Abstract
Safflower (Carthamus tinctorius L.), an important traditional Chinese medicine, is cultured widely for its pharmacological effects, but little is known regarding the genes related to the metabolic regulation of the safflower’s yellow pigment. To investigate genes related to safflor yellow biosynthesis, 454 pyrosequencing of flower RNA at different developmental stages was performed, generating large databases.In this study, we analyzed 454 sequencing data from different flowering stages in safflower. In total, 1,151,324 raw reads and 1,140,594 clean reads were produced, which were assembled into 51,591 unigenes with an average length of 679 bp and a maximum length of 5109 bp. Among the unigenes, 40,139 were in the early group, 39,768 were obtained from the full group and 28,316 were detected in both samples. With the threshold of “log2 ratio ≥ 1”, there were 34,464 differentially expressed genes, of which 18,043 were up-regulated and 16,421 were down-regulated in the early flower library. Based on the annotations of the unigenes, 281 pathways were predicted. We selected 12 putative genes and analyzed their expression levels using quantitative real time-PCR. The results were consistent with the 454 sequencing results. In addition, the expression of chalcone synthase, chalcone isomerase and anthocyanidin synthase, which are involved in safflor yellow biosynthesis and safflower yellow pigment (SYP) content, were analyzed in different flowering periods, indicating that their expression levels were related to SYP synthesis. Moreover, to further confirm the results of the 454 pyrosequencing, full-length cDNA of chalcone isomerase (CHI) and anthocyanidin synthase (ANS) were cloned from safflower petal by RACE (Rapid-amplification of cDNA ends) method according to fragment of the transcriptome.
Highlights
Safflower (Carthamus tinctorius L.), is a widely used herbal plant in the family Compositae, which are cultured in many countries worldwide
High-throughput sequencing technology has been widely used in various plants to obtain transcript coverage even without a reference genome. 454 sequencing is a reasonably low cost [28] transcriptome profiling method, and its novel and efficient high throughput approach has been used on the olive [29], Leymus chinensis [21], orchids [30], Podophyllum hexandrum [31], plum [32], Lonicera japonica Thunb. [33] and Vicia faba L. [34]
454 sequencing was applied to the safflower transcriptome for the first time, to discover the important genes in flavonoid biosynthesis, which may be related to the synthesis of Safflower yellow pigments (SYPs) during different flower developmental stages
Summary
Safflower (Carthamus tinctorius L.), is a widely used herbal plant in the family Compositae, which are cultured in many countries worldwide. The chemical components of safflower are diverse, including flavonoids [5] such as safflor yellow, alkaloids and fatty acids [6], polysaccharide and others. Safflower yellow pigments (SYPs), which are isolated from safflower petals, as flavonoid compounds, have been extensively applied in many fields, including as a medicine and natural food colorant. Hydroxysafflor Yellow A (HSYA), is the major active component of the flower and has potent and important antioxidative effects in vitro [7,8], an enormous antagonistic impact on platelet activating factor receptor [9] and vascular dementia [10], and an inhibitory effect on platelet aggregation, tumor angiogenesis, thrombosis and oxidative stress [11,12]. Related research [14] showed that the maximal inhibitory action against the proliferation of 3T3-L1 cells was 0.1 mg/L HSYA over 72 h
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