Exploration And Functional Research Of The Key Genes Related To Pharmacodynamics Substances In Baphicacanthus Cusia

Traditional Chinese Medicine(TCM)plays effect on the basis of its active substances,and the latter also directly relate to substantive connotation on the quality of TCM.Baphicacanthus cusia(Nee)Bremek,a famous-region drug in Fujian.With its leaf and stem are used to extract Indigo Naturalis(Qingdai),and its root is used as a drug ‘Nan-Ban-Lan-Gen' which has the effects of heat-clearing and detoxicating.Due to the large demand,a regardless of the quality planting just lead to lack of germplasm resources protection.Since authentic medicinal herbs play key roles for the clinical effects of TCM,it is very urgent to protect germ plasm resources.It provides unprecedented opportunities to understand organs at the molecular level with the coming of ‘Omic' era,which jumpstart the medicinal plants germplasm resources researches on the technical conditions.The development of the next generation sequencing(NGS)provides a fast,high-throughput and comprehensive interpretation of genetic information of medicinal plants,and provides an effective way to get batch sequences for species with sporadic genetic information.Indole alkaloids represented by indigo and indirubin are important active substances in B.cusia.It will provide strong support for the authentic germplasm resources protection of B.cusia under mining indole pathway genes and revealling their functions.The research contents and results are as follows.1.Successed in cultivating aseptic seedling of B.cusia,and established the rapid propagation cultivation technique to attain abundant tissue culture seedlings,which provided enough materials for trying regeneration plant system and hairy roots system of B.cusia.Meanwhile,stablished regeneration plant system of Isatis tinctoria through exploring conditions of inducing cluster buds,regenerated seedlings' proliferation,rooting and hardening-seedling,which support a complement path for the subsequent gene functions studies of B.cusia.2.Completed the transcriptome sequencing and annotation of B.cusia using Illumina Hiseq platform and established a transcriptome database which contains 293,666 unigenes.Attained 44 encoding genes of 16 enzymes of indole pathway in B.cusia through pathways annotation.B.cusia's gene database was established for the first time,and it laid a solid foundation for obtaining target genes' sequences.3.Screened the best reference genes in B.cusia and validated the optimal genes,as a result,18 S rRNA is the best gene for leaves under external stimuli,whereas UBC is the most suitable gene for different plant organs,which should facilitate future studies on gene expression in B.cusia.4.Obtained the sequences of BcSK and BcDXR from transcriptome database,and cloned their ORF using the RT-PCR technology.SMART analysis showed that there are one and three functional domains in BcSK and BcDXR,respectively.Homology alignment was analyzed using Mega3.0 and Clustal X2 programs,and the result showed both genes are close affiliation to Sesamum indicum and Nicotiana tabacum.By building a vehicle for subcellular localization and the positioning analysis showed that both genes locate in the chloroplast.Established prokaryotic expression vector of BcSK,then implemented protein expression and purification,after that,the protein activity was tested using double enzyme coupling ultraviolet spectrophotometry,and the enzymatic activity was 4.26u/g.By adopting carotenoids accumulation experiment,BcDXR was observed could increase carotenoid accumulation in E.coli pAC-BETAipi by naked eye.The gene overexpression regeneration plants and hairy roots of BcSK were attained by agrobacterium impregnation,and the expression of BcSK was 29.6 times higher than control in transgenic plants,and 38.1 times higher than control in transgenic hairy roots.Meanwhile,the content of target compounds through LC-MS method testified that BcSK accelerated constructive metabolism of shikimate pathway.The hairy roots of BcDXR were attained in the same way,then the expression of BcDXR was 14 times higher than control,also compounds' variation proved that BcDXR played an important role in tenpenoid pathway.In conclusion,this study is the first time to excavate the key enzyme genes of B.cusia,which started from tissue culture,transcriptome sequencing,stable internal gene screening to gene function study.It could provide a reference for the synthesis pathways study in B.cusia and authentic germplasm resources protection.