Application of SEUSS protein in regulation and control of plant root growth and development

文档序号：501557 发布日期：2021-05-28 浏览：24次中文

阅读说明：本技术 Seuss蛋白在调控植物根系生长发育中的应用 (Application of SEUSS protein in regulation and control of plant root growth and development ) 是由李传友翟华伟张潇月于 2019-11-08 设计创作，主要内容包括：本发明公开了SEUSS蛋白在调控植物根系生长发育中的应用,SEUSS蛋白的氨基酸序列如SEQ ID NO：3所示。实验证明,在拟南芥seu突变体中表达SEUSS基因可以改善根尖干细胞微环境,进而增强根系生长发育和提高产量。SEUSS蛋白可以调控植物根系生长发育、产量和根尖干细胞微环境。本发明具有重要的应用价值。(The invention discloses an application of SEUSS protein in regulating and controlling the growth and development of plant root systems, wherein the amino acid sequence of the SEUSS protein is shown as SEQ ID NO: 3, respectively. Experiments prove that the expression of the SEUSS gene in the Arabidopsis thaliana seu mutant can improve the microenvironment of root tip stem cells, thereby enhancing the growth and development of root systems and increasing the yield. The SEUSS protein can regulate and control the growth and development of plant root systems, the yield and the microenvironment of root tip stem cells. The invention has important application value.)

The application of the SEUSS protein is at least one of S1) -S6):

s1) regulating and controlling the plant root tip stem cell microenvironment;

s2) cultivating transgenic plants with changed root tip stem cell microenvironment;

s3) regulating and controlling the growth and development of plant roots;

s4) cultivating a transgenic plant with changed root growth and development;

s5) regulating plant yield;

s6) breeding transgenic plants with altered yield.

2. The use of claim 1, wherein: the SEUSS protein is a1) or a2) or a 3):

a1) the amino acid sequence is SEQ ID NO: 3;

a2) in SEQ ID NO: 3, the N end or/and the C end of the protein shown in the formula 3 is connected with a label to obtain a fusion protein;

a3) converting SEQ ID NO: 3, and the protein is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the specification, and is related to the microenvironment and/or root growth and development and/or yield of the root tip stem cells of the plants.

3. Use of a nucleic acid molecule encoding the SEUSS protein according to claim 1 or 2) at least one of S1) -S6):

s1) regulating and controlling the plant root tip stem cell microenvironment;

s2) cultivating transgenic plants with changed root tip stem cell microenvironment;

s3) regulating and controlling the growth and development of plant roots;

s4) cultivating a transgenic plant with changed root growth and development;

s5) regulating plant yield;

s6) breeding transgenic plants with altered yield.

4. Use according to claim 3, characterized in that: the nucleic acid molecule for encoding the SEUSS protein is a DNA molecule shown as b1) or b2) or b3) or b4) or b5) or b 6):

b1) the coding region is SEQ ID NO: 1 from the 5' end of the sequence 766-3399 site;

b2) the nucleotide sequence is SEQ ID NO: 1;

b3) the nucleotide sequence is SEQ ID NO: 2;

b4) the nucleotide sequence is SEQ ID NO: 4, or a DNA molecule as shown in the figure;

b5) a DNA molecule having 75% or more identity to the nucleotide sequence defined by b1) or b2) or b3) or b4) and encoding the sesss protein of claim 1 or 2;

b6) a DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b1) or b2) or b3) or b4) and which encodes a sesss protein as claimed in claim 1 or 2.

5. Use according to any one of claims 1 to 4, wherein:

the regulation and control of the plant root tip stem cell microenvironment is to promote the establishment of the plant root tip stem cell microenvironment or inhibit the establishment of the plant root tip stem cell microenvironment;

the cultivation of the transgenic plant with the changed root tip stem cell microenvironment is the cultivation of a transgenic plant for promoting the establishment of the root tip stem cell microenvironment or the cultivation of a transgenic plant for inhibiting the establishment of the root tip stem cell microenvironment;

the regulation and control of the growth and development of the plant root system is to promote the growth and development of the plant root system or slow down the growth and development of the plant root system;

the transgenic plant with the changed growth and development of the root system is cultured with enhanced growth and development of the root system or cultured with slowed growth and development of the root system;

the regulation and control of the plant yield is to improve the plant yield or reduce the plant yield;

the breeding of the transgenic plants with altered yield is breeding of transgenic plants with increased yield or breeding of transgenic plants with decreased yield.

6. Use according to any one of claims 1 to 5, wherein: the plant is any one of the following c1) to c 7): c1) a dicotyledonous plant; c2) a monocot plant; c3) a gramineous plant; c4) a cruciferous plant; c5) arabidopsis thaliana; c6) wild type Arabidopsis thaliana Columbia-0 subtype; c7) an Arabidopsis thaliana seu mutant.

7. A method of breeding a transgenic plant comprising the steps of: increasing the expression level and/or activity of the SEUSS protein of claim 1 or 2 in a starting plant to obtain a transgenic plant; compared with the original plant, the transgenic plant has improved root tip stem cell microenvironment and/or enhanced root growth and development and/or increased yield.

