阅读说明：本技术 调节植物类黄酮合成及紫外抗性的基因及其应用 (Gene for regulating plant flavonoid synthesis and ultraviolet resistance and application thereof ) 是由 刘宏涛 梁通 史辰 于 2018-09-10 设计创作，主要内容包括：本发明涉及调节植物类黄酮合成及紫外抗性的基因及其应用。首次研究及揭示了油菜素甾醇信号通路对植物体内类黄酮合成以及植物对紫外线的抗性的影响及其分子机制。油菜素甾醇信号通路,特别是其中的转录因子BES1,其能够负调控MYB转录因子,在此基础上调控植物体内类黄酮合成,并进而调控植物对紫外线的抗性。(The invention relates to a gene for regulating plant flavonoid synthesis and ultraviolet resistance and application thereof. The first research and the disclosure of the influence of brassinosteroid signal path on the synthesis of flavonoid in plants and the resistance of plants to ultraviolet rays and the molecular mechanism thereof. Brassinosteroid signalling pathways, particularly the transcription factor BES1 therein, are capable of negatively regulating MYB transcription factors, and on the basis of this regulate flavonoid synthesis in plants, and thus the resistance of plants to ultraviolet light.)

1. A method of modulating flavonoid synthesis in a plant or modulating plant resistance to ultraviolet light, comprising: regulating the transcription factor BES1 of brassinosteroid signaling pathway in plants, thereby regulating the synthesis of flavonoids in plants or the resistance of plants to ultraviolet rays; wherein BES1 includes homologues thereof.

2. The method of claim 1, wherein the method is selected from the group consisting of:

(a) downregulating BES1, thereby promoting flavonoid synthesis in plants or increasing plant resistance to uv light; or

(b) Up-regulating BES1, thereby inhibiting flavonoid synthesis in plants or reducing the resistance of plants to uv light.

3. The method of claim 1 or 2, wherein in (a) BES1 is down-regulated such that MYB transcription factor expression is increased, thereby promoting flavonoid synthesis in plants or increasing plant resistance to uv light; or

(b) BES1 was upregulated, resulting in reduced expression of MYB transcription factors, which in turn inhibited flavonoid synthesis in plants or reduced plant resistance to UV light;

preferably, the MYB transcription factors include MYB11, MYB12, MYB 111.

4. The method of claim 2, wherein in (a), down-regulating BES1 comprises: knocking out or silencing a gene encoding BES1, or inhibiting activity of BES1, in a plant; preferably, it comprises: BES1 is silenced by interfering molecules which specifically interfere with the expression of the coding gene of BES1, BES1 expression is inhibited by ultraviolet stress, gene editing is carried out by a CRISPR system so as to knock out the coding gene of BES1, or the coding gene of BES1 is knocked out by a homologous recombination method.

5. The method as claimed in claim 4, wherein the expression of BES1 for UV stress inhibition is UV stress with UV light having a wavelength of 280-300nm in BUV.

6. The method of claim 2, wherein in (b), the up-regulating BES1 comprises: transferring the gene encoding BES1 or an expression construct or vector containing the encoding gene into a plant; or BES1, preferably, the 698 th nucleotide of its coding region is mutated from C to T.

7. A method of modulating flavonoid synthesis in a plant or modulating plant resistance to ultraviolet light, comprising: the inhibition of MYB transcription factors by the transcription factor BES1 regulating brassinosteroid signalling pathways in plants regulates the synthesis of flavonoids or the resistance of plants to ultraviolet light in plants.

8. The method of claim 7, wherein the method is selected from the group consisting of:

(1) reducing the inhibitory effect of BES1 on MYB transcription factors, thereby promoting flavonoid synthesis in plants or improving ultraviolet resistance of plants; or

(2) Increasing the inhibitory effect of BES1 on MYB transcription factors, thereby inhibiting flavonoid synthesis in plants or reducing ultraviolet resistance of plants.

9. The method of claim 1, wherein said plant comprises: gramineae, cruciferae.

10. Use of the transcription factor BES1 of the brassinosteroid signalling pathway or a modulator thereof for modulating flavonoid synthesis in plants or modulating resistance of plants to uv light.

11. The use according to claim 10, wherein BES1 or its up-regulator inhibits flavonoid synthesis in plants or reduces uv resistance in plants;

the said BES1 down-regulator promotes the synthesis of flavonoids in plants or increases the resistance of plants to UV light.

12. Use of transcription factor BES1 of brassinosteroid signalling pathway as a molecular marker for identifying flavonoid synthesizing ability in plants or resistance of plants to ultraviolet light.

13. A method for the targeted selection of plants having enhanced or diminished flavonoid synthesizing capacity or plant resistance to ultraviolet light, said method comprising: identifying brassinosteroid signalling pathways in the test plants, in particular the expression of the transcription factor BES1 therein, which are plants with a strong flavonoid synthesizing capacity or plant resistance to uv light if the test plants have a BES1 expression lower than the average BES1 expression value of such plants; if the expression of BES1 of the test plant is higher than the average expression value of BES1 of the plants, the test plant is a plant with weak flavonoid synthesis capacity or plant resistance to ultraviolet rays.

14. A method for the targeted selection of plants having enhanced or diminished flavonoid synthesizing capacity or plant resistance to ultraviolet light, said method comprising: identifying inhibition of the MYB transcription factor by brassinosteroid signalling pathway transcription factor BES1 in the test plant, if inhibition of MYB transcription factor by BES1 in the test plant is lower than the average level of the test plant, the test plant is a plant with strong flavonoid synthesis capacity or plant resistance to ultraviolet rays; BES1 is a plant with weak flavonoid synthesizing ability or plant resistance to ultraviolet light if its inhibitory effect on MYB transcription factor is higher than the average level in this test plant.

