阅读说明：本技术 一种银杏类黄酮3′-羟化酶基因GbF3′H1及其应用 (Ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1 and application thereof ) 是由 徐立安 吴雅琼 辛月 祁铭 于 2019-09-23 设计创作，主要内容包括：本发明公开了一种银杏类黄酮3′-羟化酶基因GbF3′H1及其应用,属于分子生物学技术领域。基因GbF3′H1核苷酸序列如SEQ ID No.1所示,其表达的蛋白产物氨基酸序列如SEQ ID No.2所示。本发明通过构建基因GbF3′H1载体转化山新杨,并对转化的山新杨和非转基因型进行过量表达分析和差异代谢物分析,结果显示,与非转基因植株相比,转基因植株生长相对较慢,叶片容易沉积红色素,黄酮类化合物合成的下游产物浓度明显高于非转基因植株,表明过量表达基因GbF3′H1能够提高植物类黄酮产量,可以作为促进植物体内积累黄酮类化合物的重要参考和科学依据。(The invention discloses a ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1 and application thereof, belonging to the technical field of molecular biology. The nucleotide sequence of the gene GbF3' H1 is shown as SEQ ID No.1, and the amino acid sequence of the expressed protein product is shown as SEQ ID No. 2. According to the invention, a gene GbF3'H1 vector is constructed to transform the populus deltoids, and overexpression analysis and differential metabolite analysis are carried out on the transformed populus deltoids and non-transgenic plants, and the result shows that compared with the non-transgenic plants, the transgenic plants grow relatively slowly, red pigment is easy to deposit on leaves, the concentration of downstream products synthesized by flavonoids is obviously higher than that of the non-transgenic plants, and the overexpression gene GbF3' H1 can improve the yield of plant flavonoids, and can be used as an important reference and scientific basis for promoting the accumulation of flavonoids in plants.)

1. A ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1 has a nucleotide sequence shown as SEQ ID No. 1.

2. The expression protein of ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1 according to claim 1, wherein the amino acid sequence of the expression protein is shown as SEQ ID No. 2.

3. A vector comprising the ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1 according to claim 1.

4. The vector of claim 3, wherein the vector is a 35S: : GbF3' H1.

5. Use of the ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1 according to claim 1 or the vector according to claim 3 or 4 for increasing the yield of plant flavonoids.

6. Use according to claim 5, characterized in that it comprises the following steps: (1) constructing the vector of claim 3 or 4; (2) transforming the constructed vector into a plant or plant cell; (3) and culturing and screening to obtain plants with high plant flavonoid expression.

7. The use of claim 5 or 6, wherein the phytoflavonoid is epigallocatechin, gallocatechin or catechin.

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1 and application thereof.

Background

Ginkgo biloba (Ginkgo biloba) is the only existing species of the Ginkgoaceae family, often referred to as "Actinolitum". It originates from the biennial age about 3 hundred million years ago and has an important evolutionary position in the plant kingdom. In addition, ginkgo is a multipurpose tree species, and particularly the medicinal value is concerned. Ginkgo biloba leaf extract (GBE) has received much attention as a treatment for early Alzheimer's disease, brain dysfunction and vascular dementia. The flavonoid compounds in GBE are the most prominent pharmacological components in the current ginkgo leaves. The flavonoids are a series of C with typical characteristics₆-C₃-C₃-C₆The flavonoid skeleton compound is formed by connecting two aromatic rings through C3. The flavonoid compounds are the best known red, blue and purple anthocyanin pigments in plant tissues. In the early stages of the flavonoid pathway, chalcones were modified to flavanonols through isomerization and oxidation. In most plants, flavanonols (dihydrokaempferol, dihydroquercetin, dihydromyricetin) are a major branch point in the flavonoid biosynthetic pathway, and subsequent reactions can produce flavanonol glycosides by glycosylation, flavonols or anthocyanidins by oxidation, and flavan-3-ols (catechins, gallocatechins) by reduction.

At present, the formation and accumulation of flavonoids produced by the flavonoid biosynthetic pathway have been well studied in Arabidopsis thaliana, and related enzymes and genes have also been extensively studied in different species. Flavonoid 3 '-hydroxylase (F3' H) is a key enzyme in the flavonoid biosynthetic pathway. It is one of the important members of the cytochrome P450 subfamily and has the catalytic activity of multiple NADPH and O dependence₂A basic mono-oxygenation reaction. In the flavonoid biosynthetic pathway, F3'H hydroxylates the 3' -position of the naringin and dihydrokaempferol B rings to form flavonoids, and led the production of red cyanide-based pigments. The DNA has been separated and identified from petunia, arabidopsis, soybean, grape and other plantsThe F3' H gene was obtained. Furthermore, Shih et al (2006) overexpression of SbF 3'H from sorghum results in the conversion of most precursors of flavonol biosynthesis into 3' -hydroxylated form. Han et al (2010) detected ectopic expression of the apple callus MdF 3' H gene in Arabidopsis tt7 mutant promoted accumulation of anthocyanin. It can be seen that F3' H has broad specificity of action and has a significant impact on the synthesis of downstream flavonoid (anthocyanin) metabolites. Therefore, obtaining and analyzing key enzyme genes in the flavone synthesis pathway provides important reference and scientific basis for further understanding the molecular mechanism of accumulation of flavonoids in plants.

