Study Onfunctional Differentiation Of AaDXS Gene Familyand Molecular Mechnisim Of Low Temperatureimproving Artemisinin Production In Artemisia Annua L.

Malaria is an infectious disease which is seriously harmful to human health with an estimated number of 400 million infections and more than 1 million deaths per year around the world.Artemisia annua L. is a Chinese traditional herbal plant species that is well known as the only natural source of endoperoxide sesquiterpene lactone artemisinin for malaria treatment. The content of artemisinin in A. annua is, however, at a low level as 0.01-0.8% of dry weight. Plant secondary metabolitesengineering is one of the effective strategyto improve the artemisinin production.As a results, the functionof genesinvolving in artemisinin biosynthetic pathway, especially for members of gene family, would deepen understand the mechanism of artemisinin biosynthesis.(1)Cloning and characterization DXS gene family in A. annua The carbon skeleton of artemisinin, a sesquiterpenes compound, is derived from MVA pathway located in cytoplasm and MEP pathway located in plastid. The previous study reported that MEP pathway provided one molecular IPP to form central isoprenoid unit of FPP, which suggest that MEP pathway is the initial step involving in artemisinin biosynthesis. 1-Deoxy-D-xylulose 5-phosphate synthase(DXS), the first key enzyme in MEP pathway, can catalyze the condensation between pyruate and glyceraldehyde 3-phosphate to produce 1-Deoxy-D-xylulose 5-phosphate(DXP). In other plants such as Arabidopsis and maize, DXS is a small family constituted with 2-4 memberships. Unfortunately, the function of A. annua DXS gene family has not been report until now.In this study, RACE was performed to clone four genes coding DXS in A. annua. The amino acid alignment analysis showed that Aa DXS4 was different from the others DXS genes because of lack of the conservative amino acid binding sites for GAP and TPP. Phylogenetic tree showed all the DXS genes clustering of three branches, including Aa DXS1 belonged to DXS1 clade, Aa DXS2 and Aa DXS3 belonged to DXS2 clade, while Aa DXS4 was clustered to DXS3 clade. Actually, the DXS2 clade genes usually associated with plant secondary metabolism biosynthesis. Subcellular localization vector was constructed and introduced into tobacco protoplast, the results show that all Aa DXS genes were located in plastid byconfocal laser scanning microscope,which was consistent with the fact that proteins of MEP pathway expressed in plant plasmid.(2)Screening of the stable reference genes provided foundation for the study of DXS genes expression pattern analysisIn recent years, real-time quantitative PCR(q PCR) has been becoming a mainstream technology for gene expression profile analysis due to the higher sensitivity and specificity. In this study, q PCR was used to analysis the exprssion difference of Aa DXS family genes in different organs or plants under multi-treatments. However, the selection and validation of reference genes are essential for gene expression studies by real-time quantitative PCR. Unfortunately, few reference genes of A. annua have been estimated for real-time quantitative PCR until now. In order to select the most suitable reference gene for gene expression quantification by q PCR in A.annua, ten housekeeping genes, including 18 S ribosomal RNA(18S r RNA), actin(ACT), cyclophilin(CYP), elongation factor 1?(EF1?), glyceraldehyde 3-phosphate dehydrogenase(GAPDH), DNA-directed RNA polymerase II subunit(RP?), 60 S ribosomal protein L13(RPL13D), tubulin alpha chain(TUA), tubulin beta chain(TUB) and ubiquitin(UBQ), were used to identify reliable reference genes for normalization of q PCR data obtained from different organs of A. annua or under different treatments including cold and heat stress, methyl jasmonate(Me JA) and abscisic acid(ABA). Two Excel-based software tools, ge Norm and Norm Finder, were applied to test the expression stability among the ten candidate housekeeping genes. In order to verify the stability of the candidate reference genes, ADS gene expression levels were investigated in different organs and the expression levels of ADS, CYP71AV1 and DBR2 were surveyed under Me JA treatment, respectively.As expected, the variation in expression stability of the ten candidate reference genes tested in this study suggested there was no single reference gene that can be used for all experimental conditions in A. annua. The combination of RP?&EF1? was the most stably expressed reference genes for different organs. Under phytohormone treatment, the combination of EF1? &TUB was recommended as internal reference genes used for investigating target gene expression levels. In addition, the combination of ACT&EF1? was suitably chosen for normalization in temperature-shocked samples. However, the popular reference genes, such as CYP and 18 S r RNA had the poor performance of gene expression stability in A. annua. In order to further verify the reliability of the experimental results, RP?