CN107245497A - VIGS silencing system for identifying peony type xanthose transferase gene - Google Patents

Abstract

The invention discloses a VIGS silencing system for identifying peony yellow ketotransferase genes. The VIGS silencing system for identifying the peony yellow ketotransferase gene provided by the invention comprises: inserting a target gene into the VIGS silencing vector to obtain a recombinant vector; the target gene is a nucleotide sequence shown as a sequence 1 in a sequence table and/or a nucleotide sequence shown as a sequence 3 in the sequence table. The invention clones the conserved segments of the flavonoid ketotransferase genes PsUF3GT and PsUF5GT as target genes to construct a TRV2 vector by utilizing a homologous cloning technology, successfully establishes a VIGS silencing system, and effectively reduces the expression quantity and the total anthocyanin content of the two genes. The result of the invention has important significance for the research of the gene function of the peony and lays a foundation for analyzing the molecular mechanism of the peony stain formation.

Description

VIGS silencing system for identification of flavonoid glycosyltransferase genes in tree peony

technical field

The invention relates to the field of biotechnology, in particular to a VIGS silencing system for identifying flavonoid glycosyltransferase genes of peony.

Background technique

Peony (Paeonia suffruticosa) is a deciduous subshrub of Paeoniaceae (Paeoniaceae) Paeonia (Paeonia) peony group (Section Moutan), and is a traditional famous flower in my country. The research on the chemical basis of peony flower color formation is relatively in-depth. There are 12 kinds of anthocyanins in the petals, namely geranoid 3-glucoside (Pg3G), geranoid 3,5-diglucoside (Pg3G5G), cyanidin 3-glucoside (Cy3G), cyanidin 3,5-diglucoside (Cy3G5G), paeoniflorin 3-glucoside (Pn3G), paeoniflorin 3,5-diglucoside (Pn3G5G), cyanidin 3, 5-di-hexoside (Cy-3,5-di-hexoside), cyanidin 3-glucose-5-arabinoside (Cy3G5Ara), cyanidin 3-arabinoside (Cy3Ara), cyanidin 3 -galloyl glucoside (Cy3galloylG); paeoniflorin 3-arabinoside (Pn3Ara) and paeoniflorin 5-galloyl glucoside (Pn5galloylG), of which Pg3G, Pg3G5G, Cy3G, Cy3G5G, Pn3G and Pn3G5G are the main cyanines Glycosides (Wang L S, Shiraishi A, Hashimoto F, Aoki N, Shimizu K, SakataY. 2001. Analysis of petal anthocyanins to investigate flower coloration of Zhongyuan (Chinese) and Daikon Island (Japanese) tree peony cultivars. J PlantRes, 114: 33- 43.; Wang L S, Hashimoto F, Shiraishi A, Aoki N, Li J J, SakataY.2004.Chemical taxonomy of the Xibei tree peony from China by floralpigmentation.J Plant Res, 117:47-55.; Li Chonghui, 2010. Peony Analysis of petal flavonoids and their effects on flower color, doctoral dissertation of Institute of Botany, Chinese Academy of Sciences), cultivated varieties, especially Northwest peony varieties, have bright and large color spots at the base of petals, which not only increase the ornamental value, but are often used as varieties One of the important basis for classification, these non-spotted part anthocyanins are mainly composed of Pn3G5G, Cy3G5G and Cy3G, the content of Pn3G is very low, and almost does not contain Pg-type pigments, it is speculated that the glycosylation, methylation and high content of Cy at the base of the petals are The main cause of stains. Zhang et al. (2007) analyzed the composition of anthocyanins in the petals of 35 Northwest peony varieties. Cy3G is the main component in the spots, and Pn3G5G is the main component in the spots. The pigment content in spots is significantly higher than that in non spots (Zhang J J, Wang L S, Shu Q Y, Liu Z A, Li CH, Zhang J, Wei X L, Tian D K. 2007. Comparison of anthocyanins in non-blotches and blotches of the petals of Xibei tree peony. Sci Hortic, 114: 104-111 .). It can be seen that the glycosylation modification of anthocyanins plays an important role in the synthesis and accumulation of peony anthocyanins (Wang L S, Shiraishi A, Hashimoto F, Aoki N, Shimizu K, Sakata Y.2001. Analysis of petalanthocyanins to investigate flower coloration of Zhongyuan (Chinese) and Daikon Island (Japanese) tree peony cultivars. J Plant Res, 114: 33-43.).