15. A method of screening for a modulator that modulates flavonoid synthesis in plants or modulates uv light resistance in plants, comprising:

(1) adding the candidate substance to a system containing the brassinosteroid signalling pathway transcription factor BES1 in plants; preferably, the system comprises a brassinosteroid signalling pathway;

(2) detecting the expression or activity of the transcription factor BES1 in the system observed in (1); if the candidate substance inhibits the expression or activity of BES1, it is indicative that the candidate substance is a modulator that promotes flavonoid synthesis in plants or increases the resistance of plants to ultraviolet light; if the candidate substance increases the expression or activity of BES1, it is indicative that the candidate substance is a modulator that inhibits flavonoid synthesis in plants or reduces the resistance of plants to uv light.

16. A method of screening for a modulator that modulates flavonoid synthesis in plants or modulates uv light resistance in plants, comprising:

(a) adding the candidate substance to a system containing the brassinosteroid signalling pathway transcription factor BES1 and a MYB transcription factor in plants; preferably, the system comprises a brassinosteroid signalling pathway;

(b) observing the inhibition effect of BES1 on MYB transcription factors in the system;

wherein, if the candidate substance reduces the inhibitory effect of BES1 on MYB transcription factor, the candidate substance is a regulator for promoting flavonoid synthesis in plants or improving ultraviolet resistance of plants; if the candidate substance increases the inhibitory effect of BES1 on MYB transcription factors, it indicates that the candidate substance is a modulator that inhibits flavonoid synthesis in plants or reduces ultraviolet light resistance in plants.

17. The method of claim 16, wherein the MYB transcription factors comprise MYB11, MYB12, MYB 111; and/or

(b) Wherein said observing the inhibitory effect of BES1 on MYB transcription factors in said system comprises: the interaction of BES1 with the MYB gene promoter G-box was observed.

18. The method of claim 15 or 16, further comprising setting the control group and the test group to observe the difference between the candidate substance in the test group and the control group.

Technical Field

The invention belongs to the field of botany and molecular biology, and particularly relates to a gene for regulating plant flavonoid synthesis and ultraviolet resistance and application thereof.

Background

In the photosynthesis of plants, the main spectral regions capable of being utilized are a red light part with the wavelength of 640-660 nm and a blue light part with the wavelength of 400-500 nm. However, ultraviolet rays have no effect on photosynthesis of plants, and it is also considered that excessive ultraviolet irradiation affects the photosynthesis efficiency of plants in the prior art. During the evolution of plants, some mechanism for resisting ultraviolet stress is also obtained in vivo.

The ultraviolet UV-B (280-315nm) is a part of sunlight, can partially reach the ground surface and has important influence on the growth and development of plants. The UV-B can be used as a light signal to regulate the growth and development of plants, such as inhibiting elongation of hypocotyl, promoting accumulation of flavonoid and anthocyanin, inhibiting shade avoidance and high temperature response and the like; stress may also be imposed on the plant, such as damage to the chloroplast photosynthetic complex which inhibits photosynthesis, DNA absorption of UV-B which causes DNA damage, and the like.

As a solid growth organism, the plant is not only resistant to ultraviolet stress, but also capable of growth and development. There are relatively few studies on this aspect, and there are also few genes available for molecular design breeding. MYB transcripts have been shown to play a role in plant stress tolerance. MYB transcription factors can directly control the expression of flavonol synthesis genes, thereby promoting the accumulation of flavonoids. The flavonoid in the plant body can absorb UV-B, and can be used as a sunscreen cream to protect plant cells and enhance the resistance of the plant to ultraviolet stress. The flavonoid not only protects plants and resists ultraviolet stress, but also has important effects in human health and industrial production, has anti-inflammatory, antioxidant and anticancer effects, and can also be used as a natural product for food processing.

However, studies on UV-B induced flavonoid accumulation and resistance to UV stress are not well understood, and at least the following problems remain to be solved: 1. in the absence of UV-B, MYB gene expression is low, accumulation of flavonoids is low, and therefore how can a plant inhibit MYB expression in a normal growth state? Has found that the presence of positive regulatory factors promotes MYB gene expression, and does the presence of other factors regulate MYB gene expression? 2. How are plants balanced for normal growth and development and against uv stress? Therefore, the field needs to research these problems, and find that more genes regulating MYB are beneficial to finely and directionally regulating flavonoid accumulation; the molecular mechanism of plant balanced growth and development and ultraviolet stress resistance is clarified, and the method is favorable for cultivating high-quality varieties which resist ultraviolet stress and give consideration to yield.

Disclosure of Invention

The invention aims to provide a gene for regulating plant flavonoid synthesis and ultraviolet resistance and application thereof.

In a first aspect of the invention, there is provided a method of modulating flavonoid synthesis in a plant or modulating resistance of a plant to ultraviolet light, the method comprising: the transcription factor BES1 regulating Brassinosteroid (BR) signaling pathway in plants, thereby regulating (negatively regulating) flavonoid synthesis in plants or ultraviolet resistance of plants; wherein BES1 includes homologues thereof. Such homologues include homologous polypeptides or genes in a number of species, such as BES1 in Arabidopsis or maize, OsBZR1 homolog in rice.

In a preferred embodiment, the method is selected from: (a) downregulating BES1, thereby promoting flavonoid synthesis in plants or increasing plant resistance to uv light; or (b) up-regulating BES1, thereby inhibiting flavonoid synthesis in plants or reducing uv resistance in plants.

In another preferred example, (a) BES1 is down-regulated such that MYB transcription factor expression is increased, thereby promoting flavonoid synthesis in plants or increasing the resistance of plants to uv light; in (a) or (b), BES1 is up-regulated, resulting in reduced expression of MYB transcription factors, which in turn inhibit flavonoid synthesis in plants or reduce the resistance of plants to uv light; preferably, the MYB transcription factors include MYB11, MYB12, MYB 111.

In another preferred example, BES1 regulates MYB transcription factor expression by binding to MYB gene promoter G-box.

