CN111233990B - Ginseng CLE family polypeptide and application thereof in plant root growth regulation - Google Patents
Ginseng CLE family polypeptide and application thereof in plant root growth regulation Download PDFInfo
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Abstract
The invention discloses a ginseng CLE family polypeptide and application thereof in plant root growth regulation. The polypeptide provided by the invention: (a) PgCLE45 polypeptide: as shown in sequence 1 in the sequence table; (b) PgCLE12 polypeptide: as shown in sequence 2 of the sequence table; (c) PgCLE25 polypeptide: as shown in sequence 3 of the sequence table; (d) PgCLE27 polypeptide: as shown in sequence 4 of the sequence table. The invention also protects the application of any one of the polypeptides in inhibiting the proliferation of root tip cells of a plant, promoting the differentiation of roots of the plant, reducing the length of a root meristematic region of the plant, reducing the root length of the plant and reducing the root branch number of the plant. The ginseng CLE polypeptide is used as a plant polypeptide hormone, and has good research potential and wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a ginseng CLE family polypeptide and application thereof in plant root growth and development regulation.
Background
CLE (CLAVATA3/Embryo surrouding region-related) is the hottest plant polypeptide studied in the last decade, and is the largest plant polypeptide molecule family so far, and mainly participates in intercellular signal transduction by encoding a small segment of secreted protein, thereby maintaining the balance between proliferation and differentiation of stem cells in the process of plant growth and development. To date, CLE polypeptides have been found to play a key role in seed development, vascular bundle formation, lateral root growth, the balance between stem cell division and differentiation in the shoot apical and root apical meristems, and the like. CLE polypeptide has more in-depth research in arabidopsis and rice, while research on CLE polypeptide hormone in medicinal plants is still blank.
Ginseng is the dried root and rhizome of Araliaceae perennial herbaceous plant ginseng Panax ginseng C.A.Mey. is covered with the reputation of "the king of Baicao" and is one of the Chinese medicinal material varieties with the largest export amount in China. Because ginseng has rich root types and the market price difference of different root types is very large, the research on the growth and development of ginseng is very important. Because CLE polypeptide has important regulation and control function on root growth and development, promotes stem cell differentiation and tissue organ formation, the research on CLE in the ginseng root is helpful for explaining the growth and development of the ginseng root and provides guidance for regulating and controlling different root types. However, no report on the gene and polypeptide of the ginseng CLE exists so far, and no possible regulation effect of the polypeptide of the ginseng CLE family on plants is seen.
Disclosure of Invention
The invention aims to provide a ginseng CLE family polypeptide and application thereof in plant root growth and development regulation.
The polypeptide provided by the invention is (a), (b), (c) or (d) as follows:
(a) PgCLE45 polypeptide: as shown in sequence 1 of the sequence table (amino acid sequence: RRVRRGSDPIHN);
(b) PgCLE12 polypeptide: as shown in sequence 2 of the sequence table (amino acid sequence RLVPTGPNPLHH);
(c) PgCLE25 polypeptide: as shown in sequence 3 of the sequence table (amino acid sequence RRVPNGPDPIHN);
(d) PgCLE27 polypeptide: as shown in sequence 4 of the sequence table (amino acid sequence RRVPSCPDPLHN).
The invention also protects the application of any one of the polypeptides in inhibiting the proliferation of root tip cells of plants.
The root tip cell is an adventitious root tip cell.
The invention also protects the application of any one of the polypeptides in promoting the root differentiation of plants.
Differentiation of the root into a primary root differentiation.
The invention also protects the use of any of the above polypeptides to reduce the length of the root meristematic region of a plant.
The root meristematic region is a primary root meristematic region.
The invention also protects the application of any one of the polypeptides in reducing the root length of plants.
The root length is the primary root length.
The invention also protects the application of any one of the polypeptides in reducing the root branch number of plants.
The root branch number is the primary root branch number.
The invention also protects the application of any polypeptide in the preparation of products; the function of the product is as follows (e1) and/or (e2) and/or (e3) and/or (e4) and/or (e 5):
(e1) inhibiting proliferation of root tip cells of a plant;
(e2) promoting root differentiation of plants;
(e3) reducing the length of the root meristematic region of the plant;
(e4) reducing root length of the plant;
(e5) reducing the root branch number of the plant.
