CN108997486B

CN108997486B - Plant root development related protein and coding gene and application thereof

Info

Publication number: CN108997486B
Application number: CN201810955028.9A
Authority: CN
Inventors: 毛传澡; 蒙福宁; 向丹; 徐纪明
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2018-08-21
Filing date: 2018-08-21
Publication date: 2021-02-09
Anticipated expiration: 2038-08-21
Also published as: CN108997486A

Abstract

The invention relates to the field of biotechnology, in particular to a plant development related protein and application of a coding gene thereof in controlling plant root growth and development. Specifically, the invention discloses a protein and a gene encoding the protein; the invention also discloses the application of the gene: for the construction of transgenic plants with altered root length.

Description

Plant root development related protein and coding gene and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a plant development related protein and application of a coding gene thereof in controlling plant root growth and development.

Background

Roots are important underground organs of plants, the growth and development of the roots are strictly regulated and controlled by a plurality of genes, and some signal molecules such as plant hormones and environmental factors play an important role in regulating and controlling the growth and development of rice roots (Lehti-Shiu et al 2009). The family of receptor kinases in plants can recognize the perception and transmission of these signaling molecules and activate a series of responses downstream, thereby allowing plants to adjust their developmental status to accommodate changes in the external environment (Lehti-Shiu et al 2009). The receptor kinase LRR-RLK rich in repeated leucine is the largest subfamily of receptor kinases and plays an important role in plant growth and development, interaction with the environment and the like (Gou et al 2010). In Arabidopsis thaliana, some repeated leucine-rich receptor kinases can be used as positive regulatory factors to promote root growth and development, for example, the receptor kinase GASSHO1(GSO1) and the homologous protein GSO2 can promote root growth and development of Arabidopsis thaliana by sensing extracellular signal factor CIF to regulate formation of Kjeldahl zone (Nakayama et al 2017). Receptor kinases CEPR1 and CEPR2 and receptor kinase FERONIA promote the elongation growth of Arabidopsis roots by participating in plant nitrogen signaling pathways and energy metabolism pathways, respectively (Tabata et al 2014; Haruta et al 2014). The receptor kinase D14L in rice can promote the establishment of symbiosis between rice root and arbuscular mycorrhizal fungi (Gutjahr et al 2015).

Therefore, the separation and identification of the receptor kinase gene for the growth of rice root length is very important and necessary for further comprehensive understanding of the mechanism of the growth of rice and other monocotyledon root systems and obtaining excellent genes for cultivating new rice varieties.

Reference to the literature

BMC Genomics 11:19 (Whole Genome cloning and sequence analysis of Gou X, He K, Yang H et al (2010) Genome-side cloning and sequence analysis of leucine-rich repeat receptor-like kinase genes BMC Genomics 11: 19).

Gutjahr C, Gobbato E, Choi J et al (2015) Rice percentage of systematic arbuscular mycorrhizal fungi requirers the KARRIKIN recipient complex.science 350: 1521-.

Heese A, Hann DR, Gimenez-Ibanez S et al (2007) The receptor-like kinase SERK3/BAK1is a central regulator of amino immunity in plants Proc. Natl. Acad. Sci.104:12217-12222(Heese A, Hann DR, Gimenez-Ibanez S et al (2007) The receptor-like kinase SERK3/BAK1 plant SERK3/BAK1 as a central factor regulates The innate immunity of The plant. Proc. Acad. Sci. USA 104: 12217-12222).

Lehti-Shiu MD, Zou C, Hanada K et al (2009) evolution history and stress regulation of plant-receptor kinase/pellet genes plant physiology.150:12-26(Lehti-Shiu MD, Zou C, Hanada K et al (2009) history of evolution and regulation of stress of plant-like receptor kinase/pellet genes. plant physiology 150: 12-26).

Nakayama T, Shinohara H, Tanaka M et al (2017) A peptide hormomone required for caspase strip differentiation barrier formation in Arabidopsis roots, science 355:284-286(Nakayama T, Shinohara H, Tanaka M et al (2017) formation of the Kjeldahl band in Arabidopsis roots requires a peptide hormone. science 355: 284-286).

Tabata R, Sumida K, Yoshii T et al (2014) percentage of root-derived peptides by shoot LRR-RKs media system N-derived signalling. science 346:343-6(Tabata R, Sumida K, Yoshii T et al (2014) nitrogen-demand signal of root-derived polypeptide mediated system as perceived by plant stem LRR-RKs. science 346: 343-6).

Disclosure of Invention

The invention aims to solve the technical problem of providing a plant root development regulation related protein, and a coding gene and application thereof.

In order to solve the technical problems, the invention provides the following technical scheme:

the protein (OsRARK1) provided by the invention is derived from japonica rice Xiushui134 (Oryza sativa L.ssp. japonica acv.Xiushhui134), and is the protein of the following (a) or (b):

(a) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the protein of the sequence 1 and is derived from the protein related to the plant root development; the plant development is embodied on the characters of plant root length or the size of a specific organ and the like.

In order to facilitate the purification of OsRARK1 in (a), tags shown in Table 1 can be included but not limited to be attached to the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in the sequence 1 in the sequence table.

