A blackberry RuHCT1 gene and its expression protein and application

Abstract

The present invention discloses a blackberry RuHCT1 gene and its expression protein and application, belonging to the technical field of plant genetic engineering. Based on the blackberry fruit transcriptome data, the present invention clones and identifies a blackberry RuHCT1 gene, whose nucleotide sequence is shown in SEQ ID NO.1, and its expressed protein is shown in SEQ ID NO.2. Metabolome detection and analysis of tobacco-positive plants transgenic for blackberry RuHCT1 gene show that overexpression of the RuHCT1 gene significantly increases the content of anthocyanins in the plants, indicating that RuHCT1 is a key enzyme gene regulating the synthesis of blackberry flavonols, plays an important role in the synthesis of flavonols, and is expected to be used in the cultivation of transgenic plants with high anthocyanin content. Anthocyanins are widely used in dyes, medicines, cosmetics, etc., and are usually extracted from plants, so the present invention has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a blackberry RuHCT1 gene and an expression protein and application thereof. Background Art

[0002] Blackberry (Rubus spp.) is a shrub of the Rubus genus (Rubus) subgenus (Eubatus) of the Rosaceae family (Rasaceae), and belongs to the third generation of small fruit trees. Its fruit is an aggregate berry, rich in a variety of active substances, including vitamins, phenolic substances and a variety of mineral elements, and has a high nutritional value. Blackberry fruit also has medicinal value such as antibacterial, anti-cancer, antioxidant, protection against liver damage and protection of the cardiovascular system. Therefore, it is deeply loved by consumers and is widely used in the fields of food and health care. As an economic fruit tree, blackberry has a very high fruit utilization rate, and the by-products of fruit processing still have high-quality biologically active substances, especially anthocyanins in phenolic substances, which have extremely high economic value and research significance.

[0003] Anthocyanin is a flavonoid substance widely found in plants and is easily soluble in water. There are six common anthocyanins in plants: malva pigment, morning glory pigment, peony pigment, cyanidin, geranium pigment and delphinium pigment. They are widely found in the cell sap tissue of plant petals and fruits and the surface cells of stems and leaves, and most of them are combined with sugar to form glycosides. Due to the variability in the number and position of hydroxyl groups and sugar groups on the ring, there are many and complex types of anthocyanins, which have a variety of significant biological activities. Such as anti-inflammatory, antioxidant, anti-aging, improving pancreatic islet function, inducing tumor cell apoptosis and improving immunity. However, anthocyanins are chemically active and easily inactivated. Studies have found that acylation of the sugar group of anthocyanins with aromatic or aliphatic substituents can make anthocyanins have higher stability, color development and antioxidant activity.

[0004] Acylation is one of the most common modifications of anthocyanins. Depending on the acyl donor, there are two types of anthocyanin acyltransferases. Type I uses acyl coenzyme A (acyl-CoA) as the acyl donor and belongs to the BAHD superfamily enzyme. Type II uses acyl-activated sugars (β-acetal esters or 1-O-β-glucose esters) as the acyl donor and belongs to serine carboxypeptidases (SCPLs). Interestingly, the BAHD acyltransferase family is a class of proteins unique to plants that perform acylation modifications on plant secondary metabolites and participate in plant secondary metabolic reactions, among which hydroxycinnamoyltransferase (HCT) is an important branch of the BAHD superfamily. Some researchers have divided BAHD into five branches through phylogenetic analysis, three of which contain HCT. HCT has the dual activity of shikimate hydroxycinnamoyltransferase and quinate hydroxycinnamoyltransferase, has substrate universality, and can acylate a variety of substrates, including shikimate, quinate, 4-hydroxyphenyllactic acid, etc. In addition, HCT is one of the key enzymes in the phenylalanine metabolic pathway, and has been studied in a variety of plants such as Arabidopsis, Nicotiana, Lycopersicon, Populus, and Plagiochasma. It was found that HCT is related to the biosynthesis of plant anthocyanins, lignin, phenolic acids, and other substances.

