CN106434695B - The small G-protein Rab gene of cotton fiber length can be significantly improved - Google Patents
The small G-protein Rab gene of cotton fiber length can be significantly improved Download PDFInfo
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Abstract
The present invention discloses a kind of small G-protein Rab gene that can significantly improve cotton fiber length, belongs to biological technology application.The present invention provides the small G-protein genes of cottonGhRab11d3ORF sequence, amino acid sequence and its genome sequence in Tetraploid G. hirsutum TM-1.The present invention reports that the gene participates in the vesicle transport during cotton fiber development for the first time, positive Antisense construct, which is constructed, as target gene using this gene carries out transgene cotton functional verification, increase glucide in fiber (pectin) accumulation the result shows that being overexpressed the gene, the number of actin cytoskeleton F-actin microfilament increases, and fibre length is elongated；However, the gene expression is inhibited to reduce glucide in fiber (pectin) accumulation, the number of actin cytoskeleton F-actin microfilament is reduced, and fibre length shortens.Therefore, the overexpression gene is expected to improve fiber quality in cotton.
Description
Technical field
The present invention relates to a kind of small G-protein Rab gene of cotton, which found by cDNA differential display technique,
In upland cotton (G.hirsutum) Li Shi superbhort fiber mutant Li1 and its normal wild type material Post flowering 10 of Fibre Development
Differential expression in it fiber/ovule cell further obtains the base in upland cotton Genetic standard line TM-1 by round pcr
Because of overall length ORF sequence and genome sequence, belong to biological technology application.
Background technique
Cotton is worldwide important industrial crops, allotetraploid upland cotton account for the 90% of global sown areas of cotton with
On.Cotton fiber is that longest known to plant field is unicellular, it by be located at ovule epidermis epidermal hair former base differentiation and development and
Come.Fiber growth and morphogenesis are made of four main developmental stages: the origin of fibers phase, extend phase, secondary wall synthesis
Phase and dehydration maturity period (Basra and Malik, 1984, Wilkins and Arpat, 2005).When different Fibre Developments
The transcriptome analysis of phase is shown, in diploid cultivar, the transcript for participating in cotton fiber development is about related to 18 000 bases
Because, and in tetraploid cultivar, about 36 000 genes of transcript of cotton fiber development are participated in, comprising coming from A subgroup and D
The homeologous gene (Wilkins and Arpat, 2005) of subgroup.Therefore, it excavates with Function Identification more and Fibre Development
Related gene will be helpful to improve our understandings to cotton fiber development mechanism, meanwhile, it is also beneficial to improvement cotton fiber
Yield and quality (Guo Wangzhen etc., 2003).
Rab family belongs to one in small G-protein Ras superfamily (including five major class of Ras, Rho/Rac, Ran, Arf, Rab)
Class.First Rab gene (YPT1) is to find (Gallwitz et al., 1983) nineteen eighty-three in yeast.After 4 years,
A.Salminen and P.Novick proves that Ras albumen (Sec4) participates in vesicle transport process (Salminen and for the first time
Novick,1987).The same year, A.Tavitian and his colleagues are cloned by the cDNA library of rat brain for the first time
Homologous gene similar with SEC4/YPT gene function, and it is named as Rab (ras-likeinratbrain) (Martinez and
Goud,1998)。
Rab protein family member about 200 amino acid, all than more conservative, they have very high sequence between each other
Sequence similarity.All Rab albumen all has there are five typical conserved domain, including tetra- birds of G1, G3, G4, G5 are fast
Mono- effector binding structural domain of purine nucleotide binding domain and G2 (Takai et al., 2001, Agarwaletal.,
2009).In general, the c terminal amino acid sequence alterable height of Rab albumen, but generally with two conservative cysteine residues (CC)
Ending (Rutherford and Moore, 2002), the two very conservative cysteine residues position and albumen in film
It is had very important effect in terms of function.
