CN114621333B

CN114621333B - Transport protein of muskmelon bitter substance cucurbitacin B and application thereof

Info

Publication number: CN114621333B
Application number: CN202210277778.1A
Authority: CN
Inventors: 马永硕; 尚轶; 黄三文; 仲阳
Original assignee: Agricultural Genomics Institute at Shenzhen of CAAS
Current assignee: Agricultural Genomics Institute at Shenzhen of CAAS
Priority date: 2022-03-21
Filing date: 2022-03-21
Publication date: 2022-10-04
Anticipated expiration: 2042-03-21
Also published as: CN114621333A

Abstract

The application relates to a transport protein of a melon bitter substance cucurbitacin B and application thereof. The application discloses a molecular mechanism of melon bitter transportation for the first time, provides a theoretical basis for melon molecular assisted breeding, and simultaneously provides support for synthesizing cucurbitacin B by using a synthetic biology technology.

Description

Transport protein of muskmelon bitter substance cucurbitacin B and application thereof

Technical Field

The application relates to a transport protein of a melon bitter substance cucurbitacin B and application thereof, belonging to the technical field of biology.

Background

In practice, the fruits of cucurbitaceae plants often produce unpleasant bitter taste, which originates from cucurbitacin, a secondary metabolite, in addition to sweet, fragrant or aromatic taste or odor. Cucurbitacins are closely related to adversity and can resist the invasion of adverse environments. Cucurbitacins have been shown to enhance plant resistance to certain pests and are also associated with plants responding to mechanical damage. In addition, cucurbitacins have important significance for human health. A large number of medical researches show that the cucurbitacin has anticancer activity.

Cucurbitacins distributed in cucurbitaceae are of various types, and cucurbitacin B is mainly present in the roots and fruits of cucumis melo. Researchers measure the content of cucurbitacin B (bitter substances) in young melons and the sugar degree of mature fruits, find that the bitterness of fruits in the stage I of development is in extremely obvious positive correlation with the sugar degree of the fruits in the mature stage, and speculate that a certain conversion relationship possibly exists between the bitter substances of the melons and sugar substances. Researchers also identified cucurbitacin B synthetic gene clusters (1 OSC, 6P 450 s and 1 ACT) in melons using comparative genomics.

Because the secondary metabolites are not static in the plant body, but are transported in cells, among cells or even among different organs along with corresponding physiological needs, the transportation mechanism of cucurbitacin needs to be further explored besides the research on the synthesis regulation mechanism of cucurbitacin, so that more new ideas can be provided for plant breeding, and more powerful support is provided for efficiently synthesizing and improving cucurbitacin in vitro.

Disclosure of Invention

In order to solve the technical problems, the application provides a method for regulating and controlling synthesis or transport of cucurbitacin B, which comprises the steps of regulating and controlling the expression amount or activity of a cucurbitacin B transporter; the cucurbitacin B transporter is one of the following:

a) The amino acid sequence is shown as SEQ ID NO. 1;

b) Protein with the same function obtained by a) substituting and/or deleting and/or adding one or more amino acid residues;

c) Proteins having 80% or more than 80% homology to a) or b);

d) A fusion protein obtained by connecting a label to the N-terminal and/or the C-terminal of a), b) or C).

In one embodiment, further comprising using cucurbitacin B transporter associated biological material, being any one of the following A1) to A8):

a1 Nucleic acid molecules encoding the aforementioned proteins;

a2 An expression cassette comprising the nucleic acid molecule according to A1);

a3 A recombinant vector containing the nucleic acid molecule according to A1);

a4 A recombinant vector containing the expression cassette of A2);

a5 A recombinant microorganism containing the nucleic acid molecule according to A1);

a6 A recombinant microorganism containing the expression cassette of A2);

a7 A recombinant microorganism containing the recombinant vector of A3);

a8 A recombinant microorganism containing the recombinant vector of A4).

In one embodiment, the nucleic acid molecule according to A1) has the sequence shown in SEQ ID NO. 2.

In one embodiment, the aforementioned method further comprises regulating the synthesis gene of cucurbitacin B and/or regulatory factors thereof.

In one embodiment, the synthetic gene for cucurbitacin B is cyclase CmBi, P450 enzyme, acetylase ACT, or the like.

