Molecular Cloning, Bioinformatics Analysis and Overexpression of the Tyrosine Aminotransferase Gene in Rehmannia Glutinosa

Rehmannia glutinosa is an important medicinal plant producing many bioactive compounds such as catalpol, acteoside and so on. Tyrosine aminotransferase (TAT) is the rst key enzyme that catalyzes the reversible interconversion of tyrosine and 4-hydroxyphenylpyruvate in the tyrosine-derived branch pathway of acteoside biosynthesis. To conrm its role for acteoside accumulation, we isolated a full-length cDNA from Rehmannia glutinosa Libosch. Sequence analysis indicated that it contained a 1266 bp open reading frame, encoding a TAT of 421 amino acid residues. Multiple sequence alignment revealed that the homology of RgTAT amino acid sequence to that of Sesamum indicum(XP_011100354.1) was the highest (89.94%). Evolutionary tree showed that Sesamum indicum TAT and RgTAT were grouped together. Quantitative real-time PCR analysis indicated that the expression of RgTAT in leaves was much higher than in roots and stems,and that the expression levels of RgTAT in the tuberous roots, stems and leaves of high-acteoside cultivar BJ-3 were higher than in that of low-acteoside cultivar Wen85-5. A plant expression vector was constructed containing the RgTAT and hygromycin resistance gene (Hyg). Transgenic Rehmannia glutinosa Libosch overexpressing RgTAT was obtained via an Agrobacterium tumefaciens-mediated transformation system, in which Hyg expression was conrmed by PCR. RgTAT expression in transgenic plantlets measured by real-time quantitative PCR was 7.72 ± 0.17 times greater than its expression in the untransformed plantlets. Moreover, HPLC analysis indicated that enhanced RgTAT expression corresponded to signicantly increased acteoside for transgenic plantlets. Our results elucidate the role of RgTAT in the acteoside biosynthesis in Rehmannia glutinosa.


Introduction
Rehmannia glutinosa Libosch is a valuable medicinal and industrial crop from the Scrophulariaceae family, and widely used in Traditional Chinese medicine, pickled vegetables, beverages, liquors, and horticulture. In traditional medicine, it is prescribed to treat menopause, impotence, alopecia, and other hormone de ciencies. It is endemic to China, Japan, and Korea . In China, it is distributed in Henan, Shandong, Shanxi, Shan'xi and other provinces, but better Rehmanniae Radix is from Jiaozuo, Henan, P.R.China. called Huaidihuan, which has higher bioactive components such as catalpol and acteoside, and lower clinical dosage than that from other places.
Acteoside is a phenylethanoid glycoside, and possesses some bioactivities such as anti-in ammatory, anti-bacterial, antioxidant, anti-fungal, anti-tumor, photo-protective, liver-protective, neuro-protective properties (Alipieva et al., 2014;Zhou et al., 2016;Li et al., 2018). It was rst isolated from mullein, also found in other plant species and produced by in vitro plant culture systems (Alipieva et al., 2014). Now, commercially available verbascoside is isolated and puri ed from its native plants, for example, Herha Cistanches, but there is lack of its resource in that there are the following: (1) world-wide demand for its native plants as a herbal supplement has caused them to be over-exploited. (2) urbanization reduces the land for growing them. Therefore, it urgently requires modern biotechnologies such as gene engineering and metabolic engineering for its biosynthesis like other bioactive natural products (O'Connor 2015).
What is more, these biotechnologies need a better understanding of acteoside biosynthesis pathway.
Acteoside is synthesized via a phenylpropanoid branch pathway and a tyrosine-derived branch pathway.
Tyrosine aminotransferase (TAT) is the rst key enzyme that catalyzes the reversible interconversion of tyrosine and 4-hydroxyphenylpyruvate in the tyrosine-derived branch pathway of acteoside biosynthesis (Zhou et al., 2020). Plant TAT genes have been proposed to be important in response to abiotic stress, including drought and osmotic stresses (Wang et al., 2018) and oxidative stress (Ipson et al., 2019), and to take part in rosmarinic acid synthesis (Lu et al., 2013). In addition, the cloning of TAT genes has been reported from some plant species in NCBI. However, there are few reports on RgTAT cloning and its function in acteoside biosynthesis of Rehmannia glutinosa.
Thus, to validate the role of RgTAT for acteoside biosynthesis, we isolated it from Rehmannia glutinosa, and characterized the phylogenetic relationships among TATs from plants, examined its expression pro les in different tissues, constructed its overexpression vector transformed it into Rehmannia glutinosa via an Agrobacterium-mediated transformation system, and discovered the signi cant increase of acteoside content in transgenic Rehmannia glutinosa by its overexpression. Our studies will lay the foundation of understanding of the role of RgTAT for acteoside biosynthesis.

