Transformation With AtNHX1 Gene And Isolation Of O-methytransferase Gene In Gentiana Macrophylla

Gentiana macrophylla(Pall.)which is native to Northwestern China,is a kind of traditional Chinese medicine.Its major secondary metabolite,named as gentiopicroside,has been used in treatment of rheumatism,osteoarthritis,pains,and ulceration.Due to these features for medical purpose,the wild population and habitat of Gentiana macrophylla have been destroyed seriously.The aim of the present work is to provide a flexible way for the protection and utilization of Gentiana macrophylla via cell engineering pathway.Besides establishing the regeneration system,we also want to improve the salt tolerance of Gentiana macrophylla by transgenic method,and to isolate stress related genes from Gentiana macrophylla.Leaf segments of Gentiana macrophylla were adopted as explants.The regeneration system was established through tissue culture.The most effective concentration of 2,4-D and BA for callus induction was 1.0 mg/L respectively,and 83.89%of explants could produce calluses.The highest frequency of differentiation could reach 96.67%on the MB medium supplemented with 0.8 mg/L NAA and 0.25 mg/L KT.The application of AgNO₃ or TDZ could improve the differetiation. Morphological and histological studies confirmed the pathway of somatic embryogenesis in Gentiana macrophylla.Gentiopicroside contents of embryogenic callus,and different stages of somatic embryoids were determined by HPLC.The data revealed gentiopicroside could not be detected in embryogenic calluses.The content of gentiopicroside at globular-,heart-, torpedo-,and cotyledon-shaped stages were 0.2609%,0.3282%,0.0285%,and 0.8062%respectively(dry weight).AtNHX1 gene was introduced into Gentiana macrophylla by Agrobacterium tumefaciens-mediated transformation system.After kanamycin selection,the putative transgenic plants were tested by PCR,RT-PCR and real-time PCR.We also determined the water,proline,soluble sugar,and MDA contents of wild-type and transgenic plants grown at low(5 mmol/L)and high(200mmol/L)NaCl.The results demonstrated that the wide-type was sensitive to salinity stress,while the transgenic plants,which had improved salt tolerance,displayed the traits of halophytes.Nested primers of 3'-RACE were designed on the basis of a cloned fragment. Gentiana macrophylla was treated by SA before RNA extraction.The 3'-end of O-methytransferase was isolated,including 576 bp ORF sequence and 151 bp 3'-UTR.