Functional Verification Of The Jasmonic Acid Receptor Gene AaCOI1 In Artemisia Annua L.

Artemisinin is a new type of C15 sesquiterpene lactone compound form Artemisia annua L..Because of its unique structure and feature of the oxygen bridge,it is very obvious of treatment to cerebral malaria and anti-chloroquine malaria.The latest study found that artemisinin for the treatment of tuberculosis has a significant effect.World Health Organization(WHO)confirmed that the artemisinin combination therapy(ATCs)as the primary choice for the malaria treated,because of the malaria and mycobacterium tuberculosis need artemisinin,The latest study found that artemisinin for the resistance to viruses,anti-tumor activity,treatment of diabetes and tuberculosis and other diseases have a significant effect.So the market demand for artemisinin is very large.However,compared with the chemical synthesis method the current synthesis of artemisinin in Artemisia annua L.is higher and its less toxic than the chemical synthesis method,so artemisinin is mainly from the cultivation of Artemisia annua L.,but the content of artemisinin in wild Artemisia annua L.is very low,accounting for only 0.01%-0.8% of the dry weight of Artemisia annua L.,leading to the shortage of artemisinin,far from meeting the huge market demand.Therefore,increasing the content of artemisinin in Artemisia annua L.is the most eventful research of plant secondary metabolic research.Jas is jasmonic acid and its derivatives collectively,is an important plant signal molecules,plant growth,development,environmental changes in the defense response and plant secondary metabolite accumulation has a vital role.Studies have found that exogenous spraying of methyl jasmonate(MeJA)can make the content of artemisinin increased significantly.COI1 as the sole receptor of the jasmonic acid pathway,mainly SCFCOI1 complex formed with SCF in the plant,which plays an important role in plant growth,salt tolerance and stress resistance.In the tomato,rice and rubber plants in the JA signal pathway in the COI1 receptor-related function has been a certain study,and JA signal pathway on the biosynthesis of artemisinin has an important role in Artemisia annua L.,but for the JA signal The basic elements in the pathway are not very clear.Therefore,this study cloned the receptor COI1 of JA signaling pathway,analyzed andvalidated its biological function,and explored the role of hormone MeJA in improving the content of Artemisia annua L.In this paper,the electronic sequence of AtCOI1 in Arabidopsis thaliana was found on NCBI.The full-length of COI1 gene of Artemisia annua L.was cloned by comparing the COI1 sequence in Artemisia annua L.And the related bioinformatics analysis: The full length of AaCOI1 cDNA is 1767 bp,containing 1752 bp coding domain sequence,encoding 584 amino acids,the protein belongs to the hydrophilic protein,and rich in bright ammonia Acid repeats(LRR)and F-box domains,but there is not have the transmembrane region,complex helix region,signal peptide and other domains.The results of subcellular localization showed that AaCOI1 fusion GFP was specifically localized in the nucleus,and the results of bimolecular fluorescence complementation showed that AaCOI1 interacted with AaJAZ1 and AaJAZ5 in Artemisia annua L.in the nucleus,which was further indicating that COI1 and JAZ interact with transcription factors.Full length of the AaJAZ5 cDNA is 1144 bp,which contained 564 bp CDS,encoding 188 amino acids.Tissue expression using RT-qPCR showed that AaCOI1 and AaJAZ5 are higher expressed in the rootsand lower expressed in leaves,flowers and stems in Artemisia annua L..Meanwhile,the RT-qPCR display AaCOI1 and AaJAZ5 were induced by MeJA in Artemisia annua L.,and highest expression in 3h(P <0.05).These results further confirm that both AaCOI1 and AaJAZ5 are constituent elements of the JA signaling pathway.Finally,overexpressing AaCOI1 into hybridated Arabidopsis because of Arabidopsis thaliana of coi1-1 fruit pods,and Arabidopsis thaliana was found after MeJA treatment of sensitive to MeJA compared with mutant coi1-1,the root will become shorter and wild-type Arabidopsis thaliana,the AaCOI1-coi1-1Arabidopsis thaliana after MeJA treatment increased the content of anthocyanins.These results have demonstrated the important role of AaCOI1 in the methyl jasmonate pathway.In conclusion,our study shows that COI1 and JAZ5 have a significant effect on the jasmonic acid pathway in Artemisia annua L.,which is also highly conserved in Artemisia annua L.,and further demonstrates that the formation of artemisinin is positive response.These evidence are sufficient to confirm that the biological function of AaCOI1 is a receptor for the JA signaling pathway of Artemisia annua L..