No abstract

The artemisinic aldehyde double bond reductase (DBR2) plays an important role in the biosynthesis of the antimalarial artemisinin in Artemisia annua. Artemisinic aldehyde is reduced into dihydroartemisinic aldehyde by DBR2. Artemisinic aldehyde can also be oxidized by amorpha-4,11-diene 12-hydroxylase and/or aldehyde dehydrogenase 1 to artemisinic acid, a precursor of arteannuin B. In order to better understand the effects of DBR2 expression on the flow of artemisinic aldehyde into either artemisinin or arteannuin B, we determined the content of dihydroartemisinic aldehyde, artemisinin, artemisinic acid and arteannuin B content of A. annua varieties sorted into two chemotypes. The high artemisinin producers (HAPs), which includes the '2/39', 'Chongqing' and 'Anamed' varieties, produce more artemisinin than arteannuin B; the low artemisinin producers (LAPs), which include the 'Meise', 'Iran#8', 'Iran#14', 'Iran#24' and 'Iran#47' varieties, produce more arteannuin B than artemisinin. Quantitative PCR showed that the relative expression of DBR2 was significantly higher in the HAP varieties. We cloned and sequenced the promoter of the DBR2 gene from varieties of both the LAP and the HAP groups. There were deletions/insertions in the region just upstream of the ATG start codon in the LAP varities, which might be the reason for the different promoter activities of the HAP and LAP varieties. The relevance of promoter variation, DBR2 expression levels and artemisinin biosynthesis capabilities are discussed and a selection method for HAP varieties with a DNA marker is suggested. Furthermore, putative cis-acting regulatory elements differ between the HAP and LAP varieties.

The goal of this study is to identify characterization of expressed sequence tag (EST)-simple sequence repeats (SSR) markers from EST library of durum wheat and functional analysis of SSR-containing EST sequences for application in comparative genomics and breeding. 19,141 sequences were analyzed among which 18,937 ESTs were selected. Consistent with MISA results, 313 EST-SSRs were yielded. The final EST-SSRs were compared to the GenBank non-redundant database using BLASTX and classified based on these functions. Results indicated that the perfect EST-SSRs are the most frequent. The TTG/CTG imperfect EST-SSR had gamma-gliadin putative function that can be appropriate for durum wheat. Also, the mononucleotides and trinucleotides were the most frequent. Findings suggested that the identified EST-SSRs could be categorized into 83 types. Motifs TTG in trinucleotides and TC in dinucleotides had the highest frequency. TTG is the new motif in durum wheat identified in this study. We identified new EST-SSRs with more than trinucleotide and detected motifs that have potential to code amino acids. Arginine was the most frequent amino acid. Enzymes had the highest frequency among predicted functions. EST-SSRs have been identified in this study can be used for developing ESS-SSR-based detection tool for durum wheat in future studies and will be a useful resource for molecular breeding, genetics, genomics, and environmental stress studies. Motifs coding amino acids could be used as a new source of functional markers and biological study. In addition to, designed new PCR primer pairs are new resources for to identify useful alleles in transcription factors, storage proteins, and enzymes which incorporated them again into the cultivated material.

Artemisinin, an effective anti-malarial drug is synthesized in the specialized 10-celled biseriate glandular trichomes of some Artemisia species. In order to have an insight into artemisinin biosynthesis in species other than A. annua, five species with different artemisinin contents were investigated for the expression of key genes that influence artemisinin content. The least relative expression of the examined terpene synthase genes accompanied with very low glandular trichome density (4 No. mm−2) and absence of artemisinin content in A. khorassanica (S2) underscored the vast metabolic capacity of glandular trichomes. A. deserti (S4) with artemisinin content of 5.13 mg g−1 DW had a very high expression of Aa-ALDH1 and Aa-CYP71AV1 and low expression of Aa-DBR2. It is possible to develop plants with high artemisinin synthesis ability by downregulating Aa-ORA in S4, which may result in the reduction of Aa-ALDH1 and Aa-CYP71AV1 genes expression and effectively change the metabolic flux to favor more of artemisinin production than artemisinic acid. Based on the results, the Aa-ABCG6 transporter may be involved in trichome development. S4 had high transcript levels and larger glandular trichomes (3.46 fold) than A. annua found in Iran (S1), which may be due to the presence of more 2C-DNA (3.48 fold) in S4 than S1.