG C of 30min water-removings of seed and fiber therein, 46 DEG C drying to constant weight, with grinding
Machine grind into powder, respectively takes 0.1g, and wild type and transgenic line are respectively in triplicate.The ethyl alcohol of 3ml 80% is added, 80 DEG C mention
10mim is taken, supernatant is taken, precipitating adds the ethyl alcohol of 2ml 80%, and 80 DEG C of extraction 10mim are repeated once, last three times to merge
Together, constant volume.According to different total sugar concentrations, adjustment dilution takes 200 μ l and the anthrone sulfuric acid reaction with 800 μ l, boils
10min takes 200 μ l in OD620Spectrophotometric determination light absorption value.According to standard curve, it is converted into total sugar content, as a result such as
Shown in Fig. 6, the experimental results showed that 15 days seeds of transgene cotton Post flowering and Fiber Soluble total sugar content be above it is wild
Type cotton.
It can in sucrose transporter UmSRT1 gene cotton mature seed in [embodiment 5] specifically expressing Ustilago maydis
The detection of dissolubility sugar content
According to above-mentioned anthrone-sulphuric acid method, mature baked seed is chosen to constant weight, clays into power, it is soluble total to extract it
Sugar.As a result as shown in fig. 7, the soluble sugar content in the seed of transgenic cotton flower maturity is above wild type seeds.
[embodiment 6] transgene cotton yield factor statistics
1. the statistics of single-strain bell-forming number and every bell seed number
Single-strain bell-forming number statistics has counted wild type and each 3 plants of single plants of transgenic line when long to 135 days of cotton T1 generation
Bell number counts total bell number and plant number, single-strain bell-forming number=total bell number/plant number.Every bell seed number statistical method: harvest is normal
The cotton boll of sudden and violent peach, counts bell number, Single boll weight, every bell seed number=Single boll weight/100-grain weight * 100.
2. yield traits are investigated
Mid-September in three times, late September, mid-October harvests the cotton boll of sudden and violent peach three times, after spontaneously drying, calculates
Bell weight and seed cotton yield.In the unginned cotton of every transgenic line harvest, 100 seeds are randomly selected, hundred unginned cottons are accurately weighed
Weight, de- fiber weighs the total amount of fiber and the total amount of seed respectively again by hand, calculates the size of ginning outturn, finally takes and receive three times
The average value obtained is as final result.Single plant seed cotton heavy=mono- basal munure * Single boll weight.Gined cotton weight is fibre of the unginned cotton through mechanical lint
Weight is tieed up, ginning outturn is the ratio of gined cotton weight and seed cotton heavy, single plant gined cotton weight=mono- basal munure * Single boll weight * ginning outturn.Transgenic line
Different its Single boll weight of transformant strain dramatically increase, and single-strain bell-forming number difference is not significant, has increased slightly, and 100-grain weight is increased
Add, single bell seed number increased, and single plant seed cotton yield increases 33.43%~72.96%.Lint yield per plant increases
31.52%~65.40%.(being shown in Table two)
Two BAN:UmSRT1 transgenic line yield traits statistical result of table
| Strain | WT | BUR1 | BUR2 | BUR3 |
| Single-strain bell-forming number | 36.33±4.04a | 50.00±7.07a | 43.33±21.01a | 40.50±6.43a |
| Single boll weight (g) | 4.05±0.09a | 5.09±0.47b | 5.07±0.45b | 4.85±0.60ab |
| Every bell seed number | 23.30±0.52a | 24.51±1.58ab | 26.89±2.83ab | 27.16±2.34b |
| Ginning outturn (%) | 38.00% ± 0.01a | 37.07% ± 0.00a | 37.11% ± 0.01a | 37.61% ± 0.02a |
| 100-grain weight (g) | 10.81±0.32a | 12.50±0.23b | 11.92±0.37bc | 11.19±0.66ac |
| Single bell fiber weight (g) | 1.54±0.01a | 1.86±0.17b | 1.88±0.22b | 1.82±0.14b |
| Single plant seed cotton yield (g) | 147.16±3.15a | 254.53±23.65b | 219.50±19.59bc | 196.36±24.33c |
| Lint yield per plant (g) | 55.90±0.47a | 92.46±8.24bc | 81.60±9.76cd | 73.52±5.51d |
BUR1, BUR2, BUR3 respectively refer to the different transformant T1 of transgenic line BAN:UmSRT1 for strain, wherein in table
Different letters indicate variant between the two (P ﹤ 0.05), and the above Multiple range test is to be calculated and obtained with spss software.
