GST gene participating in transport and accumulation of isosalix integra in peony and application of GST gene

Abstract

The invention discloses a GST gene participating in transport and accumulation of isosalix integra in peony and application of the GST gene, the GST gene participating in transport and accumulation of the isosalix integra is separated and identified from P.delavayi, and after the gene is silenced in the peony, yellow petals and color spots can be promoted to be obviously lightened; overexpression in Tobacco nipponense can significantly deepen the color of petals. The gene plays an important role in flavonoid transport and accumulation, can enrich a transgenic material library of peony flower yellow traits, and provides powerful support for molecular breeding for cultivating and regulating peony flower yellow traits in the future.

Description

Technical Field

[0001] The invention relates to a molecular biology technology, in particular to a GST (Glutathione S-transferase) gene involved in the transport and accumulation of isostearin in tree peony and application thereof. Background Art

[0002] Peony (Paeonia suffruticosa) is known as the "king of flowers". After long-term cultivation and breeding, it has formed nine color systems, including red, pink, white, yellow, purple, black, green, blue, and complex colors. Among them, the number of yellow ornamental peonies is relatively small, and cultivating pure yellow peony varieties has always been one of the breeding directions of peony breeders. The common yellow peony varieties on the market all originate from Yunnan peony (Paeonia delavayi). Therefore, its flower color components are relatively clear, and the main coloring role is flavonoids. Flavonoids are mostly found in the vacuoles of plants and are divided into different types such as flavonoids, flavonols, aurones, chalcones, proanthocyanidins, and anthocyanins. The substances with higher content in yellow peonies are chalcone glycosides, flavonoid glycosides and flavonol glycosides, among which isoflavone glycosides are an important reason for the formation of its yellow color.

[0003] The biosynthesis pathway of flavonoids is highly conservative and has been studied relatively clearly. Its regulatory mechanism has also been reported in many model and non-model plants. However, the final presentation of flower color still requires the transport and accumulation of pigments. Studies have shown that GST can participate in the transport of plant flavonoids and transport multiple substrates at the same time. In the Arabidopsis mutant tt19 lacking GST transporter, the transport mechanism is destroyed, not only the location of flavonols in the cell changes, but also the content is significantly reduced; TaGSTL1 in wheat (Triticum aestivum) can selectively recognize dihydroquercetin and quercetin in vitro; VviGST1, VviGST3, VviGST4 in grapes can bind to glutathione, anthocyanin monomers, proanthocyanidin monomers and flavonol monomers; PtGSTU19 and PtGSTU20 in Populus trichocarpa can bind to multiple flavonoids in vitro; GST encoded by AtGSTF2 can bind to two unmethylated flavonols, and so on. However, GST capable of transporting isoflavone glycosides has not been found in many plants.

[0004] Hybridization between different subgroups of peony is difficult, and it usually takes a lot of time and energy to obtain the ideal variety through multiple generations of breeding. Molecular breeding is a new method for breeding new varieties. The targeted technology using transgenic technology has been widely used in crops and horticultural flowers. The molecular breeding mainly uses transgenic technology to overexpress or knock out the genes that control the relevant phenotypic traits in the target plant, so that the plant gains or loses the corresponding phenotype, and finally obtains the ideal variety. Therefore, screening the key genes that control the relevant phenotypic traits is the prerequisite and important resource for molecular breeding.

[0005] At present, GST that can transport isoflavone has not been reported, and the research focus on GST is mostly on model plants. Therefore, supplementing the role of GST gene in promoting flavonoid transport and accumulation in peony can provide a new way for the future use of biotechnology to transform flower color, which has important application value.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a GST gene involved in the transport and accumulation of isosalicylic acid in peony and its application, so as to improve the integrity of the pathway for controlling the yellow trait of flowers in peony.

[0008] The objective of the present invention is achieved through the following technical solutions:

[0009] The GST gene involved in the transport and accumulation of isoflavone in the peony of the present invention is isolated and identified from Yunnan peony (P.delavayi), and the CDS sequence of the gene is:

[0010] .