Flavonoids are an important class of secondary metabolites in plants, and usually require glycosylation modification in the last step of synthesis to increase their stability and solubility in cells and affect the color development of flowers (Tian Peng, Liu Zhanlin .2011. Glycosyltransferase superfamily [J]. 2011. Chemistry of Life, 31:732–736). Flavonoid glycosidation is completed by glycosyltransferases (UDP-glucose: flavonoid glycosyltransferases, UFGTs), and its glycosyl donor is usually nucleotide sugar (nucleotide sugar, Uridine diphosphate, UDP) such as UDP-glucose. The earliest discovery was the gene Bronze1 that controls maize melanin, which encodes flavonoid UDP-glycosyltransferase (Dooner H K, Nelson OE.1997. Controlling element-induced alterations in UDP glucose flavonoidglucosyltransferase, the enzyme specified by the bronze locus in maize. ProcNatl Acad Sci USA, 74:5623-5627.). Afterwards, found flavonoid glycosyltransferase gene (UFGT) such as the UF3GT (Schiefelbein J W, Raboy V, Fedoroff NV, Nelson Jr O E.1985.Deletions Within a defective suppressor-mutator elementin maize affect the frequency and developmental timing of its excision from the bronze locus. Proc Natl Acad Sci USA, 82: 4783-4787.) and UF5GT of gentian and grape, etc. (Yang Y Z, Labate J A, Liang Z C, Cousins P, Prins B, Preece J E, Aradhya M, Zhong GY. 2014. Multiple loss-of-function5-O-glucosyltransferase alleles revealed in Vitis vinifera, but not in other Vitis species. Theor Appl Genet, 127: 2433-2451 .). In addition, there is a glycosyltransferase in rose that first glycosylates anthocyanin 5-O and then continues to modify 3-O to form 3,5-bis-O-glycoside. Cluster analysis shows that it is closely related to anthocyanin The 3GT- and 5GT-glycosyltransferase subfamilies are distinct (Ogata J, Kanno Y, Itoh Y, Tsugawa H, Suzuki M. 2005. Plant biochemistry: anthocyanin biosynthesis in roses. Nature, 435: 757-758.; Purkayastha A , DasguptaI.2009.Virus-induced gene silencing: A versatile tool for discovery of gene functions in plants[J].Plant Physiol Biochem, 47:967-976), it can be seen that in different species, there is diversity in flavonoid glycosylation modification genes And consists of superfamily genes. Some related enzyme genes in the synthetic pathway of flavonoids (including anthocyanins) in peony petals have been cloned and expressed (Du H, Wu J, Ji K X, Zeng Q Y, Bhuiyad MW, Su S, Shu Q Y, Ren H X, Liu Z A, Wang L S.2015. Methylation mediated by ananthocyanin O-methyltransferase, is involved in purple flower coloration in Paeonia. J Exp Bot, 66: 6563-77.), while studies on flavonoid glycosyltransferase genes have not been reported yet, Therefore, it is of great significance to carry out research on the function of related genes to understand the formation of its flower color.

Regeneration and genetic transformation of tree peony are very difficult, there is no report on its genetic transformation system, and there is a technical bottleneck in the verification of gene functions of tree peony using transgenic technology. In addition, peony takes about 3 years from seedling to flowering, and it is difficult to identify the phenotype of flowering-related genes.

Virus-induced gene silencing VIGS (Virus-induced gene silencing) is a post-transcriptional gene silencing phenomenon discovered in recent years, which can cause specific degradation of endogenous mRNA sequences (Ratcliff F, Martin-HemandezA M, Baulcombe D C.2001. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J, 25: 237-245.).

Contents of the invention

In order to make up for the deficiencies in the above fields, the present invention provides a VIGS silencing system for identifying flavonoid glycosyltransferase genes of tree peony.

The VIGS silencing system for identifying the flavonoid glycosyltransferase gene of peony provided by the present invention comprises: a recombinant vector obtained by inserting a target gene into a VIGS silencing vector; the target gene is the nucleotide sequence shown in Sequence 1 in the sequence listing And/or the nucleotide sequence shown in sequence 3 in the sequence listing.

The VIGS silencing vector is a tobacco rattle virus TRV2 vector.

The invention also provides a method for constructing the VIGS silencing system for identifying the flavonoid glycosyltransferase gene of tree peony.

The construction method of the VIGS silencing system for identifying the flavonoid glycosyltransferase gene of peony provided by the present invention comprises the following steps:

(1) PCR amplification of the target gene fragment was carried out using specific primers added with BamH I restriction site and Xho I restriction site to obtain the PsUF3GT target fragment and PsUF5GT target fragment for VIGS silencing, respectively;

The nucleotide sequence of the PsUF3GT target fragment is shown in Sequence 1 in the sequence listing; the specific primers for amplifying the PsUF3GT target fragment are as follows:

3GT-VF1: AA GGATCC CATGGCTCAATGACCAAAAAGGCTAA;

3GT-VR1: TTA CTCGAGC ATTCTTCGTAAATACTCCACCGTCG;

The nucleotide sequence of the PsUF5GT target fragment is shown in sequence 3 in the sequence listing; the specific primers for amplifying the PsUF5GT target fragment are as follows:

5GT-VF1: AA GGATCC TTTGGAAAAGCAAGGAATGGTGGTG;

5GT-VR1: ACG CTCGAG TCACAAATCACCACCTTTCCCCACC;

(2) Double digestion of the PsUF3GT target fragment or the PsUF5GT target fragment and the TRV2 vector with BamH I enzyme and Xho I enzyme, after the enzyme digestion is completed, the PsUF3GT target fragment or the PsUF5GT target fragment is connected to the tobacco rattle virus TRV2 vector , after connection, transform E.coLi DH5α competent cells, screen positive single colonies, and then transform into Agrobacterium GV3101 to obtain the VIGS silencing system.