In one embodiment, the regulatory factor of the cucurbitacin B synthetic gene is CmBr, cmBt, etc. The application also provides an application of the cucurbitacin B transporter protein in regulating and controlling the expression level of a cucurbitacin B synthetic gene Bi (CmBi), wherein the cucurbitacin B transporter protein is one of the following proteins:

a) The amino acid sequence is shown as SEQ ID NO. 1;

c) Proteins having 80% or more than 80% homology to a) or b);

The application also provides a method for regulating plant bitter taste, which comprises the step of regulating and controlling the expression amount and/or activity of a cucurbitacin B transporter in a plant, wherein the cucurbitacin B transporter is one of the following proteins:

a) The amino acid sequence is shown as SEQ ID NO. 1;

c) Proteins having 80% or more than 80% homology to a) or b);

In some embodiments, modulation is a decrease or an increase.

The application also provides application of the transport protein or the related biological materials thereof in plant breeding.

In some embodiments, breeding is to breed plants with increased bitterness, or with reduced bitterness, or without bitterness.

The application also provides a method for inhibiting phytopathogens, which comprises the step of transporting cucurbitacin B synthesized in plant cells to the outside of the cells by adopting the transport protein or the related biological materials.

In one embodiment, the plant pathogenic bacteria are bacterial pathogenic bacteria, fungal pathogenic bacteria, etc., which may be present in the rhizosphere (rhizosphere pathogenic bacteria) or phyllosphere (phyllosphere pathogenic bacteria) or tissues of the fruit, etc., and which may be inhibited by cucurbitacin B.

In one embodiment, the plant of the present application is a plant of the cucurbitaceae family, such as a plant of the cucumis genus, in particular, such as cucumis melo.

The application also provides application of the transport protein or the related biological materials thereof in regulating synthesis or transport of cucurbitacin B.

Drawings

FIG. 1 shows the cluster distribution of candidate transporters and cucurbitacin B synthetic genes.

FIG. 2 shows the expression of cucurbitacin B synthetic gene and candidate transporter gene in different materials and tissues.

FIG. 3 is the interaction relationship between the candidate transporter gene CmMATE and the cucurbitacin B regulatory gene; wherein, A: a yeast single-hybrid experiment; b: a tobacco dual-luciferase activation experiment; c: gel electrophoresis migration experiments.

FIG. 4, subcellular localization analysis of candidate transporter gene CmMATE in tobacco lamina.

Figure 5, subcellular localization analysis of candidate transporter gene CmMATE in cucumber protoplasts.

Figure 6, in situ hybridization assay analysis candidate transporter gene CmMATE in root expression distribution.

FIG. 7, cucurbitacin B is enriched in the culture solution of melon seedlings.

Figure 8, the use of yeast microsome vesicle system validation of the function of CmMATE.

Figure 9 validation of CmMATE function using oocyte expression system.

FIG. 10 screening for positive hairy roots for genetic transformation using GFP as a reporter gene.

FIG. 11, phenotypic analysis of CmMATE mutants obtained using Crispr-Cas9 technology; wherein, A: comparing the contents of cucurbitacin B in roots of wild type and mutant materials and in culture solution, and sequentially setting wild type and M in each group of bar charts from left to right _CR -1、M _CR -2、M _CR -3; b: comparing the CmBi gene expression level in the wild type and the mutant material.

FIG. 12, phenotypic analysis of hairy roots of melon after overexpression of CmMATE gene; wherein, A: analyzing the content of cucurbitacin B in roots and culture solution; b: analyzing the CmBi gene expression quantity; each set of histograms in panels A and B is wild, OE-1, OE-2, OE-3, in that order from left to right.

Detailed Description

In the following examples, specific tests are exemplified, and the experimental methods used are all conventional methods unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 transport protein mining and obtaining of cucurbitacin B in melon

1. Screening of candidate genes

Using comparative genomics analysis, a transporter gene, melo3C002190, annotated as the MATE family, was found on chromosome 12 of Cucumis Melo, in clusters with the cucurbitacin B synthetic gene (FIG. 1). Next, using transcriptome data of different tissues of wild melon material and cultivated melon, it was found that the expression pattern of the Melo3C002190 gene was highly consistent with that of cucurbitacin B synthetic gene DHO and CYP87D20 (fig. 2). This co-expression pattern further enhances the accuracy of the candidate genes. We named the candidate transporter gene mlo 3C002190 as CmMATE.