In another preferred embodiment, in (a), down-regulating BES1 comprises: knocking out or silencing a gene encoding BES1, or inhibiting activity of BES1, in a plant; preferably, including (but not limited to): BES1 is silenced by interfering molecules which specifically interfere with the expression of the coding gene of BES1, BES1 expression is inhibited by ultraviolet stress, gene editing is carried out by a CRISPR system so as to knock out the coding gene of BES1, or the coding gene of BES1 is knocked out by a homologous recombination method.

In another preferred example, the expression of the UV stress inhibition BES1 is UV stress performed by UV rays with the wavelength of 280-300nm in BUV.

In another preferred embodiment, the interfering molecule is dsRNA, antisense nucleic acid, small interfering RNA, microrna, or a construct capable of expressing or forming said dsRNA, antisense nucleic acid, small interfering RNA, microrna, or a transcript thereof that encodes BES1 as a target for inhibition or silencing; preferably, the interfering molecule is dsRNA constructed from the BES1cDNA sequence.

In another preferred embodiment, (b), the upregulated BES1 includes (but is not limited to): transferring the gene encoding BES1 or an expression construct or vector containing the encoding gene into a plant; or BES1, preferably, the 698 th nucleotide of its coding region is mutated from C to T.

In another aspect of the present invention, there is provided a method of modulating flavonoid synthesis in a plant or modulating resistance of a plant to ultraviolet light, the method comprising: the inhibition of MYB transcription factors by the transcription factor BES1, which regulates the brassinosteroid signalling pathway in plants, regulates (negatively regulates) the synthesis of flavonoids in plants or the resistance of plants to ultraviolet light.

In a preferred embodiment, the method is selected from: (1) reducing the inhibition of BES1 on MYB transcription factors, thereby promoting flavonoid synthesis in plants or improving ultraviolet resistance of plants; or (2) increasing the inhibitory effect of BES1 on MYB transcription factors, thereby inhibiting flavonoid synthesis in plants or reducing ultraviolet resistance in plants.

In a preferred embodiment, the plant includes (but is not limited to): gramineae, cruciferae.

In another aspect of the present invention, there is provided the use of the transcription factor BES1 of the brassinosteroid signalling pathway or a modulator thereof for modulating flavonoid synthesis in plants or modulating the resistance of plants to uv light.

In a preferred embodiment, the BES1 or the up-regulator thereof inhibits the synthesis of flavonoids in plants or reduces the resistance of plants to ultraviolet rays; the said BES1 down-regulator promotes the synthesis of flavonoids in plants or increases the resistance of plants to UV light.

In another aspect of the present invention, there is provided the use of the transcription factor BES1 of the brassinosteroid signalling pathway as a molecular marker for the identification of flavonoid synthesizing ability in plants or the resistance of plants to ultraviolet light.

In another aspect of the present invention, there is provided a method for the targeted selection of plants having enhanced or reduced flavonoid synthesizing ability or plant resistance to ultraviolet light, the method comprising: identifying a brassinosteroid signalling pathway in a test plant, in particular the expression of the transcription factor BES1 therein, which is (potentially) a plant with a high flavonoid synthesis capacity or plant resistance to uv light if the test plant has a BES1 expression that is lower than the average BES1 expression value of the plant(s); if the test plant has a BES1 expression value that is higher than the average BES1 expression value of the plant (or plants), then it is (potentially) a plant with weak flavonoid synthesizing ability or plant resistance to ultraviolet light.

In another aspect of the present invention, there is provided a method for the targeted selection of plants having enhanced or reduced flavonoid synthesizing ability or plant resistance to ultraviolet light, the method comprising: identifying inhibition of MYB transcription factor by brassinosteroid signalling pathway transcription factor BES1 in the test plant, if inhibition of MYB transcription factor by BES1 in the test plant is lower than the average level in the plant (or the plant), then it is a plant with strong flavonoid synthesizing ability or plant resistance to ultraviolet light; BES1 is a plant with weak flavonoid synthesizing ability or plant resistance to ultraviolet light if its inhibitory effect on MYB transcription factor in the test plant is higher than the average level of the plant(s).

In another aspect of the present invention, there is provided a method of screening for a modulator that modulates flavonoid synthesis in plants or modulates resistance of plants to ultraviolet light, the method comprising: (1) adding the candidate substance to a system containing the brassinosteroid signalling pathway transcription factor BES1 in plants; preferably, the system comprises a brassinosteroid signalling pathway; (2) detecting the expression or activity of the transcription factor BES1 in the system observed in (1) in the system; if the candidate substance inhibits (preferably statistically inhibits; e.g., decreases by more than 20%, preferably by more than 50%, more preferably by more than 80%) the expression or activity of BES1, then the candidate substance is an agent that promotes flavonoid synthesis in plants or increases the resistance of plants to ultraviolet light; if the candidate substance increases (preferably statistically, e.g., promotes more than 20%, preferably more than 50%, more preferably more than 80%) the expression or activity of BES1, then the candidate substance is an agent that inhibits flavonoid synthesis in plants or reduces ultraviolet light resistance in plants.

In another aspect of the present invention, there is provided a method of screening for a modulator that modulates flavonoid synthesis in plants or modulates resistance of plants to ultraviolet light, the method comprising: (a) adding the candidate substance to a system containing the brassinosteroid signalling pathway transcription factor BES1 and a MYB transcription factor in plants; preferably, the system comprises a brassinosteroid signalling pathway; (b) observing the inhibition effect of BES1 on MYB transcription factors in the system; wherein a decrease (preferably a statistical decrease; e.g., a decrease of greater than 20%, preferably a decrease of greater than 50%, more preferably a decrease of greater than 80%) in the inhibition of MYB transcription factor by BES1 in said candidate substance indicates that the candidate substance is a modulator that promotes flavonoid synthesis in a plant or increases ultraviolet light resistance in a plant; if the candidate substance increases (preferably statistically increases; e.g., promotes greater than 20%, preferably greater than 50%, more preferably greater than 80%) the inhibitory effect of BES1 on MYB transcription factors, then the candidate substance is an agent that inhibits flavonoid synthesis in plants or reduces ultraviolet light resistance in plants.