8. The method of claim 7, wherein: the expression level and/or activity of the SEUSS protein in the starting plant is improved by introducing a nucleic acid molecule encoding the SEUSS protein into the starting plant.

9. A method of plant breeding comprising the steps of: increasing the expression level and/or activity of the SEUSS protein of claim 1 or 2 in the starting plant, thereby improving the root tip stem cell microenvironment and/or enhancing root growth and development and/or increasing yield.

10. The method of any of claims 7 to 9, wherein: the starting plant is any one of the following c1) to c 7): c1) a dicotyledonous plant; c2) a monocot plant; c3) a gramineous plant; c4) a cruciferous plant; c5) arabidopsis thaliana; c6) wild type Arabidopsis thaliana Columbia-0 subtype; c7) an Arabidopsis thaliana seu mutant.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of SEUSS protein in regulation and control of plant root system growth and development, in particular to application of SEUSS protein in regulation and control of plant root tip stem cell microenvironment.

Background

The root system is a specific vegetative organ of plants. The plants can not leave the root system for absorbing nutrients and communicating with the signal of the external environment. The normal function of the plant root system provides sufficient guarantee for the development of each organ on the ground. At present, soil and water resources are polluted to different degrees, which poses great threat to the normal growth and function maintenance of plant roots. The contents of minerals and nutrients in soil in different areas are also very different, which causes great limitation to the planting area of crops. The root system of the plant is fixed on the soil, which causes certain difficulty in studying the function of the root system. How to cultivate the plant variety with enhanced root function is a serious challenge for plant scientists at present. How to reasonably utilize a genetic engineering means to analyze the functions of genes related to the influence on root development in plants and apply the related genes to breeding practice is an urgent problem to be solved.

The mature root of the plant body basically consists of four parts, namely a root crown, a meristematic region, an elongation region and a mature region from bottom to top, and each part has a special mission. The root system of the model plant Arabidopsis is simple in structure, and the root tissue is transparent, so that the model plant Arabidopsis is a classic model for researching root development. In the root apical meristematic region, quiescent center of division (QC) cells are the tissue center of root stem cells. QC and Stem cells around the QC form a root tip Stem cell microenvironment (SCN) together, and provide a power source for continuous growth of each tissue of roots. The correct establishment of SCN and maintenance of its activity depends on the properties of QC cells. QC cells maintain stem cell activity by inhibiting self-differentiation through signal communication with surrounding stem cells. At present, the signal exchange mechanism between QC cells and the surrounding is still poorly understood. Therefore, the mutant with root development and QC function defects is screened to further analyze the function of the gene causing the mutation in the process of establishing and maintaining the root stem cell microenvironment, and then the gene is combined with breeding practice by using a genetic engineering means, thereby providing sufficient theoretical support for cultivating the variety with enhanced root system function.

Disclosure of Invention

The invention aims to improve the microenvironment of the stem cells at the root tips of plants, further enhance the growth and development of root systems and increase the yield.

The invention firstly protects the application of the SEUSS protein, which can be at least one of S1) -S6):

s1) regulating and controlling the plant root tip stem cell microenvironment;

s2) cultivating transgenic plants with changed root tip stem cell microenvironment;

s3) regulating and controlling the growth and development of plant roots;

s4) cultivating a transgenic plant with changed root growth and development;

s5) regulating plant yield;

s6) breeding transgenic plants with altered yield.

In the above application, the SEUSS protein may be a1) or a2) or a 3):

a1) the amino acid sequence is SEQ ID NO: 3;

a2) in SEQ ID NO: 3, the N end or/and the C end of the protein shown in the formula 3 is connected with a label to obtain a fusion protein;

Wherein, SEQ ID NO: 3 consists of 877 amino acid residues.

To facilitate purification of the protein in a1), the protein of SEQ ID NO: 3 to the amino terminus or carboxy terminus of the protein shown in table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

The protein according to a3), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein of a3) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a3) above can be obtained by converting the amino acid sequence of SEQ ID NO: 1 at position 766-3399 from the 5 ' end, and/or by carrying out missense mutation of one or more base pairs and/or by linking the coding sequence of the tag shown in table 1 at the 5 ' end and/or 3 ' end.

The invention also protects the application of the nucleic acid molecule for encoding the SEUSS protein, which can be at least one of S1) -S6):

s1) regulating and controlling the plant root tip stem cell microenvironment;

s2) cultivating transgenic plants with changed root tip stem cell microenvironment;

s3) regulating and controlling the growth and development of plant roots;

s4) cultivating a transgenic plant with changed root growth and development;

s5) regulating plant yield;

s6) breeding transgenic plants with altered yield.

In the above application, the nucleic acid molecule encoding the SEUSS protein can be a DNA molecule shown as b1) or b2) or b3) or b4) or b5) or b 6):

b1) the coding region is SEQ ID NO: 1 from the 5' end of the sequence 766-3399 site;

b2) the nucleotide sequence is SEQ ID NO: 1;

b3) the nucleotide sequence is SEQ ID NO: 2;

b4) the nucleotide sequence is SEQ ID NO: 4, or a DNA molecule as shown in the figure;

b5) a DNA molecule having 75% or more 75% identity to the nucleotide sequence defined by b1) or b2) or b3) or b4) and encoding said sesss protein;

b6) a DNA molecule which hybridizes with the nucleotide sequence defined by b1) or b2) or b3) or b4) under strict conditions and codes the SEUSS protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, SEQ ID NO: 1 consists of 3852 nucleotides, SEQ ID NO: 2 consists of 5062 nucleotides, SEQ ID NO: 4 consists of 5551 nucleotides, SEQ ID NO: 1 from the 5' end, the 766-3399 position of the DNA molecule encodes the amino acid sequence of SEQ ID NO: 3.

