CN101560251B - Associated protein for plant root growth and encoding gene and application thereof - Google Patents
Associated protein for plant root growth and encoding gene and application thereof Download PDFInfo
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Abstract
The invention discloses an associated protein for plant root growth and an encoding gene and application thereof. The protein is provided with one of the following amino acid residue sequences: (1) SEQ ID No.3 amino acid residue sequence in the sequence table; and (2) protein which is formed by substituting and/or deleting and/or adding one or a few SEQ ID No.3 amino acid residues in the sequence table and has associated protein activity for plant root growth. The associated protein for plant root growth and the encoding gene thereof can promote the growth of the plant root, and play great role in promoting the root system of plant, improving the yield and improving the variety of the plant (particularly crops).
Description
Technical field
The present invention relates to a kind of associated protein for plant root growth and encoding sox thereof and application, particularly relate to associated protein for plant root growth and encoding sox and their application in growth and development of plants that derives from Arabidopis thaliana.
Background technology
As higher organism, the difference of the maximum of Plants and Animals be plant must set growth, the fixing and nutritive substance of this growth draw the growth that must depend on root system of plant consumingly.So growing of root system plays crucial effects for the whole good growth of plant.No matter be monocotyledons or dicotyledons, though the structure of root system and form are different, all to form for sophisticated by root cap district, meristematic zone, elongation zone and maturation zone four parts, wherein every part is all being carried out different functions.Root cap is made up of central root cap cell and side root cap cell, and along with the continuous growth of the tip of a root, thereby root cap cell detachment has foremost played the effect of protection to the tip of a root; Central root cap cell contains a large amount of starch small graiies simultaneously, plays the effect of experiencing gravity, thereby makes the root of plant produce geotropic growth.Meristematic zone is made up of the cell that is in division stage, and the continuous division of these cells just makes the number of cell increase, and root is constantly grown.The elongation zone is made up of the cell between splitting status and differentiation state, is the transitory stage between the two.The maturation zone is made up of well differentiated cell, and the cell size and form of these differentiation and maturations no longer changes, and carries out different functions separately.
The stem cell that is called as initiating cell in the Arabidopis thaliana root meristematic tissue has produced various types of cells in every confluent monolayer cells layer through the spatial division, and these cells have been formed a vegetative clone.One in two daughter cells that initiating cell produces through division is once still keeping primitiveness, and the another one cell becomes transit-amplifyingcell (TA cell), this cell through division many times after, be differentiated to form self characteristics.The cell that is centered around the quiescent center (QC) of initiating cell central authorities seldom divides and stem cell has on every side been formed the merismatic stem cell of root habitat (stem cell niche), and QC is the organization center of this niche.QC through with the signal communication of stem cell on every side; Suppress its differentiation; The primitiveness that keeps stem cell, the understanding for these signaling molecule essence at present is very few, but from root meristematic tissue and the merismatic contrast of bud, finds; The initiating cell of root is similar to the merismatic stem cell of bud, and QC is similar to the OC cell of expressing WUS.
In the research of some two mutants of regulating and control the Arabidopis thaliana root growth and gene thereof; The similar signal transduction path of discovery CLV has also been participated in the regulation and control of root stem cell; And the isolation identification of some other two mutants; As wherein shortroot (shr) and scarecrow (scr) two mutants all to show as root short, and these two site mutation have all caused root between epidermis and endodermis, to lack a confluent monolayer cells, it is necessary that SHR and SCR express for the characteristic of keeping QC simultaneously.In addition, the PLT gene also is necessary for the establishment in root stem cell habitat.And PLT and SHR/SCR are two parallel approach in the formation of participating in regulation and control root meristematic tissue stem cell, have regulated and control merismatic formation and growth from embryo's radicle in period to the matured root in seedling stage jointly.
Summary of the invention
The purpose of this invention is to provide a kind of associated protein for plant root growth and encoding sox thereof and application.
Associated protein for plant root growth provided by the present invention, name is called ARD1, derives from Arabidopis thaliana (Arabidopsis thaliana), is (a) or protein (b) as follows:
(a) protein of forming by the amino acid residue sequence of sequence in the sequence table 3;
(b) with the amino acid residue sequence of sequence in the sequence table 3 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the root growth adjusting function by (a) deutero-protein.
Wherein, the sequence in the sequence table 3 is made up of 500 amino-acid residues.
The encoding sox of above-mentioned associated protein for plant root growth (ARD1) also belongs to protection scope of the present invention.
The cDNA gene of above-mentioned associated protein for plant root growth can have one of following nucleotide sequence:
1) dna sequence dna of sequence 1 in the sequence table;
2) polynucleotide of protein sequence shown in the sequence 3 in the code sequence tabulation;
The nucleotide sequence of the dna sequence dna hybridization that 3) under the rigorous condition of height, can limit with sequence in the sequence table 1.