Plant material
The cultivars of Rehmannia glutinosa libosch, such as low verbascoside cultivar and high verbascoside cultivar (Chai et al., 2013), grown in cultivated land in Wen County, Henan, China. Their roots, stems and leaves were used as the experimental materials. The young leaves from plant tissue culture-derived cultivar Wen 85-5 plantlets were used for RgTAT genetic transformation, which were cultured in autoclaved Murashige and Skoog (MS) basal medium.

RgTAT cloning
The full-length cDNA was cloned in three successive steps. First, RNA extraction and RT-PCR were carried out as stated by Zhou et al.(2016). Second, based on the cDNA fragment of RgTAT cloned by homology cloning method mentioned above, together with Rehmannia glutinosa SRA database in NCBI, Full-length cDNA was cloned by electronic cloning via the searching by tBLASTn and assembling with DNAMAN software of ESTs. Third, according to its coding sequence, a pair of primers were designed (Table 1). Its coding sequence (RgTAT) was ampli ed by RT-PCR (Zhou et al., 2016). The PCR product was cloned into the pMD19-Tvector (TAKARA, Japan). The pMD19-Tvectors with RgTAT were transformed into Ecoli strain DH5α, followed by PRC test and sequencing. The PCR products were fractionated on agarose gel and puri ed with OMEGA Gel Extraction Kit (Thermo Fisher Scienti c, Japan).

Construction of plant expression vector
The vector pCAMBIA1300-35S, containing the nptII (neomycin phosphotransferase) gene coding for kanamycin resistance and Hyg (R) as a plant-selectable markers, was chosen for this experiment. A fragment containing the CDS was ampli ed with the primer pair RgTAT-F with the Xbal site and RgTAT-R with the KpnI site (Table 1) under the following PCR conditions: 1 cycle of 94℃ for 5 min, 35 cycles of 94℃ for 30s, 55℃ for 30s and 72℃ for 2 min, and nally 1 cycle at 72℃ for 10 min. Then the ampli ed fragment was subcloned into the pCAMBIA1300-35S. Recombinant plasmids (pCAMBIA1300-RgTAT) were identi ed by restriction analysis of puri ed plasmid DNA, were sequenced, and then mobilized into the Agrobacterium tumefaciens strain GV3101 using the freeze-thaw method and maintained in yeast extract broth (YEB) medium, supplemented with 50 mg/L rifampicin (rif) and 50 mg/L kanamycin (kan), followed by PCR identi cation. transformed. For transformation, the young explants from 10-day-old Rehmannia glutinosa plantlets were cut into about 0.5 cm pieces and then precultured, immersed in the Agrobacterium suspension, removed, blotted, and co-cultured on a solid MS medium supplemented with 0.1mg/L NAA, 2.0mg/L6-BA and 100μmol/L AS in dark at 25 C for 2 days. After co-cultivation, the explants were washed once with sterile water and transferred to a solid callus-inducing and shoot-differentiating medium (MS basal medium supplemented with 0.1mg/L NAA, 2.0mg/L6-BA, 100μmol/LAS, Hyg (10mg/L) and cefotaxime (100μmol/L)], and cultured for 3-4 weeks under a 16h photoperiod at 25℃ to induce calli or shoot formation. Three weeks, the shoots were transferred onto a root-inducing medium containing 1 mg/L NAA and10mg/L Hyg for rooting. The regenerated plantlets with well-developed shoots and roots (To) were nally transplanted into pots and then transferred to the greenhouse.