TABLE 1 sequence of tags

The OsRARK1 in the (b) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression. The gene encoding OsRARK1 in (b) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 2 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 at the 5 'end and/or 3' end.

The coding gene of the protein also belongs to the protection scope of the invention.

The coding gene (OsRARK1) of the protein can be the DNA molecule of the following 1) or 2) or 3) or 4):

1) the coding sequence is a DNA molecule shown in a sequence 2 in a sequence table; that is, the nucleotide sequence of the coding region of the gene is set forth in SEQ ID NO: 2 is shown in the specification;

2) the DNA molecule shown in sequence 3 in the sequence table, namely, the nucleotide sequence of the genome of the gene is shown as SEQ ID NO: 3 is shown in the specification;

3) DNA molecules which hybridize under stringent conditions with the DNA sequences defined in 1) or 2) and which code for proteins of the same function;

4) DNA molecule which has more than 90% of homology with the DNA sequence limited by 1) or 2) or 3) and codes the same functional protein.

Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the genes or antisense genes thereof belong to the protection scope of the invention.

The existing plant expression vector can be used for constructing a recombinant expression vector containing the OsRARK1 gene.

The plant expression vector includes but is not limited to binary agrobacterium vector, plant microprojectile bombardment vector, etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal directs polyadenylic acid to the 3 'end of the mRNA precursor, and includes, but is not limited to, untranslated regions transcribed from the 3' end of, for example, Agrobacterium crown gall inducible (Ti) plasmid genes (e.g., nopalin synthase Nos), plant genes (e.g., soybean storage protein genes), and the like.

When the OsRARK 1is used for constructing a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before the transcription initiation nucleotide, such as but not limited to cauliflower mosaic virus (CAMV)35S promoter, and maize ubiquitin promoter (ubiquitin), and the promoters can be used alone or combined with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers having resistance (gentamicin marker, kanamycin marker, etc.), or anti-chemical agent marker genes (e.g., anti-herbicide gene), etc., which are expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

The recombinant expression vector can be specifically (I) or (II) as follows:

(I) inserting a DNA molecule shown in a sequence 2 of a sequence table into a multiple cloning site of a plasmid pCAMBIA1300-35S-EGFP to obtain a recombinant expression vector containing the gene of

claim

2 or 3;

(II) the DNA fragment of the gene shown in the sequence 2 of the sequence table is connected to a transition vector pBSSK-in the forward direction and the reverse direction and then inserted into a recombinant expression vector obtained by a plasmid pCAMBIA 1300-35S-EGFP;

(I) the plasmids pCAMBIA1300-35S-EGFP and pBSSK-in (II) were both obtained by modification in this laboratory (published).

The invention also protects a method for cultivating transgenic plants, which is characterized in that the gene or the gene fragment is connected to a transition vector pBSSK-in the forward and reverse directions and then is introduced into target plants, and the transgenic plants have changed root length compared with the target plants.

Any vector capable of guiding the expression of the exogenous gene in the plant is utilized, the OsRARK1 gene or the gene fragment provided by the invention is connected to a transition vector pBSSK-in the forward and reverse directions and then is introduced into a plant cell, and a transgenic cell line with changed root length and a transgenic plant can be obtained. The expression vector carrying the OsRARK1 gene or a part of the gene thereof can be transformed into plant cells or tissues by conventional biological methods including but not limited to Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation and the like, and the transformed plant tissues can be cultivated into plants. The plant host to be transformed may be a graminaceous plant such as rice.

When a transgenic plant having a root length greater than that of the target plant is cultivated; the method is to introduce the recombinant expression vector in the step (I) into a target plant to obtain a transgenic plant with lengthened roots. When a transgenic plant having a root length smaller than that of the target plant is cultivated; the method is characterized in that the recombinant expression vector in the step (II) is introduced into a target plant to obtain a transgenic plant with shortened roots.

The invention discovers the new functions of the protein OsRARK1 and the coding gene ORARK1 thereof, obtains a recombinant expression vector which contains the coding gene or partial segments of the coding gene and is connected to a transition vector pBSSK-in a forward direction and a reverse direction, and can obtain a transgenic plant with changed root length by transforming a target plant with the recombinant vector. Therefore, the OsRARK1 can be used as a potential molecular breeding tool to improve the yield of the plants by improving the root length of the plants.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1is the structure of OsRARK1 in example 1 and the protein map encoded by it.

FIG. 2 is a graph showing the auxin IAA-induced expression of OsRARK1 in example 2.

FIG. 3 expression of OsRARK1 in different tissues and organs of example 2;

root, stem, leaf sheath samples were taken from 7-day-old rice seedlings, YP is young ear (number represents length of young ear, unit is cm).

FIG. 4 is the phenotypic analysis of OsRARK1 overexpression transgenic plants in example 2;

a is phenotype of 7-day seedling age of WT and different OsRARK1 overexpression transgenic plants OE11, OE37 and OE51 (scale is 5 cm); b is the plant height statistics of the corresponding strain; c is the root length statistics of the corresponding strains; d is the expression level of the root gene OsRARK1 of the corresponding OsRARK1 overexpression transgenic plant; errors are expressed in SD (number of statistical individuals greater than 30) with P <0.001(T test).