The major function of Rab protein family member is responsible for intracellular albumen transport, they are intracellular vesicles transports
The essential attemperator of approach (Novick and Zerial, 1997；Brennwald,2000).Newly synthesized secretory protein
It can generally be transported from a film room of organelle to another film room (Guakan et al., 2005) by vesica, they are first
It can be transported to endoplasmic reticulum, then transported plasma membrane (Heo et al., 2005) by golgiosome, some is molten before being also sent to
Enzyme body.Even if certain macromoleculars are absorbed by plasma membrane, it can also pass through vesicle transport to endocytosis body (endosmome) and lysosome
In.Some cell surface proteins including receptor can also be sent back in plasma membrane again by interior body circulation, so endocytosis and exocytosis
Circulation (Battey et al., 1999) be all to be realized by intracellular vesicle transport, and everything all is all
For the stimulation (Koh et al., 2009) of the various stream signals of response (such as hormone).In general, vesicle transport is intracellular
Needed for various physiological functions, such as cell polarity elongation, cytokinesis, the formation of cell plates (Barr, 2009).Vesicle transport is extremely
It is few to be made of 4 steps: to be that vesica sprouts from donor membrane, followed by vesica is moved to receptor first, as vesica is docked in
On receptor membrane, vesica is merged with receptor membrane, the above process required Rab albumen participate in (Tuvim et al., 2001, Yang,
2002)。
Existing research shows that the Rab family of small G-protein is particularly significant for pollen tube polar growth.In tobacco,
The activated state of Rab11b and the overexpression of inactive mode can all inhibit the elongation of pollen tube, meanwhile, it is raw also to will affect pollen tube
Long directionality (de Graaf et al., 2005).In arabidopsis, correct tune of the RabA4d for pollen tube polar growth
Control very necessary, gene specifically expressing in pollen.Subcellular localization shows the enhanced yellow fluorescence egg of RabA4d label
The white top for being positioned at pollen tube in growth, the missing of RabA4d will lead to the destruction of polar growth and change the knot of cell wall
Structure illustrates that RabA4d plays a significant role (Szumlanski and Nielsen, 2009) in regulation pollen tube polar growth.
Summary of the invention
The object of the present invention is to provide a kind of small G-protein Rab genes (GhRab11d3) of cotton, and provide the gene
CDNAORF and genome sequence.Using this gene as target gene, transgenosis functional verification is carried out by the method for genetic engineering, with
Just it matter and is applied in production for evolving new breeds.
Another object of the present invention is to provide the albumen of the small G-protein Rab coded by said gene of above-mentioned cotton.
Another object of the present invention is to provide the application of the small G-protein Rab gene of above-mentioned cotton.
The purpose of the present invention is achieved through the following technical solutions:
A kind of cotton small G-protein Rab gene (GhRab11d3), the cDNA ORF sequence of the gene such as SEQ ID NO.1 institute
Show；Genome sequence is as shown in SEQ ID NO.2.
The albumen of above-mentioned Rab gene coding；Its amino acid sequence is as shown in SEQ ID NO.3.
Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing said gene.
Above-mentioned small G-protein Rab gene is improving cotton fiber length, improvement cotton fibre quality or is cultivating in cotton new germ plasm
Application.
Above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacterium is in high lint length, improvement cotton fiber product
Application in matter and cultivation cotton new germ plasm.
Advantages of the present invention is shown:
(1) the gene coding small G-protein that the present invention is cloned, the vesicle transport of the Rab albumen and cotton fiber elongation period
It is closely related, it is the key gene in vesicle transport approach.During vesicle transport, GhRab11d3 can be along cytoskeleton
Albumin A ctin transports macromolecular polysaccharide substance to cell membrane from endoplasmic reticulum, golgiosome, acts on finally by outlet by macromolecular
Polysaccharide material is discharged to cell wall.In the process, glucide accumulation in cell is on the one hand influenced whether, on the other hand
Influence whether quantity and form of the actin cytoskeleton F-actin microfilament in fibrocyte.Being overexpressed the gene can be with
Increase glucide in fibrocyte (pectin) accumulation, while improving actin cytoskeleton F-actin in fibrocyte
The quantity of microfilament, so that cotton fiber be promoted to extend.Thus infer, using cotton small G-protein Rab gene of the present invention as target gene into
Row transgenic research is expected to improve cotton fiber quality.
(2) gene that the present invention is cloned is more closely similar to the small G-protein Rab gene of arabidopsis from structure, in cotton
Middle forefathers are spent not study its function also.
