阅读说明：本技术 一种牡丹中参与花青苷转运和积累的gst基因及其应用 (GST gene participating in transport and accumulation of anthocyanin in peony and application thereof ) 是由 王雁 韩璐璐 周琳 于 2021-07-27 设计创作，主要内容包括：本发明公开了一种牡丹中参与花青苷转运和积累的GST基因及其应用,从牡丹(P.suffruticosa)中分离和鉴定出一个参与花青苷转运和积累的GST基因,其在花青苷转运和积累方面发挥重要作用,能够丰富牡丹花朵红色性状的转基因材料库,为未来培育和调控牡丹红色性状花朵的分子育种提供了有力支持。(The invention discloses a GST gene participating in anthocyanin transport and accumulation in peony and application thereof, wherein the GST gene participating in anthocyanin transport and accumulation is separated and identified from the peony (P.suffruticosa), plays an important role in anthocyanin transport and accumulation, can enrich a transgenic material library of peony red characters, and provides powerful support for future cultivation and regulation of molecular breeding of peony red character flowers.)

1. A GST gene involved in anthocyanin transport and accumulation in peony, wherein a GST gene involved in anthocyanin transport and accumulation is isolated and identified from peony (P.suffruticosa), and the CDS sequence of the GST gene is as follows:

ATGGTAGTTAAGGTGTATGGCTCAGCTAAAGCGGCTTGTCCACAAAGAGTGATGGTTTGCCTTCTGGAAAAGGAGGTGGAATTTGAAATTATACATGTTGATCTTGAATCTGGAGAGCACAAAAAGCCAGATTTCCTTGCTCGGCAGCCGTTTGGGCAAGTTCCAGCCATCGAGGATGGTGACTTGAAACTTTTTGAATCCAGGGCAATCATAAGATACTATGCAGCCAAGTTTGATAACCGCGGTTCAAACCTGTTGGGAACCACTTTGGAAGAGAGAGCTTTGATGGATCAATGGCTAGAAGTTGAAGCCCACAATTTCAATGATTTGGTTTACACTATTGTGCTTCAGATCGTAATTCTCCCCCGTATGGGACAAGGTACTGACTTGGCATTAGTCCGCACCTGTGAAGAAAAGCTTGAGAAAGTGCTTGATGTGTACGAGCAAAGGTTGTCAAAGAGCAGCTACCTGGCCGGAGACGATTTCACACTCGCTGATCTCAGTCATCTTCCTGGTCTCAGATACCTCATAAATGAAGCTGGAAAAGGATACCTGGTGACCGTGAGGAAGAATGTGAATGCATGGTGGGAGAATATATCAAATCGTCCTTCTTGGAAGAAATTAATGAATCTTGTTAATTAA。

2. the application of GST gene involved in anthocyanin transport and accumulation in peony of claim 1, characterized in that transgenic breeding is performed through targeted regulation and control of transgenic technology.

3. The application of the GST gene participating in anthocyanin transport and accumulation in peony of claim 2, which is used in Arabidopsis thaliana, and is overexpressed in Arabidopsis TT19 mutant to restore anthocyanin-deleted phenotype.

4. The application of the GST gene participating in anthocyanin transport and accumulation in peony as claimed in claim 2, wherein the GST gene is used in peony, and target gene silencing is targeted, so that anthocyanin accumulation in peony petals is obviously reduced, and pink petals are obviously lightened.

Technical Field

The invention relates to the technical field of biology, in particular to a GST (Glutathione S-transferase) gene participating in transferring and accumulating anthocyanin in peony and application thereof.

Background

Plant flower color is one of important ornamental characters of ornamental plants, and flower color improvement is an important breeding target of gardeners. Peony (Paeonia suffruticosa) is a fine product of the traditional famous flower in China, has the reputation of 'king in flower', forms various color systems after long-term cultivation and breeding, and is popular in domestic and foreign markets.

However, for a long time, the breeding mode of peony mainly refers to cross between varieties, the randomness and blindness in the breeding process are high, and a great amount of time and energy are usually consumed to obtain an ideal variety through multi-generation breeding. In recent years, with the rapid development of scientific technology, targeted molecular breeding technology using transgenic technology has been widely applied to crops and horticultural flowers. The molecular breeding is mainly characterized in that genes for controlling related phenotypic traits are overexpressed or knocked out in a target plant through a transgenic technology, so that the plant obtains or loses a corresponding phenotype, and an ideal variety is obtained finally. Therefore, screening key genes for controlling related phenotypic traits is a prerequisite basis and an important resource for molecular breeding.