Wherein, sequence 1 is made up of 1925 deoxynucleotides in the sequence table, and 5 of sequence 1 ' end 268-1770 position nucleotides sequence is classified encoding sequence (ORF) as in sequence table, and coding has the protein of the amino acid residue sequence of sequence 3 in the sequence table.
The genomic gene of above-mentioned associated protein for plant root growth can have one of following nucleotide sequence:
1) dna sequence dna of sequence 2 in the sequence table;
2) polynucleotide of protein sequence shown in the sequence 3 in the code sequence tabulation;
The nucleotide sequence of the dna sequence dna hybridization that 3) under the rigorous condition of height, can limit with sequence in the sequence table 2.
Wherein, Sequence 2 is made up of 4881 Nucleotide in the sequence table; Nucleotide is first exon of this genomic gene from 5 ' end 262-506 position; Nucleotide is first intron of this genomic gene from 5 ' end 507-748 position; Nucleotide is second exon of this genomic gene from 5 ' end 749-820 position; Nucleotide is second intron of this genomic gene from 5 ' end 821-916 position; Nucleotide is the 3rd exon of this genomic gene from 5 ' end 917-1093 position, and Nucleotide is the 3rd intron of this genomic gene from 5 ' end 1094-1178 position, and Nucleotide is the 4th exon of this genomic gene from 5 ' end 1179-1248 position; Nucleotide is the 4th intron of this genomic gene from 5 ' end 1249-1541 position; From 5 ' and end 1542-1829 position Nucleotide be the 5th exon of this genomic gene, Nucleotide is the 5th intron of this genomic gene from 5 ' end 1830-1937 position, from 5 ' to hold 1938-2078 position Nucleotide be the 6th each and every one exon of this genomic gene; Nucleotide is the 6th intron of this genomic gene from 5 ' end 2079-2157 position; Nucleotide is the 7th exon of this genomic gene from 5 ' end 2158-2260 position, and Nucleotide is the 7th intron of this genomic gene from 5 ' end 2261-2352 position, and Nucleotide is the 8th exon of this genomic gene from 5 ' end 2353-2499 position; Nucleotide is the 8th intron of this genomic gene from 5 ' end 2500-3085 position; Nucleotide is the 9th exon of this genomic gene from 5 ' end 3086-3132 position, and Nucleotide is the 9th intron of this genomic gene from 5 ' end 3133-3219 position, and Nucleotide is the tenth exon of this genomic gene from 5 ' end 3220-3273 position; Nucleotide is the tenth intron of this genomic gene from 5 ' end 3274-3345 position; Nucleotide is the 11 exon of this genomic gene from 5 ' end 3346-3464 position, and Nucleotide is the 11 intron of this genomic gene from 5 ' end 3465-3552 position, and Nucleotide is the 12 exon of this genomic gene from 5 ' end 3553-3688 position; Nucleotide is the 12 intron of this genomic gene from 5 ' end 3689-3766 position, and Nucleotide is the 13 exon of this genomic gene from 5 ' end 3767-4092 position.
The rigorous condition of above-mentioned height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
The expression vector, transgenic cell line and the host bacterium that contain the encoding sox of above-mentioned associated protein for plant root growth all belong to protection scope of the present invention.
Utilize plant expression vector, encoding sox (ARD1) importing vegetable cell or tissue with associated protein for plant root growth of the present invention can obtain root development enhanced plant.
When using ARD1 to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or inducible promoter.For the ease of transgenic plant cells or plant being identified and screening; Can process used plant expression vector, as adding selected marker's (gus gene, luciferase genes etc.) that can in plant, express or antibiotic marker thing (qingfengmeisu qiong affinity tag, kantlex affinity tag etc.) with resistance.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
Carry ARD1 of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated through using, and the plant transformed tissue cultivating is become plant.By the plant transformed host both can be monocotyledonss such as paddy rice, corn, wheat, also can be dicotyledonss such as tomato, Arabidopis thaliana, tobacco, cotton.
Associated protein for plant root growth of the present invention and encoding sox thereof can promote growing of roots of plants, to promoting plant root growth, improve plant biomass, in the improvement of plant (particularly farm crop) kind, play a great role.
Below in conjunction with specific embodiment the present invention is explained further details.
Description of drawings
Fig. 1 is the growing state of normal wild type plant and ard1 two mutants root
Fig. 2 is the map based cloning synoptic diagram of ARD1 gene
Fig. 3 is the upgrowth situation photo of the ard1 two mutants of commentaries on classics ARD1 gene
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and the primer is synthetic to reach examining order by the completion of the living worker's biotechnology in Shanghai ltd.
