Cloning And Functional Analysis Of BcASA And BcSGD Genes From Baphicacanthus Cusia

Baphicacanthus cusia is a perennial herb of acanthaceae,its root is taken as the traditional Chinese medicine,the"Nan-Ban-Lan-Gen",the Indigo Naturalis（Qingdai）is made from its leaf and stem,which quality is the best in Xianyou of Fujian province and known as"Jian Qingdai",it’s a famous-region drug in Fujian^[1].Previous research has indicated that Nanbanlangen contain many active ingredients,such as indole alkaloids,quinazolone alkaloid,and organic acids.Indigo and indirubin are important active substances in Indigo Naturalis.Production of indigo and indirubin are based on synthesis of indole in the shikimate pathway,the anthranilate synthase^[2]and glucosidase^[3]are play the important role in this pathway.At present,there are few studies on the molecular pathway that formation of effective substances in Baphicacanthus cusia,and less research on the function and the molecular mechanisms of key enzymes.Therefore,it is a great significance that study the functions and molecular mechanisms of anthranilate synthase and glucosidase in the synthesis of secondary metabolites to improve the formation of effective substances in Baphicacanthus cusia and increase its yield and quality.Based on the previous research of the research group,the results of this study are as follows:1.By analyzing and screening the measured transcriptome database,anthranilate synthase and glucosidase were cloned from Baphicacanthus cusia by RT-PCR,anthranilate synthase was named of BcASA gene（The accession number in Gen Bank is MH976794）,glucosidase was named of BcSGD gene（The accession number in Gen Bank is MK560201）,the lengths of their c DNA were 1110 bp and 1208 bp,respectively.2.The physicochemical properties,sequence structure,protein domain,and phylogenetic tree of the target gene were analyzed with bioinformatics methods.The results shows that both proteins encoded by these two target genes are hydrophilic proteins and located in the chloroplast.The chemical formula of BcASA protein is C₁₈₃₀H₂₉₅₃N₅₂₃O₅₄₁S₁₁,the molecular weight is 41.29 k D,and the chemical formula of BcSGD protein is C₁₉₅₆H₃₀₄₂N₅₀₀O₅₇₄S₁₄,the molecular weight is 43.20 k D.3.The plant subcellular localization vector pCAMBIA1302-BcASA and pCAMBIA1302-BcSGD were constructed,and transferred into Agrobacterium C58C1.Recombinant plasmid was transformed into tobacco epidermis by Agrobacterium-mediated leaf disc method,observed by confocal laser scanning microscope,and the results showed that both genes located in the chloroplast.4.The prokaryotic expression vector pET32a-BcASA and pET32a-BcSGD were constructed,furthermore,the optimal expression conditions of the two recombinant proteins were determined by regulate the inducing temperature and the concentration of IPTG,the results showed that the optimal expression conditions were 0.5 m M IPTG,37℃for 3 h.Moreover,two recombinant proteins were expressed in the form of inclusion boy.The target proteins were separated and purified by nickel affinity column,and verified with western blot.In addition,the enzyme activity of the purified BcSGD protein was measured,and the results indicated that the recombinant protein showed catalytic activity for p NPG,the Km and V_maxof the enzyme were 17.24 mmol/L and 5.03 nmol/min/g,respectively.5.The expression levels of BcASA and BcSGD genes in the various plant tissues of root,stem,leaf and flower of Baphicacanthus cusia are measured.The result indicates that both genes have expression in each tissue of the Baphicacanthus cusia,but the expression is different.BcASA expression is higher than BcSGD in various tissues.Moreover,both genes have the highest expression in leaf and the lowest in flower.6.Four exogenous inducing substance,100 m M methyl jasmonate（Me JA）,abscisic acid（ABA）,salicylic acid（SA）,and ethephon（ETH）,were used to treat Baphicacanthus cusia’s leaves so that investigate the effects of exogenous inducing substance on the BcASA and BcSGD in the indole metabolic pathway.Results reveal both genes were regulated by exogenous inducing substance,and the expression level is showing differentiation.The expression of BcASA gene was significantly regulated by SA,and the expression level increased by about 24 times.The expression of BcSGD gene was significantly regulated by Me JA,and the expression level increased by about 18 times.7.Established the genetic transformation system of Baphicacanthus cusia,in order to verify the in vivo function of BcASA and BcSGD genes,a large number of aseptic seedling were obtained through tissue culture,a variety of induction conditions were set,and callus induction was performed on the stems and leaves of Baphicacanthus cusia.The result showed that the best medium of induct callus was MS+6-BA 1.0mg/L+NAA 0.1 mg/L,the leaf is the optimum explant for callus formation of Baphicacanthus cusia.In addition,the BcASA and BcSGD genes were transferred to Arabidopsis,and the T0 generation seeds have been harvested.The next step of work would be the identification of transgenic lines,examination of metabolite content,purification of strain,and so on.