FIG. 5 shows the phenotypic analysis of the transgenic plants expressing OsRARK1 in example 2;

a is phenotype of 7-day seedling age of WT and different OsRARK1 suppression expression transgenic plants Ri18, Ri26 and Ri33 (scale is 5 cm); b is the plant height statistics of the corresponding strain; c is the root length statistics of the corresponding strains; d is the expression level of the corresponding OsRARK1 gene OsRARK1 for inhibiting and expressing the root of the transgenic plant; errors are expressed in SD (number of statistical individuals greater than 30) with P <0.001(T test).

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

the hydroponic medium comprises the following components:

as a result of detecting the gene expression level in the following examples, unless otherwise specified, the expression level of the target gene in Xiushui134 of a wild-type plant was 1, and the expression levels of the target genes of other plants were compared with the expression level of the target gene of the wild-type plant.

Example 1 obtaining of Gene OsRARK1

Taking wild type Xiushui134 as a material, culturing seeds in a normal nutrient solution for 7 days after dormancy breaking and germination accelerating treatment, extracting DNA and RNA of roots, carrying out reverse transcription on the obtained RNA to obtain corresponding cDNA, and carrying out PCR amplification by using the obtained DNA and cDNA as templates and using an OsRARK1 specific primer to respectively obtain the genome of the OsRARK1 and a sequence of a coded protein. The primers for amplification were as follows:

5'-ATGGATCTGCTCAGCGTCCTCCT-3'；

5'-TCATCTGCCGGCGGAGAGCTC-3',

by comparison, the OsRARK 1is found to contain 11 exons, 10 introns, 1818bp of an Open Reading Frame (ORF) region and 605 amino acids, and the molecular weight of the OsRARK 1is predicted to be 66.87KD and the isoelectric point is 5.20.

The result of predicting the structure of the protein encoded by the OsRARK1 at the NCBI website shows that the N end of the protein encoded by the gene contains an LRR (leucoine-rich repeat) structural domain, and the C end of the protein encoded by the gene contains a kinase structural domain (KD), which indicates that the protein encoded by the gene belongs to the LRR-RLK family (see figure 1).

The nucleotide sequence of the coding region of OsRARK 1is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the genome of OsRARK 1is shown as SEQ ID NO: 3 is shown in the specification; the amino acid sequence of the protein coded by the gene is shown as SEQ ID No: 1is shown.

Example 2 functional characterization of Gene OsRARK1

First, the effect of auxin on the expression of gene OsRARK1

Taking wild type Xiushui134 as a material, performing dormancy breaking and germination accelerating treatment on seeds, culturing the seeds in a normal nutrient solution for 7 days, using phytohormone IAA to treat rice seedlings for 1 hour, sampling, extracting root RNA, performing reverse transcription to obtain corresponding cDNA, and determining the expression difference of OsRARK1 under the treatment of the phytohormone IAA by using a qRT-PCR method. The primers for amplification were as follows:

5'-AAGGATTTTGAGCCAGTTGT-3'；

5'-TGCAGCTTCTTCACATGGTC-3',

the result shows that the gene OsRARK1 can be induced by the plant hormone IAA (see figure 2).

Second, expression pattern analysis of gene OsRARK1

Wild Xiushui134 rice is used as a material, cultured in a normal nutrient solution for 7 days, taken roots, stems, leaves and leaf sheaths, and then taken young ears with different development levels when the plants are about to sprout ears. All samples were snap frozen in liquid nitrogen and ground, total RNA was extracted separately and reverse transcribed to obtain the corresponding cDNA. The expression of OsRARK1 in each tissue was detected using a real-time quantitative PCR method. The results showed that the OsRARK1 gene was expressed in different tissues we examined (see FIG. 3).

Example 3 obtaining of Rice Material with OsRARK1 Gene transfer

Construction of OsRARK1 overexpression recombinant vector

mRNA of the rice 134 from Xiushui was extracted and reverse-transcribed to cDNA, and the cDNA was used as a template for PCR amplification to prepare an OsLR1 sequence (DNA shown in SEQ ID NO: 2). The primers for PCR amplification were as follows:

5'-ACGGGGGACGAGCTCATGGATCTGCTCAGCGTCCTCCT-3'，

5'-GACTCTAGAGGATCCTCTGCCGGCGGAGAGCTCGAT-3'；

the obtained PCR product is inserted between SacI and BamHI enzyme cutting sites of the laboratory modified vector pCAMBIA1300-35S-EGFP (Chen et al 2015) by using a recombinant cloning method to obtain the OsRARK1 overexpression vector. The OsRARK1 overexpression vector is verified to be correct through sequencing.