[0011] The application of the GST gene involved in the transport and accumulation of isoflavone glycosides in the above-mentioned peony is used for targeted regulation through transgenic technology to carry out transgenic breeding.

[0012] Compared with the prior art, the GST gene and its application involved in the transport and accumulation of isobarbitalin in the peony of the present invention have been verified by heterologous transformation to tobacco and by virus-induced gene silencing (VIGS) technology in peony petals, and it can participate in the transport and accumulation of flavonoids. In tobacco, overexpression of the target gene can promote the accumulation of anthocyanins in petals; in peony, by targeted silencing of the target gene, not only can the accumulation of isobarbitalin in peony petals be significantly reduced, and the yellow petals can be significantly lightened, but also the color spots can be lightened and the anthocyanin content can be reduced. Therefore, the gene can play a role in changing the color of the flower, not only can it enrich and improve the transgenic material library of the yellow trait of peony flowers, but also provide strong support for the molecular breeding of yellow trait flowers of peony in the future.

[0013] In addition to isoflavone glycosides, the gene can transport a variety of coloring substances in peony (flavonoid glycosides, flavonol glycosides and anthocyanins). The GST gene of the present invention provides a new idea for regulating the color of peony flowers by genetic engineering means and cultivating rich new varieties of peonies, which will have broad prospects in the molecular breeding of peony flower color.

[0014] Sequence Listing

[0015] CDS sequence of GST gene—PdGSTF1. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 For the identification of transgenic tobacco plants, in the figure: A: wild-type and transgenic tobacco plant phenotypes; B: relative expression of PdGSTF1; C: determination of total anthocyanin content in wild-type and transgenic tobacco plants. WT: wild-type plants; OE-1, OE-2, OE-3: transgenic plants.

[0017] Figure 2 Identification of VIGS silenced peony plants. In the figure: A: phenotype of peony petals of VIGS empty and silenced plants; B: relative expression of PdGSTF1; C: detection of total flavonol content in VIGS silenced peony petals; D: detection of total anthocyanin content in VIGS silenced peony spots. TRV: Tobacco rattle virus empty vector silenced group; TRV-PdGSTF1: target gene recombinant vector silenced group.

[0018] Figure 3 This is a graph of metabolite content in the petal area of ​​peony VIGS silencing, in which: A: comparison of isosalpin content; B: comparison of flavonoid content; C: comparison of flavonol content. TRV: tobacco rattle virus empty vector silencing group; TRV-PdGSTF1: target gene recombinant vector silencing group. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The parts of the present invention that are not described in detail are well known in the art.

[0020] The technical solution provided by the present invention specifically separates and identifies a GST gene involved in the transport and accumulation of isoflavone from Yunnan peony (P.delavayi), named PdGSTF1. Although the GST gene confirmed to have the function of flavonoid transport and accumulation plays a similar role in different plants, its CDS sequence is not the same. Therefore, by isolating and identifying the GST involved in the transport and accumulation of isoflavone in peony, the gene material library of transgenic molecular breeding of peony can be enriched, and support can be provided for the future cultivation of peonies with yellow trait flowers.

[0021] The GST gene (PdGSTF1 gene) involved in the transport and accumulation of isoflavone in the peony of the present invention is isolated and identified from the Yunnan peony (P.delavayi), and the CDS sequence of the gene is:

[0022] .

[0023] The application of the GST gene involved in the transport and accumulation of isoflavone glycosides in the above-mentioned peony is used for targeted regulation through transgenic technology to carry out transgenic breeding.

[0024] Used in tobacco, overexpression of the target gene can promote the accumulation of anthocyanins in petals.

[0025] Used in peony to carry out molecular transgenic improvement breeding of peony flower color.

[0026] By targeting and silencing the target gene, the accumulation of isoflavones in peony petals was significantly reduced, and the content of flavonoids and flavonols was also significantly reduced, making the yellow petals significantly lighter; at the same time, the color spots were faded and the anthocyanin content was reduced.

