CN115305251B

CN115305251B - Application of AP2-MYBL2 molecular module in regulating and controlling proanthocyanidin biosynthesis

Info

Publication number: CN115305251B
Application number: CN202211045250.8A
Authority: CN
Inventors: 江文波; 庞永珍
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2022-08-30
Filing date: 2022-08-30
Publication date: 2023-06-27
Anticipated expiration: 2042-08-30
Also published as: CN115305251A

Abstract

The invention provides application of an AP2-MYBL2 molecular module, an AP2-MYBL2-TT8 or an AP2-MYBL2-EGL3 protein complex in regulating and controlling biosynthesis of proanthocyanidins; the AP2 protein directly binds to the promoter of the MYBL2 gene of the direct target gene so as to activate the expression of the MYBL2 gene at the transcription level and regulate the biosynthesis of the proanthocyanidins. On the other hand, the AP2 protein forms a protein complex of AP2-MYBL2-TT8 or AP2-MYBL2-EGL3 through interaction with MYBL2 protein at the protein level, and inhibits the formation of a MYB-bHLH-WD ternary complex, thereby regulating and controlling the biosynthesis of proanthocyanidins.

Description

Application of AP2-MYBL2 molecular module in regulating and controlling proanthocyanidin biosynthesis

Technical Field

The invention belongs to the technical field of genetic engineering application, and particularly relates to application of an AP2-MYBL2 molecular module in regulating and controlling biosynthesis of proanthocyanidins.

Background

Proanthocyanidins are flavonoid secondary metabolites with important activity in plants. Not only plays an important role in the regulation of seed dormancy, longevity and germination, but also participates in regulating the biotic and abiotic stress of plants; brown cotton is the most widely used natural colored cotton at present, and the brown pigment in the fiber is proanthocyanidin; when the content of proanthocyanidins in alfalfa is higher than 2% of dry weight, the occurrence of tympanites of ruminants such as cattle, sheep and the like can be effectively prevented; in addition, it has antioxidant, antiinflammatory and anticancer effects, and is beneficial to human health. Therefore, the research on the biosynthesis and regulation of proanthocyanidins is an important research direction in the field of plant secondary metabolism.

The proanthocyanidin plays an important role in regulating and controlling the growth and development of plants and stress, and has a plurality of benefits for human health. Many prior art studies suggest that there is an unknown key gene that negatively regulates the function of TT2 or MBW ternary complex associated therewith, and thus the accumulation of proanthocyanidins, but what genes are involved therein, and the molecular mechanism of its regulation remains unclear. In CN110241124a it is disclosed that AP2 mutant seed coats are darker in color than wild type, accumulating more proanthocyanidins. However, the apparent results of the regulation of proanthocyanidin by AP2 are only relevant in the patent, but the molecular mechanism of the regulation is still unclear. The invention aims to determine target genes and key transcription factors in the AP 2-regulated proanthocyanidin biosynthesis pathway, determine a molecular mechanism of the AP2-MYBL2 molecular module for synergistically regulating the proanthocyanidin biosynthesis at the transcription level and the protein level, and elucidate a genetic network of the AP 2-regulated proanthocyanidin biosynthesis.

Disclosure of Invention

In order to solve the technical problems, the invention provides a molecular mechanism of AP2 for regulating and controlling proanthocyanidin, determines a target gene and a key transcription factor in an approach of AP2 for regulating and controlling proanthocyanidin biosynthesis, determines a molecular mechanism of an AP2-MYBL2 molecular module for synergistically regulating and controlling proanthocyanidin biosynthesis at a transcription level and a protein level, clarifies a genetic network of AP2 for regulating and controlling proanthocyanidin biosynthesis, and provides important theoretical support for improving plant proanthocyanidin biosynthesis and metabolism.

According to an aspect of the present application, there is provided the use of an AP2-MYBL2 molecular module for modulating proanthocyanidin biosynthesis.

In particular embodiments of the present application, experiments have demonstrated that MYBL2 is a direct target gene for the AP2 protein.

In a preferred embodiment of the present application, the AP2 protein binds directly to the promoter of the MYBL2 gene at the transcriptional level, thereby activating expression of the MYBL2 gene.

In a preferred embodiment of the present application, the AP2 protein activates expression of the MYBL2 gene in plants by binding to the cis-acting element M5.