The application of the VIGS silencing system for identifying peony flavonoid glycosyltransferase genes in identifying the peony flavonoid glycosyltransferase genes PsUF3GT and/or PsUF5GT also belongs to the protection scope of the present invention.

A method for identifying the flavonoid glycosyltransferase gene of tree peony also belongs to the protection scope of the present invention.

The method for identifying the flavonoid glycosyltransferase gene of peony provided by the present invention uses the VIGS silencing system.

The method comprises the steps of:

Using peony flowers with pedicels as materials, submerged in the bacterial solution containing the VIGS silencing carrier, vacuum infiltration, negative pressure treatment, and deflation;

Rinse the petals after vacuum infiltration with deionized water to remove excess bacteria;

The flower stalk is cultured in a hydroponic manner, and then the expression level of the target gene and the change of anthocyanin accumulation are detected.

The flower development period of the flowers with pedicels is full bloom or flower buds.

The duration of vacuum infiltration is 10min-15min.

The conditions for culturing the flower stalks in a hydroponic manner are as follows: soak the flower stalks in deionized water, and culture them in the dark at a temperature of 8°C and a humidity of 60% for 1 day; then transfer them to a temperature of 23-25°C 1. Cultivate under normal light for 3 days under the condition of humidity 60%.

The bacterium solution containing the recombinant vector of claim 1 or 2 is: containing the nucleotide sequence shown in sequence 1 in the sequence listing and/or the nucleoside shown in sequence 3 in the sequence listing inserted on the tobacco rattle virus TRV2 vector The recombinant vector bacterial liquid obtained from the acid sequence and the bacterial liquid containing the tobacco rattle virus TRV1 vector.

The present invention utilizes homologous cloning technology to clone the conserved segments of peony flavonoid glycosyltransferase genes PsUF3GT and PsUF5GT as target genes to construct TRV2 vectors, successfully establishes a VIGS silencing system, and effectively reduces the expression and total expression of these two genes. Anthocyanin content. The results of this study are of great significance to the study of the gene function of peony, and also lay a foundation for the analysis of the molecular mechanism of the formation of peony stains.

There are many factors that affect the silencing effect of VIGS. The first is the selection of the length and position of the silenced gene fragments. The present invention utilizes amplified fragments with a conserved segment length of 400-450 bp to silence homologous family genes, and achieves a better silencing effect. For example, when silencing a specific gene, the specificity of the sequence needs to be considered, and the method of silencing the untranslated region or specific segment can be used to ensure the silencing of the specific target gene. The invention adopts tobacco rattle virus TRV as a carrier, which can effectively silence the target gene in the growth point or meristem, and obtain a systematic silence effect. In addition, the plant material after using TRV to silence the gene is more sensitive to temperature. The present invention is carried out under the conditions of temperature control (25° C.) and humidity control (relative humidity 60%) in a light incubator, and better experimental results have been obtained. Therefore, the successful silencing of genes requires strict control over every technical link.

Aiming at the problems of difficult genetic transformation of tree peony and inability to verify gene functions, the present invention successfully silenced the PsUF3GT and PsUF5GT genes through VIGS technology, using two factors and three levels of nine experimental treatments. The best VIGS system of PsUFGTs was established. The conserved region of PsUFGTs gene was used as the target fragment, and flowers with flower stalks in S4 or S2 stage were used as materials. After vacuum infiltration for 10min-15min, they were placed in deionized water and cultured in dark for 1d (8°C). , humidity 60%); then transferred to an environment at 23-25° C. and humidity 60%, and cultured under normal light for 3 days before detection and analysis. This result laid the foundation for verifying the function of UFGTs genes in peony, and also provided research ideas and technical basis for verifying other important functional genes in peony.

Description of drawings

Figure 1 shows the nucleotide and deduced encoded amino acid sequence of the PsUF3GT target fragment; the underline indicates PSPG-box.

Figure 2 shows the nucleotide and deduced encoded amino acid sequences of the PsUF5GT target fragment; the underline indicates the characteristic sequence of UDPG.

Figure 3 is the results of gel electrophoresis detection of bacterial solution containing TRV vector; M is the molecular weight standards from top to bottom are 1000bp, 900, 800, 700, 600, 500, 400, 300, 200, 100bp respectively.

Fig. 4 Analysis of the expression level of the PsUF3GT gene in the silenced petals of different treatments; the lowercase letters indicate the difference in the degree of significance.

Fig. 5 Analysis of the expression level of PsUF5GT gene in silencing petals under different treatments; lowercase letters indicate the difference of significance degree.

