CN114015699B - 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 regulating 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 BnbHLH92 a. The gene BnbHLH92a and BnTTG1 interact to form a functional complex for inhibiting anthocyanin or procyanidine, and the BnbHLH92a and the BnTT18 promoter are combined, so that accumulation of the anthocyanin or procyanidine of the brassica napus is inhibited. The invention provides a new visual angle for revealing the transcriptional regulation of flavonoid biosynthesis pathway of brassica plants.

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 regulating anthocyanin or procyanidine in brassica plants.

Background

Rape is one of the important oil crops in the world, is not only a source of vegetable edible oil after soybean and oil palm, but also an important feed source after soybean meal, and is also an ideal raw material for developing biodiesel. With the reduction of rape planting area and the increase of the requirements of people on rapeseed oil, the improvement of the quality of the rapeseed oil, the yield and the cake quality become one of important targets of rape breeding. Under the same genetic background, yellow seeds have more advantages than black seeds, such as thinner seed coats, higher oil and protein content, lower cellulose and polyphenol content, and clearer oil. Therefore, the yellow seed trait is one of the important targets for improving the quality of rape.

Flavonoids contribute to the color formation of flowers, fruits and seeds in nature. In arabidopsis, analysis of a series of transparent seed coats (TT) shows that the seed coat color of the seeds is determined by the content of Proanthocyanidins (PAs), including more than 22 TT-type genes involved in seed coat color, revealing the mechanism of action of brassica plant yellow seed trait-related genes, such as TTG1 in cabbage, TT8 in brassica napus, brassica napus and brassica napus, but these genes also express different expression patterns between black seeds and yellow seed oil lines. In addition, TT2 (R2R 3-MYB), TT8 (basic helix-loop-helix, bHLH) and WD40 regulatory proteins can form MYB-bHLH-WDR (MBW) ternary complexes, affecting flavonoid biosynthetic pathways by modulating the abundance and pattern of structural gene expression.

MBW complexes play an important role in seed development, and are known to be involved in regulating flavonol-4-reductase (DFR), leucocyte anthocyanin dioxygenase (LDOX/TT 18), BANYULS (BAN), TT19 and TT12 in seeds, leading to changes in seed coat color. In the MBW ternary complex model, bHLH is considered a conserved core component, while transcription factors of the bHLH type are rarely reported.

However, to date, flavonoid-related genes of brassica plants have not been elucidated in great detail, it is not known how the related genes regulate transcription of flavonoid biosynthetic pathways, and there is also a lack of research into transcription factors of the bHLH type and their functional role in the anthocyanin/PAs pathway.

Disclosure of Invention

In order to solve the problems, the homologous gene BnbHLH92a of AtbHLH92 is separated by adopting an RT-PCR method, belongs to the S7 subfamily containing G-box binding proteins, and comprises a basic helix-loop-helix structural domain.

The invention aims at providing a gene involved in regulating anthocyanin/PAs pathway functions, and the specific technical scheme is as follows:

the nucleotide sequence of the BnbHLH92a gene BnbHLH92a is shown as SEQ ID NO. 1.

The second object of the present invention is to claim a recombinant plasmid comprising the above-mentioned gene BnbHLH92 a.

The invention also aims to claim engineering strains prepared by using the recombinant plasmid.

The fourth object of the invention is to claim the use of BnbHLH92a as described above for the preparation of an inhibitor for inhibiting the accumulation of anthocyanidin or procyanidins.

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

The invention aims at providing a method for inhibiting flavonoid synthesis, which comprises the following specific technical scheme:

a method for inhibiting flavone synthesis in target plant comprises over-expressing the gene BnbHLH92a in the technical scheme in the target plant.

Specifically, the method comprises the following steps:

(1) Constructing a vector comprising BnbHLH92 a;

(2) The vector is introduced into the target plant by agrobacterium-mediated methods.

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

Specifically, the target plant is a brassica plant.

Specifically, the target plant is arabidopsis thaliana.

Specifically, the BnbHLH92a is specifically expressed at the umbilicus.