II, obtaining of OsRARK1 over-expression rice

Transferring the OsRARK1 overexpression vector constructed in the first step into agrobacterium EHA105 for transforming the rice of Xiushui134, and specifically comprising the following steps:

(1) taking 500 mul of cultured bacterial liquid to a 1.5ml centrifuge tube, centrifuging at room temperature and 4000rmpAfter 2 minutes, the supernatant was removed. Preparing suspension with 30ml AAM infection bacteria solution containing 200 μmol/L acetosyringone, and obtaining final concentration OD of bacteria solution₆₀₀Is 0.01; picking out 80-120 rice calli growing to a certain size (the volume is 0.5-1 cubic centimeter), putting the rice calli into an agrobacterium tumefaciens suspension, and shaking on a horizontal shaking table for 5 minutes;

(2) taking out the callus, placing the callus on sterile filter paper, and draining the callus for 30 to 40 minutes;

(3) placing the callus on a co-culture medium with a piece of sterile filter paper, and performing dark culture at 25 ℃ for 3 days;

(4) the callus is taken out and washed with sterile water for 5 to 6 times without continuous oscillation. The cells were washed 2 times with sterile water containing 300mg/L carbenicillin sodium (Carb) and shaken for 30 minutes each on a horizontal shaker. Finally placing the mixture on sterile filter paper and draining the mixture for 2 hours;

(5) transferring the dried callus to a selection culture medium containing 300mg/L carbenicillin sodium and corresponding screening pressure for first round selection, and culturing at 28 deg.C under illumination for 14 days;

(6) transferring the initial callus with resistance callus to the culture medium containing 300mg/L carbenicillin sodium and corresponding screening pressure for the second round of selection, culturing at 28 deg.C under light until granular resistance callus grows out (about 14 days);

(7) selecting 3 to 5 yellow resistant calli from different calli, transferring into a plastic wide-mouth bottle filled with a differentiation medium, sealing with a sealing film, placing into a constant-temperature (25 ℃) culture chamber (photoperiod: 16 hours illumination), and waiting for differentiation into seedlings (about 40 days);

(8) after the seedling grows to about 3 cm, old roots and callus are cut off from the base of the seedling by scissors and placed in a rooting medium to strengthen the seedling (about 1 week). Picking out test tubes with intact root, stem and leaf of the seedling (the seedling grows to the top of the test tube, and the tube is opened in time), opening a sealing film, adding appropriate amount of distilled water or sterile water (for preventing the growth of the bacteria in the culture medium), hardening the seedling for 2-3 days, washing off agar, and transplanting the seedling to a greenhouse for growth under the water culture condition. The primer of the gibberellin-resistant gene is used for detecting the transgenic plant, and the primer sequence is as follows:

5'-ATGAAAAAGCCTGAACTCACC-3'；

5'-CTATTCCTTTGCCCTCGGACG-3',

the obtained 57T 1 positive transgenic positive rice plants were selected from three over-expressed plants (OE11, OE37 and OE51), and compared with wild type plants, the plant height of the transgenic line was not significantly changed, and the root length thereof was significantly increased (see FIGS. 4A-C).

The relative expression amount of endogenous OsRARK1 in the Xiushui134 rice and OsLR1 overexpression transgenic plants (OE11, OE37 and OE51) is analyzed by a real-time quantitative PCR method (primers: AAGGATTTTGAGCCAGTTGT and TGCAGCTTCTTCACATGGTC), and the result shows that the length of the main root of the overexpression transgenic strain of the gene OsRARK 1is positively correlated with the expression amount of OsRARK1 (see figure 4D).

Construction of OsLR1 expression-inhibiting recombinant vector

mRNA of the rice of Xiushui134 is extracted and is reversely transcribed into cDNA, and the long cDNA is used as a template to carry out PCR amplification to prepare a partial DNA sequence of OsLR 1: the partial DNA sequence is GCGGTTGACAGATTATGAAAGCCCTGGTGGGGAGGCTGCATTCTTGCGTGAAGTAGAGCTTATCAGTGTTGCAGTTCACCGGAATCTTTTAAGATTGATTGGCTTCTGTACAACACAAACAGAACGTCTACTTGTTTATCCTTTCATG;

the primers for PCR amplification were as follows:

5'-GCGGTTGACAGATTATGAAAG-3'，

5'-TTGCTGACCAATCCAATATTG-3'；

connecting the cloned PCR product with a T vector (purchased from TAKARA), and carrying out enzyme digestion on the connected plasmids respectively by using PstI, BamH I, Pst I and Sal I to obtain two fragments; both fragments were ligated together into the laboratory modified pBSSK-in vector (Chen et al.2015) in two steps. Cutting pBSSK-in with Pst I and BamH I enzyme, connecting with one fragment, cutting with Nsi I and Sal I enzyme, connecting with the other fragment; and finally, cutting the two fragments and intron by using Sac I and Sal I, and connecting the two fragments and intron into a plant binary vector pCAMBIA1300-35S-EGFP subjected to the same enzyme cutting to obtain an OsRARK1 suppression expression vector. The OsRARK1 suppression expression vector is verified to be correct through sequencing.

Fourth, the acquisition of OsLR1 inhibiting expression rice

The OsRARK1 suppression expression vector constructed in the steps is transferred into agrobacterium EHA105 and used for transforming Xiushui 143 rice to 43 strains of T1 generation positive transfer gene rice, and the specific steps are the same as the above.

Three suppression-expressing plants (Ri18, Ri26, Ri33) were selected, and the transgenic lines had no significant change in plant height and significantly shortened root length compared to the wild type (see FIGS. 5A-C).

The relative expression amount of endogenous OsRARK1 in the Xishui 134 rice and OsLR1 suppression expression transgenic plants (Ri18, Ri26 and Ri33) is analyzed by a real-time quantitative PCR method (primers: AAGGATTTTGAGCCAGTTGT and TGCAGCTTCTTCACATGGTC), and the result shows that the length of the main root of an overexpression transgenic strain of the gene OsRARK 1is positively correlated with the expression amount of OsRARK1 (see figure 5D).