2. Interaction of regulatory factors CmBr and CmBt and candidate gene CmMATE

And (3) verifying the interaction condition of the bitter major regulatory factors CmBr and CmBt and the candidate transport protein CmMATE by using a yeast single-hybrid system, a tobacco dual-luciferase activation test and a gel electrophoresis migration test.

Yeast single-hybrid assay: the promoter region of the candidate gene CmMATE was amplified by PCR using primers, and the amplification products were constructed on bait vectors of pHIS2 using In-Fusion HD Cloning Kit (Clontech) homologous recombination Kit, respectively. The transcription factors CmBr and CmBt were cloned into pGADT7 vector separately using the same method. The constructed recombinant vectors pHIS2 and pGADT7 were then co-transformed into yeast strain AH109, and the control group co-transformed unloaded pGADT7 with recombinant vector pHIS2. The transformed yeast grows on solid culture media of leucine, tryptophan double-defect type (SD-Leu-Trp) and leucine, tryptophan and histidine triple-defect type (SD-Leu-Trp-His) respectively, and a certain amount of 3-AT (sigma) can be added into the triple-defect culture media to inhibit the background expression of histidine in a control group. After culturing at 30 ℃ for 4-5 days, the results were observed. The results show that the transcription factors CmBr and CmBt can bind directly to the promoter region of the CmMATE gene (fig. 3).

Tobacco dual luciferase activation assay: the transcription factors CmBr and CmBt are respectively constructed to the downstream region of the 35S promoter of the binary vector pCAMBIA1300 by utilizing a homologous recombination method (In-Fusion HD Cloning), namely an effector protein vector. The candidate transporter gene CmMATE is constructed on a pGreen II 0800-LUC vector by the same method, and the vector is the reporter gene vector. The constructed recombinant vectors are then transformed into Agrobacterium-infected GV3101, respectively. Tobacco was subjected to transient co-injection experiments with different combinations of effector proteins and reporter genes. The control group is unloaded pCAMBIA1300 and the constructed reporter gene vector. Three days after injection, samples were taken and assayed for Firefly luciferase activity (Firefly luciferase) and renilla luciferase activity (Renillia luciferase) using the dual luciferase reporter kit (Promega). The results indicate that the transcription factors CmBr and CmBt can directly activate expression of the CmMATE gene (fig. 3).

Gel electrophoresis migration test: the transcription factors CmBr and CmBt are respectively constructed on a prokaryotic expression vector pET32 a. The constructed recombinant plasmid is transformed into escherichia coli competence BL21 (DE 3), induced expression and purification are carried out to obtain target proteins CmBr and CmBt. Then the target proteins CmBr and CmBt are respectively incubated with a DNA probe of a candidate transport protein gene CmMATE marked by biotin and electrophoresed. The results show that the transcription factors CmBr and CmBt can bind directly to the promoter region of the CmMATE gene (fig. 3).

The results show that the transcription factors CmBr and CmBt can regulate and control CmMATE gene expression and are co-regulated with a cucurbitacin B synthetic gene, so that the candidate gene is further strengthened to be probably cucurbitacin B transport protein.

Example 2 subcellular localization of candidate transporters

For the transport protein, the positioning difference of different subcellular organelle membranes is closely related to the physiological function of the transport protein. A tobacco subcellular localization system, a cucumber protoplast subcellular localization system and an in-situ hybridization test are adopted to carry out subcellular localization analysis on the candidate transport protein CmMATE.

Tobacco subcellular localization: the candidate transporter CmMATE is constructed on a pSuper1300 vector, and forms a fusion protein CmMATE-GFP with GFP. Meanwhile, a known arabidopsis thaliana cytoplasmic membrane locator protein PIP2A is constructed on a pSuper1300 vector, and forms a fusion protein PIP2A-RFP together with RFP. The recombinant vectors were then transformed into Agrobacterium-infected competent cells GV3101, respectively. Picking single clone and culturing Agrobacterium to OD ₆₀₀ 0.6-0.8, mixing agrobacterium tumefaciens containing CmMATE-GFP and PIP2A-RFP respectively, and co-infecting the epidermis under the tobacco leaf. After 2-4 days of infection, fluorescence signals were observed using a confocal fluorescence microscope (Leica) and the GFP and RFP fluorescence signals were found to coincide (FIG. 4), indicating that CmMATE was localized to the plasma membrane.