In a preferred embodiment, the MYB transcription factors include MYB11, MYB12, MYB 111; and/or (b), wherein the observation of the inhibitory effect of BES1 on MYB transcription factors in the system comprises: the interaction (binding) of BES1 with the MYB gene promoter G-box was observed.

In another preferred embodiment, the method further comprises setting the control group and the test group to observe the difference between the candidate substance in the test group and the control group.

In another preferred embodiment, the candidate substance includes (but is not limited to): brassinosteroid signaling pathways, particularly the transcription factor BES1, or interfering molecules designed from their upstream or downstream proteins, nucleic acid inhibitors, binding molecules (e.g., antibodies or ligands), small molecule compounds (e.g., hormones), and the like.

In another preferred embodiment, the system is selected from: cell system (cell culture system), subcellular system, solution system, plant tissue system, and plant organ system.

In another preferred example, the method further comprises: further cell experiments and/or transgenic experiments are carried out on the obtained potential substances, so as to further determine substances with excellent effects of regulating flavonoid synthesis in plants or regulating the resistance of the plants to ultraviolet rays from the candidate substances.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1a, c, e, Arabidopsis ultraviolet stress phenotype observations: arabidopsis thaliana species of the corresponding genotype in the figure were grown for 10 days under long-day white light, treated with broad band UV-B (Philips TL 40W/12 UV lamp, intensity 2W/m2) for 8 hours, and then returned to white light for 3 days to resume growth, and the phenotype was observed.

FIG. 1b, d, f, Arabidopsis maximum photosynthetic conversion efficiency Fv/Fm assay: the seeds of Arabidopsis thaliana were sown in the soil, grown for 10 days under long-day white light, treated with broad band UV-B (Philips TL 40W/12 UV lamp, light intensity 2W/m2) for 5 hours, and then measured for maximum photosynthetic conversion efficiency Fv/Fm with a photographic type fluorometer the next day.

FIG. 2a, Quantitative RT-PCR: col and BES1-RNAi were planted in soil, grown for 7 days in white light, sprayed with 1. mu.M BR once a day, transferred to the place under the broad band UV-B for 3 hours, samples were collected at the time points indicated in the figure, and PFG MYB gene expression was analyzed. ACT7 is a reference, labeled as the standard error of three biological replicates.

FIG. 2b, Quantitative RT-PCR: col and bes1-D-OX were grown in 1/2MS medium containing 1. mu.M BRZ for 7 days under white light, transferred to broad band UV-B for 3 hours, samples were taken at the time points indicated in the figure, and PFG MYB gene expression was analyzed. ACT7 is a reference, labeled as the standard error of three biological replicates.

FIG. 2c, HPLC assay of plant flavonoid content: arabidopsis thaliana of the corresponding genotype was grown in 1/2MS medium for 6 days under continuous white light conditions, transferred to a bouad band UV-B for two days and 0.1g each was collected, triturated and dissolved in 80% acetonitrile solution, vortexed and allowed to stand overnight at 4 ℃. After 12000 g centrifugation, the supernatant was quantified for flavonols by HPLC/DAD (Agilent 1260). HPLC analysis was performed using a C18 column (2.7X 100mm Agilent) with a flow rate of 0.8mlmin-1, an elution gradient of solution A [ water ], solution B [ methanol ] and solution D [ 0.5% formic acid ] following the following procedure (0 to 10 min, solutions B and D from 20 to 40% and 80 to 60%, respectively, 10 to 15 min, solutions B and D from 40 to 70% and 60 to 30, respectively, 15 to 20 min, solutions B and D from 70 to 90% and 30 to 10%, respectively.) DAD was used to detect UV absorption at 320 nm.

Figure 2d, DPBA staining reflects plant flavonol content: plants were grown under continuous white light for 6 days and transferred to broadband UV-B for 1 day of treatment. The treated plants were soaked in an ethanol solution containing 0.01% Triton X-100, 2.52mg/mL DPBA, rotated on a shaker for 1.5 hours, and then washed with deionized water. The fluorescence intensity at 458nm of the excitation light was observed by a fluorescence microscope. Fluorescence intensity was quantified using ImageJ.

Fig. 3a, EMSA experiment: the prokaryotically expressed BES1 protein was able to bind the PFG MYB promoter in vitro.

FIGS. 3b-d, ChIP experiments: the BES1 protein in plants was able to bind to the PFG MYB promoter. The upper part is the assay PFGMYB promoter, the red sphere represents the G-box, and the blue sphere represents the BRRE element. The ChIP-Q-PCR results are shown in the lower part. Using wild type and BES1 overexpression of BES1-Flag as material, crosslinking was preceded by treatment with 1. mu.M BR for 2 hours. The Flag antibody was used for co-immunoprecipitation and the primers covering the PFG MYB promoter were used for Q-PCR.

FIG. 3e, ChIP-Q-PCR: wild-type and BES1-RNAi were used as materials, BES1 antibody was used for co-immunoprecipitation, and Q-PCR was performed using primers representative of the PFG MYB promoter.

FIG. 3f, ChIP-Q-PCR: using wild type and BES1 overexpression BES1-Flag as material, BES1-Flag was treated with BR and BRZ, respectively, and Q-PCR was performed using primers representative of the PFG MYB promoter.

FIG. 3g-i, transient transcriptional activation assay: g, report vector and effector vector construction mode. h-i, mixing the agrobacterium transferred to the report vector with the agrobacterium transferred to the effect vector, transforming the tobacco, and detecting after three days. The graph h is the LUC/REN ratio measured, and the graph i is the LUC fluorescence photographed by CCD.

Fig. 4a, DPBA staining determination of flavonol content: plants were grown under continuous white light for 6 days and transferred to brood bandUV-B for 1 day of treatment. The treated plants were soaked in an ethanol solution containing 0.01% Triton X-100, 2.52mg/mL DPBA, rotated on a shaker for 1.5 hours, and then washed with deionized water. The fluorescence intensity at 458nm of the excitation light was observed by a fluorescence microscope. Fluorescence intensity was quantified using ImageJ.

