Chalcone synthase genes from milk thistle (Silybum marianum): isolation and expression analysis

Abstract

Silymarin is a flavonoid compound derived from milk thistle (Silybum marianum) seeds which has several pharmacological applications. Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoids; thereby, the identification of CHS encoding genes in milk thistle plant can be of great importance. In the current research, fragments of CHS genes were amplified using degenerate primers based on the conserved parts of Asteraceae CHS genes, and then cloned and sequenced. Analysis of the resultant nucleotide and deduced amino acid sequences led to the identification of two different members of CHS gene family,SmCHS1 and SmCHS2. Third member, full-length cDNA (SmCHS3) was isolated by rapid amplification of cDNA ends (RACE), whose open reading frame contained 1239 bp including exon 1 (190 bp) and exon 2 (1049 bp), encoding 63 and 349 amino acids, respectively. In silico analysis of SmCHS3 sequence contains all the conserved CHS sites and shares high homology with CHS proteins from other plants.Real-time PCR analysis indicated that SmCHS1 and SmCHS3 had the highest transcript level in petals in the early flowering stage and in the stem of five upper leaves, followed by five upper leaves in the mid-flowering stage which are most probably involved in anthocyanin and silymarin biosynthesis.

The Impact of Drought Stress on Antioxidant Responses and Accumulation of Flavonolignans in Milk Thistle (Silybum marianum (L.) Gaertn)

“…The transcription level of CHS1 was significantly higher compared with CHS2 and CHS3 ( Figure 5). The obtained results were in agreement with previously reported studies [17,21]. Overall, our findings revealed that drought stress improved the expression of the three genes (CHS1, CHS2, and CHS3), which was coordinated by a definite increase in silymarin content (23.76 mg•g −1 DM) in milk thistle.…”

“…Chalcone synthase (CHS) is a vital enzyme of the flavonoid and flavonolignans biosynthesis pathway [17,57]. Silymarin is synthesized in milk thistle plants via the phenylpropanoid pathway converting phenylalanine to 4-coumaroyl-CoA ( Figure S2).…”

“…Quantitative RT-PCR was used to analyze the relative transcript levels of three genes involved in the flavonolignan biosynthesis in milk thistle [17], namely chalcone synthase genes (CHS1, CHS2, and CHS3). The transcript levels of CHS1 increased in milk thistle leaves exposed to drought stress compared with the well-watered plants ( Figure 5A).…”

“…Recently, Zahir et al [15] stated that drought stress boosted the accumulation of the total phenolic and flavonoid substances in milk thistle plants. Biosynthesis of flavonoids in milk thistle and other plants depend on a critical enzyme, chalcone synthase (CHS) [16,17]. The activities of CHSs genes were found to be regulated by endogenous mechanisms in plant development [18,19] or by diverse exogenous stimuli.…”

Chemotaxonomic and biosynthetic relationships between flavonolignans produced by Silybum marianum populations

“…The direct precursors of the flavonolignans, the flavonoids and monolignols, are synthesized via the phenylpropanoid pathway transforming phenylalanine into 4-coumaroyl-CoA [2]. Cloning and characterization of a full-length chalcone synthase cDNA and one partial gene from S. marianum that is probably involved in flavonolignan biosynthesis have been reported recently [3]. …”