Cucumber protoplast subcellular localization: the recombinant plasmids, cmMATE-GFP and PIP2A-RFP, were co-transformed into protoplasts using the PEG-mediated transient transformation method. The specific process comprises the following steps: mixing 15 mu g of recombinant plasmid and 200 mu L of protoplast suspension in a 2mL centrifuge tube, and reversing, gently and uniformly mixing; adding 200. Mu.L, 40% PEG4000 solution (40% PEG4000,0.15M Mannitol,100mM CaCl to the centrifuge tube ₂ ) The mixture is turned over gently and mixed evenly, and is kept stand for about 8 minutes; adding 1.0mL of W5 solution, stopping reaction, and gently mixing uniformly; at low temperature centrifugationCentrifuging for 6 minutes at 80-100 x g in the machine, discarding supernatant, and repeating once; the transformed protoplasts were resuspended in W5 solution and incubated under low light for 12-24 hours. The fluorescent signals were then observed and the GFP and RFP fluorescent signals were found to coincide (fig. 5), indicating that CmMATE was localized to the plasma membrane.

In situ hybridization assay: the test is divided into three main processes of probe preparation, sheet preparation of plant material tissues and hybridization and color development. And performing PCR amplification on the specific sequence of the CmMATE gene by using a primer. The amplification product was extracted using rnase-free phenol chloroform isoamyl alcohol (25. Subsequently, using the obtained DNA as a template, sense and antisense probes labeled with digoxin were prepared using an RNA transcription polymerase kit (Promega). Two days after germination of melon seeds, young roots were used as tissue samples for in situ hybridization experiments. Young roots were placed in 4% Paraformaldehyde (PFA) fixative and evacuated for 10 minutes until the tissue material settled at the bottom of the centrifuge tube, followed by replacement with fresh fixative and fixation overnight at 4 ℃. The material is dehydrated by different ethanol concentrations, transparent by different xylene concentrations, wax-dipped in a gradient manner, embedded by a sample and the like. The embedded samples were then sectioned for subsequent hybridization imaging experiments. Tissue sections were pre-treated with ethanol at different concentrations and PBS solution containing proteinase K. Then, re-immobilization and ethanol gradient dehydration were performed using PFA, and hybridization solution was prepared for hybridization. The hybridization solution comprises the following components: 1.25 XSalt, 50% formamide, 12.5% dextran sulfate, 1.25 XDenhardt's, and 1.5mg/mL tRNA. The prepared hybridization solution is incubated with the probe, followed by hybridization. After hybridization, two washes using 5 XSSC samples followed by a 20. Mu.g/mL RNase A treatment and a 0.2 XSSC wash were performed. Finally, the hybridization was carried out at room temperature for 2 hours using digoxin-labeled antibody (1 diluted 1000), and signal collection was carried out according to the development state. The results showed that CmMATE is expressed mainly in the epidermal cells of root tips and is identical to the CmBi expression site of the cucurbitacin B synthetic gene (fig. 6).

The above results demonstrate that the candidate transporter CmMATE is a cytoplasmic membrane localization protein and is expressed predominantly in root tip epidermal cells. Presumably, this protein mediates intracellular to extracellular transport of cucurbitacin B. To further verify, we cultured melon seedlings by hydroponics and found that the liquid medium was enriched with a large amount of cucurbitacin B (fig. 7).

Example 3 Biochemical functional validation of candidate Transporter proteins

The biochemical function of CmMATE is verified by using a yeast microsome vesicle system and a toad oocyte expression system.

Yeast microsomal vesicle system: the gene CmMATE was constructed into the yeast expression vector pYES 2. The constructed recombinant plasmid was then transformed into yeast strain INVSC 1. And (3) selecting a monoclonal, culturing at 30 ℃, and inducing expression of the CmMATE protein. After the yeast cells are broken, the yeast microsomes are obtained by density gradient centrifugation and incubated with cucurbitacin B. After vacuum filtration through a PVDF filter membrane, the surface of the microsome vesicle is cleaned. Finally, the content of the microsomal vesicular cucurbitacin B was determined, and the content of the microsomal vesicular cucurbitacin B containing CmMATE protein was significantly increased compared to the control (fig. 8), indicating that CmMATE is able to mediate cucurbitacin B transport.

