Cloning, Sequence Analysis And Expression Of Wheat Uroporphybilinogen Ⅲ Synthase In E. Coli

Uroporphyringenâ…¢is the common tetrapyrrole precurser from which all chlorophylls, haems, cobalamin (vitamin B12) are derived. And Uroporphyrinogenâ…¢Synthase (UROS)is the fourth enzyme in the tetrapyrrole pathway. UROS gene is well characterized in bacteria and mammals, especially in human and murine. Medical research shows that UROS deficiency will cause Congenital Erythropoietic Porphyria (CEP).UROS is coded by hem D gene in bacteria, and hem D gene is best characterized in E.coli and B. subtilis. Until now, UROS has been purified from pea, wheat, Euglena gracilis, human erythrocyte, murine liver, et al. In addition, E. coli UROS, and UROS from human and murine had been successfully expressed and characterized in E. coli. However, there are few reports about the UROS gene in higher plants.Wheat albinism line is a natural mutant from wheat AIBIAN1. It is a low-temperature sensitive pigment mutant in which the chlorophyll biosynthesis is blocked at the Uroporphyringenâ…¢formation. According to the previous research, it is suggested that the unwonted regulation and expression of PBGD and UROS is probably related to the albinism. So, the cloning and expression of wheat UROS gene will lay a foundation for the futher study of the albinism mechanism.In this research, in cilico cloning was utilized and finally amplified the full-length CDS of wheat UROS gene by PCR. 18bp 5'UTR and 132bp 3'UTR were also amplified. The cloned sequence has been submitted to GenBank, and the Accession Number is EF160077. Compared to the contig sequence acquired from in silico cloning, the cloned sequence defaults 70bp at 3'end, while the identities between the two is 98%. Besides, the cloned sequence has 86% identities with rice UROS gene, and its identities with Arabidopsis UROS gene is lower than 70%. Nevertheless, there's not any significant sequence similarity among wheat, human, and E.coli uros. Phylogenetic analysis shows that wheat and rice are relative, higher plants are most relative to Prototheca wickerhamii. PlantS are disparate from animals. For the predicted Aa sequence, it was composed of signal peptides and mature protein.It has high sequence similarity with rice UROS and Arabidopsis UROS, but not any compared to human UROS. This suggest that the primary structure of UROS from different species are disparate. But UROS from wheat and rice are more analogical, since they are both monocotyledon. There are some highly conserved sites in UROS from various organisms despite of their sequence heterologous. These conserved sites probably play important roles in enzyme catalysis.Then, two recombined plastids were constructed-- pET40b-UROS and pET25b-UROS, in order to express wheat UROS in E.coli. Both of them were transformed to BL21(DE3)-CodonPlus. Wheat UROS has been successfully expressed in E.coli with pET25b-UROS .