FIG. 4b, Arabidopsis ultraviolet stress phenotype observations: arabidopsis thaliana species of the corresponding genotype in the figure were grown for 10 days under long-day white light, treated with broad band UV-B (Philips TL 40W/12 UV lamp, 2W/m2 light intensity) for 8 hours, and then returned to white light for 3 days of growth to observe the phenotype.

FIG. 4c, Arabidopsis maximum photosynthetic conversion efficiency Fv/Fm assay: arabidopsis seeds were sown in soil, grown for 10 days under long-day white light, treated with broad band UV-B (Philips TL 40W/12 UV lamp, light intensity 2W/m2) for 5 hours, and then measured the maximum photosynthetic conversion efficiency Fv/Fm the next day with a photographic fluorometer.

FIG. 5a, Western blot: wild type Arabidopsis thaliana Col species were grown for 7 days under continuous white light and then moved to a broad band UV-B, samples were taken at the time points in the figure and detected with BES1 antibody or Actin antibody.

FIG. 5b, Quantitative RT-PCR: col was grown in soil for 7 days under white light, transferred to broadband UV-B for 8 hours, and samples were collected at the time points indicated in the figure to analyze the expression of the BES1 gene. ACT7 is an internal reference, labeled as the standard error of three biological replicates.

FIGS. 5c-e, luciferase Pictures experiments: the BES1 promoter is used for driving and expressing luciferase transgenic material pBES1: LUC, and four conditional light treatments are respectively carried out: narrow band UV-B (NUV), broad band UV-B (BUV), broad band UV-B plus ZJB300 filters to cut off light below 300nm (BUV + ZJB300), broad band UV-B plus ZJB340 filters to cut off light below 340nm (BUV + ZJB 340). Fig. 5c is a photograph of pBES1 showing LUC signals of LUC under four light treatments, fig. 5d is a spectrum corresponding to four light conditions, and fig. 5e is a quantitative value of LUC in fig. 5 c.

FIG. 6a, corn UV stress phenotype observations: synthetic BR mutant na1 in maize and the corresponding wild type were grown for 8 days at 28 ℃ under long day white light, treated with broad band UV-B for 3 days, treated 2 hours per day, then placed back under white light to recover growth for 3 days and observed for phenotype.

FIG. 6b, maximum photosynthetic conversion efficiency Fv/Fm determination of maize: BR synthesis mutant na1 in maize and the corresponding wild type were grown for 8 days at 28 ℃ in long day white light, treated with broad band UV-B for 12 hours, and then the next day the maximum photosynthetic conversion efficiency Fv/Fm was measured with a photographic fluorometer.

FIG. 6c, observation of rice UV-stressed phenotype: BR receptor mutant d61 and the corresponding wild type in rice were grown for 25 days at 28 ℃ under long day white light, treated with broad band UV-B for 12 hours, and then returned to white light for 3 days to restore growth and observe the phenotype.

FIG. 6d, rice maximum photosynthetic conversion efficiency Fv/Fm determination: BR receptor mutant d61 in rice and the corresponding wild type were grown for 25 days at 28 ℃ in long day white light, treated with broad band UV-B for 12 hours, and then the maximum photosynthetic conversion efficiency Fv/Fm was measured the next day with a photographic fluorometer.

Detailed Description

The invention researches and discloses the influence of Brassinosteroid (BR) signal channels on flavonoid synthesis in plants and ultraviolet resistance of plants and a molecular mechanism thereof for the first time. Brassinosteroid signalling pathways, particularly the transcription factor BES1(BRI1-EMS-SUPPRESSOR 1) therein, are capable of negatively regulating MYB transcription factors, and on the basis thereof, regulating flavonoid synthesis in plants and thereby regulating the resistance of plants to ultraviolet light.

Genes, polypeptides, signal pathways and plants

As used herein, a "plant" is a plant suitable for transgenic manipulation, and may be a dicot, monocot or gymnosperm; may include crops, flower plants or forestry plants, etc. Preferably, the "plant" includes (but is not limited to): arabidopsis genus of Brassicaceae such as Arabidopsis thaliana; gramineous Oryza plants such as rice, gramineous Triticum plants such as wheat, gramineous Zea plants such as corn, etc.; chinese cabbage and pakchoi of Brassicaceae Brassica; malvaceae cotton crop, etc.

As used herein, the "Brassinosterol (BR) signaling pathway," also known as "brassinosterol cell signal transduction pathway," is characterized by increased function when phosphorylated BKI1 is detected by cell membrane surface receptor kinase BRI1 after Brassinosterol (BR) is sensed, phosphorylated BKI1 dissociates from BRI1, BRI1 interacts with BAK1 for phosphorylation. BRI1 phosphorylates BSKs and CDG, activating them. BSKs in turn phosphorylate and activate BSU1, BSU1 dephosphorylate BIN 2. The dephosphorylated BIN2 function was inhibited, thereby dephosphorylating BES1/BZR 1. Dephosphorylated BES1 is an activated form that can enrich for and bind DNA into the nucleus, activating BR-responsive gene expression. And a series of downstream events occur. Among them, BR is a known plant hormone.

In the present invention, unless otherwise specified, the related polypeptides of brassinosteroid signaling pathway or genes encoding the same (including BES1), MYB transcription factors (including MYB11, MYB12, MYB111) or genes encoding the same, and their related upstream and downstream polypeptides or genes include: the signal pathways, polypeptides or genes from a particular species listed in the examples of the invention, as well as corresponding signal pathways, homologous polypeptides or homologous genes from other species. For example, BES1 refers to a polypeptide encoded by the sequence having SEQ ID NO:1 (CDS sequence, Gene ID: 838518; AT1G19350.1), and includes variants of the sequence having the same function as the BES1 polypeptide. These variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, still more preferably 1 to 8, 1 to 5) amino acids, and addition or deletion of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. Any protein having high homology to the BES1 polypeptide (e.g., 70% or more homology to the polypeptide sequence encoded by the CDS sequence shown in SEQ ID NO: 1; preferably 80% or more homology; more preferably 90% or more homology, e.g., 95%, 98% or 99%) and having the same function as the BES1 polypeptide is also included in the present invention. Polypeptides from species other than Arabidopsis thaliana that have a high homology with the polypeptide sequence encoded by the CDS sequence of SEQ ID NO. 1 or that exert the same or similar effect in the same or similar signaling pathway are also included in the present invention.