Anthocyanin (Anthocyanin) is the most important chromogenic substance in flavonoid, is a stable compound formed by Anthocyanin after a series of modifications such as hydroxylation, methoxylation, glycosylation and acylation, and can control the color change of flower red, pink, violet, blue and the like, so that the plant can present colorful colors. At present, the synthetic pathway of anthocyanin is clearly researched and has high conservation, and the regulation mechanism is also reported in a plurality of model and non-model plants. However, transport and accumulation of anthocyanin to vacuole are required for final presentation of flower color, and gene knockout and complementation tests prove that some members of GSTs (glutathione) families are involved in transport of anthocyanin and are efficient transporters for transporting anthocyanin from endoplasmic reticulum to vacuole. The maize Bronze2(BZ2) gene is the earliest discovered GST family member associated with anthocyanin transport, and anthocyanin accumulation in the cytoplasm was detected in BZ2 deletion mutants without being transported to the vacuole; arabidopsis AtGSTF12(AtTT19) was shown to be involved in anthocyanin/procyanidin transport and accumulation; RAP in strawberry encodes a GST transporter, whose transient knockout results in reduced fruit coloring; the apple MdGSTF6 gene is proved to be involved in anthocyanin transport, and the over-expression of the apple MdGSTF6 gene in an Arabidopsis TT19 mutant can restore the anthocyanin-deleted phenotype, and the like.

Although GST has been confirmed to be involved in the transport and accumulation of anthocyanin in various plants, research is mostly focused on model plants or crops, and the research on ornamental flowers in the gene family is relatively deficient, especially in peony is rarely reported.

Therefore, the effect of supplementing GST genes in promoting anthocyanin transport and accumulation in peony is an important basis and precondition for future peony flower color molecule improvement breeding.

Disclosure of Invention

The invention aims to provide a GST gene participating in anthocyanin transport and accumulation in peony and application thereof, so as to improve the integrity of a pathway for controlling the red character of flowers in the peony.

The purpose of the invention is realized by the following technical scheme:

the GST gene involved in anthocyanin transport and accumulation is separated and identified from peony (P. suffruticosa), and the CDS sequence of the GST gene involved in anthocyanin transport and accumulation is as follows:

ATGGTAGTTAAGGTGTATGGCTCAGCTAAAGCGGCTTGTCCACAAAGAGTGATGGTTTGCCTTCTGGAAAAGGAGGTGGAATTTGAAATTATACATGTTGATCTTGAATCTGGAGAGCACAAAAAGCCAGATTTCCTTGCTCGGCAGCCGTTTGGGCAAGTTCCAGCCATCGAGGATGGTGACTTGAAACTTTTTGAATCCAGGGCAATCATAAGATACTATGCAGCCAAGTTTGATAACCGCGGTTCAAACCTGTTGGGAACCACTTTGGAAGAGAGAGCTTTGATGGATCAATGGCTAGAAGTTGAAGCCCACAATTTCAATGATTTGGTTTACACTATTGTGCTTCAGATCGTAATTCTCCCCCGTATGGGACAAGGTACTGACTTGGCATTAGTCCGCACCTGTGAAGAAAAGCTTGAGAAAGTGCTTGATGTGTACGAGCAAAGGTTGTCAAAGAGCAGCTACCTGGCCGGAGACGATTTCACACTCGCTGATCTCAGTCATCTTCCTGGTCTCAGATACCTCATAAATGAAGCTGGAAAAGGATACCTGGTGACCGTGAGGAAGAATGTGAATGCATGGTGGGAGAATATATCAAATCGTCCTTCTTGGAAGAAATTAATGAATCTTGTTAATTAA。

the application of the GST gene participating in anthocyanin transport and accumulation in peony is realized through targeted regulation and control of a transgenic technology to carry out transgenic breeding.

Compared with the prior art, the function of the GST gene participating in anthocyanin transport and accumulation in the peony is verified through heterologous transformation to an Arabidopsis TT19 mutant and through virus-induced gene silencing (VIGS) technology in peony petals, and the GST gene can participate in anthocyanin transport and accumulation. In arabidopsis, overexpression in the arabidopsis TT19 mutant restored its anthocyanin-deficient phenotype; in peony, accumulation of anthocyanin in peony petals can be obviously reduced through targeted silencing of a target gene, so that pink petals are obviously lightened. Therefore, the GST gene can enrich and perfect the transgenic material library of the peony red character, and provides powerful support for molecular breeding of peony red character flowers in the future.

Sequence listing

GST Gene-CDS sequence of PsGSTF 3.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

The technical scheme provided by the invention is that a GST gene participating in anthocyanin transport and accumulation is separated and identified from peony (P.suffruticosa), and the GST gene is named as PsGSTF 3. The CDS sequences of GSTs genes which are confirmed to have anthocyanin transport and accumulation functions are not the same in different plants although they have similar functions. Therefore, GST participating in anthocyanin transport and accumulation is separated and identified in peony, so that a gene material library for peony transgenic molecule breeding can be enriched, and support is provided for future cultivation of peony with red character flowers.