(3) real-time fluorescence quantitative PCR the result shows that the gene in upland cotton Genetic standard line TM-1 elongate fiber phase advantage
Expression, the gene is variant in different Fibre Development period expression quantity, is to start to express in origin of fibers period, quick in fiber
Elongation period (Post flowering 5,10 days) expression quantity highest, (Post flowering 15,20 and 25 days) expression quantity of secondary wall thickening period by
Gradually decline, this is the result shows that the gene and Fibre Development elongation process are closely related.
(4) the positive antisense expression vector of plant is constructed, transgenic research is carried out by receptor of land cotton material W0, to transgenosis
Material expression analysis is found: compared with wild type, it is overexpressed GhRab11d3 expression quantity in strain and significantly improves, transgenic plant
The elongated phenotype of fiber is showed, inhibits the expression of GhRab11d in expression strain to be suppressed significantly, fiber occurs in transgenic plant
The phenotype to shorten shows that the gene expression dose influences cotton fiber development.
(5) to GhRab11d3 transgenic line and non-transgenic wild type control material, choose 10 days fibers of Post flowering into
Row actin F-actin fluorescent staining analysis, the results showed that, the content for being overexpressed strain actin F-actin microfilament is aobvious
It writes and increases, on the contrary, the content of the actin F-actin microfilament of expression strain is inhibited to substantially reduce；Meanwhile cytoskeleton flesh is dynamic
The morphosis of albumen F-actin microfilament is also changed, and the actin F-actin microfilament in expression strain fiber is inhibited
Structural damage fracture shows that the gene influences actin cytoskeleton structure, and then influences Fibre Development.
(6) it to GhRab11d3 transgenic line and non-transgenic wild type control material, chooses ovule on the day of blooming and opens
10 days fibers after spending, carry out dyeing discovery using FM4-64 fluorescent dye, compared with wild type, are overexpressed and turn in GhRab11d3
In genetic material, in same day ovule protrusion of surface of either blooming or 10 days fiber tips of Post flowering are assembled more
Vesica, the glucide pectin content finally transported dramatically increase.On the contrary, inhibit in expression strain in GhRab11d3, it is identical
Developmental stage vesica quantity significantly reduces, and the glucide pectin content finally transported significantly reduces, and shows GhRab11d3 in capsule
It plays a significant role in bubble transport.
Detailed description of the invention
The Rab gene clusters of the other species of Fig. 1 GhRab11d3 and source are analyzed.
Using GhRab11d3 and its amino acid alignment of the Rab gene of other species and analysis, illustrate between different plant species
Rab albumen it is very conservative.
TcRabA4c (EOX96740.1), EgRabA4c (XP_010067891.1), PtRab (XP_002298447.1),
PeRabA4d (XP_011025048.1), VvRGP1 (XP_002264293.1), NtRGP1 (XP_016459335.1),
AtRabA4d (NP_187823.1), VvRabA4d (XP_002282798.1), TcRabA4d (EOY09425.1), AtRabA4c
(NP_199607.1), GhRab11a (AF165095.1), GhRab11b (AF165096.1)
Fig. 2 GhRab11d3 is analyzed in cotton different tissues expression characteristic.
Real-time fluorescence quantitative PCR (qRT-PCR) detects cotton small G-protein Rab gene (GhRab11d3) in cotton difference group
Knit expression characteristic.R: root；S: stem；L: leaf；Pe: petal；An: anther；Pis: gynoecium；Re: holder；Se: calyx；0d:0DPA embryo
Pearl；5d:5DPA fiber；10d:10DPA fiber；20d:20DPA fiber；25d:25DPA fiber.
Fig. 3 is excessive and inhibits the transgenic cotton floral material initiative of GhRab11d3 expression.
Transgenic plant PCR detection, shows that the positive antisense vector of the gene has been led using the detection of promoter+target gene
Enter cotton.M, P, C are respectively Marker, positive control and negative control；1-9 is transgenic plant target gene PCR detection knot
Fruit.
Expression analysis of Fig. 4 GhRab11d3 in transgene cotton.