Combining the above results, the overexpression of OsRARK1 promotes the elongation of rice roots, and the inhibition of the expression level of OsRARK1 inhibits the elongation of rice roots, which shows that OsRARK1 directly controls the growth and development of rice roots.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Sequence listing

<110> Zhejiang university

<120> a plant root development related protein and its coding gene and application

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 605

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Asp Leu Leu Ser Val Leu Leu Ile Ile Ala Ser Leu Leu Pro Phe

1 5 10 15

Ser Ala Ser Asp Arg Gln Gly Asp Ala Leu Tyr Asp Met Lys Leu Lys

20 25 30

Leu Asn Ala Thr Gly Asn Gln Leu Ser Asp Trp Asn Gln Asn Gln Val

35 40 45

Asn Pro Cys Thr Trp Asn Ser Val Ile Cys Asp Asn Asn Tyr Asn Val

50 55 60

Val Gln Val Thr Leu Ala Ser Met Gly Phe Thr Gly Val Leu Ser Pro

65 70 75 80

Arg Ile Gly Glu Leu Gln Phe Leu Asn Val Leu Ser Leu Pro Gly Asn

85 90 95

Lys Ile Thr Gly Gly Ile Pro Glu Gln Ile Gly Asn Leu Ser Ser Leu

100 105 110

Thr Ser Leu Asp Leu Glu Asp Asn Leu Leu Val Gly Pro Ile Pro Ala

115 120 125

Ser Leu Gly Gln Leu Ser Lys Leu Gln Ile Leu Ile Leu Ser Gln Asn

130 135 140

Asn Leu Asn Gly Thr Ile Pro Asp Thr Val Ala Arg Ile Ser Ser Leu

145 150 155 160

Thr Asp Ile Arg Leu Ala Tyr Asn Lys Leu Ser Gly Ser Ile Pro Gly

165 170 175

Ser Leu Phe Gln Val Ala Arg Tyr Asn Phe Ser Gly Asn Asn Leu Thr

180 185 190

Cys Gly Ala Asn Phe Leu His Pro Cys Ser Ser Ser Ile Ser Tyr Gln

195 200 205

Gly Ser Ser His Gly Ser Lys Val Gly Ile Val Leu Gly Thr Val Val

210 215 220

Gly Ala Ile Gly Ile Leu Ile Ile Gly Ala Val Phe Ile Val Cys Asn

225 230 235 240

Gly Arg Arg Lys Ser His Leu Arg Glu Val Phe Val Asp Val Ser Gly

245 250 255

Glu Asp Asp Arg Arg Ile Ala Phe Gly Gln Leu Lys Arg Phe Ala Trp

260 265 270

Arg Glu Leu Gln Leu Ala Thr Asp Ser Phe Ser Glu Lys Asn Val Leu

275 280 285

Gly Gln Gly Gly Phe Gly Lys Val Tyr Lys Gly Ala Leu Pro Asp Gly

290 295 300

Thr Lys Ile Ala Val Lys Arg Leu Thr Asp Tyr Glu Ser Pro Gly Gly

305 310 315 320

Glu Ala Ala Phe Leu Arg Glu Val Glu Leu Ile Ser Val Ala Val His

325 330 335

Arg Asn Leu Leu Arg Leu Ile Gly Phe Cys Thr Thr Gln Thr Glu Arg

340 345 350

Leu Leu Val Tyr Pro Phe Met Gln Asn Leu Ser Val Ala Tyr Arg Leu

355 360 365

Arg Glu Phe Lys Pro Gly Glu Pro Ile Leu Asp Trp Ser Ala Arg Lys

370 375 380

Arg Val Ala Ile Gly Thr Ala Arg Gly Leu Glu Tyr Leu His Glu His

385 390 395 400

Cys Asn Pro Lys Ile Ile His Arg Asp Val Lys Ala Ala Asn Val Leu

405 410 415

Leu Asp Glu Asp Phe Glu Pro Val Val Gly Asp Phe Gly Leu Ala Lys

420 425 430

Leu Val Asp Val Gln Lys Thr Ser Val Thr Thr Gln Val Arg Gly Thr

435 440 445

Met Gly His Ile Ala Pro Glu Tyr Leu Ser Thr Gly Lys Ser Ser Glu

450 455 460

Arg Thr Asp Val Phe Gly Tyr Gly Ile Met Leu Leu Glu Leu Val Thr

465 470 475 480

Gly Gln Arg Ala Ile Asp Phe Ser Arg Leu Glu Glu Glu Asp Asp Val

485 490 495

Leu Leu Leu Asp His Val Lys Lys Leu Gln Arg Glu Gly Gln Leu Gly

500 505 510

Ala Ile Val Asp Arg Asn Leu Ser Ser Asn Tyr Asp Gly Gln Glu Val

515 520 525

Glu Met Met Ile Gln Ile Ala Leu Leu Cys Thr Gln Ala Ser Pro Glu

530 535 540

Asp Arg Pro Ser Met Ser Glu Val Val Arg Met Leu Glu Gly Glu Gly

545 550 555 560

Leu Ala Glu Arg Trp Glu Glu Trp Gln Gln Val Glu Val Thr Arg Arg

565 570 575

Gln Asp Tyr Glu Arg Met Gln Gln Arg Phe Asp Trp Gly Glu Asp Ser

580 585 590

Ile Phe Asn Gln Glu Ala Ile Glu Leu Ser Ala Gly Arg

595 600 605

<210> 2

<211> 1818

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggatctgc tcagcgtcct cctaattata gcatctctgc tcccattttc agcatctgac 60