Hyg analysis of transgenic plantlets
Total genomic DNA was extracted from leaves of Rehmannia glutinosa T 0 and non-transgenic Rehmannia glutinosa plantlets. The ampli cation reaction was carried out with a pair of primers (Table1) as stated by Zhou et al (2017). The ampli ed products were separated by electrophoresis on a 0.8 % agarose gel and analyzed to rapidly screen putative to on selective media.

Gene expression analysis by real-time quantitative PCR
Total RNA isolated from T 0 transgenic Rehmannia glutinosa lines was treated with RNase-free DNase, whose quality and integrity were con rmed with agarose gel electrophoresis. Real-time quantitative PCR was performed with a pair of primers (Table1) as stated by Zhou et al (2016) using ddH 2 O as negative controls lacking the cDNA template and RgTIP41 as an internal control to normalize expression (Table1), and gene expression was quanti ed using the 2 -ΔΔCt method.

Measurement of acteoside content in transgenic Rehmannia glutinosa
Acteoside was extracted from T 0 transgenic Rehmannia glutinosa lines and wild type Rehmannia glutinosa separately. 0.2g dried samples were ground into a powder, extracted, whose nal extract was used for HPLC analysis as reported by Ouyang (2017).

Statistical analysis
Statistical analysis was conducted with SPPS software (version 19.0) as stated by Zhou et al.(2016).
Analysis of variance (ANOVA) was performed.

Results
RgTAT cloning A pair of primers (RgTAT-F1 and RgTAT-R1) were designed based on the conserved region of the TAT genes from other plant species in NCBI and used to amplify a 110-bp cDNA fragment. As a result, a speci c cDNA fragment from Rehmannia glutinosa was obtained and then sequenced; Subsequently, based on this fragment, a 1266-bp cDNA (ORF) was cloned by electronic cloning method, and then ampli ed with a pair of primers (RgTAT-F2 and RgTAT-R2), followed by harvest from the agrose gel ( Figure 1), puri cation and sequencing. The base sequence of RgTAT and the amino acid sequence of its coding protein were shown in Figure 2.
RgTAT was submitted to the GenBank database (accession number: MN648812  The recombinant vector pCAMBIA2300-35S-RgTAT could be constructed by the cloning of RgTAT into the XbaI and KpnI sites of the vector pCAMBIA2300-35S, in which RgTAT was con rmed by double digestion (Figure 7). Then, this recombinant plasmid was introduced into Agrobacterium tumefaciens strain GV3101 and veri ed by PCR with a pair of primers (Table 1) (Figure 8). Finally, RgTAT was integrated into the chromosomal DNA of Rehmannia glutinosa with an Agrobacterium-mediated transformation system.

Regeneration of transgenic plants
Our preliminary experiments for callus induction indicated that 10mg/L Hyg could completely inhibit callus induction in uninfected explants of Rehmannia glutinosa, so it was used as a selection pressure in the selection medium. After co-cultivation for 2 days (Figure 9a), Agrobacterium-infected pieces of the leaves were cultured on the callus inducing medium to induce calli formation (Figure 9b). Resistant, bright green calli were transferred to the shoot inducing selection medium (Figure 9c), and about 10% of the co-cultured explants formed shoots. When about 2.0 cm high, they were cut out of the calli, followed by their transfer to the root-inducing medium for rooting ( Figure 9d). There transgenic plants were called T0 transformants (Figure 9e). Then, they were transplanted into ower pots (Figure 9f).

Molecular veri cation of transgenic plants
The genomic DNA was isolated from the leaves of two T 0 transgenic Rehmannia glutinosa lines and untransformed controls, and used as templates for PCR. The PCR analyses showed that the expected 750 bp was ampli ed (Figure10). RgTAT mRNA expression in transgenic plant lines was measured by realtime quantitative PCR. Its levels were 7.72 ± 0.17 times in No.1 T 0 transformant, and 7.28 ± 0.13 times in No.2 T 0 transformant greater than its expression in the untransformed plants.

HPLC-based acteoside content analyses
Acteoside contents in the leaves of the untransformed Rehmannia glutinosa plantlets (CK) and individual transgenic plantlets were detected by HPLC. Two Hyg-resistant transgenic plants had variable and higher acteoside contents (one 2.89 ± 0.15% DW, the other 3.12 ± 0.16% DW) than CK (1.96 ± 0.13% DW). There was a signi cant correlation between acteoside content and relative expression level of RgTAT as R2 is equal to 0.83 (p<0.05).