In a preferred embodiment of the present application, the AP2 protein acts genetically upstream of MYBL2, regulating the biosynthesis of proanthocyanidins by activating the expression of the MYBL2 gene.

According to another aspect of the present application there is also provided the use of an AP2-MYBL2-TT8 or AP2-MYBL2-EGL3 protein complex for modulating proanthocyanidin biosynthesis.

In particular embodiments of the present application, the AP2 protein interacts with the C-terminus of the MYBL2 protein, and the N-terminus of the MYBL2 protein interacts with either the TT8 protein or the EGL3 protein.

In particular embodiments of the present application, the AP2 protein forms a protein complex of AP2-MYBL2-TT8 or AP2-MYBL2-EGL3 at the protein level by interacting with the MYBL2 protein, inhibiting the formation of a MYB-bHLH-WD ternary complex.

In particular embodiments of the present application, the AP2 protein acts genetically upstream of TT2 and TT8 in the MYB-bHLH-WD ternary complex.

The beneficial effects of the invention are as follows:

(1) The application provides application of an AP2-MYBL2 molecular module, an AP2-MYBL2-TT8 or an AP2-MYBL2-EGL3 protein complex in regulating and controlling proanthocyanidin biosynthesis. The AP2 protein directly binds to the promoter of the MYBL2 gene of the direct target gene so as to activate the expression of the MYBL2 gene at the transcription level and regulate the biosynthesis of the proanthocyanidins. On the other hand, the AP2 protein forms a protein complex of AP2-MYBL2-TT8 or AP2-MYBL2-EGL3 through interaction with MYBL2 protein at the protein level, and inhibits the formation of a MYB-bHLH-WD ternary complex, thereby regulating and controlling the biosynthesis of proanthocyanidins.

(2) The application provides a molecular mechanism of AP2 for regulating and controlling proanthocyanidin, determines a target gene and a key transcription factor in an AP 2-regulating and controlling proanthocyanidin biosynthesis pathway, determines a molecular mechanism of an AP2-MYBL2 molecular module for synergistically regulating and controlling proanthocyanidin biosynthesis at a transcription level and a protein level, clarifies a genetic network of AP2 for regulating and controlling proanthocyanidin biosynthesis, provides an important theoretical support for related variety cultivation, and provides a new solution for variety improvement.

Drawings

FIG. 1 is a graph showing the results of AP2 regulation of the expression of structural genes in the proanthocyanidin biosynthetic pathway;

FIG. 2 is the result of AP2 regulation of expression of transcriptional regulatory genes in the proanthocyanidin biosynthetic pathway;

FIG. 3 is the result of AP2 transcriptional activation of the MYBL2 gene;

FIG. 4 shows the gene expression results of MYBL2 in pods at different developmental stages in AP2 mutants;

FIG. 5 is the results of AP2 binding directly to the AT-rich element of the MYBL2 gene promoter, activating MYBL2 gene expression;

FIG. 6 is an analysis of the interaction of AP2 protein with proteins in the proanthocyanidin biosynthetic pathway in yeast;

FIG. 7 is protein interactions of AP2 and MYBL2 in tobacco;

FIG. 8 shows protein interactions of AP2 with MYBL 2C-terminus in yeast;

FIG. 9 is TT8 interacting with MYBL 2N-terminal protein in yeast;

FIG. 10 is GL3 interacting with MYBL 2C-terminal protein in yeast;

FIG. 11 is a protein interaction of EGL3 with the N-terminus of MYBL2 in yeast;

FIG. 12 is protein interactions in tobacco at the AP2 and MYBL 2C-terminus, TT8 and MYBL 2N-terminus;

FIG. 13 is the interaction of MYBL2 with GL3 or EGL3 in tobacco;

FIG. 14 is a CoIP experiment demonstrating the interaction of AP2 with MYBL2 proteins and MYBL2 with TT8, GL3 or EGL3 proteins;

FIG. 15 is a graph showing that AP2-MYBL2-TT8 or AP2-MYBL2-EGL3 was able to form protein complexes in plants by co-immunoprecipitation experiments (CoIP);

FIG. 16 is the transcriptional activity of AP2 in inhibiting MBW (TT 2-TT8-TTG 1) ternary complex by MYBL 2;

FIG. 17 is that AP2 acts genetically upstream of MYBL2, AP2 regulating the biosynthesis of proanthocyanidins by MYBL 2;

FIG. 18 is AP2 genetically acts upstream of MBW ternary complex members TT2 and TT 8;

FIG. 19 is a molecular mechanism by which AP2 regulates proanthocyanidin biosynthesis.