One, the acquisition of Arabidopis thaliana ARD1 two mutants
Screening through to an Arabidopis thaliana (Col-0) T-DNA mutant library obtains a two mutants in the root growth defective, called after ARD1; The phenotype of this two mutants is mainly: root growth is slow, and root system is undeveloped, and main root shortens; Main root is merely about 1/3 of wild-type, and is as shown in Figure 1.WT is wild-type Arabidopis thaliana (Col-0 is environmental) among Fig. 1; ARD1 is the ARD1 two mutants that above-mentioned screening obtains.
Two, with the map based cloning method obtain from two mutants ARD1 with root development genes involved ARD1
Adopt the map based cloning method from the ARD1 two mutants of above-mentioned acquisition, to separate and root development genes involved ARD1, concrete grammar may further comprise the steps:
1) F that is obtained with ARD1 two mutants (Col-0 background) and Ler (another one of Arabidopis thaliana is environmental, available from ArabidopsisBiological Resource Center) hybridization
2For segregating population is material; Therefrom having identified 2100 has with above-mentioned ARD1 mutation type surface individuals with same and carries out genetic analysis; Clone the ARD1 assignment of genes gene mapping on (T6D22) to No. 1 chromosomal BAC of Arabidopis thaliana; Be about 550Kb with the physical distance of flag F 21M12, as shown in Figure 2.Among Fig. 2, near adjacent BAC clone shown in the arrow being.
2) scope of utilizing the method for map based cloning further to dwindle goal gene, last, according to the arabidopsis gene group sequence of announcing, utilize the candidate gene of PCR method amplifying target genes scope, through sequence alignment.The final gene of confirming the place, mutational site.This gene has the nucleotide sequence of sequence 2 in the sequence table, with its called after ARD1.Sequence 2 is made up of 4881 Nucleotide in the sequence table; Nucleotide is first exon of this genomic gene from 5 ' end 262-506 position; Nucleotide is first intron of this genomic gene from 5 ' end 507-748 position; Nucleotide is second exon of this genomic gene from 5 ' end 749-820 position; Nucleotide is second intron of this genomic gene from 5 ' end 821-916 position; Nucleotide is the 3rd exon of this genomic gene from 5 ' end 917-1093 position; Nucleotide is the 3rd intron of this genomic gene from 5 ' end 1094-1178 position; Nucleotide is the 4th exon of this genomic gene from 5 ' end 1179-1248 position, and Nucleotide is the 4th intron of this genomic gene from 5 ' end 1249-1541 position, and Nucleotide is the 5th exon of this genomic gene from 5 ' end 1542-1829 position; Nucleotide is the 5th intron of this genomic gene from 5 ' end 1830-1937 position; From 5 ' and end 1938-2078 position Nucleotide be the 6th each and every one exon of this genomic gene, Nucleotide is the 6th intron of this genomic gene from 5 ' end 2079-2157 position, from 5 ' to hold 2158-2260 position Nucleotide be the 7th exon of this genomic gene; Nucleotide is the 7th intron of this genomic gene from 5 ' end 2261-2352 position; Nucleotide is the 8th exon of this genomic gene from 5 ' end 2353-2499 position, and Nucleotide is the 8th intron of this genomic gene from 5 ' end 2500-3085 position, and Nucleotide is the 9th exon of this genomic gene from 5 ' end 3086-3132 position; Nucleotide is the 9th intron of this genomic gene from 5 ' end 3133-3219 position; Nucleotide is the tenth exon of this genomic gene from 5 ' end 3220-3273 position, and Nucleotide is the tenth intron of this genomic gene from 5 ' end 3274-3345 position, and Nucleotide is the 11 exon of this genomic gene from 5 ' end 3346-3464 position; Nucleotide is the 11 intron of this genomic gene from 5 ' end 3465-3552 position; Nucleotide is the 12 exon of this genomic gene from 5 ' end 3553-3688 position, and Nucleotide is the 12 intron of this genomic gene from 5 ' end 3689-3766 position, and Nucleotide is the 13 exon of this genomic gene from 5 ' end 3767-4092 position.Its cDNA sequence is the nucleotide sequence of sequence 1 in the sequence table, and 5 of sequence 1 ' end 268-1770 position nucleotides sequence is classified encoding sequence (ORF) as in sequence table, and coding has the protein (ARD1) of the amino acid residue sequence of sequence 3 in the sequence table.
