Regulation Study Of Key Enzyme Genes And Transcripion Factor AaBBX22 In Artemisin Biosynthesis Pathway

Artemisinin content in Artemisia annua L. is very low, just 0.01~0.1% of dry leaf weight of A. annua L. The method of metabolic regulation is widely used to increase the content of artemisinin in A. annua L. So far the study has been mainly focused on over-expression of key enzymes in artemisin biosynthesis pathway. Besides, the transcription factors related with stresses are also found to play a role in the metabolic regulation of artemisinin biosynthesis.This study aims to explore the efficient strategy on increasing artemisinincontent in A. annua L., which is to overexpress two key enzymes of artemisinin biosynthesis in A. annua L. Two expression vectors featuring double T-borders, integrated with FPS-DBR2 and FPS-ALDH1, have been constructed and transformed into Artemisia annua L. respectively. Moreover, B-BOX–type zinc finger protein gene Aa BBX22 which has relation with response to stresses has been cloned from c DNA library of A. annua L.The results of the study are as follows:1. Double T-border vector integrated with FPS-DBR2 gene combination was constructed. Transgenic plants were obtained through Agrobacterium tumefaciens-mediated transformation. PCR analysis demonstrated the transgenic status of 7 independent plants. Real-time PCR showed that the expression levels of FPS-DBR2 were up-regulated in transgenic plants. HPLC-ELSD analysis showed that artemisinin contents in the transgenic plants were increased up to 3.12 fold of that of the control.2. Double T-border vector integrated with FPS-ALDH1 gene combination was constructed through site-directed mutagenesis. Transgenic plants were obtained through Agrobacterium tumefaciens-mediated transformation. PCR analysis demonstrated the transgenic status of 5 independent plants. Real-time PCR showed that the expression levels of FPS-ALDH1 were up-regulated in transgenic plants. HPLC-ELSD analysis showed that artemisinin contents in the transgenic plants were also increased, up to 1.62 fold of that of the control.3. The full length c DNA sequence of Aa BBX22 was cloned from c DNA library of A. annua L. Aa BBX22 has an open reading frame of 813 bp encoding 270 amino acids, containing two typical B-BOX bingding sites. Phylogenetic tree analysis indicated that Aa BBX22 gene had high similarity with B-BOX-type genes from Vitis vinifera and Glycine max. Real-time PCR analysis showed Aa BBX22 expressed constitutively in all tissues of A. annua with the highest expression in roots and the lowest expression in buds. The expression of Aa BBX22 could be induced by salt and drought treatments, however, ABA, MJ and cold treatments remarkably inhibited the expression of Aa BBX22. Besides wound stress could inhibits the expression of Aa BBX22 to some extent. It is speculated that that Aa BBX22 may participate in salt and drought tolerance regulation.