Discussion
Acteoside is composed of hydroxytyrosol moiety, which is synthesized from tyrosine through tyramine and dopamine, and caffeic acid moiety, which is produced from phenylalanine via a cinnamate pathway, and rhamnose moiety and glucose moiety (Qi et al., 2013). Its biosynthesis begins with the generation of phenylalanine and tyrosine precursors by the shikimate pathway (Alipieva et al., 2014), which were called phenylpropanoid pathway and tyrosine-derived pathway, respectively. The putative pathway of verbascoside biosynthesis was established in olive, and its early steps and its upstream intermediates have been known (Alipieva et al., 2014). Some key enzymes and enzymes-corresponding genes were discovered in Rehmannia glutinosa root by transcriptome sequencing (Zhou et al., 2016;Wang et al., 2017). Phenyl ammonia lyase (PAL), the rst key enzyme in the phenylpropanoid pathway, is very important for acteoside biosynthesis, and its gene was overexpressed in Rehmannia glutinosa (Xie et al., 2014). Tyrosine-derived pathway was divided into general and specialized tyrosine metabolism pathways (Xu et  Therefore, RgTAT was cloned from Rehmannia glutinosa and characterized for the rst time. Sequence alignment analysis indicated that RgTAT was homologous to TAT family members in other previously identi ed plants. Phylogenetic tree analysis revealed that RgTAT was homologous to the TATs from known plants, and the closest to the TAT from Sesamum indicum L. The genotype-speci c expression analysis showed that the expressions of RgTAT gene was greater in the roots, stems and leaves of high acteoside cultivar Beijing No.3 plants than in low verbascoside cultivar Wen85-5, suggesting its decisive roles for acteoside biosynthesis, which is similar to those validated acteoside biosynthesis-associated genes in R. glutinosa (Zhou et al., 2016;Wang et al., 2017); It is known that TyDC possesses the decisive role for acteoside biosynthesis (Zhou et al., 2016;Wang et al., 2017). Therefore, after our genotype-dependent expression analysis, we further studied the correlation of TyDC expression to TAT expression. It was found that both were positive correlation (Fig. 5), suggesting the decisive role of TAT for acteoside biosynthesis.
The Agrobacterium tumefaciens-mediated transformation system has been widely used for engineering many plant species (Liu et al., 2020). In our study, RgTAT was transferred into Rehmannia glutinosa with this transformation system, and its regeneration systems were established. PCR analysis con rmed the existence of Hyg in the transgenic plantlets. qRT-PCR analysis con rmed RgTAT expression in the transgenic plantlets was higher than in controls.
HPLC analysis revealed that acteoside content 1.5-2.0 fold higher than the control were suggesting RgTAT overexpression in the transgenic plantlets. This result demonstrated the critical role of RgTAT for acteoside biosynthesis in transgenic Rehmannia glutinosa. Our statistics result from RgTAT transgenic Rehmannia glutinosa exhibited a positive correlation between its mRNA levels and acteoside content, suggesting that the increase of RgTAT transcription lead to more acteoside synthesis.

Conclusions
We successfully cloned, spatially expressed and overexpressed RgTAT from Rehmannia glutinosa, and then established its transgenic plantlets with the increased acteoside. The results indicate the key role of RgTAT for acteoside biosynthesis pathway, and lay a foundation for future regulating RgTAT expression and enhancing acteoside content in Rehmannia glutinosa via transgenic technology and gene editing.

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Competing interests
The authors declare that they have no competing interests. Authors' contributions YZ put forward the research and revised the manuscript. JZ, DZ, and MG carried out the preparation and treatment of test materials, performed the experiment, analyzed the data, and wrote the manuscript. HD and HL helped YZ to supervise graduates and designed the experiment. All authors contributed comments to the manuscript. All authors read and approved the nal manuscript. The base sequence of RgTAT and the amino acid sequence of its coding protein *: Stop code. The numbers on the right: the number of the rst base (up) or amino acid (down) in every line.  Transformation gures a. The pieces of leaves. b. calli. c. propagated calli. d. differentiated shoots from the calli. e. regenerated plantlet. f. transplanted plantlet in a owerpot.