The Optimization Of Pinellia Ternata Tissue Culturing,and Induction Of PTA Point Mutation,and Construction Of Lemna Minor With PTA

Pinellia ternata(Thunb.)Breit,a perennial herb affiliating to Araceae,is of common traditional Chinese medicine widely being utilized in relieving a cough,diminishing inflammation,reducing phlegm,clearing damp,as latest pharmacopeia recorded.Pinellia ternata agglutinin(PTA)is a kind of lectin extracted from P.ternata,and it characters absolute specificity in biding to mannose.Accompanied with calcium oxalate crystals or scattered with cellular mucus,PTA is considered of myriad of application in anti-tumor,identifying glycosyl,responding to immune system,et al.Nowadays,researchers focus on the following tetrad domains: 1,the structure and function of PTA and the method of separating PTA from tubers more efficiently;2,the technology of expressing PTA in appropriate bioreactors and optimization in conditions of the production process;3,the research of antineoplastic,including the mechanism of antitumor,the spiecies of anti-oncology,natural medical adjuvant,anticancer activity and so on;4,transgenic bioengineering for insects resistance,and the physiological influence in insects.In this research,based on tissue culture system,we observe the change of physiological cycle in the abiotic stress.Containing beating down,recover,maturity of tuber,we discuss the modulated approach of PTA and its undiscovered adversity gene,and then we analyze the PTA gene then clone it.Reference to its conversed region on PTA sequence,we design several solitary mutation sites and double mutation sites to estimate the potential biding locus.We also remold a length of signal peptide,to bridge the end of a cysteine by disulfide linkage in hypothesis,meanwhile do RACE for PTA for the next such as silence or super express or promoter research in the future.Eventually,we build a new-type bioreactor which is duckweed from wild for the sake of expressing PTA or the other economic protein.In the result of our research,we find MS media with 3.5 mg/L 6-BA is suitable for germination of bulbils,while MS media with 0.5 mg/L NAA for the differentiation to roots.Illumination is necessary to the producing of bulbils and MS media with 1.0 mg/L 2,4-D is available to callus.We obtain the mutation gene siting on the first conversed sequence,the second sequence and the both.Also,we amplify the transformed PTA peptide,forepart PTA sequence and posteriors PTA sequence for the other research.In the end,we cultivate Lemna minor used for bioreactor.In summary,the mutant gene acquired in this experiment can be applied for detecting the activity of PTA,meanwhile judging the conversed sequence is the biding site or not via analyzing the PTA and capacity for biding to mannose.Moreover,the fraction of PTA synthesis in RACE can be utilized for gene silence and gene overexpression.At last,the duckweed bioreactor system settles a fundamental significance on the other protein expression.