CN114015699A - Application of gene BnbHLH92a in regulation of anthocyanin or procyanidine - Google Patents

Abstract

The invention relates to genetic engineering, in particular to application of a gene BnbHLH92a in regulation and control of anthocyanin or procyanidine in brassica plants. The invention discloses a gene BnbHLH92a capable of inhibiting synthesis of flavonoid compounds in brassica plants, and accumulation of anthocyanin or procyanidine of the gene. The gene BnbHLH92a interacts with BnTTG1 to form a functional complex for inhibiting anthocyanin or procyanidine, and BnbHLH92a is combined with a BnTT18 promoter, so that the accumulation of anthocyanin or procyanidine in the brassica napus is inhibited. The invention provides a new visual angle for disclosing the transcriptional regulation of the biosynthesis pathway of the brassica flavonoid.

Description

Application of gene BnbHLH92a in regulation of anthocyanin or procyanidine

Technical Field

The invention relates to genetic engineering, in particular to application of a gene BnbHLH92a in regulation and control of anthocyanin or procyanidine in brassica plants.

Background

Rape is one of the important oil crops in the world, is not only a plant edible oil source after soybeans and oil palms, but also an important feed source after soybean meal, and is an ideal raw material for developing biodiesel. Along with the reduction of the planting area of the rape and the increase of the demand of people on the rape seed oil, the improvement of the quality, the yield and the cake quality of the rape seed oil become one of the important targets of rape breeding. Yellow seeds have the advantage over black seeds of the same genetic background that they have thinner seed coats, higher oil and protein content, lower cellulose and polyphenol content and clearer oil. Therefore, the yellow seed character is one of the important targets for improving the quality of the rape.

Flavonoids contribute to the color formation of flowers, fruits and seeds in nature. In Arabidopsis thaliana, analysis of a series of clear seed coats (TT) shows that the seed coat color of seeds is determined by the content of Proanthocyanidins (PAs), wherein more than 22 TT type genes related to the seed coat color are included, and the action mechanism of related genes of yellow seed traits of Brassica plants, such as TTG1 in Chinese cabbage, TT8 in mustard type rape, Chinese cabbage and cabbage type rape and TT1 in cabbage type rape, is disclosed, but the genes are also expressed between black seed and yellow seed rape strains to show different expression patterns. In addition, TT2(R2R3-MYB), TT8 (basic helix-loop-helix, bHLH) and WD40 regulatory proteins can form a MYB-bHLH-WDR (MBW) ternary complex, and influence flavonoid biosynthetic pathways by regulating the expression abundance and pattern of structural genes.

The MBW complex plays an important role in seed development and is known to be involved in regulating dihydroflavonol-4-reductase (DFR), leukocyanin dioxygenase (LDOX/TT18), banyuls (ban), TT19 and TT12 in seeds, leading to a change in the colour of the seed coat. In the MBW ternary complex model, bHLH is considered a conserved core component, and transcription factors of the bHLH type are rarely reported.

However, to date, flavonoid-related genes of brassica have not been elucidated in great detail, and it is unknown how the related genes regulate transcription of flavonoid biosynthetic pathways, and studies on bHLH type transcription factors and their functional roles in the anthocyanin/PAs pathway are lacking.

Disclosure of Invention

In order to solve the problems, the invention adopts an RT-PCR method to separate a homologous gene BnbHLH92a of AtbHLH92, which belongs to the S7 subfamily containing G-box binding protein and comprises a basic spiral-loop-spiral structural domain.

One of the purposes of the invention is to provide a gene participating in regulating the anthocyanin/PAs pathway function, and the specific technical scheme is as follows:

the gene BnbHLH92a, wherein the nucleotide sequence of the BnbHLH92a is shown in SEQ ID NO. 1.

The second purpose of the present invention is to protect a recombinant plasmid containing the gene BnbHLH92 a.

The third purpose of the invention is to protect the engineering strain prepared by the recombinant plasmid.

The invention also aims to protect the application of the BnbHLH92a in preparing an inhibitor for inhibiting the accumulation of anthocyanin or procyanidine.

Specifically, anthocyanin or procyanidin accumulation in brassica plants is inhibited.

