Molecular Cloning And Expression Analysis Of Genes Involved In Berberine Alkaloids Biosynthesis In Corydalis Saxicola Bunt. And Microbial Transformation Of Steroidal Alkaloids In Veratrum Nigrum Linn.

Corydalis saxicola Bunting (Yanhuanglian) is one of rare medicinal plants for the treatment of hepatic disease which belongs to the Fumariaceae family. C. saxicola grows in south China, such as Guizhou, Yunnan, Szechwan and Guangxi province and only grows in limestone hills in the wild state. It was labeled as endangered plants in southern China because of its hard growth environment. C. saxicola has the effect of clearing away heat, relieve pain and arrest blood.It has been demonstrated to have many pharmacological activities, including antibacterial, antiviral, and anticancer activities. The main active constituent in C. saxicola is berberine alkaloids (e.g. berberine, dehydrocavidine), which has potential activity to treat hepatitis, hepatocirrhosis and liver cancer.(S)-tetrahydroberberine oxidase ((S)-THBO) is a key enzyme in the biosynthesis of berberine alkaloids which catalyzes the dehydrogenation of four hydrogen atoms of (S)-Tetrahydroberberine to produce berberine, the final step of berberine biosynthesis. A (S)-THB gene, designated as Cs(S)-THBO (Genbank accession No.HQ393909), was cloned from a C. saxicola cDNA library. The full-length of Cs(S)-THBO was 1127bp contains an open reading frame 699bp and predicted to encode a 232-amino acid polypeptide, with a predicted molecular mass of 25.20 kDa. Cs(S)-THBO was the first (S)-THBO gene found in C. saxicola. Real-time quantitative PCR analysis indicated that Cs(S)-THBO was constitutively expressed in roots, stems, leaves and flowers of C. saxicola, with the highest expression level in roots. The results from treatment experiment for plant defense responses revealed that expression of Cs(S)-THBO had a prominent diversity. Recombinant Cs(S)-THBO protein expressed in E. coli strain BL21 (DE3) was active. The results of feeding experiment and HPLC-DAD-ESI-MSn analysis shown that Cs(S)-THBO had the fuction of catalyzing (S)-Tetrahydroberberine to berberine but it cannot catalyze (R)-etrahydroberberine to berberine.(S)-norcoclaurine synthase, the first step of the berberine alkaloids biosynthetic pathways, catalyzes from dopamine and 4-hydroxyphenylhydroxyphenylacetaldehyde (4-HPAA)to (S)-Norcoclaurine. Based on the core sequence obtained by RT-PCR, several gene-specific primers were designed to obtain the sequences of 5'-end and 3'-end by RACE. According to the result of RACE,PCR was performed and a full-length cDNA of Cs(S)-NCS.The full-length cDNA of Cs(S)-NCS was 1003 bp containing 5'-UTR, 3'-UTRand a ORF of 621bp encoding a 207-amino acid polypeptide(Genbank accession No.JF488074).Canadine synthase catalyzes the eighth step in the biosynthesis of berberine alkaloids. The enzyme catalyzes the conversion of (S)-tetrahydrocolumbamine ((S)-THC) to (S)-tetrahydroberberine ((S)-THB) and it play an important role in the pathway of berberine. A CSN gene, designated as CsCSN (Genbank accession No.HQ393908), was cloned from a C. saxicola cDNA library. The full-length of Cs(S)-THBO was 1256bp contains 5'-UTR, 3'-UTR and an open reading frame 783bp and predicted to encode a 261-amino acid polypeptide, with a predicted molecular mass of 29.19 kDa.Part 2 Microbial transformation of steroidal alkaloids in Veratrum nigrum Linn.Veratrum Linn. (Liliaceae) is a kind of Perennial herb which characterizes by acrid and bitter in taste and cold in nature. The roots and rhizomes of several Veratrum species, such as V.nigrum L., V.dahuricum(Turcz.)Loes.f.and V.Schindleri L, are used in traditional Chinese medicine as''Li-Lu''for treatment of aphasia arising from apoplexy, dysentery, jaundice, headache, scabies and chronic malaria. The main active components in Veratrum Linn. is steroidal alkaloids. Pharmacological research indicated that steroidal alkaloids had cardic, hypotensive and antithrombotic activities , and the stilbenes had antitumor and anticoagulant function. According to the statistical data shows that there are more than 100 kind of steroidal alkaloids had isolated from Veratrum in China. However, it is well-known for V. nigrum L. that the toxic component is also steroidal alkaloids.Microbial transformation is the process by which an organism or its isolated enzyme brings out chemical change on molecules that not part of their metabolism. Microbial transformation is important in the production of commercially valuable compounts for the pharmaceutical industry. Because the well antitumor activity of steroidal alkaloids in Veratrum Linn. , we has been done some structure modification of steroidal alkaloids in Veratrum Linn.by microbial transformation with a view to obtaining more new inhibitors(low toxicity, high activity and special mode of action.) of Hedgehog pathway.Microbial transformation of veratramin by Cunninghamella echinulata in present study and obtained 4 new compounds.