Fig. 6 Analysis of anthocyanin content and components in versicolor of PsUFGTs silenced by different treatments.

detailed description

The purple-spotted peony variety 'Qinghai Lake Yinbo' (P.suffruticosa 'Qing Hai Hu YinBo') used in the present invention is planted in the peony and peony germplasm resource nursery of the Institute of Botany, Chinese Academy of Sciences. Its spots are purple-red, and non-spots are white. Anthocyanins were not detected in the non-spotted part, and there are four kinds of anthocyanins in the spotted part: Pn3G, Pn3G5G, Cy3G and Cy3G5G (the purple-spotted peony cultivar 'Qinghai Lake Yinbo' (P.suffruticosa'Qing Hai Hu Yin Bo' )’s non-patent literature is: Zhang J J, Wang L S, Shu Q Y, Liu Z A, Li C H, Zhang J, Wei X L, Tian D K.2007.Comparison of anthocyanins in non-blotches and blotches of the petals of Xibei treepeony.Sci Hortic, 114:104-111).

The VIGS carrier used in the present invention comes from tobacco rattle virus carrier (tobacco rattle virus, TRV), and TRV1/TRV2 is a gift from Professor Ma Nan of the Ornamental Plant Postharvest and Stress Physiology Laboratory of China Agricultural University (the non-patent literature that has recorded the carrier is: Tian J,Pei H X,Zhang S,Chen J W,Chen W,Yang R Y,Meng Y L,YouJ,Gao J P,Ma N.2014.TRV-GFP:a modified Tobacco rattle virus vector efficient and visualizable analysis of gene function.J Exp Bot, 65:311-322.), which has a Kanna selection marker and a 35S promoter, and pTRV2 has multiple cloning sites such as BamH I and Xho I.

The standard product cyanidin-3-glucoside (Cy3G) was purchased from French Extrasynthese company (Genay, France).

Embodiment 1,

1. Gene cloning

The extraction of petal total RNA was carried out according to the kit instructions (RNAprep Pure Plant Kit, FastQuantcDNA Tiangen Biochemical Technology (Beijing) Co., Ltd.). The upstream and downstream primers were designed according to the UF3GT and UF5GT sequences in the public database to clone the target fragment. The PsUF3GT and PsUF5GT fragments used for VIGS silencing were designed with upstream and downstream primers and added BamH I and Xho I restriction sites. The amplification primers are listed in Table 1.

Table 1 Related information of primer sequences used in the present invention

Note: The underline indicates the base restriction site.

(1) Cloned sequence analysis of PsUF3GT target fragment

The length of the PsUF3GT target fragment amplified according to the upstream and downstream primers is 413bp, and its nucleotide sequence is shown in sequence 1 in the sequence list. The PCR reaction system and procedures used are shown in Table 2 and Table 3. The amplified fragments were recovered according to the instructions using the gel extraction kit (EasyPure Quick Gel Extraction, Quanshijin Biotechnology Co., Ltd.) and connected to pEASY-T3 The vector (purchased from Quanshijin Biotechnology Co., Ltd.) was transformed into Escherichia coli competent cells (Trans1-T1Phage Resistant Chemically Competent Cell, purchased from Quanshijin Biotechnology Co., Ltd.). Positive clones were sequenced (completed by Beijing Bomaide Gene Technology Co., Ltd.). The sequence was analyzed with DNAman5.0 and online network software (http://prosite.expasy.org/mydomains/), which belongs to the Glycosyltransferase_GTB_type superfamily and contains a PSPG motif (plant secondary product glycosyltransferase box, underlined in Figure 1). The amino acid sequence of the PsUF3GT protein encoded by the PsUF3GT target fragment is shown in Sequence 2 in the sequence listing.

Table 2: PCR reaction system

Table 3: PCR Amplification Procedure

(2) PsUF5GT gene cloning and sequence analysis

The length of the cloned PsUF5GT target fragment is 418bp, and its nucleotide sequence is shown as sequence 3 in the sequence list. The PCR reaction system and procedures used are the same as those of PsUF3GT. The sequence was analyzed with DNAman5.0, and BlastP analysis could be performed with public data. It belongs to the Glycosyltransferase_GTB_type superfamily, and its amino acid has a UDPG (UDP-glycosyltransferases) characteristic sequence at 337-380, namely WcnQleVLshksvgCFLTHCGwnSsleSLvcgv.PvvafPqwaDQ (shown underlined in Figure 2) . The amino acid sequence of the PsUF5GT protein encoded by the PsUF5GT target fragment is shown in sequence 4 in the sequence listing.

2. Preparation of recombinant vector

Firstly, the bacterial liquid containing TRV2 and TRV1 (gifted by Professor Ma Nan of the Ornamental Plant Postharvest and Stress Physiology Laboratory of China Agricultural University; the non-patent literatures that have recorded TRV1/TRV2 are: Tian J, Pei H X, Zhang S, Chen J W , Chen W, Yang R Y, Meng Y L, You J, Gao J P, Ma N. 2014. TRV-GFP: a modified Tobaccorattle virus vector for efficient and visualizable analysis of gene function. J Exp Bot, 65: 311-322.)) PCR identification was carried out, the identification primers are shown in Table 1, and the amplification system and procedures are shown in Table 4 and Table 5. The amplified lengths were 500bp (TRV1) and 200bp (TRV2) respectively ( FIG. 3 ).

Table 4: PCR reaction system

The PCR amplification program is as follows in Table 5:

Table 5: PCR Amplification Procedure

Figure 3 shows the results of gel electrophoresis detection of bacterial solution containing TRV vector. The size of the amplified fragment of TRV1 is about 500bp, and the size of TRV2 is about 200bp, which confirms that the amplification is correct and can be used for follow-up research.