[0005] Currently, research on the function of HCT mainly focuses on its effect on lignin biosynthesis, and there are few reports related to anthocyanin synthesis. It has not yet been studied in blackberry. Summary of the invention

[0006] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a blackberry RuHCT1 gene. Another technical problem to be solved by the present invention is to provide an expression protein of the aforementioned gene. Finally, another technical problem to be solved by the present invention is to provide a specific application of the blackberry RuHCT1 gene.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A blackberry RuHCT1 gene, whose nucleotide sequence is shown as SEQ ID NO.1.

[0009] The amino acid sequence of the expressed protein of the blackberry RuHCT1 gene is shown in SEQ ID NO.2.

[0010] An expression box, vector, host bacteria or cell line containing the blackberry RuHCT1 gene.

[0011] Furthermore, the vector is a plant expression vector.

[0012] Furthermore, the plant expression vector is pBI121-3HA-RuHCT1.

[0013] The blackberry RuHCT1 gene is used to increase the anthocyanin content in plants.

[0014] Furthermore, the application of the blackberry RuHCT1 gene in increasing the anthocyanin content in plants comprises the following steps:

[0015] 1) Construction of the vector of blackberry RuHCT1 gene;

[0016] 2) transforming the constructed blackberry RuHCT1 gene vector into plants or plant cells;

[0017] 3) Cultivate and screen transgenic plants with increased anthocyanin content.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Based on the reference transcript set of transcriptome data of immature and mature blackberry fruits obtained by the research group in the early stage, the applicant cloned, identified, analyzed and clarified the physicochemical properties, structural characteristics and potential spatiotemporal expression patterns of the blackberry RuHCT1 gene. Metabolome detection and analysis of tobacco positive plants with RuHCT1 gene in blackberry showed that overexpression of RuHCT1 gene inhibited the synthesis of most flavonoids, and the synthesis of chlorogenic acid and lignin was also inhibited to varying degrees, but significantly increased the content of anthocyanins in the plants, indicating that RuHCT1 is a key enzyme gene regulating the synthesis of blackberry flavonols, and plays an important role in the synthesis of flavonols, and can be used to cultivate transgenic plants with high anthocyanin content. Anthocyanins have a wide range of applications in dyes, medicines, cosmetics, etc., and are usually extracted from plants. The present invention can be used to cultivate transgenic plants with high anthocyanin content, so the present invention has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Protein structure analysis diagram of RuHCT1: (a) secondary structure diagram of RuHCT1; (b) tertiary structure model diagram of RuHCT1;

[0021] Figure 2 Phylogenetic tree of BAHD superfamily genes of blackberry RuHCT1 and other species: malonyltransferases are clustered on the first main branch, alcohol acyltransferases are clustered on the second main branch, and hydroxycinnamoyltransferases are clustered on the third main branch;

[0022] Figure 3Figure 1 shows the construction of overexpression vector of RuHCT1 in tobacco and analysis of heterologous overexpression: (a) Schematic diagram of pBI121-3HA-RuHCT1 overexpression vector; (b) Diagram of transgenic tobacco growth process; c, d, e, f: Expression patterns of flavonoid biosynthesis pathway genes in control WT and RuHCT1 overexpressing tobacco; (c) RuHCT1; (d) NtCCOMT: caffeoyl-CoA3-O-methyltransferase; (e) NtCHS: chalcone synthase; (f) NtCYP98A: cytochrome P98A;

[0023] Figure 4 Digital gene expression profile of RuHCT1 in blackberry and relative expression analysis of RuHCT1; (a) Digital gene expression profile of RuHCT1 in immature and mature blackberry fruits based on transcriptome data; (b) Relative expression pattern of RuHCT1 gene in different tissues of blackberry; (c) Different developmental stages of fruit; Different letters indicate significant differences (p < 0.05);

