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.
However, research progress in the functional genes for pharmacologically active constituents has been slow. There are two major classes of pharmacologically effective components in Danshen, the hydrophilic salvianolic acids, and the lipophilic tanshinones [6]. So far, the key genes for the biosynthesis of effective components in S. miltiorrhiza, of 4-hydroxycinnamate coenzyme A ligase [6], 4-hydroxyphenylpyruvate reductase [7], cinnamic acid 4-hydroxylase [8], tyrosine aminotransferase [9], 3-hydroxy-3-methylglutaryl coenzyme A reductase [10], and phenylalanine ammonia-lyase genes [11], have been characterized and studied respectively.
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.
2. 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
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Table 1. The cultivated S. miltiorrhiza populations used in this study.
Notes: HDSI—Hengda Seed Industry, China; TDSI—Tongda Seed Industry, China; FHSI—Fenghong Seed Industry, China; SC—Self-Collected.
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Table 2. Primers designed for the cloning of the genomic SmTAT genes of the cultivated populations.
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 Advance11. 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.
3. 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 amplified 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 TableA1).
The sequence of accession EF192320.1 was used as the reference to demarcate the SmTAT gene sequences. Results showed that the full-length of genomic SmTAT gene was 2296 - 2444 bp, consisting of 6 exons with a total length of 1236 bp among all the tested populations, and 5 introns with a total length of 1060 bp for 37 populations except for W-SCHY-W-1, whose total intron length increased to 1208 bp, most of the increased 148bp distributed in introns 1 - 4; the corresponding exons or introns are equal in size for the majority (37 populations) except for W-SCHY-W-1. The total length of the SmTAT gene for the majority (37 populations) was 2296 bp, while for W-SCHY-W-1, 2444 bp (Table 3).
Nucleotide variations in the introns of the SmTAT genes
The 5 intron sequences of SmTAT of the 38 cultivated populations were aligned and compared. Results showed that there were 290 nucleotide variations with a rate of 24%, and all occurred in the population W-SCHY-W-1(Table 4). Statistics showed that there were 110 variations (11conversions, 21 transversions, 4 deletions and 74 insertions) in intron 1, 13 variations (4 transversions and 9 insertions) in intron 2, 120 variations (19 conversions, 21 transversions, 18 deletions and 62 insertions) in intron 3, 34 variations (2 conversions, 2 transversions, 30 insertions) in intron 4, and 13 variations (2 transversions, 2 transversions, 7 deletions and 2 insertions) in intron 5, accounting for variation rates of 26.3%, 15.7%, 30.9%, 45.3% and 13.5% respectively (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)
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Table 3. Structure of the genomic SmTAT genes of the cultivated S. miltiorrhiza populations.
Note: E and I for exons and introns respectively.
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Table 4. Nucleotide variation sites in introns of SmTAT genes of S. miltiorrhiza populations.
Notes: nucleotide variation sites are shown in bold; “-” represent deletions.
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Table 5. Nucleotide variation sites in exons of SmTAT sequences of S. miltiorrhiza populations.
Notes: Nucleotide variation sites are shown in bold; *: V-JS-V-2, V-HBAG-V-1, B-SC-V-2, S-HNCS-V-1, W-SXXA-V-1, Q-DZH-V-4, V-GZZY-V-1, CYSWC-DZH-V-4, V-JSSY-V-1, B-GD-V-2, S-HBAG-V-1, V-GZ-V-2 and W-GZ-V-2.
and an Arg385 that fixes the α-carboxylate of the incoming amino acid or α-ketoacid [9]. Comparison of the deduced amino acid sequences of the SmTAT of the 38 cultivated S. miltiorrhiza populations revealed that 37 populations were in consensus and 5 variations (E102D, G122A, N197K, K198E, and E299G) all occurred in population W-SCHY-W-1. Among them, N197K, K198E, and E299G were probably the most significant variations in TAT of population W-SCHY-W-1 from the remaining 37 populations.
Phylogenetic analyses of SmTAT gene and its deduced amino acid sequences
Phylogenetic trees based on the full-length SmTAT genomic sequences and the spliced 6 exons were nearly identical. Population W-SCHY-W-1 stands alone, while the other 37 populations cluster in another clade, which is branched into 2 subclades, one consisting of 13 populations (V-JS-V-2, V-HBAG-V-1, B-SC-V-2, S-HNCS-V-1, W-SXXA-V-1, Q-DZH-V-4, V-GZZY-V-1, CYSWC- DZH-V-4, V-JSSY-V-1, B-GD-V-2, S-HBAG-V-1, V-GZ-V-2, and W-GZ-V-2), and the other, of 24 populations (Figure 1(a), Figure 1(b)).
Phylogeny analyses on the combined introns sequences (from intron 1 to 5) and the deduced amino acid sequences of SmTAT showed that population W-SCHY-W-1 stands alone and all the 37 populations cluster in one clade (Figure 1(c), Figure 1(d)).
Basically, a two-clade topological structure was demonstrated in the phylogenetic trees based on the full-length SmTAT, combined introns, the spliced exons, and the deduced amino acid sequences. Population W-SCHY-W-1 is uniquely standing alone in all the phylogenetic trees.
4. 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
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Figure 1. Phylogenetic trees based on the full-length (a), the spliced exons (b), combined introns (c), and the deduced amino acid sequences (d) of SmTAT.
accelerate the breeding of this important Chinese medicinal plants.
Comparison of the deduced amino acid sequences of the SmTAT gene of the 38 the cultivated S. miltiorrhiza populations revealed that 37 populations are identical and all the 5 variations occurred in population W-SCHY-W-1, of which N197K, K198E, and E299G are probably the most significant variations in TAT of population W-SCHY-W-1.
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].
The rare white flower S. Miltiorrhiza Bge.f.alba is generally valued more over the purple flower majority S. Miltiorrhiza Bunge. The extensive variations in the genomic SmTAT gene of the white flower Danshen population W-SCHY-W-1 found in this research probably is an important genetic marker for white flower Danshen. Further elucidation of the structures and functions of SmTAT and other functional genes involved in tyrosine metabolic pathway in S. miltiorrhiza Bge.f.alba would be very beneficial in understanding its special pharmaceutical effects and for the acceleration of its breeding.
5. 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 population 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 TableA1.
Declaration of Funding
This work was funded by National Key R & D Program of China (Project/Subject SN 2017YFF0205305 and 2017YFF0210303) of the Ministry of Science and Technology of the People’s Republic of China.
Ethical Statement
This research did not involve any animal or human participants.
Appendix
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Table A1. GenBank accession numbers for the genomic SmTAT genes of the 38 cultivated populations of S. miltiorrhiza.