The fifth purpose of the invention is to provide a method for inhibiting the synthesis of flavonoid, which comprises the following specific technical scheme:

a method for inhibiting flavone synthesis in a target plant is characterized in that the gene BnbHLH92a in the technical scheme is overexpressed in the target plant.

Specifically, the method comprises the following steps:

(1) constructing a vector containing BnbHLH92 a;

(2) the vector is introduced into a target plant by an agrobacterium-mediated method.

Specifically, the BnbHLH92a inhibits the expression of a gene TT8 in a target plant.

Specifically, the target plant is a brassica plant.

Specifically, the target plant is arabidopsis thaliana.

Specifically, the BnbHLH92a is specifically expressed in the hilum.

The invention also aims to provide a method for inhibiting anthocyanin or procyanidine in a target plant by using the gene BnbHLH92a, and the specific technical scheme is as follows:

the method of BnbHLH92a of claim 1 for inhibiting anthocyanins or proanthocyanins in a plant of interest, the BnbHLH92a acting as a transcriptional repressor for inhibiting expression of BnTTG1 and BnTT 18.

Specifically, the BnbHLH92a interacts with BnTTG1 to form a functional complex for inhibiting anthocyanin or procyanidin.

Specifically, the BnbHLH92a binds to the BnTT18 promoter.

Specifically, the target plant is brassica napus.

The invention has the advantages that: the gene BnbHLH92a constructed by the invention can be used as a transcription inhibitor to destroy the synthesis of flavonoid compounds in transgenic Arabidopsis. Meanwhile, BnbHLH92a can inhibit the expression of BnTTG1 and BnTT18, the specific principle is that BnbHLH92a and BnTTG1 interact to form a functional complex for inhibiting anthocyanin or procyanidine, and BnbHLH92a is combined with a BnTT18 promoter, so that the accumulation of anthocyanin or procyanidine of the brassica napus is inhibited.

Drawings

FIG. 1 is a construction diagram of the gene BnbHLH92a of the present invention;

FIG. 2 is a subcellular localization and transcriptional activity assay of BnbHLH92 a;

FIG. 3 shows the expression pattern of BnbHLH92 a;

FIG. 4 is the BnbHLH92a negative regulation of Arabidopsis anthocyanin and PAs accumulation;

FIG. 5 is a UPLC-HESI-MS/MS map of important components in wild type and OE-92a Arabidopsis seeds;

FIG. 6 is the interaction of BnbHLH92a with BnTTG 1;

FIG. 7 shows the transcriptional regulation of BnHLH 92a on the BnTT18 promoter;

FIG. 8 is a hypothetical illustration of BnbHLH92a negatively regulating anthocyanin and PAs biosynthesis;

FIG. 9 shows BnbHLH92a overexpression T0 generation Arabidopsis thaliana screen;

FIG. 10 is the seed coat color of wild type and overexpression BnbHLH92a transgenic Arabidopsis lines;

FIG. 11 is a graph of the base peaks of the negative ion patterns of wild type and OE-92a Arabidopsis seeds;

FIG. 12 is the relative expression level of AtJAZs in OE-92a strain.

Detailed Description

The present invention is further described in detail by the following examples, which should be understood that the present invention is not limited to the particular examples described herein, but is intended to cover modifications within the spirit and scope of the present invention.

Example 1 isolation and characterization of the Gene BnbHLH92a

In this example, according to the genome annotation of Brassica napus, a nucleotide sequence of a homologous gene BnbHLH92a of AtbHLH92 was isolated by RT-PCR method, as shown in SEQ ID NO. 1. The BnbHLH92a full-length cDNA is 717bp, encodes 238 amino acids, and has a predicted molecular weight of about 27.58kDa and an isoelectric point of 10.0. In Brassica napus, 602 potential bHLHs proteins have been identified and divided into 35 subfamilies, and we found that BnbHLH92 belongs to the S7 subfamily containing the G-box binding protein. As shown in fig. 1, sequence analysis showed that BnbHLH92a contains a basic helix-loop-helix domain (fig. 1A). Previous studies have shown that LcbHLH92 is a negative regulator of the anthocyanin/PAs pathway in Leymus chinensis (Leymus chinensis). Phylogenetic analysis shows that BnbHLH92a, AtbHLH92 and LcbHLH92 belong to the same group (FIG. 1B: phylogenetic analysis for constructing BnbHLH92a and other bHLH proteins of different species by adopting the adjacent method of MEGA7.0 software), and the BnbHLH92a possibly has the function of participating in regulating the anthocyanin/PAs pathway.