[0024] Figure 5 Analysis of differentially expressed metabolites (DEMs) between tobacco control group and RuHCT1 transgenic group; heat map of correlation analysis between control, transgenic tobacco and QC samples in positive ion (a) and negative ion (b) modes;

[0025] Figure 6 Heat maps of the major differentially expressed metabolites related to phenylalanine biosynthesis in the positive (a) and negative (b) ion modes of control WT and RuHCT1-OX tobacco. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific examples. The test methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0027] Example 1: RuHCT1 gene cloning

[0028] 1. Materials and methods

[0029] The experimental material is the blackberry (Rubus spp.) 'Chester' variety, which comes from the Baima Experimental Base in Lishui District, Nanjing (119°11′E, 31°36′N). The area belongs to the subtropical monsoon climate zone, with an average annual temperature of 15.6°C, an annual rainfall of 1031.9 mm, an average sunshine of 50%, and acidic clay soil with an organic matter content of 18.67 g / kg. The roots, stems, leaves, flowers, fruiting branches and fruits (green fruits) of blackberries were picked as plant materials for expression analysis in different tissue parts. In addition, the sampling period of the fruit development stage was distinguished according to the color of the fruit (a total of 5 fruit development periods), and green fruits, green-red fruits, red fruits, red-purple fruits and purple fruits were picked respectively. All experiments were biologically repeated at least 3 times, and the collection time was June 2021, after which they were stored in liquid nitrogen and stored in a -80°C refrigerator for standby use after being brought back to the laboratory.

[0030] Total RNA from all blackberry samples was extracted using a universal plant total RNA rapid extraction kit (BioTeKe, Beijing, China) according to the instructions. Then, the total RNA from different tissues and fruit development stages of blackberry was reverse transcribed and synthesized into cDNA using the PrimeScript RT Master Mix (Perfect RealTime) kit (TaKaRa, Dalian, China).

[0031] 2. RuHCT1 gene cloning

[0032] Based on the differentially expressed RuHCT1 gene sequence screened in the blackberry transcriptome data (SRA database accession number: PRJNA701162), the open reading frame (ORF) and three-generation full-length transcript sets were obtained by BioXM2.6 software analysis for correction, and the primers were designed using Oligo 6.0 software. The primer sequences are as follows:

[0033] RuHCT1-F: 5′-ATGGCGCTTGAAGTGGAAGT-3′,

[0034] RuHCT1-R: 5′-TACTAGGTCAATTCGCGTGTT-3′.

[0035] The ORF sequence was cloned using high-fidelity PCR enzyme PrimeSTAR Max DNA Polymerase (TaKaRa, Dalian, China). The amplified product was connected to the vector according to the requirements of the pClone007 Blunt Vector Kit (TSINGKE, Nanjing, China), transferred into the competent E. coli, coated on a plate, cultured at 37°C 250rpm for 6-8h, and then potential positive colonies were selected for expansion culture. 2×T5 Super PCR Mix (Colony) (TSINGKE, Nanjing, China) was used for bacterial liquid PCR verification, and the positive bacterial liquid was sent to Qingke Biotechnology Co., Ltd. for first-generation Sanger sequencing verification. The results showed that the ORF length of the RuHCT1 gene was 1344bp (SEQ ID NO.1), encoding a protein consisting of 448 amino acids (SEQ ID NO.2).