It is to be understood that while the brassinosteroid signaling pathway (including BES1) and MYB transcription factors obtained from a particular species are preferably studied in the present invention, other polypeptides or genes obtained from other species that are highly homologous (e.g., have greater than 60%, such as 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) to said brassinosteroid signaling pathway or said MYB transcription factor are also within the contemplation of the present invention.

Method for improving plants

The invention discloses the influence of brassinosteroid signal pathways on flavonoid synthesis in plants and resistance of plants to ultraviolet rays and the molecular mechanism thereof, and has important application value in theoretical research and plant improvement. Accordingly, based on the new findings of the present inventors, the present invention provides a method for improving a plant, the method comprising: the transcription factor BES1 regulating Brassinosteroid (BR) signal channel in plant, and thus regulating the synthesis of flavonoid or ultraviolet ray resistance of plant. Or, the method comprises: the inhibition (binding) of the transcription factor BES1, which regulates the brassinosteroid signaling pathway in plants, to MYB transcription factors, in turn regulates (negatively regulates) the synthesis of flavonoids in plants or the resistance of plants to ultraviolet light.

In one aspect, the present invention provides a method for promoting flavonoid synthesis in plants or increasing ultraviolet light resistance in plants, comprising: reducing expression (including non-expression or low expression of BES1) or activity of brassinosteroid signaling pathway transcript BES1 in said plant; or reducing the inhibitory effect of BES1 on MYB transcription factors.

It is understood that, knowing the use of the brassinosterol signaling pathway, particularly the transcription factor BES1, and its mechanism of interaction with MYB transcription factors, various methods well known to those skilled in the art may be employed to modulate the expression of the BES1 or to modulate the strength of the interaction. For example, various methods known to those skilled in the art may be used to reduce or delete expression of BES1, such as delivering an expression unit (e.g., an expression vector or virus, etc.) carrying the antisense BES1 gene to a target such that the cells or plant tissues do not express or have reduced expression of BES 1.

As an embodiment of the present invention, there is provided a method of reducing expression of BES1 in a plant, the method comprising: (1) transferring an interfering molecule that interferes with the expression of BES1 into a plant cell, tissue, organ or seed to obtain a plant cell, tissue, organ or seed transformed with the interfering molecule; (2) regenerating the plant cell, tissue, organ or seed transformed with the interfering molecule obtained in step (1) into a plant. Preferably, the method further comprises: (3) selecting a plant cell, tissue or organ into which said vector has been transferred; and (4) regenerating the plant cell, tissue or organ from step (3) into a plant. For example, in a preferred embodiment of the present invention, a method for the preparation of a reagent for BES1-RNAi is provided, which allows to efficiently obtain plants with reduced expression of BES 1. Furthermore, the expression or activity of BES1 can also be down-regulated by methods of knock-out, silencing or gene editing (e.g., based on CRISPR systems).

In another aspect, the present invention provides a method of reducing flavonoid synthesis in a plant or reducing resistance of a plant to ultraviolet light, comprising: increasing expression (including overexpression of BES1) or activity of the brassinosteroid signaling pathway transcription factor BES1 in said plant; or, promote the interaction of BES1 with MYB transcription factors.

Knowing the use of the brassinosteroid signalling pathway, in particular the transcription factor BES1, various methods well known to those skilled in the art can be used to modulate the expression of the BES1 or to screen for substances that promote the interaction of BES1 with MYB transcription factors.

Applications of

In the invention, the BR signal channel is disclosed for the first time to inhibit the expression of MYB transcription factors and inhibit the synthesis of flavonoids through BES1, so that the resistance of ultraviolet stress is negatively regulated. In addition, the high-energy and low-waveband ultraviolet rays are disclosed for the first time to be capable of inhibiting the transcription of BES1, so that the inhibition on flavonoid synthesis is relieved, and the ultraviolet stress resistance of plants is enhanced. The technical scheme of the invention is beneficial to molecular design breeding, provides a theoretical basis for cultivating high-quality varieties which can resist ultraviolet stress and give consideration to yield, and can be applied to medicinal plants to improve accumulation of flavonoids.

According to the disclosure of the invention, combined with previous research of the inventor and the prior art, a new mechanism which gives consideration to ultraviolet stress resistance and vegetative growth of plants is presented: BES1 inhibits flavonoid accumulation and promotes vegetative growth in the absence of UV stress; in the presence of ultraviolet stress, BES1 transcription was inhibited, thereby relieving the inhibition of flavonoid synthesis and promoting plants against ultraviolet stress. BES1 mutant is known to exhibit reduced hypocotyl or plant height and enhanced UV stress resistance. BES1 is known to serve as a junction point for balancing vegetative growth and ultraviolet stress resistance and can be used for molecular design breeding.

According to the disclosure of the invention, firstly, BES1 inhibits MYB gene expression and regulates flavonoid synthesis, which can be applied to medicinal plants or functional crops, improves flavonoid metabolism and cultivates varieties with enriched flavonoid content. Secondly, the ultraviolet stress resistance of the plants can be enhanced by inhibiting a BR signal channel, and the ultraviolet stress resistant varieties can be cultivated by molecular design breeding based on the ultraviolet stress resistance. Thirdly, the transcription of BES1 can be specifically inhibited by low-band and high-energy ultraviolet rays, and a molecular switch can be designed on the basis of the inhibition, and gene expression can be started by the low-band and high-energy ultraviolet rays.