Detailed Description

The present invention is described in detail below with reference to specific embodiments, which are only for the purpose of facilitating understanding of the technical solutions of the present invention by those skilled in the art, and are not intended to limit the scope of the present invention.

In the present invention, the reagents, materials, equipment and the like used, if not specified, are commercially available or are commonly used in the art.

The methods of the examples, unless otherwise specified, are all conventional in the art.

EXAMPLE 1 AP2 Gene regulates expression levels of structural genes and transcriptional regulatory genes in the proanthocyanidin biosynthetic pathway

Mutants of the AP2 gene ((At 4G 36920)) AP2-6 were purchased from Arabidopsis Biological Resource Center (The Ohio State University, columbus, OH, USA). The full-length CDS sequence of the AP2 is cloned, the full-length CDS sequence of the AP2m3 is obtained through a point mutation technology, the full-length CDS sequence is cloned into a plant expression vector pB2GW7, the plant expression vector pB2GW7 is further transferred into GV3101 agrobacterium tumefaciens, the plant expression vector is transferred into an arabidopsis wild type through a flower infection method, and an over-expression plant is identified and obtained through a quantitative PCR method.

The sequences of AP2 and AP2m3 are seen at website www.arabidopsis.org. The CDS sequence of the AP2 is 17400948bp to 17403140bp of chr 4; the CDS sequence of AP2m3 was AP2, the T mutation of 17403020 to A, the A mutation of 17403023 to T, the A mutation of 17403026 to T, the A mutation of 17403029 to C, the A mutation of 17403032 to T, and the A mutation of 17403035 to T were identical in the encoded protein sequences, although the nucleotide sequences were different.

The expression levels of structural genes and transcriptional regulatory genes in the proanthocyanidin biosynthetic pathway in mutants and over-expressed materials of the AP2 gene were examined by selecting pods as materials 4 days after pollination. The research finds that: the expression level of the early structural gene in the proanthocyanidin biosynthesis pathway is not regulated and controlled by the AP2 gene; the expression level of the late key genes DFR, ANS, ANR, TT, TT19, AHA10 and the like in the proanthocyanidin synthesis pathway is obviously inhibited in the material for over-expressing the AP2 gene; the levels of gene expression of ANR, TT12, TT19 and AHA10 were significantly up-regulated in mutants of the AP2 gene (fig. 1). Meanwhile, it was also found that the expression level of TT2 gene in the transcriptional regulator closely related to proanthocyanidin biosynthesis was negatively regulated by AP2 gene, while the gene expression level of MYBL2, which inhibits proanthocyanidin biosynthesis, was significantly positively regulated by AP2 gene (FIG. 2). These results indicate that the AP2 gene may negatively regulate the biosynthesis of proanthocyanidins by positively regulating gene expression levels of MYBL2 and negatively regulating gene expression levels of TT2, thereby further affecting expression levels of structural genes or transport-related genes.

Example 2 determination of the direct target Gene of the AP2 Gene

(1) Determination of expression of the transcriptional activation MYBL2 Gene of the AP2 Gene by experiments such as transcriptional Activity analysis

In order to detect potential target genes regulated by the AP2 gene, the study cloned the promoters of the genes in the proanthocyanidin biosynthetic pathway, totaling 25 genes, as shown in Table 1, for specific sequence information see Arabidopsis thaliana https:// www.arabidopsis.org/. By detecting the activity of luciferases, the studies analyzed the regulation of their transcriptional activity by the AP2 gene. And (3) data display: the MYBL2 gene was significantly activated by the AP2 gene (fig. 3), and these results are consistent with those of fig. 2 described above.

The gene expression levels of MYBL2 in pods at different developmental stages in the mutants of the AP2 gene were further analyzed and the data showed that: the reduced level of gene expression of MYBL2 in the mutant of the AP2 gene compared to the wild type (fig. 4), also supports the expression of the AP2 gene activating MYBL2 gene. These results indicate that the AP2 gene activates gene expression of MYBL2, and that MYBL2 may be the target gene for the AP2 gene.