BnbHLH92a as activity inhibitor is located in nucleus

To study the subcellular localization of BnbHLH92a in vivo, a vector Pro35S, BnbHLH92a, which is driven by a strong promoter and fused with a GFP fluorescent protein gene, was constructed and transformed into tobacco epidermal cells. The control GFP protein showed fluorescent signals in both cytoplasm and nucleus, whereas Pro35S: BnbHLH92A-GFP fusion protein was detected only in the nucleus (FIG. 2A: BnbHLH92A protein localized in the nucleus of epidermal cells under tobacco leaf lamina), consistent with the functional localization of BnbHLH92A as a transcription factor.

To further validate whether the BnbHLH92a is a transcriptional activator or repressor, we measured its transcriptional activity in arabidopsis protoplasts using the LUC reporter system. The firefly Luciferase (LUC) reporter fused to five copies of the GAL4 DNA binding element with the minimal CaMV35S promoter, Renilla luciferase (REN) under the control of the 35S promoter was used as an internal control, and the VP16 transcriptional activation domain was used as a positive control (FIG. 2B: schematic representation of reporter and effector plasmids used in the dual-luciferase reporter assay). As shown, pBD-BnbHLH92a significantly inhibited the expression of the LUC reporter gene compared to the effect of pBD-VP 16 (FIG. 2C: analysis of the activity of Arabidopsis protoplasts transient expression on BnbHLH 92. representative results are expressed as mean. + -. SD). These results indicate that BnbHLH92a functions as a transcriptional repressor.

Spatio-temporal expression pattern of BnbHLH92a

BrassicaEDB database https:// branched. BnbHLH92a was mainly expressed in 35 days of seeds, 30, 35 and 40 days of seed coats, 30 days of episperms, 27 and 35 days of hilum, 35 and 40 days of silique, respectively (FIG. 3A: expression pattern of BnbHLH92a in different tissues and organs of Brassica napus). In order to further determine the functional role of the BnbHLH92a in the seed coat color, the expression level of the BnbHLH92a in different seed coat development stages of yellow seed and black seed rape is detected by adopting a qRT-PCR method. The expression level of BnbHLH92a in yellow seed coat is obviously higher than that in black seed coat (FIG. 3B: the expression pattern of BnbHLH92a in cabbage type rape yellow and black seed is analyzed by qRT-PCR), which indicates that it may be a negative regulator of seed coat color. In addition, in situ hybridization experiments were also performed using the BnbHLH92a probe to study the expression characteristics of the gene in developing seeds, and we found that significant signals were detected in the seed coat and hilum of yellow seeds, but not in the seed coat of black seeds (FIG. 3C-F: in situ hybridization analysis of BnbHLH92 in yellow and black seeds of Brassica napus). The specific expression of BnbHLH92a was found by in situ hybridization to be specific in the hilum (fig. 3C). In addition, the expression level of BnbHLH92a was much higher in developing seeds, seed coat, testa, petiole and pericarp than in other organs (fig. 3A), and the difference in expression level was significant in yellow and black seeds (fig. 3B). The results indicate that BnbHLH92a may play an important role in the accumulation and transport of anthocyanins/PAs.