[0036] Example 2: Analysis of RuHCT1 protein structure and physicochemical properties

[0037] DNAMAN software was used to analyze the protein sequence encoded by the RuHCT1 gene. ExPASy software (https: / / web.expasy.org) was used to predict its physical and chemical properties and protein hydrophilicity. The SOPMA online tool was used to analyze its protein secondary structure; SWISS-MODEL (https: / / swissmodel.expasy.org / ) was used to build a three-dimensional model for verification. In addition, MEGAX was used to compare the RuHCT1 protein sequence (similarity) and the phylogenetic tree was constructed by bootstrapping 1000 times using the neighbor-joining method. The details are as follows:

[0038] 1. The physicochemical properties of HCTs proteins in blackberry were analyzed using ProtParam software, and it was found that the molecular mass of RuHCT1 was 50.26 kDa, its isoelectric point was less than 7, it was an acidic protein, and its instability coefficient was greater than 40, which was a hydrophobic protein of unstable protein (Table 1). The hydrophilicity of blackberry RuHCT1 protein was predicted by ProtScale online software, indicating that it was a hydrophobic protein.

[0039] Table 1 Analysis of the physicochemical properties of RuHCT1 protein

[0040]

[0041] 2. The secondary structure of RuHCT1 protein was predicted by SOPMA online tool. The results showed that the secondary structure of RuHCT1 protein was composed of α-helix, extended chain, β-turn and random coil, among which α-helix and random coil were the main ones. SWISS-MODEL homology modeling was used to analyze the blackberry RuHCT1 protein. Its protein crystal structure model came from the BAHD family ( Figure 1 ).

[0042] 3. Analysis of phosphorylation sites and glycosylation of blackberry RuHCT1 protein

[0043] The NetPhos online tool (https: / / services.healthtech.dtu.dk / service.php?NetPhos-3.1) was used to predict the sites where the blackberry RuHCT1 protein may be phosphorylated. When the phosphorylation potential is greater than the threshold of 0.5, it indicates that the site can be phosphorylated. There are three types of phosphorylation sites: serine phosphorylation sites, threonine phosphorylation sites, and tyrosine phosphorylation sites. The phosphorylation sites of RuHCT1 are mainly serine phosphorylation sites, accounting for 46.7%. Tyrosine phosphorylation sites account for the least, at 13.3%. In summary, the blackberry RuHCT1 protein is mainly serine phosphorylated.

[0044] Table 2 Prediction of protein phosphorylation and glycosylation sites of RuHCT1

[0045]

[0046] 4. The glycosylation sites of blackberry HCT1 protein were predicted using the NetNGlyc-1.0 online tool (http: / / www.cbs.dtu.dk / services / NetNGlyc / ). When the N-glycosylation potential was greater than the threshold of 0.5, it indicated that the site could be N-glycosylated. The prediction results showed that RuHCT1 had N-glycosylation sites at the 56th, 212th, and 430th amino acids.

[0047] 5. Phylogenetic analysis of RuHCT1 and other plants

[0048] The phylogenetic tree was constructed using MEGA.7 using the NJ method. The results showed that RuHCT1 and malonyltransferases from Salvia splendens, Nicotiana tabacum, Glycine max, and Arabidopsis thaliana were clustered into one branch, alcoholacyl-transferases (AAT) were clustered into the second branch, and hydroxycinnamoyl CoAquinatetransferase (CQT) was clustered into the third branch, which was consistent with the basic characteristics of biological classification and the evolutionary branch of the BAHD family ( Figure 2 ).

[0049] Example 3: Construction of expression vector, infection of transgenic tobacco and screening of positive plants

[0050] The overexpression vector was constructed using GATEWAY technology, that is, the target fragment in the entry vector (linker-7) was transferred to the overexpression vector pBI121-des-3HA of the 35S strong promoter through LR reaction. The specific steps refer to the previous article of our laboratory [Wu Y, Wang T, Xin Y, et al., Overexpression of the GbF3′H1 gene enhanced the epigallocatechin, gallocatechin, and catechin contents in transgenic Populus, Journal of Agricultural and Food Chemistry, 2020, 68, 998-1006.DOI: 10.1021 / acs.jatc.9b07008].