The root tip cell is an adventitious root tip cell.
The root meristematic region is a primary root meristematic region.
Differentiation of the root into a primary root differentiation.
The root length is the primary root length.
The root branch number is the primary root branch number.
The invention also provides a product comprising any one of the polypeptides described above; the function of the product is as follows (e1) and/or (e2) and/or (e3) and/or (e4) and/or (e 5):
(e1) inhibiting proliferation of root tip cells of a plant;
(e2) promoting root differentiation of plants;
(e3) reducing the length of the root meristematic region of the plant;
(e4) reducing root length of the plant;
(e5) reducing the root branch number of the plant.
The root tip cell is an adventitious root tip cell.
The root meristematic region is a primary root meristematic region.
Differentiation of the root into a primary root differentiation.
The root length is the primary root length.
The root branch number is the primary root branch number.
Any of the above plants is a dicot.
The dicotyledonous plant is a ginseng plant or an arabidopsis plant.
The Panax plant can be artificially planted Ginseng radix.
The Panax plant can be radix Ginseng of Ardisia gigantea.
The arabidopsis plant may specifically be arabidopsis thaliana, e.g. colombian ecotype arabidopsis thaliana.
The invention discloses 4 ginseng CLE family functional polypeptides. The regulation and control application of the 4 ginseng CLE polypeptides in the growth and development of plant roots has the effects of inhibiting the growth of primary roots and promoting the differentiation of the primary roots. The invention fills the application blank of CLE polypeptide in medicinal plants. The ginseng CLE polypeptide is used as a plant polypeptide hormone, and has good research potential and wide application prospect.
Drawings
FIG. 1 shows the results of example 2.
FIG. 2 shows the results of example 3.
FIG. 3 shows the results of example 4.
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.
EXAMPLE 1, 4 discovery of Ginseng CLE Polypeptides
One, 4 ginseng CLE genes are obtained
A Local BLAST method is adopted, a sequence (amplification Value is 0.001) with high similarity to an Arabidopsis CLE1-CLE45 gene sequence in a ginseng transcriptome is called, after a CLE sequence specifically expressed in roots is screened, ORF Finder software is adopted to find a gene sequence with a complete ORF frame, and PCR primer design is carried out.
Grinding and crushing a ginseng root sample by using liquid nitrogen, extracting total RNA by using a TRIzol method, determining the integrity of the RNA by using 1% agarose gel electrophoresis, and determining the content of the RNA by using a nucleic acid/protein quantitative analyzer. cDNA was synthesized using Takara reverse transcription Kit (M-MLV RTase cDNA Synthesis Kit). And (3) carrying out PCR amplification by using the cDNA as a template and adopting a designed primer to obtain a PCR product.
Expression analysis of two, 4 Ginseng CLE genes
And (3) successfully comparing the nucleic acid sequence obtained in the step one with a forest ginseng transcriptome database of different-year Jilin, calling transcript expression data of the database, and carrying out heat map analysis on gene expression by using Heml software (Heatmap Illustrator, version 1.0). The following are found: different genes have respective expression characteristics.
Thirdly, discovering and synthesizing 4 ginseng CLE polypeptides
The N end of the CLE family protein is a signal peptide, and 12 polypeptides at the C end are CLE domains and are also key signal molecules for functioning. According to the structural characteristics of 12C-terminal polypeptides, 4 ginseng CLE polypeptides are obtained.
The amino acid sequences of the 4 ginseng CLE polypeptides are as follows:
pgCLE12 polypeptide (SEQ ID NO: 2 of the sequence Listing): RLVPTGPNPLHH, respectively;
pgCLE25 polypeptide (SEQ ID NO: 3 of the sequence Listing): RRVPNGPDPIHN, respectively;
pgCLE27 polypeptide (SEQ ID NO: 4 of the sequence Listing): RRVPSCPDPLHN, respectively;
PgCLE45 polypeptide (sequence 1 of sequence listing): RRVRRGSDPIHN are provided.
