CN109576284B - Multifunctional MYB transcription factor gene and application thereof - Google Patents
Multifunctional MYB transcription factor gene and application thereof Download PDFInfo
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Abstract
The MYB type transcription factor gene is GbMYB2 identified from an "activite" plant gingko, the gene sequence of the gene is shown as SEQ ID NO.1, and the encoded protein sequence is shown as SEQ ID NO. 2. Moreover, the invention discloses the application of the gene, and the encoded protein can regulate and control plant flavonoid, lignin and plant growth and development. The invention has the advantages that MYB2 transcription factor gene is cloned from ginkgo, the function of the MYB2 transcription factor gene is systematically identified, and the MYB2 transcription factor gene is found to participate in a plurality of metabolic pathways and is a multifunctional transcription factor.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a multifunctional MYB transcription factor gene and application thereof.
Background
The flavonoid is an important secondary metabolite widely existing in plants and has important significance for adapting the plants to the habitat thereof. In addition, flavonoid substances have a plurality of functions of resisting oxidation, eliminating free radicals and the like, and have important application in human medical care. The plant is a main food and medicine source for human, so that the research hotspot in the field of plant secondary metabolism is formed on how to effectively regulate and control the accumulation of flavonoid in the plant to help human to obtain better medical and edible resources. Transcription factors play an important role in many processes of cell development, and therefore, they are important candidate genes for improving bioengineering technology for regulating and controlling a series of complex traits of crops. One of the different features of transcription factors from structural genes is: under certain conditions, it can regulate one or even several functional genes of a metabolic pathway, thereby realizing the overall regulation of the relevant pathway. Therefore, studying the function of characteristic transcription factors is beneficial to the analysis of metabolic pathways and ultimately to achieve directed regulation. Plants contain many types of transcription factors, including MYB, CBF/DREB1, HSF, TGA6, BOS1, bZIP, AP2/EREBP, etc., which play important roles in plant growth and development and adaptability to the environment. MYB-type transcription factors play an important role in the regulation of flavonoid synthesis. Ginkgo biloba is a medicinal plant with abundant flavonoid, and the flavonoid is an important component of medicinal ingredients. However, there are no reports on systems for systematically studying the transcriptional factors for ginkgo flavonoid accumulation and regulation. In view of multiple functions and potential application values of MYB transcription factors, the cloning and identification of the active multifunctional MYB transcription factors of ginkgo biloba have important theoretical and application values particularly on the regulation and control of flavonoids.
Disclosure of Invention
In order to solve the problems that the function of a gingko MYB transcription factor, particularly a MYB transcription factor for regulating and controlling flavonoids, is unknown and cannot be effectively utilized, the invention provides a multifunctional MYB transcription factor gene and application thereof. The invention clones MYB transcription factor genes with regulation and control functions on the accumulation of multiple flavonoid substances from ginkgo, systematically identifies and identifies the functions of the MYB transcription factor genes, and provides important gene resources and technical methods for regulating the accumulation of flavonoid pathway substances in other plants by using biotechnology.
In order to achieve the purpose, the technical scheme provided by the invention is that the MYB transcription factor gene provided by the invention is GbMYB2, and the gene sequence of the MYB transcription factor gene is shown in SEQ ID No. 1. The flavonoid MYB transcription factor gene discovered by the invention is cloned from ginkgo, is a brand-new MYB transcription factor gene, enriches the variety of MYB transcription factors, and has important relation with the growth and disease resistance of plants due to the change of flavonoid substance accumulation. Therefore, the accumulation of flavonoids can be further changed through GbMYB2, and the application of the plant growth and disease resistance is further improved.
Furthermore, the amino acid sequence of the protein coded by the MYB transcription factor gene is shown in SEQ ID NO. 2.
Furthermore, the application of the multifunctional MYB transcription factor gene in other plants can change the accumulation of flavonoid substances in the organism through heterologous expression, particularly flavonol, anthocyanin and tannin which are flavonoid compounds with the most abundant natural content, and the invention proves that GbMYB2 can simultaneously regulate the biosynthesis of the synthesis of the three flavonoids. Simultaneously, the GbMYB2 gene also affects plant lignin, epidermal hair and growth. This is the first MYB transcription factor found in ginkgo with multiple functions.
By adopting the technical scheme, the MYB transcription factor gene is cloned from ginkgo, the function of the MYB transcription factor gene is systematically identified, the correlation between the MYB transcription factor gene and flavonoid accumulation in ginkgo is found, the accumulation of main flavonoids such as anthocyanin, flavonol, tannin and the like can be changed by overexpression in a model plant Arabidopsis, and the lignin, epidermal hair and growth and development of a transgenic plant are influenced.