Three, of the present inventionly derive from Arabidopis thaliana and transgenic functional verification root development genes involved ARD1
Genome sequence and the carrier pCAMBIA1300 of the ARD1 that obtains according to step 1; The ARD1 gene is connected into the recombinant vectors that obtains between Sma I and the Sal I restriction enzyme site of carrier pCAMBIA1300 MCS; The primer of design amplification ARD1 sequence, primer sequence is as follows:
Primer 1 (upstream primer): 5 '-
CCCGGGGTGAAGAACAAAATATTACA-3 ' (line part Nucleotide is the SmaI recognition site);
Primer 2 (downstream primer): 5 '-
GGTACCATGGCAAAATAACCTTGAGG-3 ' (line part Nucleotide is Kpn I recognition site).
The genomic dna that extracts Arabidopis thaliana kind Col-0 is as template; Under the guiding of primer 1 and primer 2, carry out pcr amplification; Reaction finishes the back PCR product is carried out the detection of 1% agarose gel electrophoresis; Reclaim the also dna fragmentation of purifying 4881bp, promptly the ARD1 genomic fragment shows that through order-checking this fragment has the nucleotide sequence of sequence 2 in the sequence table.
With the ARD1 gene fragment of above-mentioned acquisition with restriction endonuclease sma I be connected with the carrier pCAMBIA1300 that cuts through the same enzyme enzyme after Kpn I carries out double digestion; To connect product and check order, will show correct recombinant vectors called after pCAMBIA1300-ARD1 through order-checking.
PCAMBIA1300-ARD1 is transformed ARD1 two mutants SALK_009847 (available from Arabidopsis Biological Resource Center under the mediation of agrobacterium tumefaciens; ABRC); Use selected marker's Totomycin (containing 50mg/L Totomycin MS substratum) to carry out resistance screening then, screening obtains the commentaries on classics pCAMBIA1300-ARD1 plant that on the substratum that contains the 50mg/L Totomycin, can grow.Carry out the PCR Molecular Identification with the above-mentioned primer 1 commentaries on classics pCAMBIA1300-ARD1 plant that screening obtains with the primer 2 antagonism, obtain the PCR evaluation and show the correct commentaries on classics pCAMBIA1300-ARD1 plant that changes the ARD1 gene fragment over to.
Evaluation shows the correct commentaries on classics pCAMBIA1300-ARD1 plant that changes the ARD1 gene fragment over to PCR; Advance phenotype analytical; Evaluation shows that the correct commentaries on classics pCAMBIA1300-ARD1 plant (pCAMBIA1300-ARD1 among Fig. 3) that changes the ARD1 gene fragment over to sows simultaneously on the MS substratum with PCR to be about to ARD1 two mutants SALK_009847 (available from Arabidopsis Biological ResourceCenter) (ARD1 among Fig. 3); 4 ℃ of dark culturing are after 3 days; Transfer to 22 degrees centigrade illumination cultivation 8 days, as shown in Figure 3, the result shows; It is better than the upgrowth situation of not genetically modified ARD1 two mutants to change the pCAMBIA1300-ARD1 plant, shows that the sudden change of ARD1 gene influences growing of root system of plant really.
Sequence table
Claims (6)
1. associated protein for plant root growth, the protein of forming by the amino acid residue sequence of SEQ ID № .3 in the sequence table.
2. the encoding sox of the described associated protein for plant root growth of claim 1 is following 1) or 2) or 3) or 4):
1) SEQ ID № in the sequence table: 5 ' end 268-1770 position nucleotide sequence of 1;
2) SEQ ID № in the sequence table: 1 nucleotide sequence;
3) SEQ ID № in the sequence table: 2 nucleotide sequence;
4) SEQ ID № in the code sequence tabulation: the polynucleotide of 3 protein sequences.
3. the recombinant expression vector that contains the described associated protein for plant root growth encoding sox of claim 2.
4. the transgenic cell line that contains the described associated protein for plant root growth encoding sox of claim 2.
5. the host bacterium that contains the described associated protein for plant root growth encoding sox of claim 2.
6. the application of the described associated protein for plant root growth encoding sox of claim 2 in promoting plant root growth, said plant is an Arabidopis thaliana.
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| CN109929852B (en) * | 2019-04-09 | 2021-03-23 | 南京林业大学 | Liriodendron hybrid somatic embryo radicle elongation key gene LhHB9 and application thereof |
| CN112481228A (en) * | 2019-09-10 | 2021-03-12 | 山东舜丰生物科技有限公司 | Application of TPST gene in regulation and control of plant traits |
| CN112851778A (en) * | 2019-11-08 | 2021-05-28 | 中国科学院遗传与发育生物学研究所 | Application of SEUSS protein in regulation and control of plant root growth and development |
| CN111690678B (en) * | 2020-05-20 | 2023-04-07 | 山东农业大学 | Method for obtaining whole transgenic woody plant by utilizing agrobacterium rhizogenes transformation |
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