The nucleotide sequence of the invention encoding the SEUSS protein can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the SEUSS protein isolated according to the present invention, as long as they encode the SEUSS protein, are derived from and identical to the nucleotide sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes the identity to the nucleotide sequence of the present invention encoding SEQ ID NO: 3, or 80% or more, or 85% or more, or 90% or more, or 95% or more, of the nucleotide sequence of the sesss protein. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In any of the above applications, the regulating the growth of the plant root system may be promoting the growth of the plant root system or slowing the growth of the plant root system.

In any of the above applications, the controlling plant yield may be increasing plant yield or decreasing plant yield.

In any of the above applications, the regulation and control of the plant root tip stem cell microenvironment can be the establishment of a plant root tip stem cell microenvironment or the establishment of a plant root tip stem cell microenvironment inhibition.

In any of the above applications, the cultivation of the transgenic plant with the altered growth of the root system may be the cultivation of a transgenic plant with enhanced growth of the root system or the cultivation of a transgenic plant with slowed growth of the root system.

In any of the above applications, the breeding of the transgenic plants with altered yield can be breeding of transgenic plants with increased yield or breeding of transgenic plants with decreased yield.

In any of the above applications, the cultivation of the transgenic plant with the altered root tip stem cell microenvironment may be cultivation of a transgenic plant promoting establishment of the root tip stem cell microenvironment or cultivation of a transgenic plant inhibiting establishment of the root tip stem cell microenvironment.

In the use of any of the above, the plant may be any of the following c1) to c 7): c1) a dicotyledonous plant; c2) a monocot plant; c3) a gramineous plant; c4) a cruciferous plant; c5) arabidopsis thaliana; c6) wild type Arabidopsis thaliana Columbia-0 subtype; c7) an Arabidopsis thaliana seu mutant.

The invention also provides a method for cultivating transgenic plants, which comprises the following steps: improving the expression quantity and/or activity of the SEUSS protein in the starting plant to obtain a transgenic plant; compared with the original plant, the transgenic plant has improved root tip stem cell microenvironment and/or enhanced root growth and development and/or increased yield.

In the above method, the "increasing the expression level and/or activity of the SEUSS protein in the starting plant" may be achieved by a method known in the art, such as transgenosis, multicopy, and alteration of a promoter and a regulatory factor.

In the above method, the "increasing the expression level and/or activity of a SEUSS protein in a starting plant" may be carried out by introducing a nucleic acid molecule encoding the SEUSS protein into the starting plant. The nucleic acid molecule encoding the SEUSS protein can be a DNA molecule shown as b1) or b2) or b3) or b4) or b5) or b 6):

b1) the coding region is SEQ ID NO: 1 from the 5' end of the sequence 766-3399 site;

b2) the nucleotide sequence is SEQ ID NO: 1;

b3) the nucleotide sequence is SEQ ID NO: 2;

b4) the nucleotide sequence is SEQ ID NO: 4, or a DNA molecule as shown in the figure;

b5) a DNA molecule having 75% or more 75% identity to the nucleotide sequence defined by b1) or b2) or b3) or b4) and encoding said sesss protein;

b6) a DNA molecule which hybridizes with the nucleotide sequence defined by b1) or b2) or b3) or b4) under strict conditions and codes the SEUSS protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In any of the above methods, said "introducing into the starting plant a nucleic acid molecule encoding said SEUSS protein" may be effected by introducing into the starting plant a recombinant vector; the recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the SEUSS protein into an expression vector.

The recombinant vector can be specifically a recombinant plasmid pCAMBIA 1300-SEUSS. The recombinant plasmid pCAMBIA1300-SEUSS can be specifically prepared by replacing a small DNA fragment between recognition sequences of restriction enzymes SmaI and SalI of a vector pCAMBIA1300 by SEQ ID NO: 4 to obtain the recombinant plasmid.

The transgenic plants may be in particular SEUS #1, SEUS #2 and SEUS #3 as mentioned in example 2. In this case, the starting plant is Arabidopsis thaliana, specifically, an Arabidopsis thaliana seu mutant.

The invention also provides a plant breeding method, which comprises the following steps: increasing the expression level and/or activity of the SEUSS protein in the starting plant, thereby improving the microenvironment of the root tip stem cells and/or enhancing the growth and development of root systems and/or increasing the yield.

In any of the above methods, the starting plant may be any of the following c1) to c 7): c1) a dicotyledonous plant; c2) a monocot plant; c3) a gramineous plant; c4) a cruciferous plant; c5) arabidopsis thaliana; c6) wild type Arabidopsis thaliana Columbia-0 subtype; c7) an Arabidopsis thaliana seu mutant.

