Construction Of Prokaryotic Expression Vector For CDNA Encoding β-carotene Hydroxylase From NanFeng Orange(Citrus Reticulata Blanco Var. Kinokuni (Tanaka) H. H. Hu)

β-carotene hydroxylase is a non-haemachrome di-iron monooxygenase,which catalyses the conversion ofβ-carotene to zeaxanthin throughβ-cryptoxanthin.β-carotene hydroxylase exists broadly in cyanobacteria,bacterium and plants.The genes of differentβ-carotene hydroxylase in different kind of organisms have some conservatism,specially for that of the high plants and green algae which have more conservatism.β-carotene hydroxylase genes of many organisms have been cloned and expressed in cyanobacteria,bacterium and plant respectively.The purpose of our research is to clone the eDNA encodingβ-carotene hydroxylase(chy)from Nanfeng orange(Citrus reticulata Blanco vat kinokuni (Tanaka)H.H.Hu)and construct the expression vector for expressing in E.coli.1.The genome DNA and total RNA was isolated from the young leaves of Nanfeng orange.2.The gene of 13-carotene hydroxylase(CHX)with specific enzyme digestions was amplified with the specific primers which has those enzyme digestion locations by using the method of RT-PCR(reverse transcription-polymerase chain reaction). After it was recycled and purifided the gene was connected to pMD19-T vector. Then pMD19-T-CHX was transformed into E.coli DH5α..Colony PCR were done and the positive colonies from samples of bacteria were detected and the plasmid was isolated.Sequence analysis and identity of the gene with predicted eDNA encoding Nanfeng orangeβ-carotene hydroxylase registered in Genbank were done to confirm the colone of CHX was successful.3.The pMD19-T-CHX and the prokaryotic expression vector pET-20b(+)were connected after digestion and purified respectively.The recombinant plasmid pET-CHX was transformed into E.coli BL21(DE3)pLysS.We confirmed we had successfully constructed the expression vector through antibiotic screen,colony PCR,specific enzyme digestion and the sequence analysis.4.To analyse the structure ofβ-carotene hydroxylase by a series ofoniine softwares. In the experiment,the eDNA encodingβ-carotene hydroxylase was successfully cloned as well as the construction of the prokaryotic expression vector.Bioinformatic analysis ofβ-carotene hydroxylase show that it contains 311 amino acid residues.Its molecular weight is 34.7854kDa,and the theoretical pI is 9.03.TMpred analysis show that it contains four distinguished transmembrane helices which are overlap with the hydrophobic regions that predicted by ProtScale,suggesting this enzyme is a membrane protein located in thylakoid membrane.In addition,structure analysis show thatα-helix is the main secondary structure of this enzyme,and it contains many kinds of phosphorylation sites.