CN107176980A - Apple polypeptide MdCEP1PHypAnd its application - Google Patents

Abstract

The invention discloses apple polypeptide MdCEP1_P ^HypAnd its application, belong to technical field of molecular biology.The apple polypeptide MdCEP1_P ^HypAmino acid sequence as shown in SEQ.ID.NO.2.The invention also discloses above-mentioned apple polypeptide MdCEP1_P ^HypIn the regulation developmental application of plant root.Applicant of the present invention synthesizes the external source CEP small peptides containing 15 amino acid using chemical synthesis, and small peptide is carried out into hydroxylating modification, outer to arabidopsis progress to apply MdCEP1_P ^HypProcessing, Phenotypic Observation discovery, MdCEP1_P ^HypSignificantly inhibit the number of the elongation of main root and lateral root in arabidopsis.

Description

Apple Polypeptide MdCEP1PHyp and Its Application

technical field

The invention relates to apple polypeptide MdCEP1 _P ^Hyp and application thereof, belonging to the technical field of molecular biology.

Background technique

Recent research results have shown that polypeptide molecules in plants play an important role in regulating plant germination, growth, differentiation, development, signal transduction and other aspects, so they are also called polypeptide hormones. Plant polypeptide hormones are a kind of mature polypeptides with special functions formed after proteasome cleavage, their length is usually less than 20 amino acids, and their content in plants is extremely low. The most important role of plant polypeptide hormones is to participate in short-distance information transmission between cells as signaling molecules. The extracellular polypeptide hormone binds to the corresponding receptor kinase on the surface of the cell membrane in the form of ligand, activates the downstream gene and triggers the corresponding signal transduction process. So far, many types of peptide hormones in plants have been identified, including CLAVATA3 (CLV3), CLV3/EMBRYO SURROUNDING RELATED (CLE), TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF), PHYTOSULFOKINE (PSK), PLANT PEPTIDE CONTAINING SULFATED TYROSINE1 ( PSY1), C-TERMINALLY ENCODEDPEPTIDE1 (CEP1), etc. The CEP gene encodes a small peptide precursor consisting of an N-terminal secretion signal peptide, a variable domain, one or more CEP domains, and a short C-terminal extension. Studies have shown that CEPs are involved in the construction of plant root morphology, lateral root development, and root nodule formation. Overexpression of AtCEP1 in Arabidopsis inhibits the main root and reduces the number of lateral roots. Arabidopsis CEPs mutants exhibit elongated taproot phenotypes under various stress conditions. The expression of AtCEP5 in Arabidopsis is inhibited by auxin, and it binds to the downstream kinase receptor AtCEPR1, thereby inhibiting the development of main root and lateral root; in alfalfa, MtCEP1 acts on the downstream peptide receptor MtCAR2, reducing the number of lateral roots and promoting root nodulation.

Apple is an important fruit crop in the world, and the growth and development of apple roots have a significant impact on apple yield and quality. In perennial woody plants, the study of polypeptide synthesis gene CEP has not been reported yet.

Contents of the invention

One of the objectives of the present invention is to provide an apple polypeptide MdCEP1 _P ^Hyp . The applicant of the present invention synthesized an exogenous CEP small peptide containing 15 amino acids by chemical synthesis, modified the proline site of the small peptide by hydroxylation, and applied MdCEP1 _P ^Hyp to Arabidopsis thaliana, and the phenotype It was observed that MdCEP1 _P ^Hyp significantly inhibited the elongation of the main root and the number of lateral roots in Arabidopsis, revealing that the apple polypeptide MdCEP1 _P ^Hyp plays an important role in regulating auxin synthesis, transport and root system development.

The technical solution of the present invention to solve the above technical problems is as follows: Apple polypeptide MdCEP1 _P ^Hyp , the amino acid sequence is shown in SEQ.ID.NO.2.

On the basis of the above technical solutions, the present invention can also be improved as follows.

Further, the apple polypeptide MdCEP1 _P ^Hyp comes from Gala apple.

The second object of the present invention is to provide the application of the apple polypeptide MdCEP1 _P ^Hyp in regulating the development of the root system of the plant. The apple polypeptide MdCEP1 _P ^Hyp of the present invention can directly optimize the root system structure, and the shortening of the main root can inhibit the elongation of the upper part of the stem, especially in fruit trees, and can be applied to the genetic improvement of arborization rootstocks.

The technical solution of the present invention to solve the above-mentioned technical problems is as follows: the application of apple polypeptide MdCEP1 _P ^Hyp in regulating the development of plant roots.

On the basis of the above technical solutions, the present invention can also be improved as follows.

Further, the apple polypeptide MdCEP1 _P ^Hyp can reduce the length of the main root of the plant and inhibit the number of lateral roots.

The further beneficial effects of adopting the above are: inhibiting the growth of the root system, reducing photosynthetic energy consumption, and further applying photosynthetic products to the accumulation of carbon substances. At the same time, by optimizing the root system, the morphology of the aboveground parts can be adjusted, especially in fruit trees, which can be applied to the genetic improvement of arborization rootstocks.

The experimental results showed that apple polypeptide MdCEP1 _P ^Hyp affected auxin synthesis and transport, and inhibited the development of main root and lateral root.