Respectively synthesizing 4 ginseng CLE polypeptides.
Fourthly, 2 reference ginseng polypeptides are prepared
PgCLE3 polypeptide: RLSPGGPEPKHH, respectively;
PgCLE5 polypeptide: RVAPAGPNSQHN are provided.
Respectively synthesizing 2 ginseng CLE polypeptides.
Example 2 Ginseng CLE Polypeptides can regulate Arabidopsis thaliana root length and root branching
Test polypeptides: a PgCLE12 polypeptide, a PgCLE25 polypeptide, a PgCLE27 polypeptide, a PgCLE45 polypeptide, a PgCLE3 polypeptide, or a PgCLE5 polypeptide.
1. Taking Columbia ecotype arabidopsis seeds, soaking the seeds in 75% ethanol water solution for 3min, and then washing the seeds with sterilized deionized water for 10 times.
2. Grouping processing:
first group (Mock group): taking the seeds after the step 1, sowing the seeds on 1/2MS culture medium plates, and culturing for 9 days at the temperature of 22 +/-2 ℃ under illumination;
second group (PgCLE3 group): taking the seeds after the step 1, sowing the seeds on a 1/2MS culture medium plate containing 1 mu M PgCLE3 polypeptide, and culturing for 9 days at the temperature of 22 +/-2 ℃ under illumination;
third group (PgCLE5 group): taking the seeds after the step 1, sowing the seeds on a 1/2MS culture medium plate containing 1 mu M PgCLE5 polypeptide, and culturing for 9 days at the temperature of 22 +/-2 ℃ under illumination;
fourth group (PgCLE12 group): taking the seeds after the step 1, sowing the seeds on a 1/2MS culture medium plate containing 1 mu M PgCLE12 polypeptide, and culturing for 9 days at the temperature of 22 +/-2 ℃ under illumination;
fifth group (PgCLE25 group): taking the seeds after the step 1, sowing the seeds on a 1/2MS culture medium plate containing 1 mu M PgCLE25 polypeptide, and culturing for 9 days at the temperature of 22 +/-2 ℃ under illumination;
sixth group (PgCLE27 group): taking the seeds after the step 1, sowing the seeds on a 1/2MS culture medium plate containing 1 mu M PgCLE27 polypeptide, and culturing for 9 days at the temperature of 22 +/-2 ℃ under illumination;
seventh group (PgCLE45 group): taking the seeds after the step 1, sowing the seeds on a 1/2MS culture medium plate containing 1 mu M PgCLE45 polypeptide, and culturing for 9 days at the temperature of 22 +/-2 ℃ under illumination;
more than 30 seed biological replicates were set for each treatment.
3. After the completion of step 2, photographing, measuring the root length of the primary root with a vernier caliper, and counting the number of lateral roots on the primary root as the number of branch roots.
The photograph is shown in FIG. 1(a), the average of the root length of the primary root is shown in FIG. 1(b), and the average of the number of branches of the root is shown in FIG. 1 (c).
Compared with the Mock group, the PgCLE3 polypeptide and the PgCLE5 polypeptide have no influence on the primary root length and the number of root branches, the PgCLE45 polypeptide, the PgCLE12 polypeptide, the PgCLE25 polypeptide and the PgCLE27 polypeptide can obviously inhibit the primary root length, and the PgCLE45 polypeptide, the PgCLE12 polypeptide and the PgCLE25 polypeptide can obviously inhibit the number of root branches.
Example 3 Ginseng CLE Polypeptides can promote the differentiation of Arabidopsis Primary root
After completing step 2 in example 3, the primary roots of the plants were taken, and the meristematic regions of the primary roots were observed and photographed using a two-photon laser confocal microscope LSM880 and the length of the meristematic regions was examined.
The photograph is shown in FIG. 2(a), and the average length of the meristematic region of the primary root is shown in FIG. 2 (b).