&EF1? were used in combination as reference genes to examine the expression levels of ADS gene in different organs. Meanwhile, the expression levels of ADS, CYP71AV1 and DBR2 were tested by q PCR normalized with the combination of EF1? &TUB in Me JA treatment samples. The validation assays showed that EF1? and RP? were more suitable to be used as reference gene than 18 S r RNA in different organs samples, and CYP could not be used as reference gene used for normalization in Me JA treated plants.Our study will benefit future research on the expression of genes from A.annua plants under different experimental conditions.(3) Expression patterns analysis of DXS gene family in different organs and in plants under different treatmentsThe expression patterns of gene family memberships in tissue and induced materials explain partly the functional difference. According to the results of scanning the stable reference genes in A. annua, the q PCR was used to analysis the expression patterns of Aa DXS genes in different organs and leafs. Meanwhile, the abiotic induced expression patterns of Aa DXS genes were assayed by q PCR in A.annua under the treatments of Me JA, light, wounding, salt and low temperature, respectively. The results showed that the expression of Aa DXS3 was the highest level in flowers among the all detected organs. In different leafs, the highest expression level of Aa DXS3 was in apical bud and the lower expression level in mature leafs, which was consist with the expression pattern of ADS and suggested that the expression level of Aa DXS3 may be associated with the density of GSTs. The expression levels of Aa DXS2 and Aa DXS3 were induced up-regulation in Me JA treated plants and Aa DXS3 was induced more strongly than Aa DXS2. Aa DXS3 gene can quickly respond to light in 5 min,which was in line with the expression patterns of artemisinin biosynthesis related genes ADS and CYP71AV1. Aa DXS1 was the unique gene which expression level was up-regulation in wounding plants, however, the expression level of Aa DXS3 was significantly decrease. Both of Aa DXS2 and Aa DXS3 were responseto salt stress, which lead to the increase of exprssion level. The expression levels of memberships of Aa DXS gene family were decreased during the early stage of low termperature stress. However, the expression levels of Aa DXS2 and Aa DXS3 were increased significantly than control after 6 h.In order to further study the expression location of Aa DXS3 in A. annua, the promoter region of Aa DXS2 and Aa DXS3 were cloned by FPNI-PCR method. plant CARE analysis showed that p Aa DXS2 contain multiple cis-acting elements involved in light responsiveness, Me JA-responsiveness and low-temperature responsiveness. The promoter of Aa DXS3 contained cis-acting elements involved in light and Me JA responsiveness. Additionally, There are present several transcription factors binding site for MYB, b ZIP and WRKY in p Aa DXS3. Histochemical GUS Stainingshowedthat p Aa DXS3 promoter GUS expression in trichomes of transgenic Arabidopsis plants. The artemisinin biosynthesis is location in glandular secretory trichomes of A. annua. These results suggested that the function of Aa DXS3 may well provide the artemisinin precursor IPP and DMAPP.(4)The molecular mechanism of Aa DXS3 expression level up-regulation and artemisinin production improvement in A.annua plants under low temperature stress Low temperature is one of the most harmful abiotic stresses and can increase the content of secondary metabolites to enhance cold tolerance in plants.Previous publications reported that the artemisinin level was increased in Artemisia annua following a night-frost period. However, the molecular mechanism was not clear.To better understand how low temperature stress enhances artemisinin production, we investigated the effect of exogenous Me JA treatment on reducing the chill injury in A. annua. Using a time-course, we analyzed the contents of endogenous JA, artemisinin and related metabolites under the cold stress and the expression levels of JA biosynthetic genes, TFs regulating artemisinin biosynthesis and artemisinin biosynthetic genes in A. annua.We found that exogenous jasmonate effectively enhanced the freezing tolerance of A. annua. The jasmonate biosynthetic genes(LOX1, LOX2, AOC and JAR1) were induced by cold stress, leading to an increase in endogenous jasmonate in cold-treated A. annua. Increased endogenous jasmonate enhanced the expression of three jasmonate-responsive transcription factors, ERF1, ERF2, and ORA, all of which were reported to transcriptionally activate the expression of artemisinin biosynthetic genes, such as ADS, CYP71AV1, DBR2 and ALDH1. Furthermore, the expression levels of the four artemisinin biosynthetic genes were also significantly increased under cold stress. Consequently, the levels of artemisinin and related secondary metabolites, such as dihydroartemisinic acid, artemisinin B and artemisinic acid, were increased in A. annua under cold stress. Our study points to a molecular mechanism in which the production of artemisinin is regulated by cold stress in A.annua.