Cloning And Plant Expression Vector Construction Of Cinnamate-4-Hydroxylase(C4H) Gene From Astragalus Membranaceus

Radix Astragali is the dry root of Astragalus.membrcmaceus(fisch.)Bge.and Astragalus mongholicus Bge(Leguminasae Astragalus).It is one of the commonly used traditional Chinese medicine and mainly used as invigorating qi.Phenylpropanoid metabolic pathway is one of the most important secondary metabolism pathway in plants.Trans-cinnamic acid-4-hydroxylase (C4H,EC1.14.13.11),another name is trans-cinnamic acid-4-mono-oxygenase, it catalyzes trans-cinnamic acid into 4-coumaric acid salt by hydroxylation.It is the second key enzyme in phenylpropanoid metabolic pathway after L-phenylalnine ammonialyase(PAL).In order to understand the functional properties of C4H gene, we designed PCR primers that target evolutionarily conserved sequeces of C4H gene in plants and cloned full-length sequences of C4H gene in A. membranaceus by the technique of RT-PCR and RACE, analyzed characteristic of sequence, and built the plant expression vector.The results are as follows:we have first cloned full-length sequences of a novel C4H gene in A. membranaedceus by the technaque of RT-PCR and RACE,the C4H gene named Am C4H.The length of AmC4H cDNA is 1790 bp and the sequence in coding region of C4H is 1 515 bp, and the C4H include 5’untranslated region (5’UTR)of 53 bp,3’UTR of 195 bp.The putative AmC4H protein consisted of 505 amino acids with prediated molecular weight of 57.86 KDa and isoelectric point of 8.98,had the high homology with C4H of known leguminous plants.Bioinformatics analysis showed that AmC4H was a soluble and hydrophilic proteinand there were two significant hydrophobic peaks and two transmembrane peptide.The existing probability and the anchoring probability of signal peptide were 0.282 and 0.709, the cleavage site was between 24 and 25 position amino acids.The secondary structure of C4H protein prediction displayed that its secondary structure is dominated by alpha helix random coils.On the basis above, we constructed pBI121-AmC4H vector and transformed it into EHA105. This study provides a solid foundation for the study of transgenic plant.