阅读说明：本技术 一个双孢蘑菇乙烯蛋白受体Ab143539 (Agaricus bisporus ethylene protein receptor Ab143539 ) 是由 邱立友 郜熙阳 高玉千 李亚楠 张君 李涛 张朝辉 田芳 于 2019-10-31 设计创作，主要内容包括：本申请属于植物基因组技术领域,具体涉及一个双孢蘑菇基因组中的乙烯蛋白受体Ab143539。其结构中共有5个跨膜区,在第3跨膜区含有一个半胱氨酸残基,具体碱基序列如SEQ ID NO.1所示。基于结构对比结果,发明人推测Ab143539为双孢蘑菇中的乙烯受体蛋白(AbEBD2),可以结合乙烯。进一步实际验证后发现,该蛋白具有乙烯结合作用,但其结合能力明显弱于拟南芥乙烯受体ETR1。但基于这一研究结果,通过基因工程技术手段,仍然可为双孢蘑菇新品种培育、或者双孢蘑菇保鲜等技术改进奠定一定技术基础。(The application belongs to the technical field of plant genomes, and particularly relates to an ethylene protein receptor Ab143539 in an agaricus bisporus genome. The structure of the gene has 5 transmembrane regions, the 3 rd transmembrane region contains a cysteine residue, and the specific base sequence is shown as SEQ ID NO. 1. Based on the results of the structural comparison, the inventors speculate that Ab143539 is an ethylene receptor protein in agaricus bisporus (AbEBD 2), and can bind ethylene. Further practical verification shows that the protein has an ethylene binding effect, but the binding capacity of the protein is obviously weaker than that of an Arabidopsis ethylene receptor ETR 1. However, based on the research result, a certain technical basis can still be established for the cultivation of new agaricus bisporus varieties or the improvement of the agaricus bisporus preservation and other technologies through a genetic engineering technical means.)

1. An agaricus bisporus ethylene protein receptor, which is abbreviated as Ab143539, the structure of the ethylene protein receptor is provided with 5 transmembrane regions, the 3 rd transmembrane region contains a cysteine residue, and the specific base sequence is shown as SEQ ID NO. 1.

2. The method for preparing the agaricus bisporus ethylene protein receptor Ab143539 of claim 1, which is prepared by a PCR amplification method and comprises the following specific steps:

(1) extracting total RNA of agaricus bisporus, and performing reverse transcription to obtain cDNA for later use;

(2) the primer sequences for PCR amplification were designed as follows:

Type-R：5’- GGATCCAGTTTAGCACTTTGGGGGGC -3’，

Type-F：5’- GTAGTGCACATATATAAGGAAGAAC -3’；

and (2) carrying out PCR amplification by using the cDNA prepared in the step (1) as a template.

3. The use of the agaricus bisporus ethylene protein receptor Ab143539 of claim 1 in plants, wherein the protein binds ethylene.

Technical Field

The application belongs to the technical field of plant genomes, and particularly relates to an ethylene protein receptor Ab143539 in an agaricus bisporus genome.

Background

The agaricus bisporus is the edible fungus which has the widest cultivation range and the largest output and consumption in the world, and is also the edible fungus with the largest export amount in China. Physiological studies show that agaricus bisporus and higher plants are similar and can synthesize and produce ethylene, and the synthesized ethylene can further inhibit the growth of hypha and the formation of fruiting bodies and accelerate the maturation and aging of the collected fruiting bodies. However, since the related studies are still limited, it is not clear whether an ethylene signal transduction pathway similar to that of higher plants also exists in agaricus bisporus.

Studies have shown that ethylene receptors are important components of the ethylene signal transduction pathway in higher plants, originating from the bacterial two-component signal transduction system. The bacterial two-component signal transduction system consists of a histidine kinase domain (HK) and a response regulator domain (RR). The two-component signal transduction system can be developed into hybrid histidine kinase fused by HK and RR, and the two-component signal transduction system in eukaryote mostly belongs to hybrid histidine kinase. Plant ethylene receptors are transmembrane proteins, localized on the endoplasmic reticulum membrane, and the domain structures include ethylene binding domains (ethylene binding domains), GAF domains, histidine kinase domains (Hiskinase domains), and receptor effect regulators (receptor domains). For example, the ethylene binding domain of the Arabidopsis ethylene receptor ETR1 has 3 hydrophobic transmembrane domains, the GAF domain may mediate receptor protein interactions, and the histidine kinase domain exists as a homodimer. When ethylene binds to a receptor, histidine kinase is activated and the histidine residues are autophosphorylated, which can transfer phosphate groups to aspartate residues or downstream component CTR1 of receptor effect modulators, thereby activating signaling pathways.

Although some functional proteins are cloned and studied in the research of agaricus bisporus genome, the research on ethylene metabolism pathways and ethylene metabolism related genes in agaricus bisporus is still lacked, and the deep research on ethylene metabolism related proteins is obviously of great technical significance in the aspect of agaricus bisporus quality control.

Disclosure of Invention

The application aims to provide an agaricus bisporus ethylene protein receptor, thereby laying a certain technical foundation for the growth and development regulation of agaricus bisporus.

The technical solution adopted in the present application is detailed as follows.

The agaricus bisporus ethylene protein receptor has a protein sequence number of 143539 (Ab 143539 for short), has 5 transmembrane regions, contains a cysteine residue (Cys 291) in the 3 rd transmembrane region, is related to ethylene binding, and is further named as agaricus bisporus ethylene receptor protein AbEBD2, and the specific base sequence is shown as SEQ ID No. 1.