The sixth purpose of the invention is to provide a method for inhibiting anthocyanin or procyanidine in a target plant by utilizing the gene BnbHLH92a, which comprises the following specific technical scheme:

the method of inhibiting anthocyanin or procyanidin in a plant of interest by BnbHLH92a of claim 1, wherein the BnbHLH92a acts as a transcriptional repressor to inhibit expression of BnTTG1 and BnTT 18.

Specifically, the BnbHLH92a interacts with BnTTG1 to form a functional complex that inhibits anthocyanin or procyanidins.

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 thaliana. Meanwhile, bnbHLH92a can inhibit the expression of BnTTG1 and BnTT18, and the specific principle is that BnbHLH92a and BnTTG1 interact to form a functional complex for inhibiting anthocyanin or procyanidine, and BnbHLH92a and BnTT18 promoter are combined, so that accumulation of brassica napus anthocyanin or procyanidine is inhibited.

Drawings

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

FIG. 2 is a BnbHLH92a subcellular localization and transcriptional activity assay;

FIG. 3 is a representation of BnbHLH92 a;

FIG. 4 is a graph showing BnbHLH92a negatively regulating the accumulation of Arabidopsis anthocyanin and PAs;

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

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

FIG. 7 is a schematic diagram showing transcriptional regulation of the BnTT18 promoter by BnbHLH92 a;

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

FIG. 9 is a BnbHLH92a overexpressing T0 generation Arabidopsis screen;

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

FIG. 11 is a graph of negative ion mode basal peaks of wild type and OE-92a Arabidopsis seeds;

FIG. 12 shows the relative expression levels of AtJAZs in the OE-92a strain.

Detailed Description

The present invention will be further described in detail by the following examples, it being understood that the specific examples described herein are intended to be illustrative only and not to be limiting of the invention, and it will be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or substituted without departing from the structural spirit and scope of the invention, but that the modifications and substitutions fall within the scope of the invention.

Example 1 isolation and characterization of the Gene BnbHLH92a

In this example, according to the genome annotation of Brassica napus, the homologous gene BnbHLH92a of AtbHLH92 was isolated by RT-PCR, and the nucleotide sequence is shown in SEQ ID NO. 1. The BnbHLH92a full-length cDNA is 717bp, codes 238 amino acids, has a predicted molecular weight of about 27.58kDa and has an isoelectric point of 10.0. In brassica napus, 602 possible bhlh proteins have been identified and divided into 35 subfamilies, and we found that BnbHLH92 belongs to the S7 subfamily containing G-box binding proteins. As shown in FIG. 1, sequence analysis revealed that BnbHLH92a contains a basic helix-loop-helix domain (FIG. 1A). Previous studies have shown that in Leymus chinensis (Leymus chinensis), lcbHLH92 is a negative regulator of the anthocyanin/PAs pathway. Phylogenetic analysis shows that BnbHLH92a, atbHLH92 and LcbHLH92 belong to the same group (FIG. 1B: phylogenetic analysis of BnbHLH92a and other bHLH proteins of different species constructed by the adjacency method of MEGA7.0 software), indicating that BnbHLH92a may have a function of participating in modulating anthocyanin/PAs pathways.

BnbHLH92a is localized to the nucleus as an active inhibitor

To study subcellular localization of BnbHLH92a in vivo, a vector Pro35S, which is driven by a strong promoter and fused with GFP fluorescent protein gene, was constructed, bnbHLH92a, and transformed into tobacco epidermal cells. The control GFP protein showed fluorescent signals in both the cytoplasm and nucleus, whereas the Pro35S: bnbHLH92A-GFP fusion protein was detected only in the nucleus (FIG. 2A: bnbHLH92A protein was localized in the nucleus of the epidermal cells under the tobacco leaf), consistent with the functional localization of BnbHLH92A as a transcription factor.

To further verify whether BnbHLH92a is a transcriptional activator or repressor, we measured its transcriptional activity in Arabidopsis protoplasts using the LUC reporter system. Firefly Luciferase (LUC) reporter fused to five copies of GAL4 DNA binding element and the minimal CaMV35S promoter, renilla luciferase (REN) under the control of the 35S promoter was used as an internal control and VP16 transcriptional activation domain was used as a positive control (FIG. 2B: schematic of reporter and effector plasmids used in the assay of the dual luciferase reporter system). As shown, pBD-BnbHLH92a significantly inhibited LUC reporter expression compared to the effect of pBD-VP 16 (FIG. 2C: analysis of BnbHLH92 activity by Arabidopsis protoplast transient expression. Representative results are expressed as mean.+ -. SD). These results indicate that BnbHLH92a acts as a transcription repressor.

