Effects Of Light On Artemisinin Biosynthesis And The Function Study Of Cryptochrome 1 Gene In Artemisia Annua

Artemisia annua is an important medicinal plant.At present,artemisinin is the most widely used and the best anti-malarial drugs.The metabolic regulation of artemisinin has been the research focus all along.Reaserch suggested that the yield of artemisinin could significantly increase in the condition of light,salt,heavy metals and other abiotic factors.The change of light intensity and illumination time would affect the accumulation of artemisinin.Plants response to different light quality,light intensity and light cycle through the light receptor,such as phytochrome,cryptochromeandUV-Bphotoreceptorcells.Overexpressing Arabidopsis phyB in Artemisia annua could increase the synthesis of artemisinin and artemisinic acid.So it is meaningful to explore the metabolic regulation of the blue light receptor on artemisinin pathway by cloning and analyzing the A.annua CRY1 gene.In order to study the effect of light and light receptors on the metabolism regulation of artemisinin,a series of work had been carried out with the high yield artemisia species as the main research object in the aspects of light regulation,RNA-seq,cloning,phylogenetic analysis,expression analysis,and functional verification of transgenic plants.Results have been achieved as following:1.Light,especially blue and red light,participates in the regulation of of key enzyme genes expression in the artemisinin biosynthesis pathway.The 3 weeks A.annua seedlings were treated in the dark.The expression levels of ADS,CYP71AV1 and DBR2 were continuously decreased by qRT-PCR analysis.The expression level of key enzyme genes increased gradually after moving to white light.The results of the transformation from dark to monochromatic light indicated that the expression of the key enzyme genes increased in red and blue light,and the far red light had no significant effect.Therefore,light,including blue and red,involved in the regulation of the key enzyme genes expression of artemisinin.The expression of the key enzyme gene was affected by light intensity.The expression increased under strong light,the enhancement effect:80μmolm^-2s^-1>40μmolm^-2s^-1>10μmolm^-2s^-1;while low light intensity reduces the expression.2.RNA-seq showed that blue light could regulate the transcription factor related to artemisinin synthesis,and thus regulate the synthesis of artemisinin pathway.Selecting A.anuua in 40μmolm^-2s^-1 blue light treatment for 6 h and24 h as samples,while using dark environment as a control.RNA-seq was performed to obtain the total transcript of A.annua.206402 unigenes sequences were obtained.The results of GO anlysis and KEGG pathway revealed that blue light involved in the metabolic process,especially terpene metabolism.of Enrichment analysis of differentially expressed genes in KEGG pathway showed that blue light treatment could regulate the metabolism of terpenoids,and it took some time to regulate the production.Screening of transcription factors which associated with artemisinin metabolism through differential expression profile analysis.Blue light could regulate the expression of several genes involved in metabolic pathway by regulating transcription factors,thus regulating the metabolism of artemisinin.3.Cloning and analysis of A.annua CRY1Cloning AaCRY1 gene,Bioinformatics analysis showed that AaCRY1protein has high homology with other plants CRY1 protein.Overexpressing AaCRY1 in Arabidopsis thaliana cry1 mutant,the seedlings hypocotyls elongation was significantly inhibited under blue light,hypocotyls length was shortened.AaCRY1 is involved in the blue light-mediated plant photomorphogenesis.Yeast two-hybrid and subcellular co-localization showed that AaCRY1 could interact with COP1for blue light signal transduction,and then regulating gene expression.4.Overexpression of AaCRY1 gene could enhance the expression of key enzyme genes in the artemisinin biosynthetic pathway,and thus increase the content of artemisinin in transgenic plants.AaCRY1 gene was overexpressed in A.annua plants.The results of western blot showed that the AaCRY1 gene was integrated into the genome of transgenic A.annua.The results of qRT-PCR indicated that the expression of ADS,CYP71AV1 and DBR2 were significantly increased in transgenic lines.Compared with the wild type,the expression of ADS in transgenic plant increased by 7.4 times,the expression of CYP71AV1 in transgenic plant increased by 9.1 times,the expression of DBR2 in transgenic plant increased by 5.7 times.HPLC-ELSD analysis showed that the content of artemisinin in transgenic A.annua plants was 1.6 times to 2times higher than that of the control,which proved that overexpression of AaCRY1 gene can improve the content of artemisinin.