Any of the above arabidopsis seu mutants may be an arabidopsis with a loss of function of the sesss protein. In the present invention, the arabidopsis seu mutant is described in the following documents: pfleger, J., and Zambryski, P. (2004), The roll of SEUSS in auxin response and flow organic patterning development 131, 4697-. Specifically, the Arabidopsis thaliana seu mutant has 1 nucleotide substitution in the 1 st exon of the SEUSS gene (namely, the 1263 th position C of SEQ ID NO: 2 from the 5' end is mutated into T, thereby causing frame shift and early termination, and causing the loss of the function of the SEUSS protein.

Any one of the root tip stem cell microenvironments is formed by QC cells and peripheral stem cells, and can provide a power source for continuous growth of each tissue of the root system. Establishment of root tip stem cell microenvironment and maintenance of its activity depend on the characteristics of QC cells. QC cells are the tissue center of root tip stem cells, which are located in the root tip meristematic region and are inactive in division. The good root tip stem cell microenvironment is mainly expressed in the normal division of QC cells (see wild type Arabidopsis in FIG. 2), and the division limit of the QC cells is uniform; the poor root tip stem cell microenvironment (or disturbed root tip stem cell microenvironment) is mainly manifested by abnormal division of QC cells (see seu mutant in fig. 2), which is disorganized. Therefore, herein, promoting establishment of or improving the root tip stem cell microenvironment mainly refers to normal division of QC cells; the inhibition of the establishment of the plant root tip stem cell microenvironment refers to abnormal division of QC cells. Whether normal or abnormal division is based on the division of QC cells in wild plants.

Experiments prove that the expression of the SEUSS gene in the Arabidopsis thaliana seu mutant can improve the microenvironment of root tip stem cells, thereby enhancing the growth and development of root systems and increasing the yield. The SEUSS protein can regulate and control the growth and development of plant root systems, the yield and the microenvironment of root tip stem cells. The invention has important application value.

Drawings

FIG. 1 shows the growth status of seedlings of wild type Arabidopsis thaliana and of the seu mutant.

FIG. 2 is a root tip stem cell microenvironment of wild type Arabidopsis and the seu mutant.

FIG. 3 is an expression analysis of the core transcription factor WOX5 in the seu mutant.

FIG. 4 shows the effect of SEUSS transgenic Arabidopsis on the microenvironment of root tip stem cells.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The genome sequence of the SEUSS gene is shown as SEQ ID NO: 2, respectively. The nucleotide sequence of mRNA of SEUSS gene is shown as SEQ ID NO: 1 is shown. The nucleotide sequence of the SEUSS gene coding region is shown as SEQ ID NO: 1 from the 5' end by position 766-3399, encoding the amino acid sequence of SEQ ID NO: 3, or a protein of sesss.

Wild type Arabidopsis thaliana (Arabidopsis thaliana) (Columbia-0 subtype) is described in the following references: kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J.A genetic link between colored responses and flowing time through FVE in Arabidopsis thaliana. Nature genetics.2004,36: 167-.

The seu mutants are described in the following documents: pfleger, J., and Zambryski, P. (2004). The role of SEUSS in auxin response and flow organic patterning. development 131,4697-4707. specifically, The seu mutant has a 1-nucleotide substitution in exon 1 of The SEUSS gene (i.e., SEQ ID NO: 2 has a 1263-position C to T mutation from The 5' end, resulting in a frameshift and premature termination, resulting in loss of function of The SEUSS protein.

The culture conditions of Arabidopsis thaliana were: 22 ℃; 16h of light/8 h of dark; the light intensity was 12000 Lx.

Example 1 phenotype of the seu mutant and expression analysis of its core transcription factor WOX5

Phenotypes of the first, second mutant

(1) Treating Arabidopsis seeds to be tested with 70% (v/v) ethanol aqueous solution for 5min, sterilizing with 2.6% (v/v) sodium hypochlorite aqueous solution for 10min, and washing with sterilized spitting water for 5 times.

(2) After the step (1) is completed, the arabidopsis thaliana seeds to be tested are sown in a solid culture medium (MS solid culture medium or MS solid culture medium containing 20 mu M Jasmonic Acid (JA)), and vernalization is carried out for 3 days at 4 ℃.

(3) After the completion of step (2), the solid medium was cultured vertically for 6 days, and the growth state of Arabidopsis seedlings was observed.

The growth state of Arabidopsis seedlings is shown in FIG. 1(Col-0 is wild type Arabidopsis, seu is seu mutant, Control is MS solid medium, 20. mu.MJA-containing MS solid medium). The results show that compared with wild arabidopsis, the seu mutant has slow root growth, loss of geotropism, different main roots and hypersensitiveness to jasmonic acid.

(4) And (4) after the step (3) is finished, taking the root tip of the arabidopsis seedling cultured on the MS solid medium, and observing by using a laser confocal microscope (Zeiss LSM 710).

The results are shown in FIG. 2(WT is wild type Arabidopsis thaliana, and seu is a mutant of seu). The result shows that the seu mutant has a disturbed microenvironment compared with the root tip stem cell of the wild arabidopsis, and mainly shows that the QC cell is abnormally divided, and the root cap stem cell has starch granule accumulation.