cgtcaaggtg atgcactata tgatatgaag ctgaagctga atgctactgg taaccagctt 120

tctgactgga accaaaatca agttaaccca tgcacttgga attctgttat ttgtgacaac 180

aactacaatg ttgtgcaagt aacattggca tctatgggat tcactggagt tctatcacca 240

cgaattggag agcttcagtt tttgaatgtt ttgtccttgc ctggtaataa gattactggt 300

ggcatacctg agcagatcgg caacctatct agtttgacaa gtttggattt ggaagacaat 360

ttgttggttg gaccaatacc ggcttctctt ggccagcttt caaagctcca aattctgata 420

ctaagtcaaa acaatctcaa cggaactata cctgatactg tagcaagaat ctcaagcttg 480

acagacatta ggttagccta caataaactc tctggttcaa tacctggttc gctgtttcaa 540

gttgcacgtt acaacttttc tggtaataac ttgacttgtg gagcaaactt tctgcatcca 600

tgctcatcaa gtatttctta tcaaggttca tcccatggct caaaggtagg cattgtactc 660

ggaacagttg taggagcaat agggatactt ataatagggg ctgtgtttat tgtctgtaat 720

ggaaggagga aaagccatct acgagaagtt tttgtcgatg tatcaggcga ggacgatcga 780

agaattgcat ttggtcagtt gaaaagattt gcctggcgag aattacaact tgcaaccgat 840

agtttcagtg agaaaaatgt ccttggacag gggggtttcg gtaaagtgta taaaggagca 900

cttccagatg gcactaagat tgctgtaaag cggttgacag attatgaaag ccctggtggg 960

gaggctgcat tcttgcgtga agtagagctt atcagtgttg cagttcaccg gaatctttta 1020

agattgattg gcttctgtac aacacaaaca gaacgtctac ttgtttatcc tttcatgcag 1080

aatcttagtg tggcctatcg tttacgagaa ttcaaacctg gcgaaccaat attggattgg 1140

tcagcaagga aacgtgtggc aataggcaca gcacgtggac tggagtacct gcacgagcac 1200

tgtaatccca agatcataca tcgagatgtc aaggctgcca acgtcctgct tgatgaggat 1260

tttgagccag ttgttggtga ctttggcctg gcaaagttgg ttgatgtaca gaagacatca 1320

gtgacaactc aagtccgtgg aactatgggt cacattgcgc ctgaatatct gtccacaggg 1380

aagtcatcgg agagaactga tgtttttggt tatggcataa tgcttcttga gctggtcact 1440

ggacaacgcg ccatcgactt ctcacggttg gaggaagaag acgatgtgct actgcttgac 1500

catgtgaaga agctgcagag ggaagggcag ctgggcgcca tcgtggaccg gaacctgagc 1560

agcaactacg acgggcagga ggtggagatg atgatacaga tcgcgctgct gtgcacacag 1620

gcgtcgccgg aggaccggcc gtccatgtcg gaggtggtgc ggatgctgga aggggaagga 1680

ctcgcggagc ggtgggagga gtggcagcag gtggaggtga cgcggcggca ggactacgag 1740

cggatgcagc agcgcttcga ctggggcgag gactccatct tcaaccagga agccatcgag 1800

ctctccgccg gcagatga 1818

<210> 3

<211> 5462

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggatgcgatc cggaggagaa ggtcgccgaa ttccgtctta tccagagatg gggaggtaaa 60