The upstream and downstream sequences of the cloned PsUF3GT/PsUF5GT contained BamH I and Xho I restriction sites, respectively, and the bacterial solution containing the target fragment and TRV2 vector was used for plasmid extraction, using an endotoxin-free plasmid extraction kit (EndoFreePlasmind Midi Kit, purchased from From Kangwei Century Biotechnology Co., Ltd.), the extraction method was carried out according to the kit instructions. At the same time, the target fragment and TRV2 were double-digested with the above two enzymes (the endonuclease was purchased from NEB). After mixing the enzyme digestion system, place it in a 37°C water bath overnight.

Table 6: Double Enzyme Digestion System

After digestion, the PsUF3GT/PsUF5GT target fragment was ligated with the TRV2 vector using T4 DNA ligase. The ligation system is shown in Table 7.

Table 7: Connection System

After ligation, E.coLi DH5α competent cells (purchased from Kangwei Century Biotechnology Co., Ltd.) were transformed, and positive single colonies were screened with kan-resistant LB medium, and then identified by PCR and plasmid digestion. If the identification is correct, the Escherichia coli liquid containing TRV2-PsUF3GT and TRV2-PsUF5GT will be determined to be sequenced (completed by Beijing Biomed Gene Technology Co., Ltd.) to ensure the correctness of the sequence. The sequencing result was correct, and then transformed into Agrobacterium GV3101 (Agrobacterium competent cells were purchased from Beijing Bomed Gene Technology Co., Ltd.). The transformation method was the conventional freeze-thaw method. The bacterial liquid was sequenced and verified, and the verified Agrobacterium was cultured for the VIGS test.

3. Establishment of VIGS system

The VIGS test was carried out using a two-factor and three-level test system (Table 8). Factor A: flower development period, corresponding to A1 (fully open, S4); A2 (half open, S3); A3 (bud, S2). Factor B: vacuum infiltration time, respectively 10min (B1), 15min (B2) and 20min (B3). TRV2-3GT and TRV2-5GT were the target genes, and the corresponding TRV2 empty vector was the control. The transformation method refers to the literature Tian J, Pei HX, Zhang S, Chen JW, Chen W, Yang RY, Meng YL, You J, Gao JP, Ma N.2014.TRV-GFP: a modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function. J Exp Bot, 65: 311-322., using the vacuum method. The main details include that the OD ₆₀₀ of the resuspended bacteria solution is 1.0, and the OD ₆₀₀ values of the pTRV2-3/5GT, pTRV1 and pTRV2 bacterial solutions are the same. Mix the pTRV2-3/5GT and pTRV1, pTRV2 and pTRV1 bacterial solutions at a volume ratio of 1:1, and let stand in the dark for 4 hours. Peony flowers with 5cm flower stalks are cut off, randomly distributed in different treatments, submerged in the bacterial solution, vacuumed to 0.7atm, treated with negative pressure, and slowly deflated. Each treatment of three flowers was repeated 3 times. Rinse the aspirated petals with deionized water to remove excess bacterial fluid. The flower stalks were soaked in deionized water at 8°C and the humidity was about 60%, and cultured in the dark for 1 day; then transferred to 23-25°C and the humidity was about 60%, and cultivated under normal light for 3 days. Sampling was performed 4 days after infestation, and the spots and non-spots of the petals were separated. Petals were selected as RNA extraction test samples and pigment extraction samples. RNA test samples were quickly frozen in liquid nitrogen and stored at -80°C. Pigment extraction samples were freeze-dried and stored in a brown desiccator. Then detect the expression level of the target gene and the change of anthocyanin accumulation relative to the control. Since anthocyanins do not accumulate in non-spotted parts, only the composition and content of anthocyanins in spotted parts were measured after infection.

Table 8 Experimental treatment of two factors and three levels for VIGS system

4. Gene expression analysis

Gene expression analysis refers to literature (Du H, Wu J, Ji KX, Zeng QY, Bhuiyad MW, Su S, Shu QY, Ren HX, Liu ZA, Wang L S.2015. Methylation mediated by an anthocyanin O-methyltransferase, is involved in purple flower coloration in Paeonia.J ExpBot, 66:6563-77), all samples were extracted from total RNA, and reverse transcribed into cDNA, which was used as a template for fluorescent quantitative PCR. The specific method was carried out according to the instructions of the kit (FastQuant cDNA and SYBR Green, Tiangen Biochemical Technology (Beijing) Co., Ltd.) and references (Du H, Wu J, Ji KX, Zeng QY, Bhuiyad MW, Su S, Shu QY, Ren HX, Liu ZA, Wang L S.2015. Methylation mediated by an anthocyanin O-methyltransferase, is involved in purple flower coloration in Paeonia. J ExpBot, 66: 6563-77), the reaction system and program of fluorescence quantification are shown in Table 9 and Table 10. In this study, the 2- ^△△CT (Livak) method was used to calculate the relative expression of genes in real-time fluorescent quantitative PCR experiments, and PsTubulin (accession no.EF608942) was used as an internal reference gene (Du H, Wu J, Ji KX, Zeng QY, Bhuiyad MW , Su S, Shu QY, Ren HX, Liu ZA, Wang L S. 2015. Methylation mediated by an anthocyanin O-methyltransferase, is involved in purple flower coloration in Paeonia. J Exp Bot, 66: 6563-77). The expression level analysis of petal spots and non-spots was divided into 3 biological and 3 technical replicates.