The preparation method of the agaricus bisporus ethylene protein receptor Ab143539 is prepared by a PCR amplification method and comprises the following specific steps:

(1) extracting total RNA of agaricus bisporus, and performing reverse transcription to obtain cDNA for later use;

(2) the primer sequences for PCR amplification were designed as follows:

Type-R：5’- GGATCCAGTTTAGCACTTTGGGGGGC -3’，

Type-F：5’- GTAGTGCACATATATAAGGAAGAAC -3’；

and (2) carrying out PCR amplification by using the cDNA prepared in the step (1) as a template.

The agaricus bisporus ethylene protein receptor Ab143539 is applied to plants, and the protein is combined with ethylene to regulate and control the maturation process of the plants.

A method for culturing the new plant variety features that the ethylene protein receptor Ab143539 gene of Agaricus bisporus is transcribed to the genome of plant by gene engineering technique, and the ethylene is combined to regulate the growth or maturation of plant.

Studies have shown that many fungi also synthesize ethylene and thus there are multiple hybrid histidine kinases present in the fungal genome, whereas in studies on agaricus bisporus genome there are 4 hybrid histidine kinases found in agaricus bisporus genome, where the Ab143539 protein has all domain structures similar to plant ethylene receptors, namely including: 5 transmembrane regions, and a histidine kinase domain and receptor-response regulator, but no GAF domain, and a cysteine residue (Cys 291) in the 3 rd transmembrane region; the second transmembrane region of the ethylene receptor ETR1 of the prior art arabidopsis thaliana also contains a cysteine residue (Cys 65) (this residue is the amino acid necessary for ETR1 to bind ethylene). Based on these structural comparisons, the inventors speculate that Ab143539 is an ethylene receptor protein in agaricus bisporus (AbEBD 2), and can bind ethylene. Further practical verification shows that the protein has an ethylene binding effect, but the binding capacity of the protein is obviously weaker than that of an Arabidopsis ethylene receptor ETR 1. However, based on the research result, a certain technical basis can still be established for the cultivation of new agaricus bisporus varieties or the improvement of the agaricus bisporus preservation and other technologies through a genetic engineering technical means.

Drawings

FIG. 1 shows PCR amplification products of ethylene binding domains of Agaricus bisporus pseudoethylene receptor and Arabidopsis thaliana ethylene receptor and fluorescent protein gene, wherein A, Agaricus bisporus pseudoethylene receptor Ab143539 ethylene binding domain AbEBD2 and fluorescent protein gene EGFP; b, an arabidopsis ethylene receptor ethylene binding domain AtEBD1 and a fluorescent protein gene EGFP; m, Trans5K DNAladder, 1, AbEBD2, 2, EGFP, 3, AbEBD 1-EGFP; 4, AtEBD1, 5, EGFP, 6, AtEBD 1-EGFP;

FIG. 2 is a double-restriction enzyme electrophoresis diagram of an ethylene binding domain of an agaricus bisporus pseudo-ethylene receptor and an arabidopsis ethylene receptor and a fluorescent protein fusion expression vector; wherein: a, pYE-TYPE-EGFP; b, pYE-ETR 1-EGFP; m1, 1Kb Plus DNA Ladder, 1, pYE-TYPE-EGFP enzyme-cleaved, 2, pYE-TYPE-EGFP enzyme-cleaved; m2, DL15000 plus DNA Ladder, 3, pYE-ETR1-EGFP restriction enzyme, 4, pYE-ETR1-EGFP restriction enzyme;

FIG. 3 is a fluorescent microscopic observation (magnification 10X 63) of yeast transformants expressing the ethylene binding domain and fluorescent protein fusion protein: pYES2/NT/A, containing unloaded plasmid cells; pYE-TYPE-EGFP, which expresses AbEBD2-EGFP cells; pYE-ETR1-EGFP, expressing AtEBD1-EGFP cells;

FIG. 4 is a graph of the amount of ethylene bound by yeast cells expressing the ethylene binding domains of Agaricus bisporus and the Arabidopsis thaliana ethylene receptor; pYES2/NT/A, empty plasmid; AbEBD2-EGFP, agaricus bisporus ethylene receptor Ab143539 ethylene binding domain AbEBD2 and fluorescent protein gene EGFP; AtEBD1-EGFP, a fusion protein of an Arabidopsis ethylene receptor ETR1 ethylene binding domain AtEBD1 and a fluorescent protein gene EGFP; different capital letters indicate that the difference reaches a significant level (P < 0.01).

Detailed Description

The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.

Biological material:

agaricus bisporus As2796 from edible fungus institute of farm institute of Fujian province;

plasmid pEGFP-C1 was purchased from Clontech (Mountain View, CA);

saccharomyces cerevisiae INVSC1 and expression vector pYES2/NT/A were purchased from Beino Biotech, Inc. of Shanghai (Beiinuo, Shanghai, China);

culture medium:

the agaricus bisporus slant preservation and the flat culture medium are PDA culture medium;

the saccharomyces cerevisiae culture medium is an YPD culture medium, and the formula comprises 1% of yeast extract, 2% of peptone and 2% of glucose;

the culture medium for screening yeast transformant is SC-URA culture medium, and the formula comprises 6.7 g/L yeast nitrogen source base, 2 g/L uracil synthesis deficient culture medium and 20 g/L glucose.