Drawings
FIG. 1 is a graph of the cluster analysis of GbMYB2 with other species-related transcription factors in the present invention;
FIG. 2 is a multiple sequence alignment of amino acid levels of GbMYB2 with other MYB transcription factors in the invention; in the figure, the domains of R2 and R3 are marked with black lines; the domain of PLN03212 is marked with a black dashed line; possible bHLH binding domains are indicated by black boxes. Black background labeled fully conserved amino acids; regions of 75% -100% conservation are noted as dark gray backgrounds; the conservation of 50% -75% is noted as a light gray background.
FIG. 3 shows PCR identification of plants heterologously overexpressed in Arabidopsis according to GbMYB2 of the present invention.
FIG. 4 quantitative analysis of tannin in mature seeds of transgenic plants; extractable tannins were measured by DMACA and non-extractable tannins by n-butanol-hydrochloric acid.
FIG. 5 analysis of relative expression of genes involved in tannin synthesis and regulatory pathways.
FIG. 6 overexpression of GbMYB2 in Arabidopsis resulted in a decrease in anthocyanin content.
FIG. 7 quantitative analysis of expression levels of related genes in anthocyanin synthesis pathway and regulatory pathway.
FIG. 8 overexpression of GbMYB2 in Arabidopsis resulted in changes in flavonol content.
FIG. 9 changes in flavonol regulatory pathway-associated gene expression levels in transgenic plants.
FIG. 10 GbMYB2 overexpression in Arabidopsis resulted in decreased epidermal hair density.
FIG. 11 expression levels of transcription factors associated with epidermal hairs.
FIG. 12 overexpression of GbMYB2 in Arabidopsis resulted in changes in the expression levels of genes associated with the lignin synthesis pathway.
FIG. 13 is a graph showing the results of yeast two-hybrid assay for the interaction of GbMYB2 with Arabidopsis flavonoid pathway-related transcription factors. In the figure, GbMYB2 is attached to the active and binding ends, respectively, other transcription factors are attached to the active end of the vector only, double-yeast cells are cultured on two-and four-deficiency media, respectively, and the interaction of pGKT7-AD with GbMYB2-BD is used as a negative control of the system.
FIG. 14 tissue expression profiles of GbMYB2 in Ginkgo biloba were analyzed using qRT-PCR. YR, radicle; YS, caulicle; YL, young leaves; 5F-9F, fruits in different months from May to September.
FIG. 15 is a graph showing the relationship between the expression level of GbMYB2 and the total flavone accumulation in leaves at different periods. The dotted line represents the relative accumulation of total flavonoids for the corresponding month; the solid line is the relative expression level of GbMYB2 in the leaf at different times.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
The first embodiment is as follows: the invention discloses a multifunctional MYB transcription factor gene which is recorded as GbMYB2, the gene sequence of the gene is shown as SEQ ID No.1, ATG in the sequence shown as SEQ ID No.1 is an initiation codon, TGA is a termination codon, the protein coded by the gene is shown as SEQ ID No.2 in the amino acid sequence, and the application of the gene is that the gene can systematically regulate and control various compounds, particularly flavonoid compounds, through heterologous expression in other organisms.
The gene was identified and cloned in ginkgo biloba, and the following description is given of how to clone the gene from ginkgo biloba and determine that it is a MYB-type transcription factor gene, and a method of determining its use:
the first, material and method:
1.1 plant Material
The Ginkgo leaf used in this experiment was obtained from a 20cm diameter female Ginkgo tree (Ginkgo biloba L.) belonging to Beijing plantations of Chinese academy of sciences.
1.2 reagents
Reagents such as CTAB, beta-mercaptoethanol, lithium chloride, ethanol, sodium acetate, DEPC and the like have no special requirements;
preparation of DEPC-treated Water (i.e., Water from which RNase was removed by diethylpyrocarbonate): 1 per mill (V/V), keeping away from light, shaking at 37 deg.C overnight, sterilizing at 121 deg.C for 20 min, cooling, and packaging at-20 deg.C.
RNA extraction reagent CTAB solution formula (all water used for preparing the reagent is DEPC treated water): 2% CTAB (W/V); 2% polyvinylpyrrolidone (W/V); 100mM Tris-HCl (pH8.0); 25mM EDTA; 0.5g/L spermidine; 2.0M NaCl.
