Study On Rapid Propagation Of Matricaria Chamomilla L. And Genetic Transformation Of FPS Gene In Tobacco

Chamomile(Mauritania chamomile L.), an annual herbs from Asteraceae family, isone of the aromatic and medicinal plants, chamomile essential oil has been proved to haveeffects on antimicrobial, anti-inflammatory, antispasmodic, sedative, analgesic andanti-carcinogenic. The main component of the volatile oil of chamomile is chamazulene,which is a sesquiterpene compound, its biosynthetic pathway is isoprenoid metabolicpathway, whereas farnesyl pyrophosphate synthase (FPS) is one of the rate-limitingenzymes in this pathway. In the study, one side, we established rapid propagation systemof chamomile through plant tissue culture; on the other hand, we cloned FPS gene,constructed its expression vector and identified it in tobacco. These may providetheoretical evidence for establishment of genetic transformation system and breeding ofimproved variety of chamomile. The research results are as follows:1. An effieient regenearting system of Mauritania chamomile L. was established.With chamomile seeds as explant for the tissue culture in order to obtain steriled seedling.With petioles of steriled seedling as explant, effects of the same or differentphytohormones as well as their combinations on callus induction, adventitious budsinduction and adventitious roots of chamomile were respectively studied in oder to choosethe most suitable medium for the tissue culture of chamomile. The results show that withinthe scope of the study, the optimal culture medium for chamomile callus induction isMS+6-BA2.0mg/L+NAA0.3mg/L, callus induction rate is91.6%; the optimal culturemedium for chamomile adventitious bud induction is MS+6-BA1.0mg/L+NAA2.0mg/L,adventitious bud induction rate is79.4%; the optimal culture medium for chamomileadventitious roots induction is MS+6-BA0.3mg/L+NAA0.5mg/L, roots induction rate isclose to100%, steriled seedling survived easily for transplanting in the matrix vermiculite:perlite: peat (1:1:1).2. Eukaryotic expressing vector of FPS gene was constructed. According to mRNAsequence of FPS gene, the specific primers were designed. We cloned FPS gene by thespecific primers with the template of the total RNA from inflorescence of German chamomile, FPS gene was Connected to pEASY-T4Zero Cloning Vector, which wastransformed into Trans1-T1competent cells for cultivate. It turned out that1032bpfragment was obtained by RT-PCR with the total RNA of chamomile as template and thefragment encoded343amino acids. FPS gene was connected to the vector pBI121andplant expression vector pBI121-FPS was successfully constructed.3. Transformation and expression of FPS gene in Tobacco were studied. Byfreeze-thaw method, eukaryotic expressing vector of FPS gene was transformed intoAgrobacterium tumefacien. With leaves of teriled seedling of tobacco as explant, theleaves was cultivated for3d, agrobacterium tumefaciens were diluted OD6000.6andinfected leaves for10min, then leaves were dried sterilely and transferred into co-culturemedium for3d in dark. When leaves were got out of bacteriums, they were transferred intothe medium containing75mg/L Kan and400mg/L Cef for culture in illumination at25℃.When adventitious buds grown into2cm, they were transferred into the medium containingKan for taking root. Finally, we successfully got the resistant plants of tobacco. In thisstudy, we tested the resistant plants of tobacco by PCR, the results prove that2of7resistant plants of tobacco have the target gene, so the target gene has been transferred to intobacco preliminarily.