Compared with the Mock group, the PgCLE3 polypeptide and the PgCLE5 polypeptide have no influence on the differentiation of primary roots, and the PgCLE45 polypeptide, the PgCLE12 polypeptide, the PgCLE25 polypeptide and the PgCLE27 polypeptide can obviously inhibit the length of a primary root meristematic region and promote the early formation of a mature region, particularly the PgCLE45 polypeptide has the most obvious effect of promoting the differentiation.
Example 4 modulation of proliferation of root tip cells of ginseng adventitious roots by Ginseng CLE Polypeptides
In the examples, ginseng was ginseng of red horse, and the origin was Jilin Fusong. Reference documents: the research progress of the germplasm resources of the Chinese ginseng, Ma Xiao Jun, Wan Xiao quan, Xiao pegen, Hongduan, the 5 th phase of 35 th volume of 2000-5 month of China pharmaceutical journal, 289-292.
Obtaining adventitious roots of ginseng
1. Soaking the seeds of Panax Meyer in 70% ethanol water solution for 3min, soaking in 1% sodium hypochlorite solution for 30min, and washing with sterilized deionized water for more than five times.
2. Taking the seeds after the step 1, removing the seed coats, taking out the seed embryos, and placing the seed embryos in a container containing 1.0 mg.L-16-BA, and culturing to obtain aseptic seedling. The culture conditions are as follows: standing, 25 + -2 deg.C, 16h light/8 h dark.
3. Taking the aseptic seedlings obtained in the step 2, taking roots, and cutting the roots into root segments with the length of 1.0 cm.
4. Taking the root segment obtained in the step 3 (used as an explant for inducing adventitious roots), and placing the root segment in a container containing 3.0 mg.L-1MS medium plates of IBA were cultured for 4 weeks, at which time adventitious roots formed on the explants. The culture conditions are as follows: standing, culturing at 25 + -2 deg.C in dark.
5. After completing step 4, the adventitious roots were separated and placed in a container containing 3.0 mg.L-1The IBA is cultured in MS liquid medium, and subcultured every 3 weeks for 4 times continuously, and the growth of the adventitious root is rapid and stable. The culture conditions are as follows: 100 r.min-1Dark culture at 25 + -2 deg.C.
Secondly, processing ginseng adventitious roots by using ginseng CLE polypeptide
First group (Control group): placing the adventitious roots obtained in the step one in a liquid culture medium, and culturing for 30 h;
second group (PgCLE12 group): putting the adventitious roots obtained in the step one into a liquid culture medium containing 25 mu M PgCLE12 polypeptide, and culturing for 30 h;
third group (PgCLE25 group): putting the adventitious roots obtained in the step one into a liquid culture medium containing 25 mu M PgCLE25 polypeptide, and culturing for 30 h;
fourth group (PgCLE27 group): putting the adventitious roots obtained in the step one into a liquid culture medium containing 25 mu M PgCLE27 polypeptide, and culturing for 30 h;
fifth group (PgCLE45 group): and (3) putting the adventitious roots obtained in the step one into a liquid culture medium containing 25 mu M of PgCLE45 polypeptide, and culturing for 30 h.
Liquid culture medium: containing 3.0 mg.L-1MS liquid medium of IBA.
The culture conditions are as follows: 100 r.min-1(25. + -. 2) ℃ in dark.
Each set was set to 5 replicates.
After the grouping process is completed, the indefinite root is taken and adopted
EdU Alexa
647 Imaging Kit (company name: Thermo Fisher Scientific; product number: C10340), according to the instruction, proliferation of the root tip cells of adventitious roots was detected. The EdU and Hoechst33342 labeled adventitious roots were observed using a two-photon laser confocal microscope. EdU excitation light was 650nm, emission light was 670nm, Hoechst33342 excitation light was 350nm, and emission light was 461 nm. The nuclei of proliferating cells are red in color and the nuclei of other cells are blue in color.
The results are shown in FIG. 3. The cell proliferation rate of the Control group was 40.8% + -8.3%, that of the PgCLE12 group was 8.3% + -4.0%, that of the PgCLE25 group was 11.2% + -6.1%, that of the PgCLE27 group was 8.5% + -4.4%, and that of the PgCLE45 group was 7.6% + -6.0%. The result shows that the cell proliferation rate of the ginseng adventitious roots added with the ginseng CLE polypeptide is obviously reduced.