1.3 Experimental methods
1.3.1 method for extracting RNA by CTAB method
Treating the ginkgo biloba material with a mortar, pestle, spoon, etc. at a high temperature, immediately cooling the ginkgo biloba material with liquid nitrogen, ensuring that the ginkgo biloba material is always in liquid nitrogen during the grinding of the plant material.
(1) Approximately 2g of frozen material was placed in a mortar that had been conditioned and had liquid nitrogen present and rapidly ground to a fine powder.
(2) About 1ml of CTAB RNA extract was added per 100mg of plant material, vortexed and shaken for 1 minute, and lysed in a water bath at 65 ℃ for 30 minutes.
(3) 0.6 ml of chloroform was added to 1ml of CTAB extract, vortexed and shaken for 1 minute, and centrifuged at 12000rpm for 10 minutes at low temperature.
(4) The supernatant was aspirated into a 1.5 ml centrifuge tube, and 10M lithium chloride in a volume of clear 1/4 was added and the RNA was settled in a refrigerator at 4 ℃ overnight.
(5) The tubes were removed overnight at 4 degrees Celsius and centrifuged at 12000rpm at 4 degrees Celsius for 10 minutes to allow the precipitated RNA to settle.
(6) The supernatant was removed. The precipitation washing was performed by adding 1mL of 75% ethanol to 1mL of the extract, and the washing was repeated 1-2 times (4 ℃, 12000rpm, 3 mins).
(7) Centrifuging, sucking out residual ethanol by using a pipette gun, opening a tube cover, and drying the precipitate at room temperature for 5-7 minutes.
(8) The RNA was dissolved by adding 30-50. mu.L of DEPC water depending on the amount of the precipitate.
(9) Storing at-80 deg.C.
1.4 vector construction
1.4.1 plant overexpression vector construction
First, the cloned gene fragment was ligated into pENTR/D-TOPO vector in the system shown in Table 1. The ligation solution was placed in a 25 ℃ metal bath overnight. Coli competence was transformed with the ligation solution and plated on LB solid medium containing kanamycin. After the cloning and sequencing are correct, plasmids are extracted, and the target gene is connected into a plant over-expression vector pB2GW7 (shown in the system of Table 2) by utilizing an LR reaction. After positive clones are determined by PCR, plasmids are extracted, agrobacterium GV3101 competence is transformed, and the positive clones are picked for subsequent transformation of Arabidopsis.
TABLE 1 connection System of pENTR/D-TOPO vector and target Gene
TABLE 2 LR reaction System
1.4.2 Yeast two-hybrid vector construction
The yeast two-hybrid vector was constructed by the Gateway cloning system. Wherein, the method for connecting the target gene into pENTR/D-TOPO is the same as 1.4.1. And (3) connecting the pENTR/D-TOPO plasmid containing the target gene with correct sequencing into pGADKT7 or pGBKT7 vectors through LR reaction, identifying positive clone, extracting the plasmid, and using the plasmid for later-stage yeast transformation.
1.4.3 preparation and transformation of Yeast competence
(1) Picking up yeast colonies growing well on the YPAD culture medium, and culturing in 5 ml YPDA liquid culture medium at 30 ℃ overnight with shaking;
(2) inoculating 5 ml of overnight culture yeast liquid into 100 ml of YPDA liquid culture medium according to the inoculation ratio of 5 percent, and culturing until OD600nm is 0.8-1.0;
(3) centrifuging at 4 ℃ and 3000rpm for 5 minutes, removing supernatant, and collecting yeast cells;
(4) adding 20 ml of sterile water into every 100 ml of yeast liquid for cleaning; repeating the step 3;
(5) resuspend the cells with 1ml LiCl (final concentration 0.1 mol/L);
(6) transferring the mixture into a 1.5 ml sterile centrifuge tube, centrifuging the mixture at 12000rpm for 15s, and removing supernatant;
(7) repeating the step 6, and culturing the suspension cells at 30 ℃ and 200rpm for 30 minutes;
(8) for each transformation, 5. mu.l salmon sperm DNA (1. mu.g/. mu.L) was added, heat denatured at 94 ℃ for 5 minutes, and rapidly placed in an ice bath for 2 minutes;
(9) yeast competent cells were aliquoted at 50. mu.l per tube.