[0051] The overexpression vector bacterial solution containing the target fragment was sequenced and verified. After obtaining the positive clone, it was expanded with LB antibiotics containing kana for plasmid extraction. Take 150ng of the plasmid to be transformed and add it to 100μL Agrobacterium GV3101 competent cells for transformation, spread it on a medium with dual resistance to kana and rifampicin (rif), culture it at 28℃ for 2 days, randomly select single colonies, verify the bacterial solution by PCR, and store it at 4℃ for standby use. The successfully verified bacterial solution continues to expand and culture to 50mL, and the OD value of the bacterial solution is measured, preferably 0.5-0.8. Transfer the bacterial solution to a 50mL centrifuge tube, centrifuge it at 5000rpm for 10min, discard the supernatant, resuspend the bacterial solution with 40mL MS0 (4.43g / LMS+100μmol / LAS, pH5.8), and add AS to a concentration of 20mM. The specific infection of tobacco is referred to the study of Yan et al. (2021) [Yah Z, Yang H, Zhang C, et al. Functional analysis of the blackberry sucrose transporter gene RuSUT2, Russian Journal of Plant Physiology, 2021, 68, 246-253. DOI: 10.1134 / S1021443721020217]. After the transgenic tobacco grows, the leaves are taken to extract RNA, and PCR cloning is performed using the ORF cloning front primer and the overexpression vector with 3HA rear primer to screen and verify the positive transgenic plants. The primer sequences are as follows:

[0052] RuHCT1-F: 5′-ATGGCGCTTGAAGTGGAAGT-3′,

[0053] 19w-ccdB-3HA-R: 5′-TTAAAGACTAGCATAATCTGGAAC-3′.

[0054] The constructed pBI121-3HA-RuHCT1 overexpression vector and transgenic positive plants ( Figure 3 .ab).

[0055] Example 4: Fluorescence quantitative PCR analysis of RuHCT1 expression in blackberry and transgenic tobacco

[0056] GenScript Real-time PCR Primer Design-Real Time PCR Probe Design-GenScript was used to design quantitative primers (https: / / www.genscript.com / tools / real-time-pcr-taqman-primer-design-tool). The primer sequences are as follows:

[0057] RuHCT1-qRTF: 5′-CCCTAGCAGGACGAGCCCAT-3′,

[0058] RuHCT1-qRTR: 5′-GGCGTTGAGCTGGACACCAA-3′.

[0059] TB Green Premix Taq II (Tli RNASEH Plus) (Takara, Dalian, China) was used for qRT-PCR reaction. The specific operation was referred to Wu et al. (2022) [Wu Y, Zhang C, Huang Z, et al. Integrative analysis of the metabolome and transcriptome provides insights into the mechanisms of flavonoid biosynthesis in blackberry, 2022, 153, 110948. DOI: 10.1016 / J.FOODRES.2022.110948]. Ru18S was used as the internal reference gene for blackberry, and NtActin was used as the internal reference gene for transgenic tobacco seedlings for real-time fluorescence quantitative PCR. The sequences of the internal reference genes are as follows:

[0060] Ru18S-qRTF: 5′-ACGTCATCCTCCGGCAAAGC-3′,

[0061] Ru18S-qRTR: 5′-ACGACGAAGCTCGCAAGTACAC-3′.

[0062] NtActin-qRTF: 5′-CAAGGAAATCACCGCTTTGG-3′,

[0063] NtActin-qRTR: 5′-AAGGGATGCGAGGATGGA-3′.

[0064] Finally, 2 -ΔΔCTQuantitative data analysis was performed using the SPSS 24.0 statistical software program (SPSS, Chicago, IL, USA). One-way ANOVA was used to compare the mean values, and Duncan's post hoc multiple comparisons were used to calculate the significance of RuHCT1 expression levels in different tissues and at different stages of blackberry fruit (P < 0.05).

