The Cloning And Functional Study Of AaWRKY3Transcription Factor In Artemisia Annua L.

The world famous medicinal plant Artemisia annua L., producing thesesquiterpene lactoneendoperoxide artemisinin, has been used in traditionalChinese medicine for more than2,000years. Artemisinin is the centralcomponent of artemisinin combination therapy. A. annua is the only plantresource to extract artemisinin in the world, but the artimisinin productivityfrom it is very low, ranging from0.1to0.8%of the dry leaf weight. A. annuais also found to have strong tolerance to stresses. Currently more researcheshave been focused on the biosynthesis of artemisinin, but less focused on itsstress tolerance.The WRKY transcription factors are one of the biggest transcriptionregulators in plants. The most typical characteristics of them are the DNAbinding domain. This domain is called WRKY domain just after the invariantWRKYGQK amino acid sequence at the N-terminus. It contains about60residues and untypical zinc-finger structure (Cx4-5Cx22-23HxH or C_x7Cx₂₃HxC)at the C-terminus. According to the number of WRKY domain and thestructure of the zinc-finger, the WRKY transcription factors can be dividedinto three groups. There are two domains in Group I proteins and one domainin Group II and Group III. The Group III contains the zinc-fingerC_x7Cx₂₃HxC. The WRKY transcription factors have high affinity with the Wbox （TGACC/T）. The invariant amino acid sequence and the cysteine and histidine residues which tetrahedrally coordinate a zinc atom of the WRKYdomain are both required for the W-box-dependent binding activity.In many plants, some physiological processes, such as regulation ofbiosynthetic pathways, hormone signaling, abiotic stess tolerance, seed coatand trichome development and senescence are all related to WRKYtranscription factors. Moreover, the expression of some WRKY transcriptionfactors is strongly induced by wounding and pathogen infection.In this research, in order to explore the function of WRKY transcriptionfactors in artemisinin biosynthesis and the stress tolerance, AaWRKY3iscloned. The cloned gene is further analyzed by various means includingbioinformatics analysis, tissue expression analysis, yeast one hybrid and theexpression profiling analysis after hormones and stress treatments. The resultsare as follows:1. Cloning and analysis of AaWRKY3transcription factor.The cloned AaWRKY3is found to have an ORF coding324aminoacids, with molecular weight of35907.1and the pI of8.83. The sequenceis aligned with74WRKYs from Arabidopsis. The result shows that thistranscription factor has closer relationship with AtWRKY18, AtWRKY40and AtWRKY60. Further protein alignment analysis illustratesAaWRKY3belonges to Group II WRKY transcription factor.2. AaWRKY3has low relationship with artemisinin biosynthesis.Expression analysis shows that AaWRKY3has different expressionpatterns in different tissues and leaf developments in A. annua, with thehigher expression in roots and lowest expression in leaves. The expressionof AaWRKY3increased along with the development of leaves. Theseexpression patterns are different with those of the key enzyme genes such as ADS, CYP71AV1and DBR2in artemisinin biosynthetic pathways. Theyeast one hybrid experiment shows that this gene can not combine withthe Wa box of ADS promoter. All these results indicate that AaWRKY3haslow relationship with artemisinin biosynthesis.3. AaWRKY3is found to be strongly induced by abiotic stresses.It is found that after hormone and stress treatments the expression ofAaWRKY3is induced by hormone treatments, but strongly induced byabiotic stresses such as high salt, drought and the cold. It is speculated thatthis gene plays a role in abiotic stress tolerance.In summary, in this research, it is found that AaWRKY3has lowrelationship with artemisinin biosynthesis, but may play a role in abioticstress tolerance. This study provides a basis for further illucidating themechanism of stress tolerance of AaWRKY3in the future.