(10) Adding 5 microliters (about 1 mug/microliter) of the pre-transformed plasmid and 5 microliters of the heat-denatured salmon sperm DNA into each tube of the subpackaged yeast competent cells, and uniformly mixing; adding a PEG and LiCl mixed solution (the final concentration is 40% and 0.1mol/L respectively), and uniformly mixing by vortex;
(11) carrying out water bath heat shock at 42 ℃ for 40 minutes, and flicking and uniformly mixing every 5 minutes;
(12) centrifuging at 12000rpm for 15s, and removing supernatant; adding 300 microliter of sterile water suspension cells, and coating on an SD (-URA) yeast defective plate; culturing at 30 deg.C for 2-3 days, and PCR verifying positive clone after colony appears.
1.4.4 bioinformatics analysis of MYB sequences
In the experiment, the NCBI is used for comparison in a plant protein database to obtain basic information of the structure and the function of the target protein.
1.5 Induction and measurement of anthocyanins
Arabidopsis seeds were sown on MS low sugar (1%) medium and vernalized at 4 ℃ for 3-4 days. Vernalized seeds were grown for 14 days in a low light (50%), normal photoperiod (16/8) culture room. Subsequently, the batch of seedlings was transferred to a high sugar medium and cultured under high light (100%), full light (24/0) for 3 days. Finally, harvesting seedlings and storing the seedlings at-80 ℃.
The anthocyanin inducing material was ground thoroughly in a mortar and the powder was dried with a freeze dryer. Drying is generally carried out for 24 hours. The dried material was used for measurement of anthocyanins.
(1) Weighing about 20mg of the dried powder and adding 500. mu.l of 80% methanol (containing 0.1% HCl), and mixing well;
(2) performing ultrasonic treatment for 30 minutes;
(3) centrifuging at 12000rpm for 10 min, and taking the supernatant into a new centrifuge tube;
(4) sequentially adding water and chloroform with the same volume into the supernatant, and mixing uniformly for removing chlorophyll;
(5) repeating the step 3; at this time, different degrees of red color were observed in the green-removed extract;
(6) the supernatant was measured for absorbance at 530 nm; relative quantification was performed using cyanidin glycoside standards.
1.6 measurement of flavonols
Arabidopsis seeds were sown on MS medium and grown in the culture room for 6 days. And (3) taking the seedlings with consistent growth states, transferring the seedlings into an earth culture greenhouse for 21 days, and taking the overground part of the seedlings to carry out liquid nitrogen grinding and freeze drying. The dried aerial powder was the sample for later flavonol content measurement. Adding 80% methanol according to the mass-to-volume ratio of 1:5, carrying out ultrasonic treatment for 30 minutes, and soaking and extracting at 4 ℃ overnight. The next day, sonication was repeated for 30 minutes, centrifugation was performed at 12000rpm for 30 minutes, and a portion of the supernatant was subjected to detection of flavonols by HPLC.
1.7 Observation and quantification of epidermal Hair Density
Leaf epidermal hair and stem epidermal hair were observed and quantified from about 41-day-old Arabidopsis rosette leaves and first branch cauline nodes. Taking the same part of the rosette leaves of Arabidopsis thaliana in the same growth state for counting the epidermal hairs. And calculating the area of the corresponding blade by millimeter paper projection, and finally taking the total skin hair number/the blade area as the skin hair density value of the blade. Measurement of Stem epidermal Hair A stem segment between the rosette leaf and the first cauline leaf was selected for phenotype observation and measurement. The density of the stem epidermal hairs is determined by dividing the number of the epidermal hairs of the extracted stem segment in a visual field by the length of the stem segment. 6 independent plants were observed and measured for each line, and the average was the average stem epidermal hair density.
1.8 preparation of free-hand Stem sections
The base of the first flower stem of the 41-day seedling of Arabidopsis thaliana was sliced by bare hand. Two new blades are taken and arranged side by side, one side of each new blade is stuck by using a transparent adhesive tape, then the new blades are quickly cut on a fresh stem section until more than ten blades are cut, the two blades are taken away, the middle of each blade is washed by distilled water and placed on a glass slide, a cover glass is covered, and observation is carried out under a microscope.
1.9 Yeast two-hybrid method
And (3) the identified double-yeast strain is spotted on a two-lacking (-Trp-Leu) yeast culture medium, the double-yeast strain is placed in an incubator AT 30 ℃ until a bacterial colony grows up, after the growth state of the bacterial colony is confirmed to be free from variation, the bacterial colony is diluted by different gradients after the proper 3-AT concentration is confirmed, and the bacterial colony is respectively spotted on the three-lacking (-Trp-Leu-His) and the four-lacking (-Trp-Leu-His-Ade) yeast culture medium for interaction analysis.