Spatiotemporal expression pattern of BnbHLH92a

The BrassicaEDB database https:// brassica. Biodb. Org/; bnbHLH92a was expressed mainly in 35 days seed, 30 days, 35 days and 40 days seed coat, 30 days outer seed coat, 27 days and 35 days umbilicus, 35 days and 40 days pericarp, respectively (FIG. 3A: expression pattern of BnbHLH92a in different tissues and organs of Brassica napus). To further determine the functional role of BnbHLH92a in seed coat color, qRT-PCR was used to detect BnbHLH92a expression levels at different seed coat development stages of yellow and black seed oil vegetables. The expression level of BnbHLH92a in the yellow seed coats was significantly higher than that of black seed coats (FIG. 3B: analysis of BnbHLH92a expression pattern in yellow and black seeds of Brassica napus by qRT-PCR), indicating that it may be a negative regulator of seed coat color. In addition, in situ hybridization experiments were also performed using BnbHLH92a probe, and the expression profile of the gene in developing seeds was studied, and we found that a clear signal was detected in the seed coat and umbilicus of yellow seeds, but not in the seed coat of black seeds (FIG. 3C-F: in situ hybridization analysis of BnbHLH92 in yellow black seeds of Brassica napus). BnbHLH92a was found to be specifically expressed at the umbilicus by in situ hybridization (FIG. 3C). In addition, bnbHLH92a was expressed in the developing seeds, seed coats, exotesta, pedicel and pericarp in much higher amounts than the other organs (FIG. 3A), and the difference in expression amounts 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 anthocyanidins/PAs.

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

To identify the biological function of BnbHLH92a, a 35S driven over-expression vector was constructed and transformed into wild type Arabidopsis by agrobacteria mediated floral dip of recombinant plasmid pEarley gate101-BnbHLH92 a. The pEarley gate101 plasmid was screened for Basta, and was sprayed twice with 50mg/L Basta solution for one week old Arabidopsis thaliana to obtain 12 Arabidopsis thaliana strains which grew normally, DNA of the 12 Arabidopsis thaliana strains was extracted, and the DNA was detected by using the vector pre-primer and the gene post-primer F35S+ovBnBbHLH 92aR primer, and the Arabidopsis thaliana positive strain 12 strain which overexpressed BnBbHLH 92a was co-detected (FIG. 9). Subsequently, T1 and T2 transgenic arabidopsis plants were examined using the same screening assay, wherein 10 independent transgenic lines with significant changes in seed coat phenotype were generated (fig. 10). The present invention uses qRT-PCR to identify two independent strains (OE-92 a#8 and OE-92 a#31) and further analyze them. Obviously, the expression level of BnbHLH92a was significantly up-regulated in OE-92a#8 and OE-92a#31 plants (FIG. 4A: relative expression level of BnbHLH92a in OE-92a Arabidopsis), and the seed coat color was also significantly changed (FIG. 4B: DMACA staining of over-expressed OE-92a lines and wild-type control seeds). A number of studies have shown that proanthocyanidins are important metabolites determining seed coat colour in arabidopsis thaliana, and histochemical staining analysis showed a significant reduction in the content of PAs in the over-expressed BnbHLH92a arabidopsis strain compared to the wild-type control stained with DMACA reagent (fig. 4B).