Expression analysis of core transcription factor WOX5 in second and second mutant

The core transcription factor WOX5(Gene ID: 820297) is a Gene specifically expressed in QC cells, the function of which is crucial to the establishment of QC cells and the maintenance of root stem cell microenvironment, and is a common marker Gene for researching root development. Relevant studies are described in the following documents: sarkar, a.k., Luijten, m., Miyashima, s., Lenhard, m., Hashimoto, t., Nakajima, k., Scheres, b., Heidstra, r., and Laux, t. (2007) transmitted vectors guiding in Arabidopsis thaliana shaft and root cell organization 446. nature, 811-.

1. Real-time fluorescent quantitative detection of relative expression level of WOX5 gene in seu mutant

(1) And (3) taking seeds of the arabidopsis (wild type arabidopsis or seu mutant) to be detected, and culturing for 5 days to obtain the arabidopsis seedlings to be detected.

(2) And (3) after the step (1) is finished, cutting off the root tip of the arabidopsis seedling to be detected, and quickly freezing by using liquid nitrogen to obtain the root tip of the arabidopsis to be detected.

(3) And (3) after the step (2) is finished, extracting the total RNA of the root tip of the arabidopsis to be detected by adopting a Trizo1 method, and performing reverse transcription to obtain the cDNA of the arabidopsis to be detected.

(4) And (3) detecting the relative expression level of the WOX5 gene (an Actin7 gene is an internal reference gene) by taking the cDNA of the arabidopsis thaliana to be detected as a template and carrying out real-time quantitative PCR (polymerase chain reaction). The Ct value is the number of cycles that the fluorescence signal in the reaction system has undergone to reach a set threshold value. Δ Ct ═ Ct_WOX5-Ct_ActinWOX5 gene expression level was measured as a 2- Δ Ct value.

The reaction system was 20. mu.L, prepared from 10. mu.L of LSYBR Premix Ex Taq (2X), 0.5. mu.L of an aqueous solution of the forward primer (10. mu.M in concentration)0.5. mu.L of downstream primer aqueous solution (concentration is 10. mu.M), 0.5. mu.L of cDNA of Arabidopsis thaliana to be tested and 8.5. mu.L of ddH₂And (C) O.

SYBR Premix Ex Taq (2X) is a product of TAKARA.

The reaction procedure is as follows: 30s at 95 ℃; 95 ℃ for 10s, 60 ℃ for 20s, 72 ℃ for 20s, 45 cycles.

Primers for detecting WOX5 gene were 5'-GATTGTCAAGAGGAAGAGAAGGTGA-3' and 5'-AGCTTAATCGAAGATCTAATGGCG-3'.

The primers for detecting the Actin7 gene are 5'-GAGGAAGAGCATTCCCCTCGTA-3' and 5'-GAGGATAGCATGTGGAACTGAGAA-3'.

The results are shown in A in FIG. 3 (WT is wild type Arabidopsis thaliana, and seu is mutant of seu). The results show that the relative expression level of WOX5 gene in the root tip of the seu mutant is significantly reduced compared to wild type arabidopsis thaliana.

The experiments were repeated 3 times, and the results were substantially consistent.

2. Observation of pWOX5 expression of GFP marker line in seu mutant

pWOX5 GFP is described in the following references: blilou, I.A., Xu, J.A., Wildwater, M.A., Willemsen, V.A., Paponov, I.A., Friml, J.A., Heidstra, R.A., Aida, M.A., Palme, K.A., and Scheres, B.A. (2005) The PIN auxin efflux reactor network controls and patterning in Arabidopsis roots 433: 39-44. hereinafter, pWOX5: GFP is named pWOX5: GFP marker system.

(1) pWOX5, a GFP-tagged line, was hybridized with the seu mutant to give generation F1.

(2) And (3) planting F1 generations after the step (1) is completed to obtain F2 generation segregating population.

(3) And (3) after the step (2) is finished, observing the F2 generation segregation population under a fluorescence microscope, selecting plants capable of emitting fluorescence for enzyme digestion identification, and obtaining plants with the background of wild type arabidopsis thaliana and plants with the background of the seu mutant.

The enzyme digestion identification steps are as follows:

(3-1) respectively extracting genome DNA of plants capable of emitting fluorescence, and carrying out PCR amplification by using the genome DNA as a template and adopting a primer pair consisting of 5'-GAAAATGTTCCGCCTTCGAT-3' and 5'-GAATTTGCTGCGGTTCCAACT-3' to obtain a PCR amplification product of about 580 bp.

(3-2) the PCR amplification product obtained in the step (3-1) is digested with restriction enzyme BslI, and then subjected to agarose gel electrophoresis, and the following judgment is made:

if the enzyme digestion product only contains one strip and the size is 580bp, the corresponding plant capable of emitting fluorescence is the plant with the background of the seu mutant;

if the enzyme digestion product only contains two bands and the sizes of the two bands are 340bp and 240bp respectively, the corresponding plant capable of emitting fluorescence is a plant with the background of wild type arabidopsis;

if the enzyme digestion product only contains three bands and the sizes are respectively 340bp, 240bp and 580bp, the corresponding plant capable of emitting fluorescence is a heterozygote.

