阅读说明：本技术 一种利用GF14f蛋白靶向多肽配体调控水稻弱势籽粒灌浆的方法 (Method for regulating and controlling weak grain filling of rice by using GF14f protein targeted polypeptide ligand ) 是由 张志兴 陈鸿飞 林文雄 于 2020-05-27 设计创作，主要内容包括：本发明公开了一种利用GF14f蛋白靶向多肽配体溶液调控水稻弱势籽粒灌浆的方法。所述多肽配体溶液由60mg/L的14-3-3蛋白成员GF14f靶向多肽配体R18和6ml/L的表面活性剂吐温-20组成。在水稻灌浆期外源喷施GF14f蛋白靶向多肽配体溶液,多肽配体R18能与弱势籽粒中的GF14f蛋白竞争性结合,从而对灌浆过程中淀粉合成代谢起到调控作用,能够显著提高弱势籽粒千粒重及结实率。(The invention discloses a method for regulating and controlling weak rice grain filling by using a GF14f protein targeted polypeptide ligand solution. The polypeptide ligand solution consists of 60 mg/L14-3-3 protein member GF14f targeting polypeptide ligand R18 and 6ml/L surfactant Tween-20. The GF14f protein targeted polypeptide ligand solution is sprayed at an exogenous source in the grain filling period of rice, and the polypeptide ligand R18 can be competitively combined with GF14f protein in the weak grains, so that the starch anabolism is regulated and controlled in the grain filling process, and the thousand kernel weight and the setting rate of the weak grains can be obviously improved.)

1. A polypeptide ligand solution for regulating and controlling the filling of weak rice grains is characterized in that: the components of the polypeptide ligand solution comprise 60mg/L of GF14f protein targeting polypeptide ligand R18 and 6ml/L of surfactant.

2. The polypeptide ligand solution for regulating and controlling rice weak grain filling according to claim 1, wherein the polypeptide ligand solution comprises: the GF14f protein targeted polypeptide ligand R18 has the amino acid sequence as follows: PHCVPRDLSWLDLEANMCLPP are provided.

3. The polypeptide ligand solution for regulating and controlling rice weak grain filling according to claim 1, wherein the polypeptide ligand solution comprises: the surfactant is tween-20.

4. A method for regulating and controlling the grouting of rice weak grains is characterized by comprising the following steps: the polypeptide ligand solution for regulating and controlling the filling of the weak rice grains in the rice filling period is sprayed by external sources in the rice filling period, wherein the polypeptide ligand solution is the polypeptide ligand solution in the claim 1.

5. The method for regulating and controlling grouting of rice weak grains according to claim 3, wherein the method comprises the following steps: in the filling stage of rice, i.e. 5-7 days after flowering, 1-1.5L/mu of the polypeptide ligand solution for regulating and controlling filling of rice weak grains according to claim 1 is sprayed by an exogenous source.

6. The use of the polypeptide ligand solution for regulating and controlling rice weak grain filling of claim 1 in improving rice grain weight and setting percentage.

Technical Field

The invention belongs to the technical field of crop growth regulation and control, and particularly relates to a method for regulating and controlling weak rice grain filling by using a GF14f protein targeted polypeptide ligand.

Background

The grain size and the grain weight of the rice grains are closely related to the growing positions of the glumes on the spikes. The grains are grown early, the filling is fast and stable, and the grain weight is high, so that the grains are called strong grains; slow growth and development, poor filling and filling, easy to be controlled by the environment and low grain weight, which is called weak grain. The seed setting rate and the filling degree of strong and weak grains of the rice are greatly different, and some grains reach about 25 percent. A great deal of research results and production practices of predecessors prove that the weak grain filling difference and the grain weight are low, so that the exertion of the yield potential of rice is limited, the grain quality is seriously influenced, and the weak grain filling difference becomes a key limiting factor for further improving the yield potential of modern rice varieties. At the same time, this difference in grain filling is also common in other cereal crops, such as wheat and corn. The grain filling difference phenomenon is a long-standing unsolved problem in the science and production of cereal crops.

Rice grain grouting is a complex physiological and ecological process, and relates to expression regulation of a large number of genes (proteins). The applicant has shown in previous studies that the expression level of 14-3-3 protein family member GF14f protein in weak grains is significantly higher than that of strong grains, which is an important reason for poor filling of weak grains, the protein can play a role in regulating and controlling key enzymes in the starch synthesis process of weak grains through a protein interaction mechanism, and the expression level of GF14f in the grain filling period can be specifically reduced by adopting a reverse genetics method, so that the activities of three sucrose transformation and starch synthesis key enzymes of sucrose synthase (SuS), Soluble Starch Synthase (SSS) and ADPG pyrophosphorylase (AGPase) in the grain filling process can be significantly improved, and thus the filling of weak grains is facilitated. As shown by the field yield data, the yield data indicates,GF14fcompared with wild plants, The effective spike number and The grain number of each spike of The RNAi plant have no obvious difference, but The grain length of grains is increased by 12.9%, The thousand kernel weight is increased by about 10%, The setting rate of weak grains is increased by 10.85%, The yield is increased by 15.2%, and The fact that The GF14f plays an important role in The grain grouting process is proved (The Zhang et, The 14-3-3 protein GF14f novel aftertastes grains of fertility grains of rice kernels)Oryza sativaL.). The PlantJournal, 2019, 99, 344–358）。