[0027] Therefore, this gene can be used to regulate the flower color of peonies.

[0028] The following will take the overexpression of PdGSTF1 gene in tobacco and the silencing of PdGSTF1 gene in peony petals as examples to describe the implementation process of transgenic breeding using the GST gene provided by the present invention, and then verify the feasibility of the corresponding transgenic breeding process.

[0029] The specific process in which the GST gene described in the present invention participates in the transport and accumulation of isoflavone glycosides is confirmed in tobacco and peony respectively is as follows:

[0030] 1. Heterologous transformation of tobacco with PdGSTF1 gene

[0031] 1) Total RNA was extracted from the petals of Paeonia dahliae and reverse transcribed into cDNA. The complete CDS sequence was then cloned using the specific primers of PdGSTF1 and sequenced to ensure the integrity and accuracy of the sequence;

[0032] 2) The complete CDS sequence of PdGSTF1 was constructed by seamless cloning between two restriction sites (NcoI and SpeI) of the plant expression vector pCAMBIA1302 and sequenced;

[0033] 3) The constructed expression vector was transferred into Agrobacterium GV3101, spread on a YEP plate containing 50 μg / mL of kanamycin, and placed upside down in a 28°C incubator for 2-3 days. Single colonies were randomly selected for colony PCR, and the correct Agrobacterium single clone was identified and marked for later use;

[0034] 4) Pick the correct Agrobacterium single clone and inoculate it into 1.5 mL LB liquid medium containing the corresponding antibiotics, and culture it at 28°C, 200 rpm, with shaking for 24 hours;

[0035] 5) Inoculate the Agrobacterium culture at a ratio of 1% into 100 mL of LB liquid medium containing antibiotics, and culture at 28°C, 200 rpm, and shake until the OD 600 = about 1.0, 4000rpm, centrifuge for 15min, collect the bacteria;

[0036] 6) Use transformation buffer to evenly disperse the cells and resuspend them to OD 600 = about 0.6;

[0037] 7) Cut the sterile tobacco seedling leaves into thumb-sized leaf discs and infect them with Agrobacterium resuspension. After infection, transfer them to MS1 medium, wrap them with tin foil and incubate them in a 25℃ tobacco culture room in the dark for 3 days;

[0038] 8) Take out the leaves after dark culture, wash the surface bacteria with sterile water, then wash them once with sterile water containing 1000μg / mL and 500μg / mL cephalosporin, respectively, and then wash them three times with sterile water. After taking out the leaf disc, dry it with filter paper, and move the leaf disc into MS1 medium with the front side facing up;

[0039] 9) After 8-10 days of cultivation under light, callus tissue can be seen around the leaf disc, and then small buds can be differentiated; after 3 weeks of cultivation, the small buds can be cut off and transferred to MS2 medium;

[0040] 10) After growing in S2 medium for one week, the buds can be cut off and transferred to MS3 medium; after growing in MS3 medium for one week, the large buds can be cut off and transferred to rooting medium;

[0041] 11) One week after the seedlings are placed in the rooting medium, large buds can be seen taking root. After another week of growth, the rooted seedlings can be taken out of the tissue culture bottle, and the solid culture medium on the roots can be removed and then moved into a flower pot. Cover the seedlings with a light-transmitting cover to keep them moist. After the seedlings grow for 2-3 days, remove the cover. Identify the positive seedlings, culture them until they bloom, and observe the phenotype and take photos at any time.

[0042] 12) The plant growth environment is: relative humidity 60%, temperature 23°C, photoperiod 16h light, 8h dark; light intensity 80-200umol / M2 / S.

