| Baphicacanthus cusia,widely distributed in the Southwest,Southern and Eastern regions of China,is an important medicinal plant of Acanthaceae family.Indigo made from stem and leaf of Baphicacanthus cusia in Fujian has the best quality in China,and is known as “Jian Indigo naturalis”,it is the genuine medicinal of Fujian Province.The rhizoma of Baphicacanthus cusia used as medicine,called “Nan ban lan gen”,which together with indigo were included in the "Chinese Pharmacopoeia".In recent years,as the scale of the pandemic such as SARS,swine flu,avian influenza increases,and with the destruction of high-quality trueborn herb' ecological environment,those lead to the supply of the traditional Chinese medicines which origin from Baphicacanthus cusia are short of demand.Nevertheless,paying less attention to the germplasm resources protection,domestication and cultivation,the germplasm resources of Baphicacanthus cusia is facing the risk of losing,degradation and extinction.Therefore,it has becoming more and more imperative to strengthen the study on the original plant of the Chinese medicine indigo.Indigo and indirubin are the major functional components of Indigo naturalis and its original plant.Indigo and indirubin is a pair of isomer belongs to bisindole alkaloid.Indirubin is an active ingredient of Huang Dai Pian and Dang Gui Lu Hui Wan,two kinds of traditional Chinese medicine,which have beenused in the treatment of malignancies such as chronic myelogenous leukemia successfully.Currently,the studies on Baphicacanthus cusia and indigo more concentrated in the herb identification,pr ? essing technology,pharmacological activity and less in molecular biology of bioactive substances,which severely restricted the building of germplasm resources.It is imperative that elucidating the biosynthetic pathways for pharmaceutical value metabolites screening the key genes and cultivate new strains in our research work.The cDNA sequences of key genes Anthranilate synthase ? subunit(ASA)and Anthranilate synthase ? subunit(ASB)involved to the pathway of indole alkaloid biosynthesis were obtained.Anthranilate synthase has a ???? structure,? and ? subunit are eneoded by ASAand ASB.ASBtransforms glutamineinto glutamic acid and ammoniumion,which is immediately captured by ASA for the condensation with to give anthranilateThese genes of completed set enzymes in indole alkaloid pathway were systematically isolated based on the transcriptome sequencing data of Baphicacanthus cusia,and the synthesis information of the indole alkaloid come from microbial indigo metabolism.We characterized and predicted these genes' the cDNA sequences through bioinformatics techniques in the study,which will be conducive to increase the active ingredients of Baphicacanthus cusia and to provide fundamental information referring to metabolic engineering and gene improvement breeding of Baphicacanthus cusia.The content of this study includes the following aspects:(1)The full-length cDNA sequences of BcASA1,BcASA2 and BcASB genes were obtained by RT-PCR,through bioinformatics methods,.(2)Tissue-specific expression of BcASA1,BcASA2 and BcASBwere performed and the results showed that they expressed in different tissues to various degrees.BcASA1,BcASA2 and BcASB showed higher expression levels in stem than any other tissues but low levels in leaves and roots.The expression of BcASA1,BcASA2 and BcASBwas examinedat different time points(0h,1 h,2 h,4 h,8 h,12 h,24 h,36 h,48 h,72h)after MeJA,SA and ABA treatment by real-time quantitative PCR analysis indicated that all genes were also induced up-regulated by MeJA,SA and ABA.(3)Protein was purified through prokaryotic expression and its enzyme activities were also determined in vitro.BcASA1 and BcaASA2 activity were estimated through reading the amount absorbance increase of anthranilate acid at 336 nm(A336)in reaction mixture that contains the purified BcASA1 and BcASA2 protein as well as the substrates Chorismate and ammoniumchloride.Results indicated that BcASA1 and BcASA2 protein all had similarcatalytic activity(4)The Baphicacanthus cusia and Isatis tinctoria arethe main original plants of the Indigo naturalis.Due to the high efficient transformation system of Isatis tinctoria had been established,the over-expression vector of BcASA1,BcASA2 and BcASB can be constructed.Then verify their function to use the I.tinctoria genetic transformation system mediated by Agrobacterium tumefaciensC58C1 in vivo.Since we have obtained the transgenic Isatis tinctoria hairy root,the next research direction will be the phenotype and the quantities of active component of these transgenic plants... |
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