黄花蒿培养细胞中青蒿素合成代谢的体外调节

黄花蒿培养细胞通过两步培养积累青蒿素．第１步在含有０．２～０．４ｍｇ／Ｌ６－苄基氨基嘌呤（６－ＢＡ）和３～４ｍｇ／Ｌ吲哚乙酸（ＩＡＡ）的Ｎ６培养基中进行细胞的增殖培养，第２步将培养好的细胞转入含０．２～０．４ｍｇ／Ｌ６－ＢＡ和０．２～０．４ｍｇ／ＬＩＡＡ的改良Ｎ６培养基中进行青蒿素的合成．青蒿素的合成量为１９０μｇ／ｇ干细胞左右．当在第２步培养中加入青蒿素合成前体青蒿酸，青蒿素合成量比仅靠激素诱导提高了３倍多．青蒿素的合成途径是植物固醇合成途径的分支途径，当在青蒿素合成过程即第２步培养中加入固醇生物合成抑制剂双氯苯咪唑和氯化氯胆碱处理，可使代谢向合成青蒿素的方向移动，青蒿素合成量明显提高．经２００ｍｇ／Ｌ氯化氯胆碱处理２ｄ，黄花蒿细胞合成青蒿素量为３７２μｇ／ｇ干细胞；经２０ｍｇ／Ｌ双氯苯咪唑处理４ｄ，黄花蒿细胞合成青蒿素量为１５４０μｇ／ｇ干细胞，比靠激素诱导提高了８倍多，与诱导脱分化细胞的黄花蒿叶中所含的青蒿素（３０００μｇ／ｇ干细胞）处于同一个数量级．以上结果表明：在通过植物激素调节可以合成青蒿素的黄花蒿培养细胞中，缺乏青蒿素合成前体是青蒿素合成量低的重要原因．因此，在青蒿素合成的过程中通过体外调节，

Artificial Regulation of Artemisinin Biosynthesis Metabolite in Cultured Cells of Artemisia annua L.

Artemisinin can be biosynthesized in cultured cells of Artemisia annua by plant hormone regulation during suspension culture. Two step culture system was established: First, the cells grew in N 6 medium with 0 2～0 4 mg/L 6 BA and 3～4 mg/L IAA for proliferation. Then, the cells cultured by the above method were inoculated to the modified N 6 medium containing 0 2～0 4 mg/L 6 BA and 0 2～0 4 mg/L IAA for artemisinin biosynthesis. The artemisinin productivity was about 190 μg/g dry cell. While arteannuic acid, direct precursor of artemisinin biosynthesis, was fed during the course of artemisinin biosynthesis of cultured cells, artemisinin productivity increased more than 3 times compared with that relying on plant hormone regulation mentioned above. The result demonstrated the productivity of artemisinin depends on the amount of its precursor in cultured cells which possessed capability of artemisinin biosynthesis by hormone regulation. Another attempt was made to increase the accumulation of artemisinin by using various inhibitors of sterol biosynthesis. The pathway of artemisinin biosynthesis shared mevalonate pathway with that of plant sterol biosynthesis before the formation of farnesyl pyrophosphate, and free sterol inhibited the synthesis of co precursor of sterol and artemisinin. It appeared reasonable that the production of artemisinin might be increased as sterol inhibitors inhibited one or more enzyme in the mevalonate pathway after the formation of farnesyl pyrophosphate which resulted in a shunt toward artemisinin production rather than sterol production, and the feedback regulation of free sterol might be eliminated because of no more sterol. The effects of two sterol inhibitors, miconazole and chlorocholine chloride (CCC), on artemisinin biosynthesis were obtained. While the two inhibitors were added to the medium during artemisinin biosynthesis of cultured cells, respectively, cultured cells treated with 200 mg/L CCC for 2 days accumulated artemisinin 372 μg/g dry cell whereas cells treated with 20 mg/L miconazole for 4 days produced artemisinin 1 540 μg/g dry cell.