(4) And (3) harvesting seeds of a plant with the background of the wild type Arabidopsis and a plant with the background of the seu mutant, and sequentially obtaining seeds of the wild type F2 generation positive Arabidopsis and seeds of the mutant F2 generation positive Arabidopsis.

(5) And (4) planting the wild type F2 generation positive Arabidopsis seeds of different lines obtained in the step (4), observing under a fluorescence microscope, and if the seeds can emit fluorescence, determining the corresponding wild type-F2 generation positive seedlings as wild type-F2 generation homozygous positive seedlings. And (4) planting the positive arabidopsis seeds of the mutant F2 generation of different strains obtained in the step (4), observing under a fluorescence microscope, and if the seeds can emit fluorescence, determining that the corresponding positive seedling of the mutant-F2 generation is the homozygous positive seedling of the mutant-F2 generation.

(6) And respectively taking the root tips of the mutant-F2 generation homozygous positive seedlings and the wild type-F2 generation homozygous positive seedlings which grow for 5 days, and observing the root tips under a laser confocal microscope.

The detection results are shown in B in FIG. 3 (WT is a wild-type-F2 generation homozygous positive seedling, and seu is a mutant-F2 generation homozygous positive seedling). The result shows that compared with the wild type-F2 generation homozygous positive seedling, the fluorescence expression level of the root tip of the mutant-F2 generation homozygous positive seedling is obviously reduced.

Example 2 obtaining of transgenic SEUSS Arabidopsis thaliana and its influence on microenvironment of root tip Stem cells

Construction of recombinant plasmid pCAMBIA1300-SEUSS

1. Taking the genome DNA of a wild type arabidopsis seedling as a template, and adopting a primer 1: 5' -ACGCGTCGACTATTCAGCAATGGTTCAT-3' (underlined recognition site for the restriction enzyme SalI) and primer 2: 5' -TCCCCCGGGATCGCGTTCCAATCAAAATTGTTG-3' (recognition sites for the restriction enzyme SmaI are underlined) was subjected to PCR amplification, and a DNA fragment of about 5551bp was recovered.

2. The vector pCAMBIA1300 was digested with restriction enzymes SmaI and SalI, and the vector backbone of about 9kb was recovered.

3. The DNA fragment recovered in step 1 was digested with restriction enzymes SmaI and SalI, and the digested product was recovered.

4. And connecting the enzyme digestion product with a vector skeleton to obtain the recombinant plasmid pCAMBIA 1300-SEUSS.

The recombinant plasmid pCAMBIA1300-SEUSS was sequenced. The sequencing result shows that the recombinant plasmid pCAMBIA1300-SEUSS is a plasmid obtained by replacing a small DNA fragment between recognition sequences of restriction enzymes SmaI and SalI of the vector pCAMBIA1300 by SEQ ID NO: 4 to obtain the recombinant plasmid. SEQ ID NO: 4, from the 5' end, positions 1 to 943 are the promoter of the SEUSS gene.

The recombinant plasmid pCAMBIA1300-SEUSS expresses SEQ ID NO: 3, or a protein of sesss.

II, acquisition of GV3101/pCAMBIA1300-SEUSS

The recombinant plasmid pCAMBIA1300-SEUSS is introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium, which is named as GV3101/pCAMBIA 1300-SEUSS.

Thirdly, obtaining of SEUSS transgenic arabidopsis

1. GV3101/pCAMBIA1300-SEUSS was transformed into the seu mutant by the Arabidopsis inflorescence floral dip transformation method (described in Clough, S.J., and Bent, A.F. Floraldip: expressed method for Agrobacterium-mediated transformation of Arabidopsis thaliana plant J. (1998)16, 735-743.), obtaining T3101/pCAMBIA 1300-SEUSS₁Transgenic SEUSS gene Arabidopsis seeds.

2. Will T₁Transfer SThe EUSS gene Arabidopsis seeds are sown on an MS solid culture medium containing 50mg/L hygromycin, and the Arabidopsis capable of normally growing (resistant seedlings) is T₁Transgenic SEUSS gene positive seedling, T₁The seeds received by the SEUSS gene-transferred positive seedlings are T₂Transgenic SEUSS gene Arabidopsis seeds.

3. T of different strains₂Transgenic SEUSS gene Arabidopsis seeds are sown on a solid culture medium containing 50mg/L hygromycin MS for screening, and if the ratio of the number of Arabidopsis capable of normally growing (resistant seedlings) to the number of Arabidopsis incapable of normally growing (non-resistant seedlings) in a certain strain is 3: 1, the strain is a strain with SEUSS gene inserted into one copy, and the seeds received by the resistant seedlings in the strain are T₃Transgenic SEUSS gene Arabidopsis seeds.

4. Will T₃The transgenic SEUSS gene Arabidopsis seeds are sown on an MS solid culture medium containing 50mg/L hygromycin again for screening, and the transgenic SEUSS gene Arabidopsis seeds are resistant seedlings, namely T₃The transgenic SEUSS gene Arabidopsis thaliana is homozygous for the generation. 3 of them are T₃The generation-homozygous SEUSS gene-transferred Arabidopsis strains are named SEUS #1, SEUS #2 and SEUS #3 respectively, and subsequent experiments are carried out.