Screening method

After knowing the brassinosteroid signalling pathway, the effect on flavonoid synthesis in plants and plant resistance to ultraviolet light and their molecular mechanisms, one can screen for substances or potential substances that can directionally regulate flavonoid synthesis in plants and plant resistance to ultraviolet light by modulating this signalling pathway, particularly BES1, based on this new finding.

Accordingly, the present invention provides a method of screening for a modulator that modulates flavonoid synthesis in plants or modulates resistance of plants to ultraviolet light, the method comprising: selecting a substance which specifically regulates (inhibits) the brassinosteroid signal pathway transcription factor BES1 in plants by taking the brassinosteroid signal pathway transcription factor BES1 in the plants as a screening target point, wherein the substance is a regulator for promoting the synthesis of flavonoid in the plants or improving the resistance of the plants to ultraviolet rays; alternatively, a substance is selected which specifically up-regulates (increases) brassinosteroid signalling pathway transcription factor BES1 in plants, said substance being a modulator which inhibits the synthesis of flavonoids in plants or reduces the resistance of plants to UV light.

The present invention provides a method of screening for a modulator that modulates flavonoid synthesis in plants or modulates resistance of plants to ultraviolet light, the method comprising: selecting a substance which specifically regulates (inhibits) the inhibitory action of a brassinosteroid signal pathway transcription factor BES1 on MYB transcription factors in plants by taking the inhibitory action (combination) of the brassinosteroid signal pathway transcription factor BES1 on the MYB transcription factors as a screening target, wherein the substance is a regulator for promoting the synthesis of flavonoids in plants or improving the resistance of the plants to ultraviolet rays; alternatively, a substance is selected that specifically upregulates this inhibitory effect, said substance being a modulator that inhibits flavonoid synthesis in plants or reduces the resistance of plants to ultraviolet light.

Methods for targeting proteins or specific regions thereof to screen for substances that act on the target are well known to those skilled in the art and all of these methods can be used in the present invention. The candidate substance may be selected from: peptides, polymeric peptides, peptidomimetics, non-peptidic compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, nucleic acid sequences, and the like. Depending on the kind of substance to be screened, it is clear to the skilled person how to select a suitable screening method.

In the present invention, the interaction between proteins and the strength of the interaction can be detected by various techniques known to those skilled in the art, such as GST-sink technique (GST-Pull Down), phage display technique, yeast two-hybrid system or co-immunoprecipitation technique.

Through large-scale screening, a class of potential substances which specifically act on brassinosteroid signal pathways, particularly BES1, or act on a BES1 and MYB transcription factor interaction complex and have a regulating effect on flavonoid synthesis or metabolism can be obtained.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally according to conventional conditions, such as those described in J. SammBruk et al, molecular cloning experiments, south, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Experimental Material

BES1 knockdown BES1-RNAi plants: amplifying cDNA containing a BES1 coding region, connecting BES1cDNA to two multiple cloning sites of a pHANNIBAL vector in opposite directions by using an enzyme digestion connection method, connecting a promoter and a downstream expression sequence of the vector to a plant expression vector, transforming a wild type Arabidopsis thaliana Col, and selecting a BES1-RNAi transgenic material;

BES1 gain-of-function point mutations BES1-D and BES 1-D-OX: the functional point mutation of BES1 is known to be the mutation of 698 th nucleotide in the coding region from C to T. BES1 was amplified using primers containing this point mutation, clone BES1-D was cloned, and then expression BES1-D was driven using the BES1 endogenous promoter to obtain BES1-D transgenic plants. Expression was driven by the 35S promoter to bes1-D, resulting in bes1-D-OX transgenic plants.

3. Transgenic plant BES 1-Flag: the CDS sequence of the coding region of BES1 is cloned, the 35S promoter is used for driving expression of BES1, and a Flag tag is fused, the Arabidopsis wild type Col is transformed, and a 35S-BES1-Flag transgenic plant is obtained.

4. Transgenic plant pBES1: LUC: the BES1 promoter is cloned to drive and express the firefly luciferase LUC coding gene, and the Arabidopsis wild Col is transformed to obtain a pBES1 LUC transgenic plant.

5. Arabidopsis BR synthetic mutant det 2: after the Arabidopsis thaliana wild type seed Col is subjected to EMS mutagenesis, a mutant which is subjected to continuous photomorphogenesis in the dark is screened and named as det2, and then map-based cloning is carried out to determine a gene. The det2 gene is mutated, and the mutant can be reproduced by a CRISPR method.

6. Arabidopsis BR receptor mutant BR 1: after the Arabidopsis thaliana wild type seed Col is subjected to EMS mutagenesis, a BR insensitive mutant is screened and named as bri1, and then the gene is determined by map-based cloning. The BR receptor of the mutant is mutated, and the mutant can be further reproduced by a CRISPR method.

7. Arabidopsis PFG MYB triple mutant MYB11 MYB12 MYB 111: three T-DNA mutants, SALK-077068, MYB12-1f, GABI-Kat291D01 (these three materials were purchased from Arabidopsis center for biological resources ABRC), inserted into PFG MYB, were separately cross-polymerized to identify pure and triple mutations.

8. Maize BR synthetic mutant na1 was purchased from Maize COOP.

9. Rice BR receptor mutant d 61: the rice is mutagenized by N-methyl-N-nisourea, a dwarf mutant is screened and named as d61, the phenotypic analysis suggests that the dwarf mutant is a BR signal mutant, the BR receptor gene mutation is further analyzed and found, and the d61 is proved to be a BR receptor mutant through complementary verification. Further the mutants can be reproduced with the CRISPR method.

10, uvr 8: purchased from the arabidopsis thaliana bio-resource center ABRC.