In recent years, with the development of biotechnology, small artificial molecules such as polypeptide ligands (PA) have become effective technologies that directly affect protein functions. Peptide ligands are short (8-20 amino acids) peptides displaying a peptide loop in an inert scaffold protein with a high specific binding affinity for a given target. Peptide ligands can specifically bind to and inactivate target proteins and therefore act as strong competitive inhibitors. They can specifically block the formation of protein-protein interactions and interfere with gene function without altering protein levels or structure. The essence of polypeptide ligand technology is to specifically inhibit a target protein at the protein level, thereby rendering it biologically nonfunctional in vivo. The method of utilizing the polypeptide ligand to specifically bind the target protein can specifically interfere the interested plant gene on the protein level, and is helpful for accurately understanding the biological function of the target gene in the origin and the morphogenesis of complex characters. The screened polypeptide ligand of the target protein can play a role in regulating the growth and stress resistance of crops by spraying and regulating in vitro, and the strategy has been widely applied to the disease resistance and growth and development of crops, for example, the targeted polypeptide ligand of the tomato spotted wilt virus TSWV sprayed from an external source can obviously enhance the ability of tomato to resist the spotted wilt disease.

Aiming at the expression mode and the functions of a 14-3-3 protein family member GF14f in the grain filling stage of rice, the invention screens a polypeptide ligand fragment R18 capable of being combined with GF14f in a targeted manner, and the polypeptide ligand fragment is externally sprayed on grains in the grain filling stage and acts on GF14f in a targeted manner, so that the thousand kernel weight and the setting rate of weak grains are obviously improved, the targeted regulation and control on the weak grain filling are realized, and relevant research reports are not found at home and abroad.

Disclosure of Invention

The invention provides a method for regulating and controlling weak rice grain filling by using a GF14f protein targeted polypeptide ligand, wherein the polypeptide ligand can be competitively combined with a 14-3-3 protein member GF14f protein in grains in a targeted manner, so that the polypeptide ligand is not combined with interacting protein, thereby regulating and controlling starch anabolism in the rice grain filling process and achieving the purpose of improving thousand kernel weight and setting percentage of the weak grains.

A polypeptide ligand solution for regulating and controlling weak grain filling of rice comprises the following components: 60mg/L of GF14f protein targeted polypeptide ligand R18 and 6ml/L of surfactant.

The GF14f protein targeting polypeptide ligand R18 has an amino acid sequence of PHCVPRDLSWLDLEANMCLPP.

The surfactant is Tween-20.

A method for regulating and controlling the filling of weak rice grains comprises the following steps: and the polypeptide ligand solution for regulating and controlling the filling of the rice weak grains is sprayed from an external source in the filling period of the rice.

Preferably, in the filling period of rice, namely 5-7 days after flowering, 1-1.5L/mu of the polypeptide ligand solution for regulating and controlling filling of rice weak grains is sprayed by an exogenous source.

The polypeptide ligand solution for regulating and controlling the filling of the weak rice grains is applied to the improvement of the grain weight and the setting percentage of the rice.

The invention has the following beneficial effects:

1. the 14-3-3 protein family member GF14f protein polypeptide ligand R18 is an effective component of a spraying agent, and has simple components and no pollution.

2. The polypeptide ligand can be combined with GF14f protein in grains in a targeted mode, and is combined with the targeted protein GF14f through competitive interaction, so that the polypeptide ligand is not combined with the interacted protein, and the function of the protein is regulated and controlled under the condition that the protein expression is not influenced.

Description of the drawings:

fig. 1 is a schematic diagram of molecular docking of GF14f protein targeting polypeptide ligand R18 with GF14 f. A: polypeptide ligand R18 and GF14f protein docking scheme; b: schematic representation of the binding amino acid sites of the GF14f protein to the polypeptide ligand R18.

Fig. 2 is a diagram of confocal laser fluorescence imaging of interaction between a GF14f protein-targeted polypeptide ligand R18 and a GF14f protein.

FIG. 3 shows the result of competitive pull-down experiments between GF14f protein targeting polypeptide ligand R18 and GF14f protein and its starch synthesis related interacting protein. (a) The method comprises the following steps The result of a competitive pull-down experiment of a GF14f protein targeting polypeptide ligand R18, GF14f protein and sucrose synthase (SuS); (b) the method comprises the following steps The result of competitive pull-down experiments of GF14f protein targeting polypeptide ligand R18, GF14f protein and Soluble Starch Synthase (SSS); (c) the result of a competitive pull-down experiment of the GF14f protein targeting polypeptide ligand R18, the GF14f protein and ADPG pyrophosphorylase (AGPase).

FIG. 4 shows the influence of an exogenous GF14f protein targeting polypeptide ligand R18 on the enzyme activity of sucrose synthase (SuS) of rice kernels.

Fig. 5 shows the influence of exogenous spray GF14f protein targeting polypeptide ligand R18 on the enzymatic activities of kernel sucrose synthase (SuS), Soluble Starch Synthase (SSS), and ADPG pyrophosphorylase (AGPase).

Fig. 6 shows the influence of exogenous spraying of GF14f protein targeting polypeptide ligand R18 on the setting rate and thousand kernel weight of weak grains in the mature period.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the following examples are only examples of the present invention and do not represent the scope of the present invention defined by the claims.