| Artemisia annua is an important medicinal plant, and the regulatory mechanism of artemisinin is always the focused research area in A. annua. Previous studies have shown that AP2/ERF transcription factors play important roles in the regulation of secondary metabolism. Artemisinin is produced and stored in glandular secretory trichomes present on aerial surfaces of the plant. So if there are trichome-specific AP2/ERF transcription factors, and whether the genes directly or indirectly regulate the artemisinin biosynthetic pathway remain unknown. In addition, jasmonic acid (JA) is an important signaling molecule in plants. Recent studies have found that the treatment of exogenous JA promote the formation of trichomes and biosynthesis of the antimalarial compound artemisinin in A. annua. However, the significance and function of endogenous JA in trichome formation and second metabolic regulation of A. annua remain unknown. To understand the mechanisms of metabolic regulation in artemisinin, the high artemisinin cultivar of A. annua is used in this study. Through the studies including gene cloning, evolution analysis, expression analysis, promoter analysis, functional study of transgenic plants and gray mildew resistance of transgenic A. annua plants, the main results were achieved as listed below.1. AaOBA is a trichome-specific transcriptional factor, which is a positive regulator in the biosynthesis of artemisinin and disease resistance to B. cinerea.Seven AP2/ERF transcription factors were cloned through the screening of cDNA libriary and RACE library and analyzed in A. annua. Real-time quantitative PCR (RT-Q-PCR) showed that one of the transcription factors, named AaORA, had similar expression pattern as those of ADS, CYP71AV1and DBR2in different tissues of A. annua. AaORA targeted to the nuclei and the cytoplasm, and was responsive to methyl jasmonate (MeJA) and wound treatments. To examine the expression pattern of AaORA in details, the1193-bp promoter sequence of AaORA was cloned by genomic walking. GUS staining of transgenic A. annua plants containing GUS gene driven by AaORA promoter showed that AaORA was a trichome-specific transcription factor, which expressed in both glandular secretory trichomes (GSTs) and non-glandular T-shaped trichomes (TSTs) of A. annua. In AaORA-overexpressing transgenic lines, dihydroartemisinic acid and artemisinin levels were increased by22-35%and40-53%, respectively. In RNAi transgenic lines, dihydroartemisinic acid and artemisinin contents were decreased to63-41%and64-52%of the control level, respectively. The up-regulated or down-regulated expression levels of AaORA in A. annua could regulate the expression levels of AaERF1, ADS, CYP71AV1and DBR2, significantly. The up-regulated or down-regulated expression levels of those genes resulted in significant increase or decrease of artemisinin and dihydroartemisinic acid. Yeast one hybrid showed that AaORA could bind to the promoter of ADS. And transient expression assay in N.benthamiana showed that AaORA could induce the promoter activities of ADS. All above results demonstrated that AaORA was a positive regulator in the biosynthesis of artemisinin.Overexpression of AaORA in Arabidopsis could greatly increase the transcript levels of defense marker genes PLANT DEFENSIN1.2(PDEF1.2), HEVEIN-LIKE PROTEIN (HEL) and BASIC CHITINASE (CniB). Six days after inoculation with Botrytis cinerea, the wild-type plants were dry and died, while most of the AaORA-overexpressing plants grew well. Six days after inoculation with B. cinerea, all the three independent RNAi lines showed more serious disease symptoms than the vector control line. All above results demonstrated that AaORA was a positive regulator of disease resistance to B. cinerea.2. Overexpression of AaAOC gene increased the content of endogenous Jasmonic acid, the density of gland trichomes and the expression levels of genes in artemisinin biosynthetic pathway.The jasmonate biosynthetic pathway genes AaAOC and AaAOS from A. annua were cloned by RACE. The2276bp AaAOC-promoter was cloned by genomic walking. Several elements such as W-box, G-box and ABRE-box existed in the promoter which involved in hormone and stress responsiveness. The expression of AaAOC can be induced vigorously by different hormones and stresses, too. The AaAOC promoter-GUS fusion study showed that AaAOC expressed ubiquitiously in all organs of transgenic A. annua plants, which agreed well with the results of RT-PCR and RT-Q-PCR. In AaAOC-overexpressing transgenic lines, the content of endogenous JA was increased and was2to4.7-fold of the control, and the density of gland trichomes was also increased and was1.5to1.8-fold of the control. RT-Q-PCR showed that the expression levels of FPS, CYP71AV1and DBR2were increased significantly in AaAOC-overexpressing transgenic lines, resulting in significant increase of artemisinin, dihydroartemisinic acid and artemisinic acid in AaAOC-overexpressing transgenic lines.3. Overexpression of ADS, CYP71AV1and CPR genes promoted the metabolic flux to flow towards the biosynthesis of artemisinin, and effectively increased the artemisinin content in transgenic A. annua plants.Overexpression of ADS, CYP71AV1and CPR genes in A. annua was firstly achieved in this study. Southern blot analysis revealed the low-copy insertions existed in the genomes of transgenic A. annua plants. RT-Q-PCR analysis showed that the expression level of ADS, CYP71AV1and CPR genes were increased and HPLC analyses showed that the artemisinin contents were significantly increased in the transgenics plants. One of the transgenic plants, ACR16, was found to contain2.4-fold artemisinin (15.1mg/g DW) of the control plants (pCAMBIA2300+transgenic plants). Our study demonstrated that overexpression of ADS, CYP71AV1and CPR genes together in A. annua could effectively increase the artemisinin content in transgenic A. annua plants. |
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