SEQUENCE LISTING
<110> institute of traditional Chinese medicine of Chinese academy of traditional Chinese medicine
<120> ginseng CLE family polypeptide and application thereof in plant root growth and development regulation
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Claims (2)
1. Use of a polypeptide for inhibiting proliferation of root tip cells of a plant of the genus Panax;
the polypeptide is (a), (b), (c) or (d) as follows:
(a) PgCLE45 polypeptide: as shown in sequence 1 in the sequence table;
(b) PgCLE12 polypeptide: as shown in sequence 2 of the sequence table;
(c) PgCLE25 polypeptide: as shown in sequence 3 of the sequence table;
(d) PgCLE27 polypeptide: as shown in sequence 4 of the sequence table.
2. The application of the polypeptide in preparing products;
the polypeptide is (a), (b), (c) or (d) as follows:
(a) PgCLE45 polypeptide: as shown in sequence 1 in the sequence table;
(b) PgCLE12 polypeptide: as shown in sequence 2 of the sequence table;
(c) PgCLE25 polypeptide: as shown in sequence 3 of the sequence table;
(d) PgCLE27 polypeptide: as shown in sequence 4 of the sequence table;
the product has the function of inhibiting the proliferation of root tip cells of the Panax plant.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012000047A1 (en) * | 2010-06-30 | 2012-01-05 | The University Of Queensland | Soybean nodulation regulatory peptides and methods of use |
| CN103088054A (en) * | 2012-12-29 | 2013-05-08 | 中国科学院植物研究所 | Method for interfering plant endogenous CLE family polypeptide hormone and antagonist polypeptide for small molecule polypeptide hormone |
| CN110184277A (en) * | 2019-05-20 | 2019-08-30 | 中国科学院植物研究所 | Application of the CLE25 gene in the starting of regulation plant phloem and growth course |
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| MX2012010600A (en) * | 2010-03-18 | 2012-10-05 | Basf Plant Science Co Gmbh | Plants having enhanced yield-related traits and method for making the same. |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012000047A1 (en) * | 2010-06-30 | 2012-01-05 | The University Of Queensland | Soybean nodulation regulatory peptides and methods of use |
| CN103088054A (en) * | 2012-12-29 | 2013-05-08 | 中国科学院植物研究所 | Method for interfering plant endogenous CLE family polypeptide hormone and antagonist polypeptide for small molecule polypeptide hormone |
| CN110184277A (en) * | 2019-05-20 | 2019-08-30 | 中国科学院植物研究所 | Application of the CLE25 gene in the starting of regulation plant phloem and growth course |
Non-Patent Citations (7)
| Title |
|---|
| Border sequences of Medicago truncatula CLE36 are specifically cleaved by endoproteases common to the extracellular fluids of Medicago and soybean;Michael A. Djordjevic等;《Journal of Experimental Botany》;20110601;第62卷(第13期);Table 1、Table 2、Firgue 1 * |
| CLE peptides and their signaling pathways in plant development;Yasuka L. Yamaguchi等;《Journal of Experimental Botany》;20160526;第67卷(第16期);Table 1 * |
| FON2 SPARE1 Redundantly Regulates Floral Meristem Maintenance with FLORAL ORGAN NUMBER2 in Rice;Takuya Suzaki等;《PLOS GENETICS》;20091016;第5卷(第10期);Figure 3 * |
| Perception of root-active CLE peptides requires CORYNE function in the phloem vasculature;Ora Hazak等;《EMBO reports》;20170612;第18卷(第8期);第1377页右栏第5段 * |
| Search for nodulation-related CLE genes in the genome of Glycine max;Virginie Mortier等;《Journal of Experimental Botany》;20110127;第62卷(第8期);Table 1 * |
| 植物CLE家族基因的鉴定和聚类;张哲;《中国博士学位论文电子期刊网》;20200115(第01期);图3-8、表S4、第84-89页 * |
| 植物多肽信号分子CLE 家族;高丽;《生物技术通报》;20141106(第11期);第14-23页 * |
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