Quantitative qPCR analysis GhRab11d3 gene turns base in 3 different expression vectors (35S-SC, RDL-SC, RDL-ASC)
Because of the expression in 5 days, 10 days and 15 days fibers of strain Post flowering.W0 is receptor control, and 35S-SC and RDL-SC show target base
Because of Overexpression vector, RDL-ASC shows that target gene inhibits expression vector, and 35S represents composition type expression promoter, and RDL is represented
Fibre Development predominant expression promoter.28,347,372 and 303 be 35S-SC transgenic line；56,210 and 73 turn for RDL-SC
Genetic material；98,217 and 111 be RDL-ASC transgenic line.It is T3 for transgenic homozygous strain.
Fig. 5 transgenic plant and the fibre length of receptor W0 compare.
In transgenosis pure lines and W0, the phenotypic analysis of Post flowering 10,15,20 days and mature period fibre length.Table
The bright fibre length for being overexpressed plant is significantly elongated, and the fibre length of expression plant is inhibited significantly to shorten.
The interaction of Fig. 6 Rab11d3 albumen and four Actin albumen.
The interaction of Rab11d3 and ACTa, ACTb, ACT2, ACT4 in yeast is verified.
Actin F-actin microfilament fluorescent staining is analyzed in 10 days fibers of Fig. 7 transgenic plant and receptor W0 Post flowering.
Show that the content for being overexpressed actin F-actin microfilament in strain fiber dramatically increases, on the contrary, inhibiting expression strain
The content of actin F-actin microfilament substantially reduces in series fiber；Meanwhile actin cytoskeleton F-actin microfilament
Morphosis is also changed, and the actin filament structural damage fracture in expression strain fiber is inhibited.
Fig. 8 transgenic line and wild type are analyzed on the day of blooming with vesica fluorescent staining in 10 days fibers of Post flowering.
It is dyed using FM4-64, compared with wild type, the ovule on the day of GhRab11d3 is overexpressed and either blooms in material
In protrusion of surface or 10 days fiber tips of Post flowering assemble more vesicas, on the contrary, inhibiting expression strain in GhRab11d3
Middle vesica quantity significantly reduces.Scale=50 μm.
The pectin content measurement analysis in Post flowering 5 days and 10 days fibers of Fig. 9 transgenic line and wild type
It compared with wild type, is overexpressed in material in GhRab11d3, either Post flowering 5 days or Post flowering 10 days, fruit
Glue content obviously increases, on the contrary, inhibiting in expression strain in GhRab11d3, pectin content is significantly reduced.
Specific embodiment
(1) cotton small G-protein gene GhRab11d3 full length sequence obtains:
This experiment utilizes cDNA differential display technique, in upland cotton (G.hirsutum) Li Shi superbhort fiber mutant Li1
And its in the ovule fiber composite sample comparative analysis of the normal wild type material Post flowering 10DPA of Fibre Development, it was found that
One in Li1 wild type, the extremely significant highly expressed est sequence than in superbhort fiber mutant, further clone obtain overall length
Gene.By homologous comparison, Rab albumen is encoded, is further searched in Rab family gene, is named as GhRab11d3
(as shown in Figure 1).Specific naming rule is: containing 57 Rab protein gene family members in arabidopsis altogether.This 57 Rab
Protein family member is divided into eight major class, be respectively designated as RabA, RabB, RabC, RabD, RabE, RabF, RabG and
RabH.In this eight major class, each major class is divided into several groups again, wherein RabA points are six group of RabA1-RabA6, RabB
It is divided into mono- group of RabB1, it is two group of RabC1 and RabC2 that RabC, which is divided to, and it is two group of RabD1 and RabD2 that RabD, which is divided to, and RabE divides
For mono- group of RabE1, it is two group of RabF1 and RabF2 that RabF, which is divided to, and RabG points are tri- group of RabG1-RabG3, and RabH points are
Mono- group of RabH1, in this way, eight major class of RabA-RabH of arabidopsis is divided into 18 groups again.Compared to arabidopsis, Lei Mengdeshi cotton
There are 87 Rab protein gene family members in database, is also accurately divided into eight major class and (names and advise according to initial Rab albumen
Then, respectively correspond and be named as eight large family of Rab11, Rab2, Rab18, Rab1, Rab8, Rab5, Rab7 and Rab6), but only
17 groups are uniquely the RabG1 group of RabG in arabidopsis than the group that arabidopsis lacks.
