Tyrosine Aminotransferase Gene ( SmTAT ) Revealed Genetic Diversity and Phylogeny of Cultivated Danshen ( Salvia miltiorrhiza ) Populations

Chinese traditional medicine Danshen is the radix of the perennial herbs of Salvia miltiorrhiza Bunge, which has a variety of pharmacological effects and is traditionally and extensively applied clinically to treat cardiovascular dis-orders. In this research, the genomic genes for tyrosine aminotransferase (TAT) of 38 cultivated populations of Danshen in China were cloned and bioinformatic analyses were conducted to reveal its genetic diversity and phylogeny. The full-length SmTAT was 2296 - 2444 bp including 6 exons (encoding 411 amino acids) and 5 introns. Overall, the SmTAT genes in cultivated Danshen populations are highly conserved with a relative low level of genetic diversity. The spliced exons (1236 bp) had 23 SNP variations with a rate of 1.86%, of which 22 occurred in the white flower S. miltiorrhiza Bge.f.alba population (W-SCHY-W-1) and led to 5 amino acid variations. The entire 290 SNP variations with a rate of 24% in the 5 introns occurred exclusively in W-SCHY-W-1. Phylogenetic trees based on the full-length, combined introns, the spliced exons, and the deduced amino acid sequences of SmTAT all showed a two-clade basic structure with W-SCHY-W-1


Introduction
Chinese traditional herbal medicine, Danshen, is the radix of perennial herbs of Salvia miltiorrhiza Bunge of the family Labiatae. It has been traditionally and extensively used in clinical practice to treat various ailments such as cardiovascular, cerebrovascular, hyperlipidemia, and acute ischemic stroke diseases [1] [2] [3] [4] [5].
Danshen owns abundant germplasm resources and many cultivated populations in China. In recent years, with the increase in market demand, the often-chaotic introduction of varieties in field cultivation and the nonstandard field management cause some confusion and result in poor quality of the herbal medicine. Extensive researches have been conducted on its cultivation, germplasm resources protection, and molecular identification.
The biosynthetic pathway of rosmarinic acid consists of two parallel phenylalanine and tyrosine branches [12]. Tyrosine aminotransferase (TAT) (EC 2.6.1.5) is the rate-limiting step in the tyrosine branch, which catalyzes the formation of 4-hydroxyphenylpyruvate from tyrosine and ultimately the biosynthesis of salvianolic acids. Various other structurally diverse natural compounds are also derived from the tyrosine metabolic pathway, among which tocopherols, plastoquinone, and ubiquinone are essential to plant survival [13].
So far, our knowledge of the plant tyrosine metabolism pathway remains rudimentary, and genes encoding the pathway enzymes have not been fully defined, despite that the tyrosine aminotransferase genes have been cloned in a few other plants such as Coleus blumei (AJ458993), Arabidopsis thaliana [14], Glycine max (AAY21813), Medicago truncatula (DQ006809) as well as S. miltiorrhiza, and functionally studied by overexpression of single gene and coexpression of several in S. miltiorrhiza hairy root cultures [15]. The structure and the genetic diversity of tyrosine aminotransferase genes among the various cultivated populations of S. miltiorrhiza are still unknown.
In this research, the genomic tyrosine aminotransferase genes of the 38 cultivated populations of S. miltiorrhiza from the major cultivation regions of China, were for the first time cloned by walking technology, and its sequences were analyzed bioinformatically to understand the genomic structure, genetic diversity, and phylogeny of the cultivated S. miltiorrhiza populations.

Materials and Methods
Plant materials Seeds of 38 cultivated Danshen populations were collected from three major seed industries representing more than 30 regions of China (Table 1); uniform seeds preliminarily selected according to size, color and shape were used for sowing in Southwest University Agricultural Station, Chongqing; and morphologically representative single plants of each population were used for extraction of genomic DNAs.
Primer design Two pairs of primers for cloning of the genomic SmTAT genes of cultivated S. miltiorrhiza populations were designed based on the reference accession (EF192320.1) with Primer 5 ( Table 2).

Genomic DNA extraction
Total leaf genomic DNAs were extracted with Qiagen DNeasy Plant Mini Kit (Multi Sciences, Hangzhou, China) according to the manufacturer's instructions. The purity was assessed by agarose gel electrophoresis followed by Goldview staining, and the quantity was determined spectrophotometrically by Shimadzu UV mini-1240. Purified genomic DNAs were dissolved in 10 mmol/L Tris-HCl buffer and stored at −70˚C.
PCR amplification About 1.0μg genomic DNA templates were amplified with primer pairs TAT-FP1/RP1 and TAT-FP2/RP2 respectively in a reaction mixture of 50 μL: 1.1 × T3 Super PCR Mix 36.0 -44 μL, 10 μmol/L primers each 2.0 μL (final concentration 0.4 μM) in Biometra TGRADIENT thermocycler (Biometra GmbH, Germany) with the programme: initial-denaturation at 98˚C for 3 min followed by 35 cycles of denaturation at 98˚C for 10 s, annealing at 55˚C/60˚C for 10 s and elongation at 72˚C for 5 -15 s, and a final extension at 72˚C for 2 min.
Amplified products were electrophoresed in 1% agarose gel and visualized with Goldview stain. And after recovery and purification, they were bidirectionally sequenced by dideoxy chain termination with ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, USA) and manually spliced and checked.
Sequence data processing The BLAST confirmed two segments of the SmTAT gene sequences of the 38 cultivated populations of S. miltiorrhiza were spliced with Vector NTI Ad-vance11. The spliced whole sequences were manually checked to ensure the quality of sequences and BLAST analyzed to confirm the gene of interest. The spliced whole SmTAT gene sequences were deposited in GenBank. Sequences were aligned with Vector NTI Advance11 to identify the nucleotide variation sites. Phylogenetic trees were constructed with MEGA X based on Neighbor-Joining (NJ) with a bootstrap value of 1000.

