Transcriptome Sequencing Of Huperzia Serrata And Mining Of Genes Related To Hup A Biosynthesi

Huperzia serrata(Thunb.)Trev,also known as Qian Ceng Ta,is a perennial herbaceous fern of Huperziaceae.It has a long history of clinical application in Chinese folk medicine for pain relief and treatment of swelling,detoxification.The alkaloid of huperzine A(Hup A),isolated from H.serrata,is an efficient,reversible and highly selective acetylcholinesterase inhibitor(AChEI),it can be used to treat senile dementia,myasthenia gravis,and improve human learning and memory capabilities.Now,Hup A has become one of the most effective drugs for treating Alzheimer’s disease at home and abroad.The content of Hup A in H.serrata is extremely low,only around one in ten thousandth,further more,H.serrata is dwarf and growing slowly.In this case,the traditional extraction and separation methods are difficult to meet market demand.Another way to produce Hup A is the organic synthesis method,however,the method is very complicated since multiple steps are involved,with low yields and challengs in preparation of optically pure products.With the development of biotechnology in recent years,the biosynthetic pathway and the genes which regulate and modify the biosynthesis of intermediates have been elucidated.Under this circumstance,it is possible to achieve a directional synthesis or large-scale production of the target product.As an example,reconstruction biosynthesis pathways of taxol,artemisinic acid and indole-type alkaloids in engineered systems have been completed in large-scale production.It is thus not strange to see that elucidating the key enzymes,involved in the biosynthesis of Hup A is now attracted much attention.In this study,transcriptome technologies have been applied to mine the key enzyme genes,involved in the biosynthesis of Lycopodium alkaloids.The following results have been achieved:1.Applying LCMS-IT-TOF to analyze Lycopodium alkaloids from root,stem and leaf of H.serrata,10 outstanding compound peaks were identified as Lycopodium alkaloids.After being compared with the standard substance(Hup A,B and C),it was determined that 7 out of the ten peaks represents Huperzine A(Hup A),Huperzine B(Hup B),Huperzine C(Hup C),N-Methylhuperzine A,Huperzine M(Hup M),Huperzimine and Lycoposerramine C(or 11α-Hydroxyfawcettidine),respectively2.With UPLC-Q-Trap technology,the mentioned seven compounds were further analyzed in order to discover the alkaloid’s regularities of distribution in H.serrata’s root,stem and leaf,as well as its provinces of origin(Hunan,Fujian,Yunnan).The result shows the distributions of Hup A,Hup B and Hup M have the same tendency:leaf>stem>root,and Yunnan>Fujian>Hunan;while the distribution of Hup C has a different tendency:root>stem>leaf,it is the only one that has this opposite distribution tendency among all 7 alkaloids.In P.cryptomerianus,the contents of Hup A,Hup B and Hup M are high,yet there’s barely any Hup C;there’s almost no Lycoposerramine C(or 11α-Hydroxyfawcettidine)in the root,while it has a presence in leaves.All the other compounds are with no distribution difference among different parts of plants,nor among different origin of plants.3.The root,stem and leaf samples of H.serrata from Hunan,Fujian and Yunnan and the leaf of P.cryptomerianus were also analyzed by transcriptome sequencing.A total of 789,749 transcripts,398,420 unigenes of H.serrata,and total 310,668 transcripts and 232,716 unigenes of Phlegmariurus cryptomerianus were obtained.Functional annotation and expression difference analysis were conducted on obtained unigenes with Bioinformatics technology.4.According to the biosynthetic pathway of Hup A proposed by the literature isotope labeling experiment,the relevant genes(enzymes)that may be involved in the biosynthesis of Lycopodium alkaloids were screened:1).LDC and CAO sequences from pubilished literatures,along with 10 novel LDC sequences,were found from the unigenes of H.serrata;4 novel LDC sequences and CAO sequences were found from unigenes of Phlegmariurus cryptomerianus.2).Using keywords of "alkaloid","polyketide","chalcone","P450",and"HMG" to search in Gene Similarity Table of H.serrata-P.cryptomerianus,13,10,3,49 and 10 related genes were found,respectively.3).Conducting different analysis between alkaloid’s distribution in roots,stems,and leaves,and the amount of expression in the transcriptome data,7 of Ⅲ polyketide synthase,12 of P450 enzymes,and 12 of coenzyme A related enzymes were found.4).Searching "isomerase" in differentially expressed genes of root and leaf from Hunan,Fujian,and Yunnan,33 results found in Fujian samples,23 results were found in Yunnan samples,and 37 results were found in Hunan samples.There are 11 identical genes.In summary,using alkaloid distribution-transcriptome data correlation analysis,this study has found several genes that might participate in the biosynthesis of Lycopodium alkaloids.It will provide reference for clone and authentidation of key enzyme in alkaloid biosynthesis in the future.