Further study show that RabA, RabB, RabC, RabD, RabE, RabF, RabG and RabH eight in arabidopsis are big
Family respectively corresponds eight large family of Rab11, Rab2, Rab18, Rab1, Rab8, Rab5, Rab7 and Rab6 in Lei Mengdeshi cotton.
By the Rab albumen and arabidopsis clustering of Lei Mengdeshi cotton, clear ownership of 17 groups in 8 major class, in Arab
After number plus small English alphabet is distinguished, so it is named as Rab11a-Rab11f, Rab2a, Rab18a-Rab18b,
17 group such as Rab1a-Rab1b, Rab8a, Rab5a-Rab5b, Rab7b-Rab7c and Rab6a, each group it is different at
Sequence of positions arrangement between member according still further to chromosome from small to large, from top to bottom, respectively by suitable behind small English alphabet
Sequence adds Arabic numerals, completes the name of all 87 Rab protein gene family members of Lei Mengdeshi cotton.
In our current research, genetic homology highest in the RabA4 class of our target gene and arabidopsis, with cotton
Rab11d class is corresponding, and further analysis finds that the gene is located at the third position of all 13 chromosomes in this group,
Finally it is named as GhRab11d3.
According to existing sequence, design PCR amplification primer, expanded respectively from upland cotton TM-1 the gene ORF overall length and
Genome sequence.Amplification the primer is shown in Table 1.
Table 1: amplification the primer
(2) bioinformatic analysis of cotton small G-protein gene GhRab11d3
BLAST analyzes (http://www.ncbi.nlm.nih.gov/blast) discovery, the amino of GhRab11d3 coding
Acid sequence and arabidopsis small G-protein Rab gene (AtRabA4d) have very high homology (77%).In the website NCBI CD
In Search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), which is guarded
Structural domain prediction discovery, GhRab11d3 have the structural domain of complete small G-protein Rab gene, and 4 guanylic acids combine
Two cysteine residues of structural domain and an effector binding structural domain and C-terminal.
(3) quantitative RT PCR analysis of cotton small G-protein gene GhRab11d3
Design the special primer of GhRab11d3:
F:5’-CCAATGATGAACAAGAAGAAT-3’
R:5’-CTATGGCTATGATGATGACG-3’
In different tissues organ, quantitative RT-PCR detection is carried out.The result shows that the gene is in root, stem, leaf and floral organ
In have expressed, in each different times differential expression of Fibre Development, begin to express in origin of fibers (blooming first 3 days),
As Fibre Development expression quantity is gradually increased, in fiber rapid elongation phase (Post flowering 5 and 10 day) expression quantity highest, subsequent expression
Amount is gradually reduced (as shown in Figure 2).
(4) the transgenosis functional verification of cotton small G-protein gene GhRab11d3
PBI121 is a kind of traditional plant binary expression vector, and promoter is 35S promoter.It constructs based on this
The sense expression vector of 35S promoter, the insertion position of sense fragment be 35S-P (promoter) and Nos-T (terminator) it
Between.
The pCAMBIA2301 being transformed is a kind of traditional plant binary expression vector, and promoter is RDL promoter.
The positive antisense binary vector of RDL promoter is constructed based on this, and the insertion position of positive antisense fragments is RDL-P (promoter)
Between Nos-T (terminator).
Positive Antisense construct is constructed with genes of SEQ NO ID.1 sequence segment of the present invention, after the completion of building,
Functional verification is carried out by Agrobacterium-mediated transformation cotton, obtains 35S promoter justice transgenic plant and RDL promoter
Positive antisense transgenic plant (as shown in Figure 3).Three above vector transgene plant has identified (as shown in Figure 4) by PCR.
Expression analysis finds that in just vector transgene plant, the expression of the gene is significantly increased, and fibre length is elongated, and antisense
In vector transgene plant, the expression of the gene is suppressed significantly, and fibre length shortens (as shown in Figure 5).Therefore make the gene
Function relevant to cotton fiber development is directly verified.