Example 2 overexpression of BnbHLH92a inhibits anthocyanin and PA in transgenic Arabidopsis thaliana

To identify the biological function of BnbHLH92a, a 35S driven overexpression vector was constructed and transformed into wild type Arabidopsis by floral dipping of the recombinant plasmid pEarleyGate101-BnbHLH92a by Agrobacterium mediated method. The screening label of the pEarleyGate101 plasmid is Basta, 50mg/L Basta solution is sprayed twice to arabidopsis thaliana with one week age, 12 arabidopsis thaliana plants growing normally are screened, DNA of the 12 arabidopsis thaliana plants is extracted, a vector front primer and a gene rear primer F35S + ovBnbHLH92aR primer are used for detection, and 12 arabidopsis thaliana positive strains of over-expression BnbHLH92a are detected together (figure 9). Subsequently, transgenic arabidopsis plants of T1 and T2 generations were tested using the same screening and identification method, in which 10 independent transgenic lines with significantly changed seed coat phenotype were generated (fig. 10). The invention adopts qRT-PCR method to identify two independent strains (OE-92a #8 and OE-92a #31) and further analyze. Obviously, the expression level of BnbHLH92a in OE-92a #8 and OE-92a #31 plants is significantly up-regulated (FIG. 4A: relative expression level of BnbHLH92a in OE-92a Arabidopsis), and the seed coat color is also significantly changed (FIG. 4B: DMACA staining of over-expressed OE-92a strains and wild type control seeds). Numerous studies have shown that proanthocyanidins are important metabolites determining seed coat color in arabidopsis, and histochemical staining analysis showed a significant reduction in the content of PAs in the lines of arabidopsis overexpressing BnbHLH92a compared to the wild-type controls stained with DMACA reagent (fig. 4B).

Furthermore, quantitative analysis showed that anthocyanin (FIG. 4C: anthocyanin levels of WT and different transgenic lines) and PAs (soluble and insoluble PAs) were detected in transgenic lines of Arabidopsis overexpressing BnbHLH92a in comparison to wild type (FIG. 4D: soluble proanthocyanidin levels of WT and different transgenic lines; FIG. 4E: insoluble proanthocyanidin levels of WT and different transgenic lines). To investigate in detail the relationship of BnbHLH92a to the flavonoid biosynthetic pathway, transgenic lines were further analyzed for the type and content of polyphenols and flavonoids by UPLC-HESI-MS/MS (FIG. 11). The results show that the BnbHLH92a-OE has significant difference with the content of flavonoid compounds and metabolic derivatives thereof in wild plants, in particular, epicatechin and proanthocyanidin (Table 1: UPLC-HESI-MS/MS identifies significant difference flavonoid compounds (mu g g-1FW) in wild type and OE-92a Arabidopsis seed extracts, and figure 5 (A) [ DP2] -1: (B) [ DP2] -2; (C) [ DP3] -1; (D) [ DP3] -2; (E) [ DP4 ]; (F) epicatechin shows that BnbHLH92a may play an important role in regulating anthocyanin and PAs accumulation.

TABLE 1 UPLC-HESI-MS/MS identification of differentially significant flavonoids (μ g g-1FW) in wild-type and OE-92a Arabidopsis seed extracts

To further determine the influence of BnbHLH92a on flavonoid biosynthetic pathway genes, the expression level of structural genes and regulatory genes in BnbHLH92a-OE strain was determined by qRT-PCR method. Consistent with the reduction of polyphenols and flavonoids in strains of BnbHLH92a OE, the sustained down-regulation of the expression of flavonoid biosynthetic genes (including AtTT6, AtDFR/TT3, AtTT18/LDOX, AtBAN/ANR, AtTT8, and AtTTG1) in strains of BnbHLH92a-OE compared to controls (FIG. 4F: qRT-PCR analysis of flavonoid biosynthetic genes of strains of BnbHLH92 a-OE) indicated that BnbHLH92a has a negative regulatory effect on flavonoid biosynthesis in strains of BnbHLH92 a-OE. In one aspect, the expression levels of AtJAZ3, AtJAZ5, AtJAZ6, AtJAZ8, AtJAZ10, and AtJAZ12 were significantly higher in the BnbHLH92a-OE strain than the control (fig. 12). Our results also demonstrated that bHLH92 protein inhibited the expression of TT8 by activating the JAZ gene. On the other hand, we found that AtTTG1 in BnbHLH92a-OE strain was also significantly reduced compared with wild type control (FIG. 4E), which indicates that BnbHLH92a not only indirectly inhibited the expression of TT8, but also could be directly involved in MBW model, and a new mechanism inhibited the synthesis pathway of Arabidopsis flavonoids.