The CDS sequence of the PsGSTF3 gene isolated and identified in the present invention is:

ATGGTAGTTAAGGTGTATGGCTCAGCTAAAGCGGCTTGTCCACAAAGAGTGATGGTTTGCCTTCTGGAAAAGGAGGTGGAATTTGAAATTATACATGTTGATCTTGAATCTGGAGAGCACAAAAAGCCAGATTTCCTTGCTCGGCAGCCGTTTGGGCAAGTTCCAGCCATCGAGGATGGTGACTTGAAACTTTTTGAATCCAGGGCAATCATAAGATACTATGCAGCCAAGTTTGATAACCGCGGTTCAAACCTGTTGGGAACCACTTTGGAAGAGAGAGCTTTGATGGATCAATGGCTAGAAGTTGAAGCCCACAATTTCAATGATTTGGTTTACACTATTGTGCTTCAGATCGTAATTCTCCCCCGTATGGGACAAGGTACTGACTTGGCATTAGTCCGCACCTGTGAAGAAAAGCTTGAGAAAGTGCTTGATGTGTACGAGCAAAGGTTGTCAAAGAGCAGCTACCTGGCCGGAGACGATTTCACACTCGCTGATCTCAGTCATCTTCCTGGTCTCAGATACCTCATAAATGAAGCTGGAAAAGGATACCTGGTGACCGTGAGGAAGAATGTGAATGCATGGTGGGAGAATATATCAAATCGTCCTTCTTGGAAGAAATTAATGAATCTTGTTAATTAA。

the application of the GST gene participating in anthocyanin transport and accumulation in peony is realized through targeted regulation and control of a transgenic technology to carry out transgenic breeding.

Used in Arabidopsis thaliana, overexpression in Arabidopsis TT19 mutant can restore its anthocyanin-deficient phenotype.

When the anthocyanin-containing peony flower bud is used in peony, the accumulation of anthocyanin in peony petals is obviously reduced through targeted silencing of a target gene, and pink petals are obviously lightened.

The implementation process of transgenic breeding by using the GST gene provided by the invention is described below by taking the overexpression of the PsGSTF3 gene in an Arabidopsis TT19 mutant and the silencing of the PsGSTF3 gene in peony petals as examples respectively, so that the feasibility of the corresponding transgenic breeding process is verified.

The specific process that the function of the GST gene participating in anthocyanin transport and accumulation is respectively proved in Arabidopsis and peony is as follows:

first, the PsGSTF3 gene heterologously transforms the Arabidopsis TT19 mutant

1) Extracting total RNA from peony petals, and performing reverse transcription to obtain cDNA. Then, a complete CDS sequence is obtained by cloning by using a specific primer of PsGSTF3, and sequencing is carried out to ensure the integrity and accuracy of the sequence;

2) constructing a complete CDS sequence of PsGSTF3 between two enzyme cutting sites (Hind3 and XbaI) of plant expression vector PHB by a seamless cloning mode, and sequencing;

3) the constructed expression vector is transferred into agrobacterium GV3101, spread on LB plate containing kanamycin 50 mug/mL, and cultured in 28 deg.C incubator for 2-3 days. Randomly selecting a single colony, performing colony PCR, and identifying a correct agrobacterium tumefaciens monoclonal as a marker for later use;

4) selecting correct agrobacterium tumefaciens monoclonal to be inoculated into 1.5mL of LB liquid culture medium containing corresponding antibiotics, carrying out shaking culture at 28 ℃ and 200rpm for 24 hours;

5) inoculating the underswing agrobacterium culture into 100mL LB liquid culture medium containing antibiotics according to the proportion of 1%, performing shaking culture until OD600 is about 1.0 at 28 ℃ and 200rpm, centrifuging for 15min at 4000rpm, and collecting thalli;

6) blowing and beating the thalli uniformly by using a transformation Buffer, and suspending until the OD600 is about 1.0;

7) inverting all inflorescences into the bacterial liquid suspended by the transformation Buffer in advance for about 30 seconds, and repeatedly transforming for 7 days according to the method;

8) after 2-3 weeks, pouring less nutrient solution as much as possible to accelerate aging, collecting mature seeds in paper bags, and drying in a dryer for 7 days for later use;

9) the sterilized seeds were spotted on 1/2MS medium (containing screening antibiotics: 30 μ g/mL HYG) plate, sealed;

10) vernalizing for 48h in a refrigerator at 4 ℃, and putting into a climatic chamber to start germination and growth. The plant growth environment is as follows: the relative humidity is 60%, the temperature is 23 ℃, the illumination period is 16h, and the illumination period is 8 h; the illumination intensity is 80-200 umol/M2/S;

11) observing after 8-15 days, distinguishing positive seedlings, transplanting the positive seedlings into planting soil, observing the phenotype at any time, and taking pictures.