(2) The MYBL2 gene is a direct target gene of the AP2 gene, and the AP2 protein is directly combined with a promoter of the MYBL2 gene so as to activate the expression of the MYBL2 gene

TABLE 1

Gene name	Gene number	Promoter length before the gene start codon ATGThe degree (bp),
			CHS	At5G13930	1328
CHI	At3G55120	1126
			CHIL	At5G05270	1726
F3H	At3G51240	1123
			F3'H	At5G07990	1912
DFR	At5G42800	1208
			ANS	At4G22880	1097
ANR	At1G61720	689
			TT12	At3G59030	1701
TT19	At5G17220	1264
			AHA10	At1G17260	618
TT9	At3G28430	1845
			TT10	At5G48100	1190
TT15	At1G43620	1372
			TT2	At5G35550	2245
MYB5	At3G13540	918
			TT8	At4G09820	1501
GL3	At5G41315	1210
			EGL3	At1G63650	1233
TTG1	At5G24520	1090
			MYBL2	At1G71030		974
TT1	At1G34790	1392
			TT16	At5G23260	1497
TTG2	At2G37260	1490
			STK	At4G09960	1595

method and procedure for transcriptional activation experiments: the CDS full-length sequence of AP2m3 and all gene promoter sequences are cloned into pENTR/SD/D-TOPO respectively, and then gene sequencing proves that all sequences are correct; then pENTR-AP2m3 is further cloned into a p2GW7 effect vector, all pENTR-promoters are respectively cloned into a p2GW7-LUC reporter gene vector, and plasmids are extracted by utilizing an endotoxin-free mass extraction kit. Protoplasts of 4-week-old Arabidopsis leaves were isolated, 10. Mu.g of effector plasmid, 6. Mu.g of reporter plasmid and 1. Mu.g of reference plasmid were mixed, transferred into protoplasts by PEG method, and cultured in the dark at 25℃for 16-20 hours in an incubator, and the protoplasts were collected and assayed for luciferase activity. Experimental results show that the MYBL2 gene can be a target gene directly regulated and controlled by the AP2 gene. Previous studies (Dinh TT, girke T, liu X, yant L, schmid M, chen X (2012) The floral homeotic protein APETALA2 recognizes and acts through an AT-rich sequence element. Development) have also shown that AP2 protein inhibits or activates expression of downstream target genes by binding to the cis-acting element AT-rich. Thus, the study first analyzed to find that the MYBL2 gene promoter region shares 5 AT-rich elements (TTTGTT or AACAMA) (FIG. 5 a), then mutated to CCCCC, respectively, resulting in a series of mutated promoters, and analyzed how these mutations affect the regulation of the transcriptional activity of the AP2 gene by detecting the activity of luciferases. The data studied show that: the cis-acting element M5 (AACAAA) is a key site for the AP2 gene to regulate MYBL2 (fig. 5 b).

Further through the ChIP test, the method and the steps of the ChIP test: firstly, constructing a carrier for over-expressing AP2m3 and fusing Myc, transforming Arabidopsis by an inflorescence infection method, and identifying by qRT-PCR to obtain a positive transgenic strain; harvesting Arabidopsis pods 3-5 days after flower opening, soaking in 1% formaldehyde solution, and vacuumizing for 15 minutes; experiments were then performed using ChIP-grade Myc antibodies, following standard chromatin immunoprecipitation experimental procedures, and the results demonstrated that the AP2 protein activated expression of the MYBL2 gene in plants by binding to the cis-element M5 (fig. 5 c). These data indicate that the MYBL2 gene is a direct target gene for the AP2 gene.

Example 3 determination of proteins that interact with AP2 protein

(1) Determination of AP2 interacting proteins by Yeast two-hybrid experiments

To investigate whether there is a protein interaction between the AP2 protein and transcription factors in the proanthocyanidin biosynthetic pathway, 11 transcription factors involved in proanthocyanidin biosynthesis and the CDS sequence of the protein were cloned and constructed on pGADT7 vector. Meanwhile, the CDS sequence of AP2 was constructed on pGBKT7 vector. Experimental method and procedure for yeast two-hybrid: preparing a competent yeast strain AH109, transferring the constructed bait plasmid pGBKT7 into yeast by using a LiCl-PEG method, and screening in an SD/-Trp culture medium to obtain positive clones; then culturing on SD/-Trp/-His culture medium with different concentrations of 3-AT for 3-5 days to determine the optimal concentration of 3-AT to be 15mM/L; preparing AH109 competence containing bait plasmid, transferring a prey plasmid pGADT7 into yeast by using a LiCl-PEG method, and screening in SD/-Trp/-Leu culture medium to obtain positive clones; finally, protein interactions were identified on SD/-Trp/-Leu/-His/-Ade medium containing 15 mM/L3-AT. The experimental results show that: AP2 interacted with MYBL2 strongly and AP2 interacted with TT2 and MYB5 weakly (FIG. 6).