Furthermore, quantitative analysis showed that the levels of anthocyanin (anthocyanin levels in FIG. 4C: WT and different transgenic lines) and PAs (soluble and insoluble PAs) were significantly reduced in the overexpressed BnbHLH92a Arabidopsis transgenic lines relative to the wild-type (soluble proanthocyanidin levels in FIG. 4D: WT and different transgenic lines; insoluble proanthocyanidin levels in FIG. 4E: WT and different transgenic lines). To investigate in detail the relationship of BnbHLH92a to flavonoid biosynthetic pathways, the species and content of polyphenols and flavonoids in transgenic lines were further analyzed by UPLC-HESI-MS/MS (FIG. 11). The results show that there is a significant difference in the levels of flavonoids and their metabolic derivatives in BnbHLH92a-OE and wild-type plants, especially epicatechin and proanthocyanidin (Table 1: UPLC-HESI-MS/MS identifies the differential significant flavonoids (μ g g-1 FW) in wild-type and OE-92a Arabidopsis seed extracts), FIG. 5 (A) [ DP2] -1, (B) [ DP2] -2, (C) [ DP3] -1, (D) [ DP3] -2, (E) [ DP4]; (F) epicatechin), indicating that BnbHLH92a may play an important role in regulating anthocyanin and PAs accumulation.

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

To further determine the effect of BnbHLH92a on flavonoid biosynthetic pathway genes, qRT-PCR was used to detect the expression levels of structural and regulatory genes in BnbHLH92a-OE strains. Consistent with the reduction of polyphenols and flavonoids in BnbHLH92a OE lines, expression levels of flavonoid biosynthetic genes (including AtTT6, atDFR/TT3, atTT18/LDOX, atBAN/ANR, atTT8 and AtTTG 1) in BnbHLH92a-OE lines were continuously down-regulated as compared to controls (FIG. 4F: qRT-PCR analysis of flavonoid biosynthetic genes in BnbHLH92a-OE lines), indicating negative regulation of flavonoid biosynthesis in BnbHLH92a-OE lines. On the one hand, in the BnbHLH92a-OE strain, the expression levels of AtJAZ3, atJAZ5, atJAZ6, atJAZ8, atJAZ10 and AtJAZ12 were significantly higher than the control (FIG. 12). Our results also confirm that bHLH92 protein inhibits TT8 expression by activating the JAZ gene. On the other hand, we found that AtTTG1 was also significantly reduced in the BnbHLH92a-OE strain compared to the wild-type control (fig. 4E), suggesting that BnbHLH92a not only indirectly inhibited TT8 expression, but may also directly participate in the MBW model, inhibiting the arabidopsis flavonoid synthesis pathway with a new mechanism.

BnbHLH92a interacts with BnTTG1, negatively regulating anthocyanin/PAs accumulation

In the present invention, bnbHLH92a overexpression significantly down-regulated the mRNA levels of the AtDFR/TT3, atTT18/LDOX, atBAN/ANR, atTT8, atTTG1 and JAZ genes (FIG. 4F). Thus, we further tested the potential interactions of BnbHLH92a and BnTTG1, with both BnTTG1a and BnTTG1b members in canola. In the yeast two-hybrid (Y2H) assay, there was an interaction between BnbHLH92a and BnTTG1 (FIG. 6A: the yeast two-hybrid assay in which BnbHLH92a interacted with BnTTG 1). Meanwhile, interactions observed in yeast two-hybrid experiments were further identified using the two-molecule fluorescence complementation method (BiFC). First, subcellular localization analysis was performed on BnTTG1 using tobacco epidermal cells. Control GFP protein was distributed throughout the cells (FIG. 6B: subcellular localization of BnTTG1-GFP fusion protein in epidermal cells under tobacco leaf), while Pro35S: bnTTG1a fusion protein was aggregated in the cytoplasm and nucleus (FIG. 6B), while Pro35S: bnTTG1B fusion protein was localized in the nucleus (FIG. 6B), consistent with published results for TTG1 localization 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. After mixing the BnbHLH92a-cYFP and nYFP-BnTTG1a or BnbHLH92a-cYFP and nYFP-BnTTG1b plasmids, yellow fluorescence was detected in the separately co-transfected tobacco epidermal cells (FIG. 6C: biFC analysis of the interaction of BnbHLH92a and BnTTG1a in tobacco leaf lower epidermal cells; FIG. 6D: biFC analysis of the interaction of BnbHLH92a and BnTTG1b in tobacco leaf lower epidermal cells). These results indicate that BnbHLH92a is capable of interacting with BnTTG1 in vivo to form functional complexes that inhibit anthocyanin or procyanidins.