[0065] 1. Analysis of heterologous overexpression of RuHCT1 in tobacco

[0066] Three relatively vigorous transgenic lines were selected for expression level detection, and it was found that RuHCT1 was highly expressed in all transgenic plants ( Figure 3 .c), the transgenic lines RuHCT-1, RuHCT-2, and RuHCT-3 were upregulated by 184, 123, and 2606 times, respectively, compared with non-transgenic tobacco. In addition, to explore the effect of RuHCT1 heterologous expression on key enzyme genes in the tobacco flavonoid biosynthesis pathway, we detected the expression levels of tobacco HCT downstream genes caffeoyl CoA 3-O-methyltransferase (CCOMT), chalcone synthase (CHS), and cytochrome P98A (CYP98A). The results showed that CCOMT ( Figure 3 .d)、CHS( Figure 3 .e) and CYP98A( Figure 3 The expression levels of CCOMT in the three transgenic lines (RuHCT-1, RuHCT-2, and RuHCT-3) were significantly lower than those in wild-type tobacco, respectively. The expression levels of NtCHS were downregulated by 19.6, 12.9, and 29.4 times, respectively. The expression patterns of NtCYP98A were downregulated by 5.1, 5.3, and 9.3 times, respectively.

[0067] 2. Digital gene expression profile of RuHCT1 in blackberry and relative expression analysis of RuHCT1

[0068] Based on the previous transcriptome data of blackberry fruit, the FPKM values ​​of RuHCT1 in immature and mature blackberry fruits were analyzed by digital gene expression profiling ( Figure 4 .a), the results showed that RuHCT1 was expressed at a higher level than in immature fruit. RT-qPCR experiments were used to analyze the expression levels of RuHCT1 in roots, stems, leaves, flowers, fruiting branches and fruits of blackberry at different developmental stages. The results showed that the expression level of RuHCT1 in roots and fruiting branches was higher, significantly higher than that in other tissues, and the expression level in stems and immature fruit was significantly lower than that in other tissues ( Figure 4 .b). However, as the fruit develops, it is found that the RuHCT1 gene shows significant differences in the fruit development process. The expression level increases in the early stage of fruit development, then gradually decreases, and increases sharply to reach a peak during the fruit ripening period. The expression level of RuHCT1 gene in purple fruit (fully ripe fruit) is significantly higher than that in other periods ( Figure 4 .c). The relative expression trend of RuHCT1 in red and purple fruits is consistent with the results of previous transcriptome data, indicating that the results of this experiment are reliable.

[0069] Example 5: Metabolome analysis in transgenic tobacco

[0070] 1. LC-MS / MS analysis method

[0071] Leaves of three non-transgenic tobacco strains and three transgenic tobacco strains were selected and added with pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v), respectively. Low-temperature ultrasonic extraction was performed for 30 min, standing at -20°C for 10 min, centrifuged at 14000g and 4°C for 20 min, the supernatant was vacuum dried, and for mass spectrometry analysis, 100 μL of acetonitrile aqueous solution (acetonitrile: water = 1:1, v / v) was added and vortexed for re-dissolution, centrifuged at 14000g and 4°C for 15 min, and the supernatant was sampled and analyzed.

[0072] The samples were separated using an Agilent 1290 Infinity LC ultra high performance liquid chromatography system (UHPLC) HILIC column; column temperature 25°C; flow rate 0.5 mL / min; injection volume 2 μL; mobile phase composition A: water + 25 mM ammonium acetate + 25 mM ammonia, B: acetonitrile; the gradient elution program was as follows: 0--0.5 min, 95% B; 0.5-7 min, B linearly changed from 95% to 65%; 7-8 min, B linearly changed from 65% to 40%; 8-9 min, B was maintained at 40%; 9-9.1 min, B linearly changed from 40% to 95%; 9.1-12 min, B was maintained at 95%; the sample was placed in an automatic sampler at 4°C during the entire analysis process.