Table 9 Fluorescent quantitative PCR amplification system

Table 10 Fluorescent quantitative PCR amplification program

Results: Analysis of gene expression after silencing UFGTs in petals

The PsUF3GT and PsUF5GT gene fragments with amplified conserved region fragment lengths of 413bp and 418bp were used to construct TRV2 vectors for silencing the target gene. Through 9 experiments with two factors and three levels, the optimal treatment of UFGT gene silencing was found, and the gene expression after silencing was analyzed (Figure 4 and Figure 5). By analyzing the expression of PsUF3GT and PsUF5GT genes in spots and non-spots of different treatments, it was found that the expression of PsUF3GT gene among the nine treatments decreased the most in the spots of treatment 2, which was lower than that of control treatment 1, treatment 6 and treatment 8 respectively. 65.0%, 45.0%, and 71.0%. The expression level of PsUF5GT decreased the most in the non-plaques of treatment 7, which was 15.9% and 85.0% lower than the control treatment 1 and 8, respectively.

5. Determination of spot anthocyanins

Anthocyanins were extracted from the dried piebald by weighing (Li CH, Du H, Wang LS, Shu QY, Zheng YR, Xu YJ, Zhang JJ, Zhang J, Yang RZ, Ge Y X.2009.Flavonoid composition and antioxidant activity of tree peony (Paeonia Section Moutan) Yellow Flowers. JAgric Food Chem, 57:8496–8503.), that is, weigh 0.1-0.2g of dried petals, use 0.2% methanol solution of formic acid to sonicate for 20min, and place in the dark for 2h at 12,000rpm Centrifuge for 5 minutes to fully extract, and repeat 3 times. Before analysis by HPLC, take 1 mL of the extract and filter it into a sample bottle with a 0.22 μm nylon microporous filter. The analysis system used was Dionex HPLC-DAD system, and the chromatographic column was TSK gel ODS-80Ts QA, 4.6mm (inner diameter)×150mm (column length) (Tosoh Corporation, Japan). The qualitative and quantitative analysis of anthocyanins refer to (Zhang JJ, Wang LS, Shu QY, Liu ZA, Li CH, Zhang J, WeiX L, Tian D K.2007.Comparison of anthocyanins in non-blotches and blotches of the petals of Xibei tree peony. Sci Hortic, 114: 104-111.; Li CH, Du H, Wang LS, Shu QY, Zheng YR, Xu YJ, Zhang JJ, Zhang J, Yang RZ, Ge Y X. 2009. Flavonoid composition and antioxidant activity of tree peony (Paeonia Section Moutan) Yellow Flowers. J Agric Food Chem, 57: 8496–8503.), that is, liquid chromatography analysis conditions: chromatographic column TSKgel ODS-80Ts QA, 4.6mm (inner diameter) × 150mm (length); The column temperature was 35°C, the flow rate was 0.8mL/min, and the injection volume was 10μL. Specific phase A: 10% methanol aqueous solution; phase B: 1% formic acid water; phase C: pure methanol; phase D: formic acid: acetonitrile (0.1:99.9V/V) solution. Linear gradient elution program: 0-50min, 5%-20% of phase B, 95%-80% of phase C; 50-60min, 20%-5% of phase B, 80%-95% of phase C. The flow rate is 0.8mL/min, the column temperature is 35°C, the injection volume is 10μL, and the detection wavelength is 525nm (analysis of anthocyanins). HPLC-ESI-MS ² analysis was performed using an Agilent 1100LC/MSD Trap VL liquid-mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). Mass spectrometry conditions: electrospray ionization (ESI), ion trap analyzer, capillary voltage 3.5kV, nebulizer pressure 241.3kPa, drying temperature 350°C, drying gas (N2) flow rate 6.0mL/min. The results were analyzed with LC/MSD Trap software (V5.2). HPLC-ESI-MS/MS ² analysis was performed using an Agilent 1100LC/MSD Trap VL liquid-mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). The standard product is cyanidin 3-O-glucoside (Cy3G), and the anthocyanin content is calculated by the standard product semi-quantitative method (Wang LS, Shiraishi A, Hashimoto F, Aoki N, Shimizu K, Sakata Y.2001.Analysis of petal anthocyanins to investigate flower coloration of Zhongyuan (Chinese) and Daikon Island (Japanese) tree peony cultivars.J Plant Res, 114:33–43.), that is, use different concentrations including 0.015625mg/ml, 0.03125mg/ml, 0.0625mg/ml ml, 0.125mg/ml, 0.025mg/mL, 0.05mg/mL, 1.0mg/mL standard products, the standard curve formula is: anthocyanin content TA (total anthocyanin) [mAU] = 0.0012 [Cy3G (μg /mL)] + 0.0121 (R ² =0.994). The anthocyanin content is expressed in mg/g dry petals (DW). The results were analyzed with Chameleon software (Ver. 6.60).