aaggaggggg atccacctaa ccaaccaagt tgcgtcgcgc ctaaaaaaaa tggccgcacg 120

cgctccgccc cggcagcacc ggtcaattcc tgctgcttcc tcgagtcaac tgctccagcg 180

cagcgcccca cccacgcctt ttacccgcgc gccagcggcg gcggcggcgg cggcggggtg 240

aggaggtgaa gtgggagagg cggcgtggac ctgcgacgac ctgcttgctg cccagcctgc 300

gagggcaagc cactggtagg tagtacgtgc ttctctcttc agtcttcact gtctgtgtgt 360

gtgtgaattg gaatttggaa gtgctggagg taagcagtaa gagaaggatc agatttggag 420

aggctgaatc tctgattctc tctggtgttt aaagagtttg ttcaattcga attgtgggtt 480

taattttggt gccgtctctc actgggaggg atcactggat cacagttgtg ctgcttctgg 540

gaacgttttg gttaattcta tcaggtctgt agttcttcat gatttttttt tgaatggatg 600

tagttcttca tgatagcttc tagaatatct gagtcttgct aatattcctt ttttttttaa 660

tagcaagaga atttagtggt gctgacatac taaaaattag aaggtttatg tgctgtatgg 720

gcatgttcag atattttgat ttgttaggca aatttagata tttttaacca tggcgacctt 780

ttggtttggg ggcataattg ggagcttggt tttgcttcct tttttttgaa cggaagtttt 840

gcttgtttca tgcgttctct ttggaaataa taattctagt acgttttgct gtgatgaatt 900

caagcctcta ttttagtata ccgatgaaac aattgtgcat ttcaaggcaa cttttgcttt 960

tcagcataac taacagcata acagtgaaca gatagtataa taataacagt aactacaaat 1020

tctgctttgc ctatcactca atcttgtatg aacctctttt cttatccaat aatattttgt 1080

ctatcttata ggtgccatac tggactggct tgcttgttaa ctgggttagc cagggactcc 1140

ggtattatac tactatgatc agttcgtgta ctttttttcg ccgggcaagt ttccccaagg 1200

aactcttttg atgctgtaat tataactgaa gagtcatgga tctgctcagc gtcctcctaa 1260

ttatagcatc tctgctccca ttttcagcat ctgaccgtca aggtttattt acatgcctat 1320

catttatgta gttattcaag ctattttgga gtgcttccac aatcatgagt ttgcaaatac 1380

ttttgggtaa taaataaatt tcttttattt ccttaggtga tgcactatat gatatgaagc 1440

tgaagctgaa tgctactggt aaccagcttt ctgactggaa ccaaaatcaa gttaacccat 1500

gcacttggaa ttctgttatt tgtgacaaca actacaatgt tgtgcaagtg taagtttatt 1560

ttgttgatgt tttgcttcac ttttattttc tctccatttt gagttaattc tatatttacg 1620

tatctcttaa acttatatgg cataatagtt cttgatttat gttccacatt cattaccagt 1680

gagctccaga agcatctgtt tgagcacatg attatgattt ttttgatgca cttgcacatg 1740

ctatctgcac ttctttgatg cacgagcaca tttcataatt gaactatatg aaaatggaaa 1800

aatcatacaa catgctgttt ctttaaatct tcttcacacc atctgttatt tctggcaaaa 1860

caacatctag atggttctgt tgaccccgtt atgcatgttc catatctagg ttttttgttt 1920

gttccaagtt ggtgtgcctg atgacagatg ccctcctgca gaacattggc atctatggga 1980

ttcactggag ttctatcacc acgaattgga gagcttcagt ttttgaatgt tttgtaagtg 2040

tacttgtgtt gattgttgga atttctctca gatatttggt ggtagttgtg atgaaatagc 2100

tactgtgatc ctagaggaaa ccagaagctg gtattatgtg agaagtgcca actgttagct 2160

atttctgtgc ttgctgccca aagatttggt tgtcttagtt accatgtact gtggtcttag 2220

actccagttt ttgctaacca ctgtagtgca tgtcattgca ggtccttgcc tggtaataag 2280

attactggtg gcatacctga gcagatcggc aacctatcta gtttgacaag tttggatttg 2340

gaagacaatt tgttggttgg accaataccg gcttctcttg gccagctttc aaagctccaa 2400

attctgtatg tacttgaacc ttagtccttt gttttatgaa tgtataatga tttaacctgc 2460

tcaccttttt atctcttaaa caggatacta agtcaaaaca atctcaacgg aactatacct 2520

gatactgtag caagaatctc aagcttgaca gacatgcaag gactcataca tgatttctgt 2580

tgacaacagt ccatgcctga attatttgac taacatattt tttttcttat gattgcagta 2640

ggttagccta caataaactc tctggttcaa tacctggttc gctgtttcaa gttgcacgtt 2700

acaagtactg ttggatcact gtgacttgta tactcatgtt ttttcaagtt tgagctttga 2760

gttctgacaa atgttaattt tctttacagc ttttctggta ataacttgac ttgtggagca 2820

aactttctgc atccatgctc atcaagtatt tcttatcaag gtaggaagca attttttccc 2880

tgttcaacat tgcacatctg tcatacagca tccattggat gcctgtcaga attttcgctg 2940

gaatatgcta attctatctg tttaataggt tcatcccatg gctcaaaggt aggcattgta 3000

ctcggaacag ttgtaggagc aatagggata cttataatag gggctgtgtt tattgtctgt 3060

aatggaagga ggaaaagcca tctacgagaa gtttttgtcg atgtatcagg tagtatttta 3120

cttcatgatt ttctttctat ccattcattc aactaaacct tttcctacag tcttgtttat 3180

tattttattg aaacaaagat gtaaattatg ccgcaaaatt ttatcatgcg acccccaaaa 3240

caaaaacagt gcaagcagag ataactcaca catgttggtt tatttctaaa ctatggtttc 3300

caggttgtgt atgtcaacac agctattttt actggcagtt tcgcttactg ctccatgttt 3360

aataaataat cttggtgaag tattttggtc aaaaaaattt atggcaaaat aagaaaaaaa 3420