Fourth, molecular identification

The Arabidopsis seed to be tested is T of SEUS #1₃T of seed generation, SEUS #2₃Seed generation, SEUS # 3T₃Generation seed or wild type arabidopsis seed.

1. Taking an arabidopsis seed to be detected, soaking the arabidopsis seed in 70% (v/v) ethanol water solution for 30s, and washing the arabidopsis seed with sterile water for 3 times; then spread on MS solid medium and vernalize at 4 ℃ for 3 days.

2. And (3) after the step 1 is finished, taking the to-be-detected arabidopsis seeds, and culturing for 7 days to obtain to-be-detected arabidopsis seedlings.

3. Extracting genome DNA of the arabidopsis seedling to be detected, taking the genome DNA as a template, and performing PCR amplification by adopting a primer pair consisting of 5'-GGTAAAGGAGAAGAACTTTTCA-3' and 5'-GAATTCTTATTTGTATAGTTCA-3' to obtain a PCR amplification product; then, the following judgment is made: if some PCR amplification product contains DNA segment of about 720bp, the arabidopsis seedling to be detected corresponding to the PCR amplification product is a positive seedling.

The results show that T of SEUS #1₃T of seed generation, SEUS #2₃Generation of seed and T of SEUS #3₃The seedlings obtained by the generation of seeds are all positive seedlings.

Influence of microenvironment of root tip stem cells

The Arabidopsis seed to be tested is T of SEUS #1₃T of seed generation, SEUS #2₃Seed generation, SEUS # 3T₃Seed generations, wild type arabidopsis seeds or seu mutant seeds.

2. And (3) after the step 1 is finished, taking the to-be-detected arabidopsis seeds, and culturing for 5 days to obtain to-be-detected arabidopsis seedlings.

3. And (3) after the step (2) is finished, observing the root tip of the arabidopsis thaliana seedling to be detected under a laser confocal microscope.

The results of the partial assays are shown in FIG. 4(WT is wild type Arabidopsis thaliana, seu is mutant, pCAMBIA1300-SEUSSseu is SEUS # 1). The results show that the microenvironment of the root tip stem cells of the seu mutant is disordered, and the microenvironment of the root tip stem cells of the seu #1, the seu #2, the seu #3 and the wild type arabidopsis thaliana are normal. Therefore, the introduction of the SEUSS gene into the seu mutant can restore the microenvironment of the root tip stem cells from disorder to normal.

The results show that the SEUSS protein can promote the establishment and maintenance of the microenvironment of the stem cells at the root tips of the plants, further promote the growth and development of root systems and improve the yield of the plants.

<110> institute of genetics and developmental biology of Chinese academy of sciences

Application of SEUSS protein in regulation and control of plant root growth and development