Data analysis method:

The sequences in this study were analyzed using DNAMAN5.0. Fluorescent quantitative PCR data were analyzed by software SPSS13.0v (SPSS Inc., Chicago, IL) for significant difference analysis (P<0.05), and Excel 2007 software was used to draw charts.

Results: Determination and Analysis of Anthocyanin Content in Silenced Versicolor

On the basis of analyzing the expression levels in the petal spots and non-spots of different treatments after gene silencing, the components and contents of anthocyanins in the nine treatment spots were determined (Figure 6). The detection results were compared with the fluorescence quantitative results. Basically the same, that is: the total amount of 4 kinds of anthocyanins in treatment 2 decreased by 24.2% compared with control treatment 1, among which Pn3G accounted for 20.0% of the decrease, followed by Cy3G, Pn3G5G, Cy3G5G, which were 2.3%, 1.4% and 0.1% respectively. %; 3G type glycosides decreased by 92.2%, 3G5G type decreased by 7.8%. Total anthocyanins in treatment 7 decreased by 28.2% compared with control treatment 8, among which Pn3G accounted for 12.5% of the largest decrease, followed by Pn3G5G, Cy3G5G, Cy3G decreased by 9.3%, 6.1% and 0.01% respectively; 3G5G type glycosides decreased by 54.9% % while 3G-type glycosides decreased by 45.1%.

<110> Institute of Botany, Chinese Academy of Sciences

<120> VIGS silencing system for identification of flavonoid glycosyltransferase gene in tree peony