aagtagcatg caatatgcaa agctgtcaag tgtttagcac cttcagtaat tttctgataa 3480

aagatatgga aggttgtttc taaggtagcg tttcaattta caaatttagg attaggatat 3540

gcataggctg catttacatt tagtcaagag taattttagt taactaatct gtaacttatg 3600

tccccaggcg aggacgatcg aagaattgca tttggtcagt tgaaaagatt tgcctggcga 3660

gaattacaac ttgcaaccga tagtttcagt gagaaaaatg tccttggaca ggggggtttc 3720

ggtaaagtgt ataaaggagc acttccagat ggcactaaga ttgctgtaaa gcggttgaca 3780

gattatgaaa gccctggtgg ggaggctgca ttcttgcgtg aagtagagct tatcagtgtt 3840

gcagttcacc ggaatctttt aagattgatt ggcttctgta caacacaaac agaacgtcta 3900

cttgtttatc ctttcatgca gaatcttagt gtggcctatc gtttacgagg tactttctat 3960

aactgatcaa taatttgttt cgttcttcca attttatgct tctactactg tactataaag 4020

tttctcggat acttcactat tgtgcctatg gattatgcaa ctgatacttg tagtgctttt 4080

aatttacgct tgttgggtaa tatggtgcat gtgcactatt gatttcaaca tcgcatatgc 4140

tcactttctt atgtttagat tcaatattgg aagatttcaa tatgtacctc attgtgttta 4200

acgatgcaga attcaaacct ggcgaaccaa tattggattg gtcagcaagg aaacgtgtgg 4260

caataggcac agcacgtgga ctggagtacc tgcacgagca ctgtaatccc aagatcatac 4320

atcgagatgt caaggctgcc aacgtcctgc ttgatgagga ttttgagcca gttgttggtg 4380

actttggcct ggcaaagttg gttgatgtac agaagacatc agtgacaact caagtccgtg 4440

gaactatggg tcacattgcg cctgaatatc tgtccacagg gaagtcatcg gagagaactg 4500

atgtttttgg ttatggcata atgcttcttg agctggtcac tggacaacgc gccatcgact 4560

tctcacggtt ggaggaagaa gacgatgtgc tactgcttga ccatgtgagt ctactcttgc 4620

gaagctggaa caaaattatc ctagcctctg atagccatgt gttgacataa ttgaacgaat 4680

tttttttgaa actaactgtg ccagatagtg cgaaatttca tactgatata gcaagaaaaa 4740

ttataaaaat acagcatgag aggctataat cagcaggaaa agaaaaaaaa caaaataatt 4800

gttgctctca ttgaacatca tattcattct gaagcctgca ccgcttgaat catcagtgtc 4860

atgatcatgc ttgctggttg ctgctgaaaa tgtgcaggtg aagaagctgc agagggaagg 4920

gcagctgggc gccatcgtgg accggaacct gagcagcaac tacgacgggc aggaggtgga 4980

gatgatgata cagatcgcgc tgctgtgcac acaggcgtcg ccggaggacc ggccgtccat 5040

gtcggaggtg gtgcggatgc tggaagggga aggactcgcg gagcggtggg aggagtggca 5100

gcaggtggag gtgacgcggc ggcaggacta cgagcggatg cagcagcgct tcgactgggg 5160

cgaggactcc atcttcaacc aggaagccat cgagctctcc gccggcagat gacgactgcg 5220

tacgtcctga tgcatgtcac ttcccaatcc cagtgttaat agatatacat gtatgtatat 5280

gtatggttgg ctcttagtta ccatagggtt ggttatgtca ctaaagaacc catgtcaagc 5340

ctccatttgt gtagaggaaa acagctagag acgcatgtgt tcagagttca gacttcagaa 5400

gtcccaagtc gcatctgctt ggcgaagccg agccacattt tctaccatca tacgagaaaa 5460

ga 5462

Claims

1. Use of a gene characterized by: for constructing a transgenic plant having an altered root length;

the gene is any one of the following 1) to 3):

1) the nucleotide sequence of the coding region of the gene is shown as SEQ ID NO: 2 is shown in the specification;

2) the nucleotide sequence of the genome of the gene is shown as SEQ ID NO: 3 is shown in the specification;

3) the DNA molecule is hybridized with the DNA sequence defined in 1) or 2) under strict conditions and encodes the same functional protein.

2. Use according to claim 1, characterized in that:

introducing the recombinant expression vector I into a target plant to obtain a transgenic plant with a longer root length;

the recombinant expression vector I is as follows: converting SEQ ID NO: 2 into the multiple cloning site of the plasmid pCAMBIA1300-35S-EGFPOsRARK1(ii) overexpressing the recombinant vector.

3. Use according to claim 1, characterized in that:

introducing the recombinant expression vector II into a target plant to obtain a transgenic plant with shortened root length;

the recombinant expression vector II comprises:

a section of DNA is connected to a transition vector pBSSK-in a forward direction and a reverse direction and then inserted into a plasmid pCAMBIA1300-35S-EGFPOsRARK1Inhibiting the expression of the recombinant vector;

the nucleotide sequence of the DNA is as follows: GCGGTTGACAGATTATGAAAGCCCTGGTGGGGAGGCTGCATTCTTGCGTGAAGTAGAGCTTATCAGTGTTGCAGTTCACCGGAATCTTTTAAGATTGATTGGCTTCTGTACAACACAAACAGAACGTCTACTTGTTTATCCTTTCATG are provided.