BnbHLH92a binds directly to the BnTT18 promoter and inhibits its expression

The results indicate that BnbHLH92a is a negative regulator affecting flavonoid biosynthesis. Among the enzymes encoding flavonoid biosynthesis, DFR, TT18 and BAN belong to Late Biosynthetic Genes (LBGs) that are regulated by MBW ternary complexes consisting of three transcription factors, namely (R2R 3-MYB, bHLH and WD 40). To study this process in more detail, we used the bHLH-type transcription factor recognition site cis-regulatory element G-box to search for the BnbHLH92a targeting gene, we found multiple cis-regulatory elements G boxes in the BnTT18 promoter region (FIG. 7A: typical and core G box elements are distributed in the promoter region of BnTT 18), indicating that these structural genes may be directly regulated by BnbHLH92 a. As expected, bnbHLH92a can bind directly to BnTT18 promoter in vivo by yeast single hybridization (Y1H) (FIG. 7B: yeast single hybridization experiments. Binding screening was performed on SD-Leu-Ura medium in the presence of AbA 250. Empty vector and BnTT18 promoter (pGADT 7+BnTT18pro) served as negative controls). A dual luciferase reporter assay was further performed to determine this hypothesis. The dual luciferase reporter plasmid contained the BnTT18 promoter sequence fused to LUC, REN was driven by CaMV35S promoter as an internal control, and the plasmid containing the full-length cDNA sequence of BnbHLH92a served as an effector (FIG. 7C: schematic diagram of reporter and effector plasmids used in dual luciferase reporter assays). As expected, the data show that the activity of the BnTT18 promoter is significantly reduced by the BnbHLH92a (fig. 7D: dual luciferase reporter gene analysis indicates that the BnbHLH92a can inhibit the activity of the BnTT18 promoter). In conclusion, bnbHLH92a may bind directly to the BnTT18 promoter, negatively affecting the flavonoid synthesis pathway of Brassica napus. BnbHLH92a may result in reduced anthocyanin/PAs accumulation by modulating BnTTG 1.BnbHLH92a was found to bind directly to the promoter region of BnTT18 by Y1H and double luciferase experiments (FIG. 7). Correspondingly, we also noted a significant reduction in transcript redundancy of AtTT18 in the BnbHLH92a-OE strain. These data indicate 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 BnbHLH92a is used as a negative regulator of anthocyanin/PAs biosynthesis pathway, but also provides molecular evidence for that BnbHLH92a can directly inhibit the expression levels of BnTTG1 and BnTT18, so that the accumulation of anthocyanin/PAs of brassica napus is reduced. Thus, we provided 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 potential mechanisms of seed coat colors and provides a theoretical basis for genetic improvement of brassica napus.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Sequence listing

<110> university of southwest

<120> application of BnbHLH92a to regulation of anthocyanin or procyanidine

<130> 2021

<160> 1

<170> SIPOSequenceListing 1.0

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<211> 717

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<213> Artificial sequence (BnbHLH 92 a)

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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 (8)

1. Use of BnbHLH92a, the nucleotide sequence of which is shown in seq id No.1, for the preparation of an inhibitor of anthocyanin or procyanidin accumulation in arabidopsis thaliana and brassica napus.
2. 2. The method for inhibiting anthocyanin or procyanidin in a target plant by BnbHLH92a according to claim 1, wherein the BnbHLH92a is used as a transcription inhibitor to inhibit the expression of BnTTG1 and BnTT18, and the target plant is Arabidopsis thaliana and Brassica napus.
3. 3. The method of claim 2, wherein the BnbHLH92a interacts with BnTTG1 to form a functional complex that inhibits anthocyanin or procyanidins.
4. 4. The method of claim 2, wherein the BnbHLH92a binds to the BnTT18 promoter.
5. 5. The method according to claim 2, characterized in that the specific steps comprise:

   (1) Constructing a vector comprising BnbHLH92 a;

   (2) The vector is introduced into the target plant by agrobacterium-mediated methods.
6. 6. A method of inhibiting flavonoid synthesis in a plant of interest, wherein the BnbHLH92a of claim 1 is overexpressed in a plant of interest that is an arabidopsis and brassica napus inhibitor.
7. 7. The method of claim 6, wherein the BnbHLH92a inhibits expression of the gene TT8 in the plant of interest.
8. 8. The method of claim 6, wherein the target plant is a brassica plant.