[0043] 2. Silencing the PdGSTF1 gene in peony petals using virus-induced gene silencing (VIGS)

[0044] 1) Total RNA was extracted from the petals of Paeonia dahliae, reverse transcribed into cDNA, and the 276 bp non-conserved domain base sequence of the PdGSTF1 gene was selected to design upstream and downstream specific primers, the target sequence was cloned, and sequencing was performed to ensure the accuracy of the sequence;

[0045] 2) The 276 bp base sequence of the PdGSTF1 gene was constructed by seamless cloning between two restriction sites (EcoRI and BamHI) of the plant expression vector pTRV2-GFP and sequenced;

[0046] 3) The constructed expression vector was transferred into Agrobacterium GV3101, spread on YEP (cannabinoids 50 μg / mL, rifampicin 25 μg / mL) plates, and placed upside down in a 28°C incubator for 2-3 days. Single colonies were randomly selected for colony PCR, and the correct Agrobacterium single clones were identified and marked for later use;

[0047] 4) Pick the correct Agrobacterium monoclone and inoculate it into 5 mL LB liquid medium containing the corresponding antibiotics, shake it at 28°C, 200 rpm for 16-20 hours, until the bacterial liquid is clear and not whitish;

[0048] 5) Inoculate the Agrobacterium culture in 100 mL LB liquid medium (100 μg / mL of kanamycin, 25 μg / mL of rifampicin, 10 mM MES, 200 uM AS) at a ratio of 1%, shake overnight at 28°C and 200 rpm, and incubate until the bacterial solution reaches OD 600 =1.8, centrifuge at 5000rpm for 10min, and collect the cells;

[0049] 6) Use Agrobacterium infection buffer (MES10mM; AS100uM; MgCl 2 10mM; sterile water as solvent) to suspend the cells and adjust the OD 600 = about 1.0; pTRV1 was mixed with equal volumes of Agrobacterium infection buffer containing pTRV2-GFP (control) and pTRV2-GFP-PdGSTF1, and Silwet L-77 was added to the two mixed infection buffers at a final concentration of 0.01%, and the mixture was allowed to stand at room temperature at 24°C for 4 hours in the dark for later use;

[0050] 7) The specific operation of infection is as follows: the test material is the peony variety 'Haihuang', select the unopened and tight buds, use a syringe to inject the bacterial solution into the buds, calyx and flower stems, and the amount of bacterial solution used for each bud is about 3mL. Hang a sign to indicate the important information such as the treatment group in detail;

[0051] 8) After the injection is completed, wrap it with a black plastic bag and shade it. Remove the shade bag after 24 hours and let it grow in a normal environment;

[0052] 9) After about 7 days, the phenotype will appear and take pictures and test the pigment.

[0053] It can be seen from the above implementation scheme that the present invention can enrich the transgenic material library that promotes the transport and accumulation of flavonoids in peony flowers, and the present invention can be used to target and regulate the yellow trait of peony flowers through transgenic technology.

[0054] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

[0055]

Claims

1. A GST gene involved in the transport and accumulation of isoflavone glycosides in tree peony, characterized in that: A GST gene involved in the transport and accumulation of isoflavone glycosides was isolated and identified from P. delavayi. The CDS sequence of the gene is: .

2. An application of the GST gene involved in the transport and accumulation of isoflavone glycosides in peony according to claim 1, characterized in that: Transgenic breeding is carried out through targeted regulation of transgenic technology.

3. The use of the GST gene involved in the transport and accumulation of isoflavone glycosides in peony according to claim 2, characterized in that: Used in tobacco, overexpression of the target gene can promote the accumulation of anthocyanins in petals.

4. The use of the GST gene involved in the transport and accumulation of isoflavone glycosides in peony according to claim 2, characterized in that: Used in peony to carry out molecular transgenic improvement breeding of peony flower color.

5. The use of the GST gene involved in the transport and accumulation of isoflavone in peony according to claim 4, characterized in that: By targeting and silencing the target gene, the accumulation of isoflavones in peony petals was significantly reduced, and the content of flavonoids and flavonols was also significantly reduced, making the yellow petals significantly lighter; at the same time, the color spots were faded and the anthocyanin content was reduced.

Images