BnbHLH92a interacts with BnTTG1 to negatively regulate the accumulation of anthocyanin/PAs

In the present invention, BnbHLH92a overexpression significantly down-regulated the mRNA levels of AtDFR/TT3, AtTT18/LDOX, AtBAN/ANR, AtTT8, AtTTG1, and JAZ genes (FIG. 4F). Therefore, we further tested the potential interaction of BnHLH 92a and BnTTG1, with BnTTG1 having both BnTTG1a and BnTTG1b members in oilseed rape. In the yeast two-hybrid (Y2H) experiment, there was an interaction between BnbHLH92a and BnTTG1 (FIG. 6A: yeast two-hybrid experiment in which BnbHLH92a and BnTTG1 interact). Meanwhile, the interaction observed in the yeast two-hybrid experiment was further identified using a bimolecular fluorescence complementation method (BiFC). First, the tobacco epidermal cells were used to perform subcellular localization analysis of BnTTG 1. The control GFP protein was distributed throughout the cell (FIG. 6B: subcellular localization of BnTTG1-GFP fusion protein in tobacco leaf subepithelial cells), whereas the Pro35S: BnTTG1a fusion protein was concentrated in the cytoplasm and nucleus (FIG. 6B), whereas the Pro35S: BnTTG1B fusion protein was localized in the nucleus (FIG. 6B), consistent with published results for localization of TTG1 to Arabidopsis nuclei and cytoplasm. BnbHLH92a was fused to the c-terminus of Yellow Fluorescent Protein (YFP), and BnTTG1a and BnTTG1b were fused to the n-terminus of YFP protein, respectively. Yellow fluorescence was detected in the respectively co-transfected tobacco epidermal cells after mixing the plasmids BnbHLH92a-cYFP and nYFP-BnTTG1a or BnbHLH92a-cYFP and nYFP-BnTTG1b (FIG. 6C: BiFC analysis of the interaction of BnbHLH92a and BnTTG1a in the epidermal cells under tobacco leaf; FIG. 6D: BiFC analysis of the interaction of BnbHLH92a and BnTTG1b in the epidermal cells under tobacco leaf). These results indicate that BnbHLH92a is able to interact with BnTTG1 in vivo to form functional complexes that inhibit anthocyanidins or procyanidins.

BnbHLH92a directly binds to BnTT18 promoter and inhibits the expression thereof

The results indicate that BnbHLH92a is a negative regulator that affects flavonoid biosynthesis. Among the enzymes encoding flavonoid biosynthesis, DFR, TT18 and BAN belong to the Late Biosynthetic Genes (LBGs) which are regulated by the MBW ternary complex consisting of three transcription factors, i.e. (R2R3-MYB, bHLH and WD 40). To investigate this process in more detail, we used the bHLH type transcription factor recognition site cis-regulatory element G-box to search for the BnbHLH92 a-targeted gene We found multiple cis-regulatory elements G box in the BnTT18 promoter region (FIG. 7A: typical and core G box elements are distributed in the promoter region of BnTT 18), suggesting that these structural genes may be directly regulated by BnHLH 92 a. As expected, BnbHLH92a could bind directly to the BnTT18 promoter in vivo by yeast single-hybrid (Y1H) (FIG. 7B: yeast single-hybrid experiments. binding screen on SD-Leu-Ura medium in the presence of AbA 250. empty vector and BnTT18 promoter (pGADT7+ BnTT18pro) as negative controls). A dual luciferase reporter assay was further performed to confirm this hypothesis. The bifluorinase reporter plasmid contained the BnTT18 promoter sequence fused to LUC, REN driven by CaMV35S promoter as an internal control, and a plasmid containing the full-length cDNA sequence of BnbHLH92a as the effector (FIG. 7C: schematic representation of reporter and effector plasmids used in the dual luciferase reporter assay). As expected, the data showed that the activity of the BnTT18 promoter was significantly reduced by BnHLH 92a (FIG. 7D: dual luciferase reporter gene analysis shows that BnHLH 92a can inhibit the activity of the BnTT18 promoter). In conclusion, the BnbHLH92a can be directly combined with the BnTT18 promoter to have negative influence on the cabbage type rape flavonoid synthetic pathway. BnbHLH92a may lead to reduced anthocyanin/PAs accumulation by regulating BnTTG 1. The BnbHLH92a could be found to bind directly to the promoter region of BnTT18 by Y1H and dual luciferase experiments (FIG. 7). Accordingly, we also noted a significant reduction in transcript abundance of AtTT18 in the BnbHLH92a-OE strain. These data suggest that the negative feedback mechanism of BnbHLH92a may be an important gene regulatory network for anthocyanin/PAs biosynthesis during rape seed development.

