Chitosan and its oligosaccharides, a promising option for sustainable crop production- a review

TRICHOME AND ARTEMISININ REGULATOR 1 Is Required for Trichome Development and Artemisinin Biosynthesis in Artemisia annua

Amorpha-4,11-diene synthase (ADS) and Cyt P450 monooxygenase (CYP71AV1) in Artemisia annua L. are two key enzymes involved in the biosynthesis of artemisinin. The promoters of ADS and CYP71AV1 contain E-box elements, which are putative binding sites for basic helix-loop-helix (bHLH) transcription factors. This study successfully isolated a bHLH transcription factor gene from A. annua, designated as AabHLH1, from a cDNA library of the glandular secretory trichomes (GSTs) in which artemisinin is synthesized and sequestered. AabHLH1 encodes a protein of 650 amino acids containing one putative bHLH domain. AabHLH1 and ADS genes were strongly induced by ABA and the fungal elicitor, chitosan. The transient expression analysis of the AabHLH1-green fluorescent protein (GFP) reporter gene revealed that AabHLH1 was targeted to nuclei. Biochemical analysis demonstrated that the AabHLH1 protein was capable of binding to the E-box cis-elements, present in both ADS and CYP71AV1 promoters, and possessed transactivation activity in yeast. In addition, transient co-transformation of AabHLH1 and CYP71AV1Pro::GUS in A. annua leaves showed a significant activation of the expression of the GUS (β-glucuronidase) gene in transformed A. annua, but mutation of the E-boxes resulted in abolition of activation, suggesting that the E-box is important for the CYP71AV1 promoter activity. Furthermore, transient expression of AabHLH1 in A. annua leaves increased transcript levels of the genes involved in artemisinin biosynthesis, such as ADS, CYP71AV1 and HMGR. These results suggest that AabHLH1 can positively regulate the biosynthesis of artemisinin.

/pdf/cloning-and-characterization-of-aabhlh1-a-bhlh-transcription-21eskeohqi.pdf

Cloning and characterization of AabHLH1, a bHLH transcription factor that positively regulates artemisinin biosynthesis in Artemisia annua

Malaria is still an eminent threat to major parts of the world population mainly in sub-Saharan Africa. Researchers around the world continuously seek novel solutions to either eliminate or treat the disease. Artemisinin, isolated from the Chinese medicinal herb Artemisia annua, is the active ingredient in artemisinin-based combination therapies (ACTs) used to treat the disease. However, naturally artemisinin is produced in small quantities, which leads to a shortage of global supply. Due to its complex structure, it is difficult chemically synthesise. Thus to date, A. annua remains as the main commercial source of artemisinin. Current advances in genetic and metabolic engineering drives to more diverse approaches and developments on improving in planta production of artemisinin, both in A. annua and in other plants. In this review, we describe efforts in bioengineering to obtain a higher production of artemisinin in A. annua and stable heterologous in planta systems. The current progress and advancements provides hope for significantly improved production in plants.

/pdf/a-review-of-biotechnological-artemisinin-production-in-13vtn5szbs.pdf

A Review of Biotechnological Artemisinin Production in Plants

Artemisinin, the endoperoxide sesquiterpene lactone, is an effective antimalarial drug isolated from the Chinese medicinal plant Artemisia annua L. Due to its effectiveness against multi-drug-resistant cerebral malaria, it becomes the essential components of the artemisinin-based combination therapies which are recommended by the World Health Organization as the preferred choice for malaria tropica treatments. To date, plant A. annua is still the main commercial source of artemisinin. Although semi-synthesis of artemisinin via artemisinic acid in yeast is feasible at present, another promising approach to reduce the price of artemisinin is using plant metabolic engineering to obtain a higher content of artemisinin in transgenic plants. In the past years, an Agrobacterium-mediated transformation system of A. annua has been established by which a number of genes related to artemisinin biosynthesis have been successfully transferred into A. annua plants. In this review, the progress on increasing artemisinin content in A. annua by transgenic approach and its future prospect are summarized and discussed.

Transgenic approach to increase artemisinin content in Artemisia annua L.

Artemisinin, a sesquiterpene lactone endoperoxide derived from Artemisia annua L. (Asteraceae), is the most effective antimalarial drug. We used two methods: genome walking and thermal asymmetric interlaced polymerase chain reaction, to isolate the unknown 5'-flanking sequence of the cyp71av1 gene. The subsequent sequence analysis using bioinformatics software revealed that there are several cis-acting elements inside the cyp71av1 promoter. The 5'-rapid amplification of the cDNA ends method was used to determine the transcription start site of the cyp71av1 gene. We then mapped it at the 18 base upstream of the ATG initiation codon. For simple functional characterization, we built fusion vectors between the 5'-deletion promoter and the gas reporter gene. The expression levels of the transferred vectors into A. annua L. were analyzed by the transient expression way. The beta-glucuronidase assay results indicated that deletion of the region to -1551 bp did not lead to much damage in the GUS activity, whereas further deletion, to -1155 bp, resulted in a 5.5-fold reduction of GUS activity. In stabilized transgenic A. annua L. seedlings we observed that GUS expression was restricted to trichomes, which means that the promoter of the cyp71av1 gene is trichome-specific. Compared with the constitutive CaMV 35S promoter, which can express genes throughout the plant, influence on the trichome system through the trichome-specific expression promoter merely imperils plant growth. In addition, the promoter of the cyp71av1 gene contains several binding sites for transcription factors, which implies that the cyp71av1 promoter responds to more than one form of stimulation.

Cloning and characterization of trichome-specific promoter of cpr71av1 gene involved in artemisinin biosynthesis in Artemisia annua L.

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Cloning and characterization of trichome-specific promoter of cpr71av1 gene involved in artemisinin biosynthesis in Artemisia annua L.