Second, result in
2.1 screening and cloning of the GbMYB2 Gene
In the invention, a transcriptome database is searched by inputting a MYB keyword in a ginkgo leaf transcriptome sequencing database, and then sequence splicing and cloning are carried out according to a search result, so that 1 MYB is successfully obtained finally. Subsequently, experiments were carried out on the transcription factor for complementation of Arabidopsis related mutants and overexpression of wild type, respectively. It was found that overexpressed plants of GbMYB2 exhibited a phenotype of flavonoid-related metabolite changes.
2.2 sequence analysis of the GbMYB2 Gene
According to the invention, preliminary function prediction is carried out on GbMYB2 through an Arabidopsis protein database in NCBI. The results show that GbMYB2 has relatively high sequence similarity to AtMYB4, AtMYB34, AtMYB6, AtMYB3, AtMYB7, and AtMYB5 in arabidopsis, with the greatest range of similarity to AtMYB4, with a gene similarity interval of up to 67%, and a homology of 47% in this interval (table 3). Similar genes in Arabidopsis thaliana listed in Table 3 are mostly MYB transcription factors related to the phenylpropane pathway. Therefore, the present invention speculates that GbMYB2 may be a phenylpropane pathway-related transcription factor. According to the invention, GbMYB2 and other known flavonoid metabolic pathway and epidermal hair pathway related transcription factors are subjected to cluster analysis, and the clustering is found to be independent and close to the epidermal hair negative control transcription factor and the anthocyanin activated transcription factor in the flavonoid pathway (figure 1).
In addition, the invention also selects the reported transcription factor protein sequence to carry out multi-sequence alignment together with GbMYB 2. The results show that GbMYB2 has obvious R2 and R3 characteristic domains (FIG. 2). However, the domain not common in the sequence of GbMYB2 was found by NCBI prediction for PLN03212 (fig. 2).
TABLE 3 sequence similarity List of transcription factor proteins in GbMYB2 and Arabidopsis
2.3 identification of plants overexpressing GbMYB2 in Arabidopsis
In order to research the in-vivo function of GbMYB2, GbMYB2 is overexpressed in wild type Arabidopsis, RT-PCR verification shows that 6 transgenic seedlings with high expression quantity are obtained in total, and strains with relatively high expression quantity are selected and named as OE1(overexpression line 1) and OE 2. Through fluorescent quantitative PCR detection, the expression level of GbMYB2 in the transgenic plant is improved by about 80000 to 120000 times compared with that in the wild type (FIG. 3).
2.4GbMYB2 overexpressing plants exhibiting flavonoid phenotypes and down-regulating related genes
In order to explore the in vivo function of GbMYB2, the invention makes phenotypic observation on homozygote of transgenic Arabidopsis, and finds that the transgenic line has several phenotypes: the seed coat color is lightened, the anthocyanin accumulation is reduced, the flavonol content is reduced, the epidermal hair density is reduced, the root dysplasia is comprehensively phenotypic, and different phenotypes have different expression degrees in different strains.
2.4.1 reduction of tannin content in seed coat of transgenic plants
First, the color of the seed coat of mature seeds of the transgenic plants is changed from dark brown of the wild type to light yellow or light brown. Tannin is considered to be a major influencing substance of seed coat color of arabidopsis thaliana seeds at present. Therefore, the present invention measured the content of tannin in transgenic arabidopsis seeds. The results show that the soluble tannins of the transgenic plants are reduced by 90 to 60 percent compared with the tannins of the wild plants, while the insoluble tannins are not changed significantly (figure 4).
In order to explore the reason of tannin reduction in the seed coat of the transgenic plant, the invention utilizes the pods pollinated for 4 days to carry out quantitative analysis on the genes of tannin synthesis and regulation pathways. The results show that key genes of the tannin synthesis pathway (such as ANR) are not significantly changed, whereas the expression level of TT8 (a bHLH type transcription factor) in the tannin regulatory pathway is significantly down-regulated by about 93% in the transgenic lines (fig. 5). It is thus understood that the effect of GbMYB2 on tannins in arabidopsis seed coats may affect the expression of transcription factors involved in their synthesis.
2.4.2 transgenic plants with reduced anthocyanin accumulation associated with downregulated Gene expression levels
The invention discovers that the anthocyanin content of the basal part of the rosette leaves and the basal part of the first flower stem of the transgenic plant is reduced and even completely disappears. In order to more accurately observe and quantify the change of anthocyanin in transgenic arabidopsis, the transgenic arabidopsis and wild arabidopsis are treated by high-sugar high-light induction. Under the induction condition, the wild arabidopsis shows dark purple red, which indicates that a large amount of anthocyanin is enriched; however, in the transgenic plants, there was no significant accumulation of anthocyanin and the whole seedlings still appeared bright green. Subsequently, the anthocyanin content of the transgenic plants is determined, and the result shows that the anthocyanin content of the transgenic plants is only about 10% of that of the wild plants (figure 6). Experimental results prove that the transfer of GbMYB2 seriously inhibits the synthesis and accumulation of anthocyanin in Arabidopsis.