In conclusion, the invention not only further proves that the BnbHLH92a is used as a negative regulatory factor of an anthocyanin/PAs biosynthetic pathway, but also provides molecular evidence for the BnbHLH92a which can directly inhibit the expression levels of BnTTG1 and BnTT18 so as to reduce the accumulation of anthocyanin/PAs in Brassica napus. Therefore, we provide a hypothetical model of the involvement of BnbHLH92a in the regulation of the anthocyanin/PAs biosynthetic pathway (fig. 8). The result of the invention provides a theoretical basis for deep understanding of the potential mechanism of seed coat color and genetic improvement of brassica napus.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> university of southwest

Application of <120> gene BnbHLH92a in regulation of anthocyanin or procyanidine

<130> 2021

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 717

<212> DNA

<213> Artificial sequence (BnbHLH92a)

<400> 1

atggattttt tttctctaga ttctgttttc caagaagaag gagaaaattt ctgggatatg 60

atcgccggtg acgtctccgg cgatggtgac ggtgacaaaa ccgtaggtgt accaaacaga 120

agcgccttca ggtcatacgt gagggaccat gaacagagga gggtatcgtc ttcatcgacg 180

gtgaacgtga agaggagaat ggtgaatctt ctgagaaaaa attgggagga gaagaaaatt 240

gtagcagtgc cggggaagga gagatgccgg cgacatatga tgaaagagag aacgagaaga 300

gagaaacaaa aacagagtta cttagctctc caatctctat tacctgccac taagagtgat 360

aaaaattcga ttgttgaaaa ggccgttgat cagattagga aactagaagg attaaagaaa 420

gaactagaga gaaaaatgaa tgtgttggag gcaaaatcag cacgggatca tgatgaaatg 480

aatggaaaaa aggttaggtt taatgtacaa gaacctttgt cggggatcga ttcagttgta 540

gaagttcttc agtgtcttaa atcaatgggg acaaatctca atacggtcca agccaatttc 600

tctccacacg agttctcagc gaccatgaac atcgagactc agataagagg agaagaggtg 660

gaaaaaagag tacagaaaag actccaggaa actgaatgga aactcctttt gttttga 717

Claims (10)

1. The gene BnbHLH92a is characterized in that the nucleotide sequence of the BnbHLH92a is shown in SEQ ID NO. 1.

2. A recombinant plasmid comprising the gene BnbHLH92a according to claim 1.

3. The use of the BnbHLH92a of claim 1 for the preparation of an inhibitor for inhibiting the accumulation of anthocyanidins or procyanidins.

4. A method for inhibiting flavonoid synthesis in a target plant, which comprises overexpressing BnbHLH92a in the target plant according to claim 1.

5. The method according to claim 4, characterized by the specific steps comprising:

(1) constructing a vector containing BnbHLH92 a;

(2) the vector is introduced into a target plant by an agrobacterium-mediated method.

6. The method of claim 4, wherein the BnbHLH92a inhibits the expression of gene TT8 in a target plant.

7. The method of claim 4, wherein the target plant is a Brassica plant.

8. The method of BnbHLH92a for inhibiting anthocyanin or procyanidin in a target plant of claim 1, wherein the BnbHLH92a acts as a transcriptional repressor that inhibits the expression of BnTTG1 and BnTT 18.

9. The method as claimed in claim 8, wherein the BnbHLH92a interacts with BnTTG1 to form a functional complex that inhibits anthocyanidins or procyanidins.

10. The method of claim 8, wherein the BnbHLH92a binds to the BnTT18 promoter.

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