Results and Analyses
Structural features of the SmTAT genes of the cultivated S. miltiorrhiza populations The SmTAT genes of the 38 cultivated populations were successfully ampli-fied by the walking primers designed (TAT-FP1/RP1 and TAT-FP2/RP2). Agarose gel electrophoresis showed distinct single bands of about 700 bp and 2000 bp respectively with the two primer pairs.
All the obtained SmTAT gene sequences of the 38 cultivated populations of S. miltiorrhiza were BLAST confirmed and submitted to GenBank (Accession numbers shown in Table A1).
The sequence of accession EF192320.1 was used as the reference to demarcate  (Table 4).
Nucleotide variation in exons of the SmTAT genes Alignment of the spliced 6 exon sequences of all the 38 cultivated S. miltiorrhiza populations showed that there were 23 nucleotide variation sites, of a variation rate of 1.86%, among which 12 conversions, 11 transversions. Most variations (22) occurred in population W-SCHY-W-1 and were distributed mainly in exons 2-4 ( Table 5).

Amino acid variations in the deduced amino acid sequences of SmTAT
The spliced exon sequences of the SmTAT gene were 1236 bp in length with a complete reading frame of 1233 bp encoding 411 amino acid residues. The deduced amino acid sequences are highly conserved. All showed the aminotransferases family-I pyridoxal-phosphate attachment site (SLSKRWLVPGWRLG)

Discussion
The spliced exon sequences of the SmTAT gene were 1236 bp in length with an ORF of 1233 bp encoding 411 amino acid residues, consistent with the similar report [9]. The deduced amino acid sequences of SmTAT are highly conserved. All showed the aminotransferases family-I pyridoxal-phosphate attachment site and an Arg385 that fixes the α-carboxylate of the incoming amino acid or α-ketoacid. The high conservation and overall low level of diversity of SmTAT genes demonstrated in this research suggests the necessity to further conserve its wild resources and to identify novel genetic resources materials as well as to Natural Resources The white flower S. miltiorrhiza Bge.f.alba is a varietae or forma of S. miltiorrhiza Bge. Usually, the flower color of S. miltiorrhiza Bge is purple, while that of S. miltiorrhiza Bge.f.alba is white. There have been comparative reports of the medicinal value of the purple and white flower Danshen. One report showed two more bioactive ingredients in S. miltiorrhiza Bge.f.alba [16]. Another found that the contents of some trace element in white flower Danshen were higher than those in purple flower Danshen [17]. Still another showed that the phenolic acids contents in white flower Danshen were about two times higher than those in purple flower Danshen [18]. Also found is that most parts of S. miltiorrhiza Bge. f. alba plant had higher contents of bioactives than S. miltiorrhiza Bge [19]. The white flower S. miltiorrhiza Bge. f. alba had special pharmacological effect for treatment of thromboangiitisobiterans [19]. The crude drug of S. miltiorrhiza Bge.f. alba was found to increase cerebral blood flow significantly, reduce neuronal apoptosis, and promote neuronal regeneration in rats with cerebral ischemia/reperfusion impairment [20]. Bge.f.alba would be very beneficial in understanding its special pharmaceutical effects and for the acceleration of its breeding.

Conclusions
The successfully walking technologically cloned full-length genomic SmTAT was about 2296 bp and consisted of 6 exons and 5 introns. The spliced exon sequence of SmTAT gene was 1236 bp in length, encoding a complete reading frame of 411 amino acids. All the 23 SNP variation sites (1.86%) occurred in the white flower W-SCHY-W-1 population. The only 5 amino acid variations were located in population W-SCHY-W-1.
The 5 introns of SmTAT had 290 SNP variation sites, which were located in W-SCHY-W-1only, with a variation rate of 24% far greater than that in the spliced exons, indicating the faster evolution of the introns. Phylogenetic trees based on the full-length genomic SmTAT, the spliced exons, combined introns, and the deduced amino acid sequences all showed a two-clade structure with population W-SCHY-W-1 standing alone, which represented a special popula-tion as regard to the TAT gene. The uniquely extensive variation in the genomic SmTAT gene of W-SCHY-W-1 is probably an important genetic marker for the white flower Dansen and a valuable molecular breeding target.
Further comparative and functional studies on SmTAT in relation to both the elucidation of plant tyrosine metabolism and the biosynthesis of the pharmacological ingredients in the currently cultivated S. miltiorrhiza Bge populations especially S. miltiorrhiza Bge.f.alba would be very revealing and valuable in SmTAT based molecular breeding.

Data Availability Statement
All the SmTAT sequence data of the 38 cultivated populations of Danshen has been submitted in GenBank and accession numbers has been provided in Table  A1.

Ethical Statement
This research did not involve any animal or human participants.

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.