In order to research the cause of anthocyanin reduction, the invention utilizes 17-day seedlings to analyze the anthocyanin synthesis and regulation pathway related gene expression level. Experiments prove that F3H and ANS in the anthocyanin synthesis pathway are remarkably reduced by about 90% and 85% respectively, and the expression level of DFR is reduced by about 97%. However, the anthocyanin synthesis transcription factor, such as PAP1, was not significantly changed (fig. 7). Therefore, unlike the tannin reduction principle, the anthocyanin synthesis is reduced mainly because structural genes of the anthocyanin synthesis pathway are significantly down-regulated, and related transcription factors are not significantly changed.
2.4.3 transgenic plants are downregulated in expression levels of genes associated with reduced flavonol content
Under the condition that tannin and anthocyanin are remarkably reduced, the content of flavonol of the other branch of the flavonoid pathway of arabidopsis is measured. The results showed that total flavonol glycosides were reduced by about 75-50% in the over-expressed plants compared to wild type (fig. 8). Among them, K3R7R and K3G7R decreased by 88% -67% and 75% -50%, respectively, and K3GR7R decreased by 50% (fig. 8). Experimental results prove that the overexpression of GbMYB2 in Arabidopsis severely inhibits the synthesis and accumulation of flavonol in Arabidopsis.
The invention utilizes 21-day rosette leaves to analyze the expression quantity of related genes. Experiments prove that the expression levels of the structural genes CHS, CHI, F3H, F3' H and FLS in the synthesis pathway of flavonol are remarkably reduced (FIG. 9). It is known that structural genes of the flavonol synthesis pathway are significantly down-regulated.
2.4.4 transgenic plants with reduced epidermal hair density associated with the down-regulated expression level of genes
The invention observes the epidermal hair of the transgenic plant. The results show that the density of rosette leaf epidermal hair of the transgenic plants is reduced by about 60 percent compared with the wild type. The invention also carries out statistics on the density of the epidermal hairs at the base of the first flower stem, and the result shows that the density of the epidermal hairs is reduced by about 95-80% (figure 10). On the first stem segment of the transgenic plants, the density of epidermal hairs is very low, some hairs even appear hairless, and the density of leaf epidermal hairs is relatively reduced less. Overexpression of GbMYB2 in Arabidopsis results in a decrease in rosette leaf and stem epidermal hair density in the plant.
The invention carries out quantitative analysis on partial genes of epidermal hair generation pathway. Quantitative materials were selected from 21 day rosette leaves and the base of the first cauline. Leaf quantification results show that the expression levels of GL1, GL2 and GL3 are reduced remarkably, and the expression levels of related genes in transgenic plants are only about 13.3%, 3.3% and 4.0% of wild type; the expression level of TTG2 is slightly reduced; the expression levels of EGL3 and TTG1 did not change significantly (FIG. 11). Similarly, the expression level of the relevant gene at the stem base was as follows: the expression levels of GL2 and TTG2 in the transgenic plants are respectively between 1/180 and 1/5 of wild type; GL1 was significantly down-regulated in the partial strain. However, unlike leaves, the expression level of the stem base GL3 of transgenic Arabidopsis was not significantly different from that of wild type. EGL3 and TTG1 were expressed in the same way as leaves, and their expression level at the base of stem of transgenic plant was not significantly different from that of wild type (FIG. 11). Therefore, GbMYB2 can regulate epidermal hair density by inhibiting expression of epidermal hair-related transcription factors (GL1, GL2 and GL3) in Arabidopsis thaliana.
2.5 Lignin pathway-associated Gene expression levels in transgenic plants
The invention also studies the change of lignin content in transgenic plants. The transgenic plants are found to be short, and the base parts of the transgenic plants and the first flower stems of the wild type 41-day seedlings are sliced and stained with lignin, so that the lignin content in the transgenic plants is found to be reduced. In addition, quantitative detection of genes associated with the lignin pathway revealed that the expression levels of PAL and HCT of major genes of the lignin synthesis pathway were decreased (FIG. 12). Thus, it was demonstrated that GbMYB2 affects the accumulation of lignin, resulting in dwarfed plant growth.