Table 2: transgenosis target gene detects the primer on genome and expression
(5) the interaction albumen of cotton small G-protein gene GhRab11d3 excavates
After building bait carrier GhRab11d3-pGBKT7, we screen one from cotton fiber Yeast expression library
Actin sequence relevant to cytoskeleton.In cotton gene group sequence, it has been found that have 5 Actin genes in Fibre Development
Phase predominant expression.And in this five Actin genes, it is completely the same after amino acid sequence there are two translating into, so, we
The gene that wherein four Actin genes are verified as subsequent experimental only is selected, is respectively designated as ACTa, ACTb, ACT2 and ACT4.
In order to verify GhACT full-length gene whether with bait GhRab11d3-pGBKT7 exist each other effect, we by GhACTa,
GhACTb, GhACT2 and GhACT4 gene are cloned into respectively on pGADT7 carrier, are finally successfully built into carrier GhACTa-
PGADT7, GhACTb-pGADT7, GhACT2-pGADT7 and GhACT4-pGADT7.
Further by carrier GhRab11d3-pGBKT7 respectively with GhACTa-pGADT7, GhACTb-pGADT7, GhACT2-
For pGADT7, GhACT4-pGADT7 cotransformation into strains A H109, screening for the first time lacks training three of the 3-AT added with 20mM
It supports and is carried out on base SD/-His/-Leu/-Trp, meanwhile, it is real that same conversion is also done on two scarce culture medium SD/-His/-Leu
It tests, transformation efficiency is checked with this.By the positive colony screened for the first time in four scarce culture medium SD/- being coated on X- α-Gal
It crosses on Ade/-His/-Leu/-Trp, and further confirms that whether the two is real positive colony.Positive and feminine gender is right
According to growth is then converted first on two scarce culture medium SD/-His/-Leu, later, while three in the 3-AT added with 20mM are lacked
Culture medium is bred with dibbling on the four scarce culture mediums of X- α-Gal is coated with.
It is double miscellaneous it is demonstrated experimentally that Rab11d3 albumen and the four Actin albumen in elongate fiber phase predominant expression by yeast
There are interaction (as shown in Figure 6), show that Rab11d3 protein function is related to fiber skeleton albumen.
(6) cell in 10 days fibers of cotton small G-protein gene GhRab11d3 transgenic line and wild type Post flowering
The dyeing of skeletal actin F-actin microfilament
10 days cotton fibers after big Tanaka takes away and spends, are fixed 10-20min with 4% paraformaldehyde immediately, use PBS
(PH=7.0) buffer rinses 2-3 times, and 10 minutes every time, cotton fiber cell is placed in the Alexa of of containing 1U
It dyes 60-90 minutes in PBS (PH=7.0) buffer of phalloidin in room temperature, is rinsed with PBS (PH=7.0) buffer
2-3 times by fibrocyte Alexa remained on surfacePhalloidin is cleaned.By clean cotton ovule and fibre
Dimension, which is placed under laser co-focusing (LSM710, Zeiss), to be observed.Green florescent signal analysis is found: being overexpressed in strain fiber
The content of actin F-actin microfilament dramatically increases, and the actin F-actin microfilament in inhibition expression strain fiber
Content substantially reduces (as shown in Figure 7).Meanwhile the morphosis of cytoskeletal protein microfilament is also changed, and expression is inhibited
The impaired fracture of actin F-actin Subfilament Structure in its fiber of strain.