(2) Determination of AP2 interacting proteins by bimolecular fluorescence complementation assay (BIFC)

To further confirm the above protein interactions, the study constructs the AP2 gene into vector pBI-2YN-CAT of BIFC experiments and constructs MYBL2, TT2 and MYB5 into pBI-2YC-CAT, respectively. Experimental method and procedure for bimolecular fluorescence complementation experiment (BIFC): the constructed vector is transferred into agrobacterium GV 3101. Positive clones were obtained by conventional PCR identification, and cultured at 28℃until OD600 was 0.5-0.8; centrifuging at 5000rpm for 10min to collect bacterial liquid, and then re-suspending bacterial cells with MES buffer (MES final concentration of 0.05M/L, sodium phosphate final concentration of 2mM/L, acetosyringone final concentration of 0.1mM/L and 0.5% anhydrous glucose, pH of 5.6) until OD600 is 0.5-0.8; buchner's smoke grown for 4 weeks was injected and grown for 36-48 hours, and fluorescent signals generated by protein interactions were observed with a laser confocal microscope. A strong protein-interacting fluorescent signal was detected in the Nicotiana benthamiana nuclei co-expressing the AP2 gene with the MYBL2 gene (FIG. 7), whereas no distinct fluorescent signal was detected between AP2 and TT2 or MYB 5. Thus, the experimental results of BIFC confirm that AP2 protein interacts with MYBL2 protein in plants.

Example 4 determination of interaction of AP2 with MYBL2 protein

(1) Determination of protein interaction Domains between AP2, MYBL2 and TT8/GL3/EGL3

To further ascertain the interaction of AP2 with MYBL2 protein, AP2N (front 870bp of CDS containing AP2 gene) or AP2C (rear 435bp of CDS containing AP2 gene) was constructed onto pGBKT7 vector, MYBL2N (front 270bp of CDS containing MYBL2 gene), MYBL2C (rear 318bp of CDS containing MYBL2 gene), MYBL2dC (front 546bp of CDS containing MYBL2 gene), MYBL2dN (rear 42bp of CDS containing MYBL2 gene), MYBL2NE (front 423bp of CDS containing MYBL2 gene) and MYBL2CE (rear 165bp of CDS containing MYBL2 gene) onto pGBKT7 vector. The experimental procedure and procedure for yeast two-hybrid was as above, but the optimum 3-AT concentration was 2mM/L. The results of the yeast two-hybrid experiments show that the AP2 protein interacts with the MYBL 2C-terminal protein in yeast (FIG. 8).

To analyze protein interactions of MYBL2 and TT8, GL3 or EGL3 in yeast, CDS of MYBL2 was constructed on pGBKT7 vector, CDS of TT8, GL3 and EGL3 were constructed on pGADT7 vector, respectively. The experimental procedure and procedure for yeast two-hybrid was as above, but the optimum 3-AT concentration was 2mM/L. Further analysis of their interactions, it was found that TT8 protein and EGL3 protein interacted with MYBL 2N-terminus in yeast, while GL3 protein interacted with MYBL 2C-terminus in yeast (FIGS. 9-11).

Subsequently, the full-length CDSs of TT8, GL3 and EGL3 genes were respectively constructed to vectors pBI-2YN-CAT of BIFC experiments, and MYBL2C (the latter 318bp of CDS containing MYBL2 gene) and MYBL2N (the first 270bp of CDS containing MYBL2 gene) were respectively constructed to pBI-2YC-CAT. The interaction of the AP2 protein with the C-terminus of MYBL2 in tobacco in plants was further confirmed by BIFC experiments (experimental methods and procedures are the same as above), the interaction of the TT8 protein with the N-terminus of MYBL2 in tobacco, and the interaction of the GL3 protein or EGL3 protein with the MYBL2 protein in tobacco (FIGS. 12-13).