2.6GbMYB2 interacting with Arabidopsis thaliana flavonoid metabolic pathway transcription factor
In order to research the action mechanism of GbMYB2 and flavonoid pathway-related transcription factors, GbMYB2 and Arabidopsis flavonoid metabolic pathway-related transcription factors (AtTT8, AtTT2, AtTTG1, AtTTG2, AtMYB111, AtMYB113, AtMYB114, AtGL3, AtEGL3 and AtPAP1) are respectively connected into pGADKT7 and pGBKT7 to perform yeast two-hybrid experiments. The results show that GbMYB2 has strong interactions with AtTT8, AtTT2, AtTTG1, attmyb 113, attgl 3 and AtEGL3, but no interactions with AtTTG2, attmyb 111 and attmyb 114 (fig. 13). This suggests that GbMYB2 is capable of interacting with many transcription factors of the flavonoid metabolic pathway, thereby down-regulating the individual branches of the flavonoid pathway as a whole, resulting in reduced accumulation of the relevant end products. In addition, GbMYB2 is able to interact with itself. This suggests that GbMYB2 may inhibit flavonoid metabolic pathways in Arabidopsis by forming homodimers or multimers, which then interact with transcription factors associated with flavonoid synthetic pathways, respectively.
2.7GbMYB2 is associated with the accumulation of ginko flavonoids
The invention constructs a tissue expression profile of GbMYB 2. The results show that although GBMYB2 is expressed in all roots, stems, leaves and fruits of ginkgo biloba, its expression level in leaves is much higher than other tissues, and therefore GBMYB2 is mainly expressed in leaves (fig. 14). The accumulation of flavonoids is extremely susceptible to the environment, and the relationship between the accumulation of flavonoids and the expression level of GbMYB2 is researched. The invention collects leaves in different months, and summarizes and analyzes the relationship between the total flavone content and GbMYB2 expression quantity. The results show that GbMYB2 shows a negative correlation with the change in total flavone content in some periods (fig. 15). The expression level of GbMYB2 steadily increases from 3 months to 6 months, while the content of flavonoids in leaves rapidly decreases from 3 months and increases back to 8 months after 5 months; the expression quantity of GbMYB2 rapidly decreases from 6 months to 7 months, and increases back from 7 months to 9 months; total flavonoids slightly increased from month 9 to 10, while GbMYB2 rapidly decreased in this time. From this, it can be seen that to some extent, GbMYB2 has a negative correlation with the accumulation of total flavonoids. Therefore, GbMYB2 is presumed to be capable of carrying out negative control on the accumulation of ginkgo total flavonoids in the ginkgo body.
Third, the final conclusion
1. The gingko GbMYB2 gene encodes a multifunctional MYB transcription factor, and the expression level of the GbMYB2 gene in gingko is related to the accumulation level of flavonoids. GbMYB2 is a key transcription factor for regulating and controlling ginkgo flavonoid biosynthesis.
2. Ginkgo accumulates abundant flavonoid compounds, and is a model plant for researching flavonoid regulation. GbMYB2 can be expressed in model plants, and the expressed transcription factor can regulate multiple metabolic pathways, so that candidate genes and beneficial references are provided for producing active flavonoids by plant metabolic engineering.