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<170> PatentIn version 3.5

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<211> 3852

<212> DNA

<213> Arabidopsis thaliana

<400> 1

gattgtgatt acaaaaaaaa aacatttcta gggcatgtca ttgacttcct ttgaagaaga 60

agaagaagag agagagagac tgaaaaagaa aaagtaagaa agggaatgaa ataaatccga 120

tcgaatacaa ttttgacgtc cgcaaggaca ctattctctc tcttctcatc ttcttctttc 180

tctcttctct ctctctctct acagcgaaga acaggacaca aattactctg tgaagcttaa 240

aaaggtataa tttttaataa aacccagaaa ctacaatttc taggcggcag agaaaatttg 300

tttctacttc tttttgtttc tctttgtctt ctcgtctctt tatccccaaa cccatcaata 360

aaaaacccta aaagacttca aatttcttca agattttgtt catcggaagt tttcagattt 420

ttagttgtgt aaagtctctt cctttttgtt cgattctctt tgttttgagt cttatcagag 480

acttattctt caattccacc tctgggtggt gagagttgga aatggagtag atttgcttaa 540

tttagatctg taagtaatca acagacagtg ttgtttggtt ctagaatgtt cgatacggaa 600

tctcatcact cagattcatc tctttccgtg gtgtttacga ctctgaagct tatcattgct 660

cagaaagaat acgtctttgc tcctacacag agctgagttt tataaagtcg tcagttttgt 720

ttcttgcttt ctttgttgat tgtggagttg tgaattagta agagtatggt accatcagag 780

ccgcctaatc ctgttggggg tggtgaaaat gttccgcctt cgatattagg aggacaagga 840

ggagctcctc ttccttctca accagcattt ccttcacttg tctctccgcg tactcagttt 900

ggtaacaata tgagtatgag tatgcttggg aatgctccaa atatatcttc tcttctcaat 960

aatcagtctt ttgtaaatgg tatccctggt tctatgattt ctatggatac aagtggtgct 1020

gagtctgacc cgatgtctaa cgtcgggttt agtggtttgt cgtcttttaa tgcgtcgagt 1080

atggtgtctc cgcgctcatc aggtcaagtt cagggtcagc agttttcgaa tgtttcggct 1140

aaccagttgt tggctgagca acaacggaat aagaaaatgg agacgcagag ttttcaacat 1200

ggtcagcagc agtcaatgca gcagcagttt tcgacagtgc gtggtggtgg attagcaggt 1260

gtgggacctg ttaagatgga gcctggtcag gtttcgaatg atcagcagca tggtcaagta 1320

caacagcagc aacagaaaat gttgagaaac ctagggtcag ttaagttgga accgcagcaa 1380

attcaggcca tgagaaattt ggcccaagtg aaaatggaac ctcaacattc tgagcagtca 1440

ttgtttctcc aacaacagca gaggcaacaa cagcaacaac aacaacaaca atttcttcaa 1500

atgccggggc aatctccgca ggctcaaatg aacatatttc agcagcagag acttatgcaa 1560

cttcaacaac agcaactctt gaaatctatg cctcaacagc gtcctcaatt accacagcag 1620

ttccaacagc agaatttacc tctaaggcca cctctgaaac cagtgtatga acctggcatg 1680

ggtgctcagc gtcttacaca gtatatgtac agacaacaac ataggcctga agacaataat 1740

atcgagttct ggagaaaatt tgtagctgag tactttgctc ctaatgccaa aaagagatgg 1800

tgcgtctcta tgtatggcag tggcaggcaa acaacaggcg ttttccctca ggatgtgtgg 1860

cactgtgaga tatgtaaccg aaagcctgga cgtggttttg aggcaaccgc cgaagtcctt 1920

ccgcggctgt ttaagattaa gtatgagagt gggacgttag aagaactgtt atatgtagat 1980

atgccaaggg agtcccagaa ttcatctggc caaattgtcc tggagtatgc aaaagcaaca 2040

caagagagtg tctttgagca tcttcgggtt gttcgtgatg gccaacttcg aatagtcttc 2100

tcgccagatc ttaagatatt ctcctgggaa ttttgtgctc ggcggcatga agagcttatt 2160

ccacgaagac ttttgatacc gcaggttagt cagcttggat cggcagctca gaaatatcaa 2220

caagctgctc aaaatgcaac aacagattct gctcttccgg agctacaaaa taattgcaat 2280

atgtttgttg catctgctag acaattggca aaggccctgg aagtaccact tgtgaatgat 2340

ttgggataca caaagagata tgttcggtgt ttgcagatct cagaggtggt aaatagtatg 2400

aaggacctga tagattatag cagagaaaca cgaacaggac caatcgagag tttagccaag 2460

tttcctcgga gaacaggccc ttcatctgca ctgcctggtc cttctcctca gcaagccagc 2520

gaccagctta ggcagcagca gcaacaacaa cagcagcagc agcaacagca acaacaacaa 2580

caacaacaac agcagcagca gcaaacagtt tcccagaata caaacagtga tcaaagtagt 2640

aggcaagttg cactaatgca gggtaatcca agcaatggtg taaattatgc ctttaatgca 2700

gcctctgcat ccacttccac cagcagcatc gcagggctca tccaccagaa ttcaatgaag 2760

ggaagacatc agaatgctgc ttacaatcct ccaaacagcc cctatggagg aaactctgtt 2820

cagatgcaat cacctagttc ctcgggtacc atggtgccat catcatcgca gcaacaacac 2880

aacctgccaa catttcagtc tccaacatcc tcatctaata acaataatcc ctctcaaaac 2940

gggataccat ctgttaatca catgggttcc acaaactcac cagcaatgca acaggcaggt 3000

gaggttgatg gaaacgagtc tagctcggtg cagaagatac tgaatgaaat cctgatgaac 3060

aaccaagctc ataataatag ctcaggagga agcatggttg ggcatgggtc tttcgggaat 3120

gatgggaagg gtcaagctaa tgtaaatagt tctggtgttt tactgatgaa tggccaagtg 3180

aacaacaaca acaacacaaa tattggaggt gctggtgggt ttggtggtgg gattggtcaa 3240

tccatggcag caaacggaat caataatata aacggtaaca atagtctcat gaacggaaga 3300

gttgggatga tggtgcggga tccaaacggt caacaggatt taggaaacca acttttagga 3360

gcagtgaatg gtttcaacaa ttttgattgg aacgcgtgaa atgaagaaga ggaagaagag 3420

acactgaaag ggtaatgttc tgcttgcgca gcatcacaca tcttcaaact caggataagg 3480

ataggccaat aagccgtgat attgtgctta agaaaggaag agtcaaatct ttgtatcaat 3540

aactggtttc taggtcacct ccacaatcac caataccatc tcttgagtta ctgtttcttt 3600

ttttactttg gggagttttt cttatgcttt atagtttata atgtacatct ttctatcggt 3660

gtgtgttcct tagggcacat catcagggag gcaatgaatc aagctctatc cttcatttat 3720

ctctctggtc tcttccatgt gttaaccaaa cttccctaaa ttctgaccac aacgttaaat 3780

atgtgcaatg ctcttgaacg atgctgatcg gtcatgattt tttgttgtta gtgcaatgaa 3840

ataccacaag cc 3852

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<211> 5062

<212> DNA

<213> Arabidopsis thaliana

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