(2) Demonstration of the interaction of AP2 protein with MYBL2 protein by immunoprecipitation experiments (CoIP), and interaction of MYBL2 protein with TT8 protein, GL3 protein or EGL3 protein

In order to further examine whether the AP2 protein interacts with MYBL2 protein in a plant body and whether the MYBL2 protein interacts with TT8 protein, GL3 protein or EGL3 protein in the plant body, research and construction of vectors in which the AP2m3 protein is fused with Myc, the MYBL2 protein is fused with Flag and the TT8/GL3/EGL3 protein is respectively fused with HA are performed. Experimental method and procedure for co-immunoprecipitation experiment (CoIP): the constructed vector is transferred into agrobacterium GV 3101. Positive clones were obtained by conventional PCR identification, and cultured at 28℃until OD600 was 0.5-0.8; centrifuging at 5000rpm for 10min to collect bacterial liquid, and then re-suspending bacterial cells with MES buffer (MES final concentration of 0.05M/L, sodium phosphate final concentration of 2mM/L, acetosyringone final concentration of 0.1mM/L and 0.5% anhydrous glucose, pH of 5.6) until OD600 is 0.5-0.8; buchner cigarettes were injected for 4 weeks and grown for a further 36-48 hours. The tobacco leaves were harvested, ground to a fine powder in liquid nitrogen, and then protein was extracted according to the protein immunoprecipitation kit (magnetic bead method) from Biyun Tian, inc., and immunoprecipitated. Finally, immunoprecipitated proteins were detected according to conventional western blot assay methods and procedures. In tobacco leaves co-transformed with AP2m3-Myc and MYBL2-Flag, MYBL2-Flag could be successfully immunoprecipitated with Myc antibody, indicating that AP2 protein interacted with MYBL2 protein in plant protein (FIG. 14 a). CoIP experiments also demonstrated that MYBL2 protein interacted with TT8 protein, GL3 protein or EGL3 protein in vivo (FIGS. 14 b-d).

(3) Demonstration of the formation of protein complexes by Co-immunoprecipitation experiments (CoIP) with AP2-MYBL2-TT8 or AP2-MYBL2-EGL3

The above data indicate that the AP2 protein is capable of interacting with the C-terminus of MYBL2, while the N-terminus of MYBL2 interacts with either the TT8 protein or the EGL3 protein. Thus, studies speculated that either AP2-MYBL2-TT8 or AP2-MYBL2-EGL3 may form protein complexes in plants. CoIP experiments (methods and procedures are as above) also showed that AP2-MYBL2-TT8 or AP2-MYBL2-EGL3 forms protein complexes in plants (FIGS. 15 a-b), whereas AP2-MYBL2-GL3 is not able to form protein complexes in plants (FIG. 15 c).

(4) Inhibition of transcriptional activity of MBW ternary complexes by AP2 via MYBL2

The above results indicate that AP2-MYBL2-TT8 or AP2-MYBL2-EGL3 forms protein complexes in plants. MBW (MYB-bHLH-WD, wherein MYB comprises TT2 and MYB5, TT2 is a major gene, bHLH comprises TT8, GL3 and EGL3, WD is TTG 1) ternary complex directly regulates and controls transcription of key genes in the biosynthesis pathway of proanthocyanidins, and plays a very important role in the biosynthesis process of proanthocyanidins. Whether the AP2-MYBL2-TT8 or AP2-MYBL2-EGL3 protein complex affects the transcriptional activity of the MBW (TT 2-TT8-TTG 1) ternary complex. To answer this scientific question, studies analyzed their expression of the first branching enzyme ANR that regulates proanthocyanidin biosynthesis. Transcriptional activation experiments (experimental methods and procedures are the same above), and the data indicate that AP2 cannot inhibit activation of the ANR gene by the ternary complex in the absence of MYBL 2; when both AP2 and MYBL2 were present, activation of the ANR gene by the ternary complex was significantly inhibited (fig. 16). These results indicate that AP2 inhibits the transcriptional activity of MBW ternary complexes by MYBL 2.