[0073] 2. Metabolite Statistical Analysis

[0074] In order to achieve higher metabolite coverage and better detection effect, two modes were used for metabolite detection: positive ion mode (POS) and negative ion mode (NEG). In the subsequent data analysis process, the positive and negative ion models were analyzed separately. Regarding the metabolome analysis of transgenic tobacco, unsupervised dimensionality reduction principal component analysis (PCA) was applied to all samples. PCA is a statistical program that can preliminarily visualize the differences between samples from different groups. It was performed by R language gmodels (v2.18.1). Partial least squares discriminant analysis (PLS-DA) is a supervised dimensionality reduction method that better distinguishes the metabolomic characteristics of the two groups by screening variables related to class membership. [ E, Saccenti E, Smilde AK, et al. Double-check: validation of diagnostic statistics for PLS-DA models in metabolomics studies. Metabolomics, 2012, 8, S3-S16. DOI: 10.1007 / s11306-011-0330-3], applied to the control group using the R package ropls (http: / / www.r-project.org / ). Orthogonal projection to latent structure discriminant analysis (OPLS-DA) [Wu Y, Wang T, Xin Y, et al. Overexpression of theGbF3′H1 gene enhanced the epigallocatechin, gallocatechin, and catechin contents in transgenic Populus, Journal of Agricultural and Food Chemistry, 2020, 68, 998-1006.DOI: 10.1021 / acs.jafc.9b07008] is an extension of PLS-DA that incorporates the orthogonal signal correction (OSC) filter into the PLS model. The OPLS-DA model was further validated by cross-validation and permutation test. For cross-validation, the data were divided into seven subsets, and each subset was then used as a validation set. R2 represents the total variation in the data matrix explained by the model. The predictive ability (Q 2 ) value to verify the OPLS-DA model in metabolomics. 2 When the value is greater than 0.4, it is considered to be an acceptable prediction model. 2 When the value is greater than 0.9, it is considered to be a good prediction model.

[0075] 3. Analysis of differentially abundant metabolites

[0076] The projection score variable importance (VIP) of the (O)PLS model was applied to rank the metabolites that best distinguished the two groups. In addition, the T test was also used as a univariate analysis to screen differential metabolites. T-tests with a P value <0.05 and VIP≥1 were considered to be differential metabolites between the two groups. The abundance of differential metabolites in the same group was normalized using z-score. The VIP scores of OPLS-DA were then used to plot [Yoon D, Kim YJ, Lee WK, et al. Metabolic changes in serum metabolome of beagle dogs fed black ginseng. Metabolites, 2020, 10(12): 517. DOI: 10.3390 / METAB010120517]. The Pearson correlation coefficient and p-value were calculated using the R function. The closer the correlation coefficient is to 1, the higher the similarity of metabolic abundance. The correlation was significant when p≤0.05 [Rao G, Sui J, Zhang J. Metabolomics reveals significant variations in metabolites and correlations regarding the maturation of walnuts (Juglans regia L.). Biology Open, 2016, 5(6): 829-826. DOI: 10.1242 / bio.017863]. The abundance of differential metabolites was normalized by z-score and hierarchical clustering was performed using the R package pheatmap to show the cumulative differences between the two groups. Z-score (standard score) is based on the relative content of metabolites and is used to measure the relative content of metabolites at the same level. The calculation formula is as follows:

[0077] z=(x-μ) / σ.

[0078] where x is the abundance of the metabolite, μ is the mean abundance of the metabolite in each sample, and σ is the standard deviation [Chen X, Xie CS, Sun LX, et al. Longitudinal Metabolomics Profiling of Parkinson′sDisease-Related alpha-Synuclein A53T Transgenic Mice. PLOS ONE. 2015, 10(8): e0136612. DOI: 10.1371 / journfl.pone.0136612].