(7) ovule and Post flowering 10 on the day of cotton small G-protein gene GhRab11d3 transgenic line and wild type are bloomed
Vesica fluorescent staining in its fiber
10 days cotton fiber cells of ovule and Post flowering on the day of crop field takes away flower, it is glimmering to be placed in 7.5mg/L FM4-64 at once
It is dyed in photoinitiator dye, room temperature dyes 2 hours, is rinsed 2-3 times, 5 minutes every time, ovule and fiber surface is remained with DMSO
FM4-64 dye liquor is cleaned.Clean cotton ovule and fiber are placed under laser co-focusing (LSM710, Zeiss) and are observed.To red
Color fluorescence signal analysis is found, compared with wild type, the ovule surface on the day of GhRab11d3 is overexpressed and either blooms in material
In protrusion or 10 days fiber tips of Post flowering are populated with more vesicas, on the contrary, inhibiting in expression strain in GhRab11d3
Vesica quantity significantly reduces (as shown in Figure 8).The power of vesicle transport ability eventually leads to vesicle transport polysaccharose substance to carefully
Pectin content on cell wall generates variation.It chooses transgenic line and compares W0 in the fiber sample of 5DPA and 10DPA, use
The pectin content reagent extracts kit (micromethod, 100/48 samples of pipe) of Suzhou Ke Ming Bioisystech Co., Ltd, specific method
With reference to its pectin content kit specification.Pass through measurement, it has been found that: in 5DPA, turning the 303 of 35S-SC is its pectin content
It is significantly improved than W0, turning the 210 of RDL-SC is the extremely significant increase of its pectin content ratio W0, and turns the pectin of 98 systems of RDL-ASC
Content then reduction more extremely significant than W0.In 10DPA, turn 303 systems of 35S-SC and turn the 210 of RDL-SC to be its pectin content and W0 phase
Than, it is all extremely significant increase, and turning the 98 of RDL-ASC is its pectin content compared with W0, then is extremely significant reduction (such as Fig. 9 institute
Show).
<110>Agricultural University Of Nanjing
<120>the small G-protein Rab gene of cotton fiber length can be significantly improved
<160> 3
<210> 1
<211> 690
<212> DNA
<213>upland cotton (Gossypium hirsutum)
<220>
<221>cotton small G-protein Rab gene ORF sequence
<400> 1
atgtctagtt tgcaaagggg tttcgatcaa aagatagatt acgtgttcaa agtggtgcta 60
ataggagact ctgctgtggg gaaatcgcag ctcttatccc gcttcacaag aaacgaattc 120
aatatcgatt ccaaggccac catcggcgtc gaattccaaa ctaaaacttt gcttatcgat 180
cataaatccg tcaaggctca gatttgggat actgctggtc aagaaaggta ccgggcggta 240
acgagtgcat actacagagg agcagtgggg gcaatgctgg tgtatgacat aactaagcgt 300
cagtcatttg atcatgttgc caagtggtta gaggaattgc gtggacatgc tgataaaaac 360
attgtgatta tgcttgtggg caacaagtct gaccttgcct cccttagagc tgttcccata 420
gaggatgcta aggaattcgc ccaaagggaa agcctcttct tcatggagac ttcagccctt 480
gaggccacta atgtcgaatc ggctttcctt actgttgtaa cagagattta ccgagtcatc 540
agcaagaaaa acctggtagc caatgatgaa caagaagaat ccgggggtaa tgcatcactt 600
ctcaagggaa ctaccattgt tgtccctggt ccccaaccac aatctggttc tggaggaaag 660
agtttcagtt gctgtgcgtc atcatcatag 690
<210> 2
<211> 958
<212> DNA
<213>upland cotton (Gossypium hirsutum)
<220>
<221>cotton small G-protein Rab gene group sequence
<400> 2
atgtctagtt tgcaaagggg tttcgatcaa aagatagatt acgtgttcaa agtggtgcta 60
ataggagact ctgctgtggg gaaatcgcag ctcttatccc gcttcacaag aaacgaattc 120
aatatcgatt ccaaggccac catcggcgtc gaattccaaa ctaaaacttt gcttatcgat 180
cataaatccg tcaaggctca gatttgggat actgctggtc aagaaaggta tataaacttt 240
atacatacaa acccttttcc tattactcat tttactgcta tttgtttatg ggaattggga 300
cgatgccctt tatcaaccat taaacatatc tatgttgcta aaaagcttga gactttggat 360
