Cloning And Genetic Transformation The Key Genes For Artemisinin Synthesis Of CYP71AV1 And ALDH1

Artemisinin is a sesquiterpene lactone compound extracted from Artemisia annua.The secretory glandular hairs of Artemisia annua are the place where artemisinin is synthesized and stored.They are mainly distributed on the tender leaves and flower buds of Artemisia annua.The density,size and cell metabolism level of secretory glandular hair are positively correlated with artemisinin content.AaCYP71AV1 and AaALDH1 are the key enzyme genes for artemisinin synthesis.In this study,the promoters and gene sequences of Artemisia annua AaCYP71AV1 and AaALDH1 were cloned to construct a constitutive plant expression vector 35S::AaCYP71AV1,35S::AaALDH1 and GUS reporter vector of AaCYP71AV1 and AaALDH1 gene promoters.Using the leaf discs of Artemisia annua with the key enzyme gene iaa M for auxin synthesis obtained in the early stage of the laboratory as the recipient material,the bivalent genetic transformation study was carried out to promote gene overexpression of Artemisia annua AaCYP71AV1 and AaALDH1 in Artemisinin biosynthesis pathway based on promotion of auxin synthesis in the glandular hairs of the univalent transfection of iaa M gene.It is hoped that while increasing the growth rate of glandular hair cells and activating cell metabolism,more precursors of artemisinin synthesis can be obtained to effectively increase the content of artemisinin.The study has successfully established a bivalent transgenic Artemisia annua genetic transformation system with endogenous artemisinin synthase gene and exogenous auxin synthase gene.The size,density and artemisinin content of the glandular hairs in the leaves of the obtained transgenic plants were significantly increased.The main findings are as follows:1.According to the AaCYP71AV1 and AaALDH1 gene promoter sequences and CDS sequence information of Artemisia annua included in Genbank,the promoter sequences of AaCYP71AV1 and AaALDH1 genes and the c DNA sequences of AaCYP71AV1 and AaALDH1 genes were cloned by PCR and RT-PCR techniques.AaCYP71AV1Pro::GUS,AaALDH1Pro::GUS reporter vector and plant expression vectors of 35S::AaCYP71AV1 and 35S::AaALDH1 were respectively constructed according to the above promoters and genes after sequencing analysis.2.Using the leaf disc transformation method mediated by Agrobacterium to transform the GUS reporter vector(AaCYP71AV1Pro::GUS,AaALDH1Pro::GUS)with Artemisia annua variety(428-A)as the recipient to obtain transgenic regenerated plants.GUS histochemical staining results showed that the GUS reporter gene driven by the above two promoters had high expression activity in the glandular hairs of Artemisia annua leaves,confirming the specific expression of AaALDH1 and AaCYP71AV1 genes in the glandular hair cells of Artemisia annua.It further shows that glandular hair cells are the key part of artemisinin synthesis and storage.3.Using the monovalent transgenic GTpro::iaa M Artemisia annua as the recipient,the plant expression vector(35S::AaCYP71AV1,35S::AaALDH1)was used for the bivalent genetic transformation of Artemisia annua through Agrobacterium-mediated leaf disc transformation.PCR molecular identification of regenerated plants with the obtained bivalent transgenes(GTpro::iaa M/35S::AaCYP71AV1,GTpro::iaa M/35S::AaALDH1)showed that AaCYP71AV1 and AaALDH1 genes had been successfully transformed and integrated into the genome of Artemisia annua.4.The expression of AaALDH1 and AaCYP71AV1 genes in transgenic Artemisia annua were detected by RT-PCR semi-quantitative and q RT-PCR fluorescence quantitative analysis.The results showed that the expression of AaALDH1 and AaCYP71AV1 genes in the bivalent transgenic Artemisia annua plants was significantly higher than that in the control plants and the monovalent receptor transgenic plants.The results suggest that overexpression of the artemisinin synthase gene is expected to promote the synthesis of artemisinin while the AaALDH1 and AaCYP71AV1 gene expression levels in the monovalent receptor transgenic plants have no significant change from the control plants,which indicating that the specific expression of auxin synthase gene iaa M in glandular trichomes cells has no significant effect on the expression of artemisinin synthase gene.5.According to observation of the glandular trichomes of Artemisia annua leaves by fluorescence microscope and scanning electron microscope,the density of secretory glandular trichomes and the size of apical cells in the leaves of the monovalent receptor transgenic plants and the two bivalent transgenic plants were significantly larger than those of the control plants,and the differences were statistically significant.6.The artemisinin content of the transgenic Artemisia annua plants was detected by HPLC.The results showed that the artemisinin content of the two bivalent transgenic Artemisia annua plants were 27.77mg/g and 28.19mg/g,which were 2.3times and 2.4 times higher than the control plants.The artemisinin content of the monovalent receptor transgenic GTpro::iaa M plant was 15.52mg/g,which was 1.3times higher than that of the control plant.In summary,the specific expression of the iaa M gene in the glandular trichomes of Artemisia annua can effectively increase the concentration of auxin during the development of glandular trichomes,thus promoting the growth rate of glandular trichomes cells and activating cell metabolism,and thus increasing the artemisinin content.On this basis,the overexpression of AaALDH1 and AaCYP71AV1 genes can promote the effective synthesis and accumulation of artemisinin precursors in the artemisinin biosynthesis pathway,and ultimately increase the content of artemisinin.