Disclosure of Invention

Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a ginkgo flavonoid 3 '-hydroxylase GbF3' H1 gene. The invention also aims to provide application of the ginkgo flavonoid 3 '-hydroxylase GbF3' H1 gene.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1 has a nucleotide sequence shown as SEQ ID No. 1.

The amino acid sequence of the expression protein of the ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1 is shown as SEQ ID No. 2.

The vector of the ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1.

Preferably, the carrier is 35S: : GbF3' H1.

The ginkgo flavonoid 3 '-hydroxylase GbF3' H1 gene or the carrier is applied to improving the yield of plant flavonoids.

Preferably, the application comprises the following steps: (1) constructing the vector; (2) transforming the constructed vector into a plant or plant cell; (3) and culturing and screening to obtain plants with high plant flavonoid expression.

Preferably, the plant flavonoid is epigallocatechin, gallocatechin or catechin.

Has the advantages that: compared with the prior art, the invention has the advantages that:

(1) the cDNA sequence of the ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1 is obtained by first cloning, the phylogenetic position of the ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1 and the expression characteristics in ginkgo are revealed through bioinformatics analysis and space-time expression analysis, and the result shows that the gene expression level is higher in 4 and 9 months of ginkgo neutralization;

(2) the invention carries out subcellular localization and in-vivo plant function analysis of the ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1, and phenotype observation and metabolite content determination analysis of transgenic poplar and non-transgenic poplar. The GbF3' H1-GFP protein emits GFP signals mainly in cytoplasm, and provides clues for the role of the protein in plant biological processes. qRT-PCR analysis shows that the expression level of the gene of the transgenic poplar can reach 5162 times of that of non-transgenic seedlings at most. In addition, compared with a non-transgenic plant, the transformed plant grows slowly, the red pigment is easy to deposit on leaves, and the concentration of a downstream product synthesized by the flavonoid compound is obviously higher than that of the non-transgenic plant, so that the yield of the plant flavonoid can be improved by the over-expression gene GbF3' H1, and therefore, the gene can be used as an important reference and scientific basis for promoting the accumulation of the flavonoid compound in a plant body.

Drawings

FIG. 1 is a diagram of structural features and phylogenetic analysis of the GbF3' H1 protein; A. the deduced sequence alignment of GbF3'H1 with other species F3' Hs proteins, the Genbank accession numbers of these proteins in the sequence are as follows: taxus chinensis (ATG29929.1), Picea chinensis (ABR16821.1), Corchorus olitorius (OMO79261.1), Epimedium sagittatum (ADE80941.1), Cephalotus folliculularis (GAV84063.1), Viviaamurensis (ACN38268.1), Vitis vinifera (BAE47003.1), Cichorium intybus (ACN65825.1), Chromolaena odorata (AEA06595.1), Cosmos subphreneus (ACO35752.1), and Dahlia pinnata (ADB 77826.1); B. constructing a molecular phylogenetic tree of the F3' Hs protein by adopting a maximum likelihood method;

FIG. 2 is a diagram of spatiotemporal expression rule of gene GbF3' H1 detected by qRT-PCR; r, S, L, K, B and P are root, stem, leaf, nut, bud and petiole, the time expression patterns 4, 5, 6, 7, 8, 9 and 10 represent 4 months, 5 months, 6 months, 7 months, 8 months, 9 months and 10 months respectively; error bars indicate the deviation of three biological replicates, with the mean difference between different letters being statistically significant (p < 0.05);

FIG. 3 is a graph of a subcellular localization and overexpression analysis of GbF3' H1 protein; gbf3' H1 protein subcellular localization analysis: GFP: green fluorescence; auto: autofluorescence; merged 1: GFP + Auto; merged 2: merged 1+ bright. Scale: expression level of 10 μm.b.gbf 3' H1 gene in transgenic plants; WT: non-transgenic poplar, L1-L8: 1-8 of transgenic strains;

FIG. 4 is a graph of growth status and phenotypic changes of transgenic shoots;

FIG. 5 is a graph showing the content of 3 important differential flavonoid metabolites in the non-transgenic poplar and the GbF3' H1 transgenic poplar groups.

Detailed Description

The invention is further described with reference to specific examples. The molecular biological experiments, which are not specifically described in the following examples, can be performed by methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or methods conventional in the art, or according to kits and product instructions.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Materials: tissue expression experiment of the gene of ginkgo is carried out by taking 1-year-old ginkgo seedlings (leaves, stems, roots and petioles) and 25-year-old ginkgo (kernels and buds) as materials. The 1-year-old ginkgo seedlings are taken as test materials, and leaves are collected once a month from 4 months to 10 months at different leaf development stages. After collection, the plant material was snap frozen in liquid nitrogen and placed in an ultra-low temperature freezer at-80 ℃. Three biological replicates were performed per sampling point. In addition, tissue culture seedlings of clonal Populus davidiana (Populus davidiana x Populus bolliana) were grown under 16h light and 8h dark photoperiod and under 25 ℃ (day) and 18 ℃ (night).