[embodiment 7] transgene cotton phenotypic analysis
Mid-October, compared with taking mature transgene cotton to carry out phenotype with wild type, it was found that transgene cotton
Cotton boll, seed increase (Fig. 8).Take the seed with fiber in the middle part of mature cotton boll, with comb among seed to two sides by fiber
Comb is straight, respectively takes 3 samples and wild type it was found that fiber is shortened (Fig. 9).From three materials of wild type and transgenosis
In respectively randomly select 20 cotton seeds, manual lint weighs total fiber weight W1, takes the fiber of 6 beam 1.5mg or so respectively,
And make in order, the weight W2 of every beam is weighed, in total 24 beam, 10min is boiled into the water, as standby in 45% acetic acid after cooling
With.Using dustless filter paper suck dry moisture, every intercept is dispersed in 6 drop pure water by 3 sections of clip 1-2mm long intercept respectively, is shown
Micro mirror is observed and is taken a picture.The quantity of fiber in each drop is settled using Photoshop tally function, is calculated and is taken the flat of every bundle fiber
Equal fiber count N2.Finally obtain mature fibers quantity N1 on every seed.N1=(W1/20)/(W2/N2) is calculated as the result is shown
Mature fibers quantity is apparently higher than wild type (Figure 10) on every seed of transgenic line.
The above detailed description of the present invention is not intended to limit the present invention, and those skilled in the art can do according to the present invention
Various modifications and change out, as long as it does not depart from the spirit of the invention, should belong to model defined in appended claims of the present invention
It encloses.
Bibliography:
A kind of plant expression vector of the such as Pei Yan and its application in improvement of cotton fiber traits: China,
CN102296085A[P].2011-12-28.
International competitiveness analysis [J] modern times commerce and trade industry of Zhang Yanna, Wu Pingping Cotton in China, 2013,25 (11):
66-68.
Miller PA,Rawlings JQ(1967)Selection for increased lint yield and
correlated responses in upland cotton,Gossypium hirsutum L.Crop Sci 7(6):637–
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Norbert Sauer.Molecular physiology of higher plant sucrose
transporters[J].Febs letters 2007,581(12):2309-2317.
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Claims (6)
1. a kind of plant expression vector of specifically expressing Ustilago maydis sucrose transporter UmSRT1 gene includes at least jade
Chinese sorghum smut sucrose transporter UmSRT1 gene and specific promoter, the UmSRT1 gene order forward direction are connected to starting
After son, for the nucleotide sequence of the UmSRT1 gene as shown in SEQ ID No.4, the promoter is BAN promoter.
2. plant expression vector described in claim 1, wherein the BAN specificity that the specific promoter is arabidopsis starts
Son.
3. the Agrobacterium-mediated Transformation body containing the plant expression vector as described in claim any one of 1-2.
4. as the described in any item plant expression vectors of claim 1-2 are improving the application in output of cotton.
5. a kind of preparation method of the transgene cotton of any one of 1-2 containing claim plant expression vector, including it is following
Step:
1) Ustilago maydis sucrose transporter UmSRT1 gene is prepared；
2) Ustilago maydis sucrose transporter gene UmSRT1 gene is operably connected with BAN promoter, is constructed
Plant expression vector comprising Ustilago maydis sucrose transporter UmSRT1 gene and BAN promoter；
3) plant expression vector is converted into host, obtains transformant；
4) host is converted into plant, obtains the transgene cotton of specifically expressing sucrose transporter UmSRT1 gene.
6. a kind of method for improving output of cotton, including by specifically expressing Ustilago maydis sucrose transporter UmSRT1
The plant expression vector of gene is integrated into the step in cotton gene group, the nucleotide sequence of the UmSRT1 gene such as SEQ ID
Shown in No.4.
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