tagaggattg gtttctcttt gatttctgac tgtatttggt cagtcataat tgagcttgat 420
aataggtgaa actatgtata tatatatatg tacggtatgt atgatatgtt gataatggtg 480
gttgatggaa cacaggtacc gggcggtaac gagtgcatac tacagaggag cagtgggggc 540
aatgctggtg tatgacataa ctaagcgtca gtcatttgat catgttgcca agtggttaga 600
ggaattgcgt ggacatgctg ataaaaacat tgtgattatg cttgtgggca acaagtctga 660
ccttgcctcc cttagagctg ttcccataga ggatgctaag gaattcgccc aaagggaaag 720
cctcttcttc atggagactt cagcccttga ggccactaat gtcgaatcgg ctttccttac 780
tgttgtaaca gagatttacc gagtcatcag caagaaaaac ctggtagcca atgatgaaca 840
agaagaatcc gggggtaatg catcacttct caagggaact accattgttg tccctggtcc 900
ccaaccacaa tctggttctg gaggaaagag tttcagttgc tgtgcgtcat catcatag 958
<210> 3
<211> 229
<212> PRT
<213>upland cotton (Gossypium hirsutum)
<220>
<221>cotton small G-protein amino acid sequence
<400> 3
Met Ser Ser Leu Gln Arg Gly Phe Asp Gln Lys Ile Asp Tyr Val Phe
1 5 10 15
Lys Val Val Leu Ile Gly Asp Ser Ala Val Gly Lys Ser Gln Leu Leu
20 25 30
Ser Arg Phe Thr Arg Asn Glu Phe Asn Ile Asp Ser Lys Ala Thr Ile
35 40 45
Gly Val Glu Phe Gln Thr Lys Thr Leu Leu Ile Asp His Lys Ser Val
50 55 60
Lys Ala Gln Ile Trp Asp Thr Ala Gly Gln Glu Arg Tyr Arg Ala Val
65 70 75 80
Thr Ser Ala Tyr Tyr Arg Gly Ala Val Gly Ala Met Leu Val Tyr Asp
85 90 95
Ile Thr Lys Arg Gln Ser Phe Asp His Val Ala Lys Trp Leu Glu Glu
100 105 110
Leu Arg Gly His Ala Asp Lys Asn Ile Val Ile Met Leu Val Gly Asn
115 120 125
Lys Ser Asp Leu Ala Ser Leu Arg Ala Val Pro Ile Glu Asp Ala Lys
130 135 140
Glu Phe Ala Gln Arg Glu Ser Leu Phe Phe Met Glu Thr Ser Ala Leu
145 150 155 160
Glu Ala Thr Asn Val Glu Ser Ala Phe Leu Thr Val Val Thr Glu Ile
165 170 175
Tyr Arg Val Ile Ser Lys Lys Asn Leu Val Ala Asn Asp Glu Gln Glu
180 185 190
Glu Ser Gly Gly Asn Ala Ser Leu Leu Lys Gly Thr Thr Ile Val Val
195 200 205
Pro Gly Pro Gln Pro Gln Ser Gly Ser Gly Gly Lys Ser Phe Ser Cys
210 215 220
Cys Ala Ser Ser Ser
225
Claims (6)
1. a kind of method for improving cotton fiber quality, which is characterized in that overexpression cotton small G-protein Rab base in cotton
Cause, the ORF sequence of the gene is as shown in SEQ ID NO.1.
2. the method according to claim 1, wherein the genome sequence of the cotton small G-protein Rab gene is such as
Shown in SEQ ID NO.2.
3. cotton small G-protein Rab gene is improving cotton fiber length or is improving the application in cotton fibre quality, the gene
ORF sequence is as shown in SEQ ID NO.1.
4. application according to claim 3, which is characterized in that the genome sequence of the cotton small G-protein Rab gene is such as
Shown in SEQ ID NO.2.
5. recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing cotton small G-protein Rab gene are improving cotton
Application in fibre length, improvement cotton fibre quality, the ORF sequence of the gene is as shown in SEQ ID NO.1.
6. application according to claim 5, which is characterized in that the genome sequence of the cotton small G-protein Rab gene is such as
Shown in SEQ ID NO.2.
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| CN201610974555.5A CN106434695B (en) | 2016-11-03 | 2016-11-03 | The small G-protein Rab gene of cotton fiber length can be significantly improved |
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Non-Patent Citations (3)
| Title |
|---|
| Comparative Proteomic Analysis Provides New Insights into the Fiber Elongating Process in Cotton;Yi-Wei Yang,et al;《Journal of Proteome Research》;20080930;第7卷;4623–4637 |
| PREDICTED: Gossypium hirsutum ras-related protein RABA4c-like(LOC107930116),mRNA;Genbank;《Genbank XM_016861686》;20160518 |
| 植物小G蛋白功能的最新研究进展;石峰;《热带农业科学》;20090731;第29卷(第7期);69-75 |
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