Secondly, silencing PsGSTF3 gene in peony petals by using virus-induced gene silencing (VIGS) technology

1) Extracting total RNA from peony petals, carrying out reverse transcription to obtain cDNA, selecting a 272bp non-conservative domain base sequence of PsGSTF3 gene, designing upstream and downstream specific primers, cloning a target sequence, and sequencing to ensure the accuracy of the sequence;

2) the 272bp base sequence of the PsGSTF3 gene is constructed between two enzyme cutting sites (BamHI and PSTI) of a plant expression vector pTRV2-GFP by a seamless cloning mode, and sequencing is carried out;

3) the constructed expression vector was transferred to Agrobacterium GV3101, spread on LB (kanamycin 50. mu.g/mL, rifampicin 25. mu.g/mL) plates, and cultured in 28 ℃ incubator for 2-3 days while inverted. Randomly selecting a single colony, performing colony PCR, and identifying a correct agrobacterium tumefaciens monoclonal as a marker for later use;

4) selecting correct agrobacterium tumefaciens for single cloning, inoculating the agrobacterium tumefaciens to 5mL of LB liquid culture medium containing corresponding antibiotics, shaking at 28 ℃ and 200rpm for 16-20h, and allowing the liquid to be completely separated without whitening;

5) inoculating the agrobacteria with small shake according to 1% ratio, adding into 100mL LB liquid medium (kana 100. mu.g/mL, rifampicin 25. mu.g/mL, MES 10mM, AS 200uM), shaking overnight at 28 deg.C and 200rpm, taking out when the bacterial liquid concentration reaches OD600 ═ 1.8, 5000rpm, centrifuging for 10min, and collecting thallus;

6) infection with agrobacterium buffer (MES 10 mM; AS 100 uM; MgC 1210 mM; sterile water as a solvent) and adjusting the OD600 to about 1.0; respectively mixing pTRV1 with an agrobacterium infection buffer solution containing pTRV2-GFP (control) and pTRV2-GFP-PsGSTF3 in equal volume, respectively adding Silwet L-77 with the final concentration of 0.01% into the two mixed infection buffer solutions, and standing for 4h at room temperature of 24 ℃ under a dark condition for later use;

7) the specific infection operation is as follows: the test material is peony 'Zhao powder', unopened and compact buds are selected, bacteria liquid is injected into the buds, calyces and flower stems by using an injector, the usage amount of the bacteria liquid of each bud is about 3mL, and important information such as treatment groups and the like is marked in detail by hanging cards;

8) after the injection is finished, wrapping the injection with a black plastic bag, shading, taking down the shading bag after 24h, and growing in a normal environment;

9) after about 7 days, phenotype can appear, and photographing and GFP fluorescence detection are carried out.

According to the implementation scheme, the transgenic material library for promoting anthocyanin transport and accumulation in the peony can be enriched, and the red character of the peony can be controlled in a targeted manner by utilizing the transgenic technology.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Sequence listing

<110> forestry research institute of China forestry science research institute

<120> GST gene participating in anthocyanin transport and accumulation in peony

<160>1

<170> SIPOSequenceListing 1.0

<210>1

<211>642

<212>DNA

<213> peony (P. suffruticosa)

<400>1

ATGGTAGTTAAGGTGTATGGCTCAGCTAAAGCGGCTTGTCCACAAAGAGTGATGGTTTGC 60

CTTCTGGAAAAGGAGGTGGAATTTGAAATTATACATGTTGATCTTGAATCTGGAGAGCAC 120

AAAAAGCCAGATTTCCTTGCTCGGCAGCCGTTTGGGCAAGTTCCAGCCATCGAGGATGGT 180

GACTTGAAACTTTTTGAATCCAGGGCAATCATAAGATACTATGCAGCCAAGTTTGATAAC 240

CGCGGTTCAAACCTGTTGGGAACCACTTTGGAAGAGAGAGCTTTGATGGATCAATGGCTA 300

GAAGTTGAAGCCCACAATTTCAATGATTTGGTTTACACTATTGTGCTTCAGATCGTAATT 360

CTCCCCCGTATGGGACAAGGTACTGACTTGGCATTAGTCCGCACCTGTGAAGAAAAGCTT 420

GAGAAAGTGCTTGATGTGTACGAGCAAAGGTTGTCAAAGAGCAGCTACCTGGCCGGAGAC 480

GATTTCACACTCGCTGATCTCAGTCATCTTCCTGGTCTCAGATACCTCATAAATGAAGCT 540

GGAAAAGGATACCTGGTGACCGTGAGGAAGAATGTGAATGCATGGTGGGAGAATATATCA 600

AATCGTCCTTCTTGGAAGAAATTAATGAATCTTGTTAATTAA 642