NovoFunction Of PtCRTISO In Cis-ABA To Trans-ABA Isomerization And Studies On Instrumental Analytical Methodplogy Of Plant Secondary Metabolites And All-type Phytohormones

Poplar is an important species for afforestation and plain greening.Poplar is a typical representative of poplar basic application research,production and cultivation.However,its cloning and function of carotenoid isomerase CRTISO,especially the transformation of cis-ABA and trans-ABA isomers,have not been reported.In this research,CRTISO has been found as the enzyme during the process of cis-ABA to trans-ABA,which was known as the key enzyme in the synthesis of beta-carotenoid.In order to verify the function,six kinds of expression vectors were built and the protein was purified after expressed in E.coli.While the protein expressed from E.coli was inclusion body,eukaryotic expressive vector was built to express the CRTISO in pichia pastoris.After purified and concentration,the CRTISO protein expressed in pichia pastoris was collected for enzyme reaction.The result showed that the CRTISO could catalyze the cis-ABA to trans-ABA.Then CRTISO's plant expression vector was constructed and the CRTISO was transformed in Nicotiana tabacum L.The determination of content of cisltrans ABA in transgenetic tabacum showed that the CRTISO really could catalyze the cis-ABA to trans-ABA.This work has focused on the following 5 parts for the CRTISO:Part ?:Gene cloning and therotic Modelling:PtCRTISO gene has been first cloned and gene sequence has been registrated on the Genbank at NCBI website(Genbank No KX 227459.1).Based on the PtCRTISO sequence,the bioinformatics analysis hase been applied and obtained the toal length of the sequence is 1842 bp,encoding 613 amino acids,sequene isentity reaches 84%,putative protein moleculer weight is 67.4KDalton,Secondary structure shows 16 alpha helix and 20 belta turns.Using Discuvery Studio software suite to molecular modeling,the results showed that the CRTSIO protein could docking cis-ABA and trans-ABA.The data peovide the therotic results that the protein could bind the cis-ABA and trans-ABA and might catalysis isomerization of cis-ABA to trans-ABA.Part ?:Based one possible therotic docking results,in vitro prokaryotic overexpression vector has been constructed for overexpression to obtain the soluble enzyme protein to reseach its activity.6 types of overexpression vectors has been tried and all of them the inclusion body without soluable protein for the activity studies.Part ?:In order to obtain the soluable protein for its enzymatic activety studies,eukarytic yeast overexpression vector has been instroduced and constructed and finaly soluable protein has been obtained.The CRTISO really DO have the cis-ABA to trans-ABA isomerization activety in vitro campare to boiled enzyme and buffer controls.Cis-ABA could photoisomerized to trans-ABA but its ratio stoped at 1:1 and never over the 1:1.The CRTISO catalysis Cis-ABA isomerized to trans-ABA,its ratio could reach and over 1:1?The in vitro CRTISO enzymatic isomeriaation of cis-ABA to trans-ABA activity has been proved.Part ?:Based on the moleculer modeling and in vitro activety verification,the pant expression binary vector pBin 121-PtCRTISO vector has been constructed for plant transformation,to prove the CRTISO in vivo activity.By meant of Agrobacteria medied method CRTISO transgenic tobacco has been obtained.Natrual grown poplar leaves have been harvested at varied seasons(in Spring,summer,autum and winter).Samples are under applied for qPCR and HPLC-MS ABA quanty analysis,the result shows cis-ABA to trans-ABA ratio and qPCR results of four seasons matches the regulation and proved the in vivo activit of CRTISO.The results might imply a new mechanism of ABA isomerization regulation ABA physiological activity during the plant growth and development.Part ?:The research methods used in this study include analytical testing methods and molecular construction expression methods.Among the several methods,according to different secondary metabolites,a different kinds of analysis method is established,for example in view of the plant hormones to establish performance liquid chromatography(HPLC)and mass spectrometry instrument analysis method,in view of the lignin and soluble monosaccharide establish gas chromatography and mass spectrometry instrument analysis method,in view of the phenolic acid establish a high performance liquid chromatography(HPLC)with diode arrays spectrometer detection method,and so on.These methods are not only considering the nature of the target compounds and the complexity of the plant samples,but also considering the applicability of the method in a larger scope.For the different experimental conditions in different laboratories,researchers can choose different ways to study.These methods provide an important reference for related topics.Molecular building method is using the traditional construction of prokaryotic expression vector and relatively novel construction of eukaryotic expression vectors.The CRTISO expressed by the prokaryotic was inclusion body,while the protein expressed by eukaryotic cell yeast could be effective for protein exocytosis,and this method could improve the expression level of inclusion protein expressed by E.coli,and make it possible to measure enzyme activity in vitro.This expression method by yeast could lay the solid foundation for subsequent protein function research.In this study,expression vectors were also constructed for plants to infect model plant tabacum,which played an important role in verifying the functions of target genes in plants.This study has established the methods that could be applied to a wide range,also worked on the key enzyme of plant hormone metabolism pathways,set up effective protein expression system,verified the enzyme activity conjecture,and verified the function of the target gene in the plants.These methods were made up an important part of plant hormones metabolism pathways,and the important basis for the further research of plant hormones.