The operation methods not specifically described in the present embodiment are conventional techniques, and thus are not explained in detail herein.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> a multifunctional MYB transcription factor gene and uses thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 921
<212> DNA
<213> GbMYB2 Gene sequence (Ginkgo biloba)
<400> 1
atgggtcgct ctccaatgtg ttcaaaagta ggtttaggtc tgaacaaagg agcttggact 60
gtggaggagg ataacctact catcaaatat tttcaaactc atgatgaagg tggtggctgg 120
aaatctgttc cgaagaaagc aggcctgaaa cggtgtggaa agagctgcag gttgcgctgg 180
atgaattacc tccgacctaa catcaagaga ggaaatattt ctcccgacga ggaagacctg 240
ataatcagac tccatggtct ccttgggaat agatgggcac taattgcagg aagaattcct 300
ggccgaaccg ataacgagat taagaattat tggtacacta ctttgagcaa gagagtggct 360
ttgaaaggaa atgaagccaa agagcataag acatatccaa tgaaacggag ccgaggtcat 420
tctgcctgca agcagttaat catgccagac agcaatacca aaatgcaaga cctgttgtca 480
gcttctccta cgagaaaaga attagaagta tcgacaaatc aaagtctatc agagtccgtc 540
gtttctaaca ctgacgatgt tagaactgat tcaaatgtcc aatccggttc ccccggtctt 600
caagaaatac gtgcaagccg aatattttta ccctctttcc gaggcaacca ggtttcttct 660
cattcagaac ttatggctcc tgcaaatcct atgattgaat caaacataga cagaaaattg 720
ttctcattag tcgacgatta tctgtctgtc tccacagaac ttagcctcgg cttttctgga 780
atgaattgct ccgtatcgaa gttcagtact aattctcata atctctacct catgggcagc 840
tcactatcaa ataccgatca tcatatgcca tgtggagacc agagcgtatg tagagatcag 900
tatcgttgga tgaatacatg a 921
<210> 3
<211> 306
<212> PRT
<213> protein encoded by GbMYB2 gene (Ginkgo biloba)
<400> 3
Met Gly Arg Ser Pro Met Cys Ser Lys Val Gly Leu Gly Leu Asn Lys
1 5 10 15
Gly Ala Trp Thr Val Glu Glu Asp Asn Leu Leu Ile Lys Tyr Phe Gln
20 25 30
Thr His Asp Glu Gly Gly Gly Trp Lys Ser Val Pro Lys Lys Ala Gly
35 40 45
Leu Lys Arg Cys Gly Lys Ser Cys Arg Leu Arg Trp Met Asn Tyr Leu
50 55 60
Arg Pro Asn Ile Lys Arg Gly Asn Ile Ser Pro Asp Glu Glu Asp Leu
65 70 75 80
Ile Ile Arg Leu His Gly Leu Leu Gly Asn Arg Trp Ala Leu Ile Ala
85 90 95
Gly Arg Ile Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Tyr Trp Tyr
100 105 110
Thr Thr Leu Ser Lys Arg Val Ala Leu Lys Gly Asn Glu Ala Lys Glu
115 120 125
His Lys Thr Tyr Pro Met Lys Arg Ser Arg Gly His Ser Ala Cys Lys
130 135 140
Gln Leu Ile Met Pro Asp Ser Asn Thr Lys Met Gln Asp Leu Leu Ser
145 150 155 160
Ala Ser Pro Thr Arg Lys Glu Leu Glu Val Ser Thr Asn Gln Ser Leu
165 170 175
Ser Glu Ser Val Val Ser Asn Thr Asp Asp Val Arg Thr Asp Ser Asn
180 185 190
Val Gln Ser Gly Ser Pro Gly Leu Gln Glu Ile Arg Ala Ser Arg Ile
195 200 205
Phe Leu Pro Ser Phe Arg Gly Asn Gln Val Ser Ser His Ser Glu Leu
210 215 220
Met Ala Pro Ala Asn Pro Met Ile Glu Ser Asn Ile Asp Arg Lys Leu
225 230 235 240
Phe Ser Leu Val Asp Asp Tyr Leu Ser Val Ser Thr Glu Leu Ser Leu
245 250 255
Gly Phe Ser Gly Met Asn Cys Ser Val Ser Lys Phe Ser Thr Asn Ser
260 265 270
His Asn Leu Tyr Leu Met Gly Ser Ser Leu Ser Asn Thr Asp His His
275 280 285
Met Pro Cys Gly Asp Gln Ser Val Cys Arg Asp Gln Tyr Arg Trp Met
290 295 300
Asn Thr
305
Claims (1)
1. The application of a MYB transcription factor GbMYB2 is characterized in that the gene sequence of GbMYB2 is shown as SEQ ID No. 1; the application is that GbMYB2 is overexpressed in arabidopsis thaliana, so that the content of soluble tannin in seed coats is reduced, the accumulation of anthocyanin is reduced under the condition of adversity stress, total flavonol glucoside is reduced, the synthesis and accumulation of flavonol are inhibited, the density of leaf epidermal hair is reduced, or the accumulation of lignin is reduced, so that the growth of plants is short.
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| CN112080507B (en) * | 2020-09-04 | 2022-04-08 | 扬州大学 | Key gene GbMYB4 for regulating and controlling ginkgo flavonoid synthesis, protein expressed by gene GbMYB4, vector and application of gene GbMYB4 |
| CN112079911B (en) * | 2020-09-04 | 2022-04-08 | 扬州大学 | Key gene GbMYB6 for promoting synthesis of ginkgo flavonoids, and protein, vector and application of key gene GbMYB6 for expression |
| CN112280787B (en) * | 2020-11-10 | 2021-09-21 | 中国科学院华南植物园 | Glycyrrhiza MYB1 gene, and encoded protein and application thereof |
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