<130> P170440/ZWY

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 413

<212>DNA

<213> Artificial Sequence

<220>

<223> PsUF3GT

<400> 1

catggctcaa tgaccaaaag gctgaatccg tcgcgtacat cagctttggc acggctgcga 60

ctccacctcc aaccgagatt ttagctatag cagaagcatt agaagcaagt ggggttgcct 120

ttttatggtc acttaaggac catttaaatg tgcatttgcc aaaagggttt ttagacaaaa 180

caagagcatg tggaatggtt gtgccatggg ctcctcaatt acaaatacta gcacatggtg 240

ctgttggggt gtttataacg cattgtggtt ggaactcggt actagagagt atcggaggtg 300

gagtgcctat gatttgtagg ccattcttcg gcgaccaaaa gttgaacgcg tgtatggtgg 360

aggacgtgtg ggaaattggt gtgaaaatcg acggtggagt atttacgaag aat 413

<210> 2

<211> 136

<212> PRT

<213> Artificial Sequence

<220>

<223> PsUF3GT

<400> 2

Trp Leu Asn Asp Gln Lys Ala Glu Ser Val Ala Tyr Ile Ser Phe Gly

1 5 10 15

Thr Ala Ala Thr Pro Pro Pro Thr Glu Ile Leu Ala Ile Ala Glu Ala

20 25 30

Leu Glu Ala Ser Gly Val Ala Phe Leu Trp Ser Leu Lys Asp His Leu

35 40 45

Asn Val His Leu Pro Lys Gly Phe Leu Asp Lys Thr Arg Ala Cys Gly

50 55 60

Met Val Val Pro Trp Ala Pro Gln Leu Gln Ile Leu Ala His Gly Ala

65 70 75 80

Val Gly Val Phe Ile Thr His Cys Gly Trp Asn Ser Val Leu Glu Ser

85 90 95

Ile Gly Gly Gly Val Pro Met Ile Cys Arg Pro Phe Phe Gly Asp Gln

100 105 110

Lys Leu Asn Ala Cys Met Val Glu Asp Val Trp Glu Ile Gly Val Lys

115 120 125

Ile Asp Gly Gly Val Phe Thr Lys

130 135

<210> 3

<211> 418

<212>DNA

<213> Artificial Sequence

<220>

<223> PsUF5GT

<400> 3

tttggaaaag caaggaatgg tggtgccatg gtgtaatcaa ttagaggttc tgtctcataa 60

gtcagtgggt tgttttctga cacattgtgg gtggaattcg agtttggaga gtttggtttg 120

tggggtgccg gtggtggcgt ttcctcagtg ggcggatcaa gccaccaatg ccaagctgat 180

tgaagatgtg tggaagactg gggtgagaat ggtggtgaat gaagatggtg tggttgaagg 240

ttgtgagatt aagagatgtt tggaaatggt tatgggaggt ggagagagag gggaggaaat 300

gagaaggaat gttgagaaat ggaaggagtt ggcgagggaa gctgtcaagg atggtgagtc 360

ttctgacaaa aatctaaaag cttttgtcaa tgaggtgggg aaaggtggtg atttgtga 418

<210> 4

<211> 138

<212> PRT

<213> Artificial Sequence

<220>

<223> PsUF5GT

<400> 4

Leu Glu Lys Gln Gly Met Val Val Pro Trp Cys Asn Gln Leu Glu Val

1 5 10 15

Leu Ser His Lys Ser Val Gly Cys Phe Leu Thr His Cys Gly Trp Asn

20 25 30

Ser Ser Leu Glu Ser Leu Val Cys Gly Val Pro Val Val Ala Phe Pro

35 40 45

Gln Trp Ala Asp Gln Ala Thr Asn Ala Lys Leu Ile Glu Asp Val Trp

50 55 60

Lys Thr Gly Val Arg Met Val Val Asn Glu Asp Gly Val Val Glu Gly

65 70 75 80

Cys Glu Ile Lys Arg Cys Leu Glu Met Val Met Gly Gly Gly Glu Arg

85 90 95

Gly Glu Glu Met Arg Arg Asn Val Glu Lys Trp Lys Glu Leu Ala Arg

100 105 110

Glu Ala Val Lys Asp Gly Glu Ser Ser Asp Lys Asn Leu Lys Ala Phe

115 120 125

Val Asn Glu Val Gly Lys Gly Gly Asp Leu

130 135

Claims (10)

1. identify the VIGS silencing systems of tree peony flavonoids glycosyltransferase gene, it is characterised in that：Comprising：Carried in VIGS silences The recombinant vector that target gene is obtained is inserted on body；The target gene be sequence table in nucleotide sequence shown in sequence 1 and/or Nucleotide sequence shown in sequence 3 in sequence table.

2. identify the VIGS silencing systems of tree peony flavonoids glycosyltransferase gene, it is characterised in that：The VIGS silent carriers For Tobacco rattle virus TRV2 carriers.

3. the construction method of the VIGS silencing systems of tree peony flavonoids glycosyltransferase gene is identified described in claim 2, including Following steps：

(1) target gene fragment is carried out using the specific primer that with the addition of BamH I restriction enzyme sites and Xho I restriction enzyme sites PCR is expanded, and respectively obtains PsUF3GT targets fragment, the PsUF5GT target fragments for VIGS silences；

The nucleotide sequence of the PsUF3GT targets fragment is as shown in sequence 1 in sequence table；Expand the PsUF3GT targets fragment Specific primer is as follows：

3GT-VF1：AAGGATCCCATGGCTCAATGACCAAAAGGCTAA；

3GT-VR1：TTACTCGAGCATTCTTCGTAAATACTCCACCGTCG；

The nucleotide sequence of the PsUF5GT targets fragment is as shown in sequence 3 in sequence table；Expand the PsUF5GT targets fragment Specific primer is as follows：

5GT-VF1：AAGGATCCTTTGGAAAAGCAAGGAATGGTGGTG；

5GT-VR1：ACGCTCGAGTCACAAATCACCACCTTTCCCCACC；

(2) the PsUF3GT targets fragment or PsUF5GT targets fragment and TRV2 carriers are carried out with BamH I enzymes and Xho I enzymes double PsUF3GT targets fragment or PsUF5GT targets fragment, are attached by digestion after the completion of digestion with Tobacco rattle virus TRV2 carriers, Transformed E .coLi DH5 α competent cells after connection, the positive single bacterium colony of screening, are then transformed into Agrobacterium GV3101, i.e., Obtain the VIGS silencing systems.

4. the VIGS silencing systems of the identification tree peony flavonoids glycosyltransferase gene described in claim 1 or 2 are in identification tree peony Application in flavonoids glycosyltransferase gene PsUF3GT and/or PsUF5GT.

5. a kind of method for identifying tree peony flavonoids glycosyltransferase gene, it is characterised in that：Described in usage right requirement 1 or 2 VIGS silencing systems.

6. method according to claim 5, it is characterised in that：Methods described comprises the following steps：

Using flower of the tree peony with anthocaulus as material, it is immersed in the bacterium solution containing recombinant vector described in claim 1 or 2, vacuum Infiltration, Negative pressure is deflated；

Petal after vacuum infiltration is rinsed with deionized water, unnecessary bacterium solution is removed；

The anthocaulus is cultivated using water planting mode, afterwards the expression of testing goal gene and anthocyanin accumulation Change.

7. method according to claim 5, it is characterised in that：The flower growth period of the flower of the band anthocaulus be standard-sized sheet or Petal.

8. method according to claim 5, it is characterised in that：The duration for vacuumizing infiltration is 10min-15min.

9. method according to claim 5, it is characterised in that：The anthocaulus is used into the condition that water planting mode is cultivated For：Anthocaulus is dipped in deionized water, temperature be 8 DEG C, light culture 1 day under conditions of humidity 60%；Temperature is transferred to afterwards For normal illumination culture under conditions of 23-25 DEG C, humidity 60% 3 days.

10. method according to claim 5, it is characterised in that：It is described to contain recombinant vector described in claim 1 or 2 Bacterium solution is：Contain nucleotide sequence and/or sequence table shown in sequence 1 in the insetion sequence table on Tobacco rattle virus TRV2 carriers The recombinant vector bacterium solution that nucleotide sequence shown in middle sequence 3 is obtained and the bacterium solution containing Tobacco rattle virus TRV1 carriers.