(5) Genetically AP2 acts on the upstream of MYBL2 and MBW ternary complex, regulating biosynthesis of proanthocyanidins by MYBL2

AP2 regulates the biosynthesis of proanthocyanidins via MYBL 2. In order to further analyze the relation between AP2 and ternary complex of MYBL2 and MBW genetically, the study obtained mutants of MYBL2 gene MYBL2-1 and MYBL2-2, and over-expressed strain of MYBL2 OE H3 and MYBL2 OE H6, and mutants of TT2 and TT8 genes, which were then genetically hybridized with mutant AP2-6 of AP2 gene and over-expressed strain AP2m3 OE, respectively, to further obtain homozygotes. Qualitatively and quantitatively, the proanthocyanidin content was analyzed (fig. 17), and compared with the wild type Col, the seed coats of the MYBL2 OE H3 and MYBL2 OE H6 lines were both lighter in color, and the proanthocyanidin content was also significantly lower than that of the wild type. When the MYBL2 gene is overexpressed in the AP2-6 mutant, the content of the proanthocyanidin is close to that of MYBL2 over-expression material; on the other hand, when the MYBL2 gene was mutated in the AP2 overexpressing material, resulting in a proanthocyanidin content close to that of the MYBL2 mutant, these results indicate that: AP2 acts genetically upstream of MYBL2, regulating biosynthesis of proanthocyanidins by MYBL 2.

Meanwhile, studies have also analyzed the relationship of the ternary complex of AP2 and MBW genetically. The results showed that the double mutants AP2-6/TT2 and AP2-6/TT8 had similar proanthocyanidin content in the single mutants TT2 or TT8, respectively (FIG. 18). Thus, AP2 acts genetically upstream of MBW ternary complex members TT2 and TT 8.

According to all the above findings, the molecular mechanism by which AP2 regulates proanthocyanidin biosynthesis includes two aspects: on the one hand, the AP2 can activate the expression of MYBL2 genes at the transcription level by directly combining with a promoter region of MYBL 2; on the other hand, AP2 can directly interact with MYBL2 at protein level, and by interaction of MYBL2 protein with TT8 or EGL3 protein, AP2-MYBL2-TT8 or AP2-MYBL2-EGL3 protein complex is formed, which disrupts MBW ternary complex formation, and finally inhibits expression of late genes (LBGs) DFR, ANS, ANR, TT, TT19, AHA10, etc. in primary pigment biosynthesis pathway, and inhibits proanthocyanidin biosynthesis (fig. 19).

The AP2-MYBL2 molecular module is used as a new important candidate gene for improving plant proanthocyanidin biosynthesis, and can be used for increasing or reducing the proanthocyanidin content in plant breeding work; in order to increase the content of proanthocyanidins, the MBW protein complex is more stable by mutating an AP2-MYBL2 molecular module, so that the accumulation of proanthocyanidins is promoted; in order to reduce the content of proanthocyanidins, the expression of the MYBL2 gene can be further positively regulated by the AP2 through increasing the expression of the AP2 gene, or the expression of the AP2-MYBL2 molecular module gene can be simultaneously increased, so that more AP2 proteins are generated, more MYBL2 proteins are cooperatively generated, more AP2-MYBL2-TT8 or AP2-MYBL2-EGL3 protein complexes are further generated in plants, the formation of MBW protein complexes is inhibited, and finally the content of proanthocyanidins is reduced.

The foregoing is merely exemplary of the present application, and the scope of the present application is not limited to the specific embodiments, but is defined by the claims of the present application. Various modifications and changes may be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

Application of AP2-MYBL2 molecular module in regulating and controlling proanthocyanidin biosynthesis.
2. The use according to claim 1, wherein the AP2 protein binds directly to the promoter of the MYBL2 gene at the transcriptional level, thereby activating expression of the MYBL2 gene.
3. The use according to claim 1, wherein the MYBL2 gene is a direct target gene for the AP2 protein.
4. The use according to claim 1, wherein the AP2 protein activates expression of the MYBL2 gene by binding to the cis-acting element M5 in plants.
5. The use according to claim 1, wherein the AP2 protein acts genetically upstream of MYBL2, regulating the biosynthesis of proanthocyanidins by activating the expression of the MYBL2 gene.
Application of AP2-MYBL2-TT8 or AP2-MYBL2-EGL3 protein complex in regulating biosynthesis of proanthocyanidins.
7. The use of claim 6, wherein the AP2 protein interacts with the C-terminus of the MYBL2 protein and the N-terminus of the MYBL2 protein interacts with the TT8 protein or EGL3 protein.
8. The use according to claim 6, wherein the AP2 protein forms a protein complex of AP2-MYBL2-TT8 or AP2-MYBL2-EGL3 at the protein level by interacting with the MYBL2 protein, inhibiting the formation of MYB-bHLH-WD ternary complex.
9. The use according to claim 8, wherein the AP2 protein acts genetically upstream of TT2 and TT8 in MYB-bHLH-WD ternary complex.