The beneficial effects of the present invention are:

1. External application of MdCEP1 _P ^Hyp to Arabidopsis thaliana showed a phenotype of inhibited main root length and lateral root number; revealing that the apple polypeptide MdCEP1 _P ^Hyp plays an important role in regulating auxin synthesis, transport and root system development.

2. The apple polypeptide MdCEP1 _P ^Hyp of the present invention can directly optimize the root system structure, and the shortening of the main root can inhibit the elongation of the aboveground stem, especially in fruit trees, and can be applied to the genetic improvement of arborization rootstocks.

Description of drawings

Figure 1 is the CEP polypeptide sequence in the MdCEP1 protein.

Figure 2 is the phenotype observation of root development of Arabidopsis thaliana treated with different concentrations of MdCEP1 _P ^Hyp . From left to right, the concentrations of MdCEP1 _P ^Hyp are 0 μM, 1 μM and 10 μM, respectively.

Figure 3 is the phenotype observation of Arabidopsis thaliana treated with MdCEP1 _P ^Hyp level, from left to right, control and MdCEP1 _P ^Hyp treatment respectively.

detailed description

The principles and features of the present invention are described below in conjunction with the accompanying drawings, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

Example 1 Apple MdCEP1 amino acid sequence analysis

1. The MdCEP1 gene comes from Gala apple. The coding region of MdCEP1 gene contains 366 bp of nucleotides, encoding 121 amino acids. Using the database of the NCBI website (http://www.ncbi.nlm.nih.gov/) and the analysis software of the expasy website (http://www.expasy.org/), the functional domains and The conserved domains were compared and analyzed, and it was found that the MdCEP1 predicted protein contained a unique CEP domain (103-117 amino acids) of the CEP family, as shown in Figure 1 .

Example 2 Synthesis and modification of CEP domain in apple MdCEP1 gene

1. In order to further study the function of the CEP domain, 15 amino acids of the CEP domain were chemically synthesized by Sangon Bioengineering Co., Ltd. (DFRTPAPGHSPGVGH).

2. Carry out hydroxylation modification (DFR{Hyp}TAPGHS{Hyp}GVGH) on the two Pro residues of the synthetic polypeptide to obtain MdCEP1 _P ^Hyp .

Example 3 Applying MdCEP1 _P ^Hyp to Arabidopsis

Disinfect Arabidopsis thaliana seeds with 70% alcohol for 3 minutes, 4% sodium hypochlorite for 8-10 minutes (shaking several times during the period), rinse with sterilized water for 5 times, and place them in 4°C for vernalization for two days , to obtain vernalized Arabidopsis seeds.

1. Vertical treatment culture of Arabidopsis thaliana: sow the vernalized Arabidopsis seeds on the seed germination medium (directly spread on the surface), vertical light culture (25-28°C, 16h long-day/8h short-day) , 3d), select seedlings with consistent growth. Transplanted onto MS medium containing 0 μmol L ^-1 , 1 μmol L ^-1 and 10 μmol L ^-1 MdCEP1 _P ^Hyp respectively, and continued vertical light culture (25-28°C, 16h long-day/8h short-day, 10d), then observe and take pictures.

2. Arabidopsis thaliana horizontal treatment culture: sow the vernalized Arabidopsis seeds in MdCEP1 _P ^Hyp MS medium containing 0 μmol L ^-1 and 1 μmol L ^-1 respectively, and put them into the light incubator for horizontal cultivation (25-28°C, 16h long day/8h short day, 30d), then observe and take pictures.

The results of exogenous MdCEP1 _P ^Hyp showed that the exogenous _{MdCEP1 P} ^Hyp treatment significantly affected the root development of Arabidopsis thaliana, as shown in Figure 2, showing that the main root length and the number of lateral roots of Arabidopsis were severely inhibited, and the The inhibitory effect was more obvious with the increase of ^Hyp concentration.

The results of exogenous MdCEP1 _P ^Hyp showed that after long-term horizontal cultivation, the exogenous MdCEP1 _P ^Hyp treatment significantly affected the growth of Arabidopsis, mainly showing the phenotype of inhibited plant growth, as shown in Figure 3.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

sequence listing

〈110〉Shandong Agricultural University

<120> Apple polypeptide MdCEP1PHyp and its application

<160>2

〈170〉PatentIn version 3.5

<210>1

〈211〉15

<212> PRT

<213> Apple

<400>1

DFRTPAPGHSPGVGH 15

<210>2

〈211〉15

<212> PRT

<213> Apple

<400>1

DFRPHypTAPGHSPHypGVGH 15

Claims (4)

1. apple polypeptide MdCEP1_P ^Hyp, it is characterised in that the amino acid sequence is as shown in SEQ.ID.NO.2.

2. apple polypeptide MdCEP1 according to claim 1_P ^Hyp, it is characterised in that the apple polypeptide MdCEP1_P ^HypCome From loud, high-pitched sound apple.

3. a kind of apple polypeptide MdCEP1 of regulation and control root system development as claimed in claim 1_P ^HypIt is developmental in regulation plant root Using.

4. application according to claim 3, the apple polypeptide MdCEP1_P ^HypPlant main root length can be reduced and suppress side Radical mesh.