title: Cloning and characterization of trichome-specific promoter of cpr71av1 gene involved in artemisinin biosynthesis in Artemisia annua L.
abstract: 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.
authors.affiliation: Plant Biotechnology Research Center, School of Agriculture and Biology, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Yueyue
authors.initials: Y
authors.lastname: Wang
authors.forename: Ke
authors.initials: K
authors.lastname: Yang
authors.forename: Fuyuan
authors.initials: F
authors.lastname: Jing
authors.forename: Meiya
authors.initials: M
authors.lastname: Li
authors.forename: Ting
authors.initials: T
authors.lastname: Deng
authors.forename: Runze
authors.initials: R
authors.lastname: Huang
authors.forename: Boshi
authors.initials: B
authors.lastname: Wang
authors.forename: Guofeng
authors.initials: G
authors.lastname: Wang
authors.forename: Xiaofen
authors.initials: X
authors.lastname: Sun
authors.forename: Ke-Xuan
authors.initials: KX
authors.lastname: Tang
mesh_terms: D000962:Antimalarials / Q000378:metabolism* / Q000494:pharmacology; D031172:Artemisia annua / Q000235:genetics / Q000378:metabolism*; D037621:Artemisinins / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000096:biosynthesis; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D017930:Genes, Reporter; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy* / Q000469:parasitology; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000187:drug effects / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D024363:Transcription Initiation Site; D014170:Transformation, Genetic
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D018076:DNA, Complementary; C031327:artemisinin; D005966:Glucuronidase