[0079] 4. Experimental results

[0080] Overexpression of RuHCT1 caused significant changes in the transcriptional level of transgenic tobacco, so the metabolic levels of transgenic and non-transgenic tobacco leaves were investigated. Through non-targeted LC-MS determination, it was found that a total of 14,384 metabolites were detected in positive ion mode, of which 797 metabolites could be qualitatively identified; a total of 11,442 metabolites were detected in negative ion mode, of which 503 could be qualitatively identified. By calculating the correlation between wild-type, transgenic tobacco and QC samples, the results presented the degree of variation in metabolite composition and abundance between samples in the form of a heat map ( Figure 5 .a). In both positive and negative ion modes, the correlation within the three transgenic tobacco lines was high ( Figure 5 .a, b), indicating that the experimental results are more reliable.

[0081] In order to accurately and fully find the differential metabolites in the leaves of transgenic plants and non-transgenic plants, PLS-DA model and OPLS-DA model were established. After statistical analysis of the data, it was confirmed that the parameters of PLS-DA model and OPLS-DA model were reasonable and stable, and could be used for metabolomics analysis. In addition, the OPLS-DA model was verified by permutation, and it was found that the OPLS-DA model had good reliability. Therefore, the OPLS-DA results were selected for the subsequent differential metabolite screening analysis.

[0082] 5. Biosynthesis of phenylalanine in tobacco

[0083] To investigate the effect of overexpression of RuHCT1 gene on related metabolites in phenylalanine metabolic pathway, the differential metabolites of phenylalanine and downstream pathways in tobacco of transgenic HCT group and WT group were analyzed. Figure 6 The results showed that in the positive ion mode, three types of differential metabolites were detected in transgenic tobacco, including flavonoids, anthocyanins and lignins ( Figure 6.a). Among them, the contents of flavonoid compounds nobiletin, phlorizin, luteolin 7-glucoside, apigenin 7-glucoside, and neohesperidin were lower than those of non-transgenic plants, while the contents of kaempferol 3-O-rutinoside and naringenin-7-O-glucoside were significantly upregulated. The content of anthocyanidin (Cyanidin) increased in transgenic plants, while the content of lignin derivatives (Lignin) decreased. In the negative ion mode, two types of differential metabolites, flavonoids and organic oxides, were detected ( Figure 6 .b), among which the contents of flavonoid compounds catechingallate, (+)-catechin, ligustroflavone, and quercetin3-glucoside were down-regulated in transgenic plants. It was also found that the content of quercitrin was down-regulated in RuHCT1-1 and RuHCT1-3 lines, but significantly up-regulated in RuHCT1-2; the content of astragalin was up-regulated in RuHCT1-1 and RuHCT1-2 lines, but significantly down-regulated in RuHCT1-3 lines. The content of organic oxide chlorogenic acid was slightly up-regulated in RuHCT1-1 lines, but significantly down-regulated in RuHCT1-2 and RuHCT1-3 lines. Overall, the content of most flavonoids detected in transgenic tobacco decreased, the content of lignin and chlorogenic acid also decreased, and the content of anthocyanins increased. This shows that the overexpression of the RuHCT1 gene inhibited the synthesis of most flavonoids, and the synthesis of chlorogenic acid and lignin was also inhibited to varying degrees, but significantly increased the content of anthocyanins in the plants.

Claims

1. A blackberry RuHCT1 gene, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expression protein of the blackberry RuHCT1 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

3. An expression cassette, vector or host bacteria containing the blackberry RuHCT1 gene according to claim 1; the vector is a plant expression vector; the plant expression vector is pBI121-3HA-RuHCT1.

4. Use of the blackberry RuHCT1 gene according to claim 1 in increasing the anthocyanin content in plants; the plant is tobacco.

5. The use of the blackberry RuHCT1 gene in increasing anthocyanin content in plants according to claim 4, characterized in that: The following steps are involved: 1) Construction of the blackberry RuHCT1 gene vector; 2) Transform the constructed blackberry RuHCT1 gene vector into tobacco; 3) Cultivate and screen transgenic tobacco with increased anthocyanin content.

Images