Experimental methods

1. Ultraviolet stress phenotype observation

The seeds of Arabidopsis thaliana were sown in the soil, grown for 10 days under sunshine white light, treated with Broad band UV-B (Philips TL 40W/12 UV lamp tube with light intensity of 2W/m2) for 8 hours, and then returned to white light to recover the growth for 3 days, and the phenotype was observed.

2. Measurement of maximum photosynthetic conversion efficiency Fv/Fm of plant

Arabidopsis seeds were sown in soil, grown for 10 days under long-day white light, treated with broad band UV-B (Philips TL 40W/12 UV lamp, light intensity 2W/m2) for 5 hours, and then the photosynthetic parameters of the plants were measured the next day with a photographic fluorometer.

HPLC analysis

Arabidopsis thaliana of the corresponding genotype was grown in 1/2MS medium for 6 days under continuous white light conditions, transferred to a broadband UV-B for two days and 0.1g each sample was collected, triturated and dissolved in 80% acetonitrile solution, vortexed and allowed to stand overnight at 4 ℃. After 12000 g centrifugation, the supernatant was subjected to HPLC/DAD (Agilent1260) to quantitate the flavonols. HPLC analysis was performed using a C18 column (2.7X 100mm Agilent) with a flow rate of 0.8ml min-1, an elution gradient of solution A [ water ], solution B [ methanol ] and solution D [ 0.5% formic acid ] following the procedure (0 to 10 min, solutions B and D from 20 to 40% and 80 to 60%, respectively, 10 to 15 min, solutions B and D from 40 to 70% and 60 to 30, respectively, 15 to 20 min, solutions B and D from 70 to 90% and 30 to 10%, respectively.) DAD was used to detect UV absorption at 320 nm.

DPBA dyeing

Plants were grown under continuous white light for 6 days and transferred to broad band UV-B for 1 day of treatment. The treated plants were soaked in an ethanol solution containing 0.01% Triton X-100, 2.52mg/mL DPBA, rotated on a shaker for 1.5 hours, and then washed with deionized water. The fluorescence intensity at 458nm of the excitation light was observed by a fluorescence microscope.

5. Real-time fluorescent Quantitative polymerase chain reaction (Quantitative RT-PCR)

Taking cDNA as a template, and carrying out a reaction by using SYBR Green qPCR mix of Takara, wherein the reaction is carried out in an MX3000(Stratagene) system, and the operation program is as follows: 95 ℃ for 30 sec; 95 ℃ for 5 sec; 60 ℃ for 30 sec; the fluorescence signal acquisition was performed for 40 cycles during the procedure. The Quantitative RT-PCR result uses an active 7 gene primer as an internal reference, and has two technical repeats and more than 2 biological repeats.

6. Chromatin co-immunoprecipitation ChIP

Planting Pro35S BES1-Flag and Col-0 wild type in a long-day artificial climate chamber, and collecting Arabidopsis seedlings with the seedling age of about 12 days, wherein the mass is 2 g; then fixing and crosslinking by formaldehyde; extracting cell nucleus and ultrasonic crushing; protein A/G agarose beads combined with Flag antibody are used for immunizing and co-precipitating protein-DNA compound, and rotary incubation is carried out overnight; eluting and de-crosslinking; purifying digestive protein and DNA; and (4) carrying out quantitative PCR analysis.

7. Gel retardation assay (EMSA)

1) Expressing and purifying BES1 protein by using a prokaryotic system;

2) preparing a probe: the PFG MYB promoter was chosen to contain the G-box sequence, two oligonucleotides were synthesized complementary and then annealed to form a double-stranded oligonucleotide, ligated into a T-vector (pEASY-blunt, CB111, Transgene). After the connection of the target sequence is verified by sequencing, the plasmid is used as a template, a Cy 5-labeled M13 forward and reverse primer is used for PCR amplification of the target sequence, and a PCR product is recovered by alcohol precipitation and is used as a labeled probe. And (3) carrying out PCR amplification on a target sequence by using unlabeled M13 forward and reverse primers, and recovering alcohol precipitation to obtain the cold probe.

3) Reaction: mu.L of binding buffer [25mM HEPES (pH 7.5), 40mM KCl, 3mM DTT, 10% glycerol, 0.1mM EDTA, 0.5mg/mL BSA, 0.5mg/mL poly-Glutamate ] was prepared, and 15ng of probe and 200ng of protein were added and reacted on ice for 30 min.

4) And (3) detection: 10 mu L of reaction product is directly loaded to 6 percent non-denaturing polyacrylamide gel electrophoresis and is electrophoresed under the condition of 4 ℃ and the constant pressure of 100v is 1-2 h. Cy 5-labeled probe in the gel was detected directly with Starion FLA-9000(FujiFilm, Japan).

8. Transient transcriptional activation assay (Dual-LUC assay)

The PFG MYB promoter (Gene ID of MYB 11: 825435, the promoter of which adopts a region 2Kb upstream of the initiation codon, Gene ID of MYB 12: 819359, the promoter of which adopts a region 2Kb upstream of the initiation codon, and Gene ID of MYB 111: 834993, the promoter of which adopts a region 2Kb upstream of the initiation codon) is amplified and connected into a pGreen II 0800-LUC reporter Gene vector by an enzyme digestion connection method, and the vector has 35S driving RENILLA and a target Gene promoter driving LUC expression. Agrobacterium containing the reporter vector and the effector vector (BES1-Flag or BES1-D-Flag) were mixed, cotransformed into tobacco, and the expression of the reporter gene was examined three days later. The LUC/REN ratio was calculated by quantifying the fluorescence signal using a chemiluminescence detection system luminometer (GloMax 20/20, Promega) using the dual fluorescence reporter assay kit from Promega. Or the LUC signal is directly acquired using a cold CCD.

9. Luciferase LUC photography

Transgenic plant pBES1, LUC species, was grown in 1/2MS medium for 7 days under continuous white light, then transferred to corresponding UV light for 8 hours, samples were collected for 0, 2, 4, and 8 hours, respectively, 2.5mM fluorescein substrate was added, and LUC signal was collected using cold CCD.