Toad oocyte expression system: the gene CmMATE is constructed on an oocyte expression vector pGEMHE. cRNA preparation was then accomplished using the mMessage mMachine Transcription Kit (Invitrogen) in vitro Transcription Kit. Injecting cRNA into each oocyte, standing at 18 ℃ for 48 hours, and injecting cucurbitacin B into each oocyte after protein expression. After incubation for 1 hour, cucurbitacin B in oocyte culture solution was extracted and its content was determined. The content of cucurbitacin B in oocyte culture fluid containing CmMATE protein was significantly increased compared to the control (fig. 9), indicating that cucurbitacin B can be transported out of the cell by the CmMATE protein.

The above results demonstrate that CmMATE protein can directly transport cucurbitacin B.

Example 4 genetic functional verification of candidate Transporter proteins

To further validate the function of the candidate transporter CmMATE, we established an agrobacterium rhizogenes-mediated melon root system and screened genetically transformed positive roots using GFP as a reporter gene (fig. 1)0). Firstly, a gene editing technology of CRISPR-Cas9 is utilized to obtain CmMATE mutants of three muskmelon hairy roots, and the mutants are named as M _CR -1，M _CR -2，M _CR -3. Compared with wild materials, after the mutation function of the CmMATE gene is inactivated, the content of cucurbitacin B which is discharged outside the hairy roots of the melon to the outside of the roots is obviously reduced, and meanwhile, the expression level of a cucurbitacin B synthetic gene CmBi is also obviously reduced, so that the content of the whole cucurbitacin B in the roots of the melon is obviously reduced (figure 11). On the contrary, in the muskmelon hairy roots with the CmMATE gene over-expression (OE-1, OE-2 and OE-3), the ability of discharging cucurbitacin B outside is obviously increased, the cucurbitacin B content in the culture solution outside the roots is obviously accumulated, the expression level of the CmBi gene is also obviously improved, and the whole cucurbitacin B content in the muskmelon roots is obviously increased (figure 12). The results show that the CmMATE gene participates in the transportation of cucurbitacin B from cells in roots to the outside of the roots in melon plants, and the gene also has a regulating effect on the synthesis of cucurbitacin B.

While the present application has been described in considerable detail with reference to certain preferred versions thereof, it is not intended to limit the scope of the invention to the exact forms disclosed.

SEQUENCE LISTING

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Claims

1. A method for regulating synthesis or transport of cucurbitacin B, which is characterized in that: comprises the step of regulating and controlling the expression quantity or activity of cucurbitacin B transporter; the cucurbitacin B transporter is one of the following proteins:

a) The amino acid sequence is shown as SEQ ID NO. 1;

b) A fusion protein obtained by connecting a label to the N-terminal and/or C-terminal of a).

2. The method of claim 1, wherein: further comprising using a cucurbitacin B transporter-associated biomaterial being any one of the following A1) to A8):

a1 A nucleic acid molecule encoding the protein of claim 1;

a3 A recombinant vector containing the nucleic acid molecule according to A1);

a4 A recombinant vector containing the expression cassette of A2);

a6 A recombinant microorganism containing the expression cassette of A2);

a7 A recombinant microorganism containing the recombinant vector of A3);

a8 A recombinant microorganism containing the recombinant vector of A4).

3. The method of claim 2, wherein: a1 The sequence of the nucleic acid molecule is shown as SEQ ID NO. 2.

4. A method according to any one of claims 1 to 3, wherein: further comprises regulation and control of a cucurbitacin B synthetic gene and/or a regulatory factor of the cucurbitacin B synthetic gene.

5. The method of claim 4, wherein: the modulation is a decrease or an increase.

6. The application of the cucurbitacin B transporter in improving the expression level of a cucurbitacin B synthetic gene Bi is characterized in that the cucurbitacin B transporter is one of the following proteins:

a) The amino acid sequence is shown as SEQ ID NO. 1;

7. A method for regulating bitter taste of plants, which is characterized by comprising the following steps: the method comprises the step of regulating and controlling the expression quantity and/or activity of a cucurbitacin B transporter in a plant, wherein the cucurbitacin B transporter is one of the following proteins:

a) The amino acid sequence is shown as SEQ ID NO. 1;

b) A fusion protein obtained by connecting a label to the N end and/or the C end of a);

the plant is a melon.

8. The method of any one of claims 1-3,7, wherein: the modulation is a decrease or an increase.