| The soybean (Glycine max (L.) Merr.) is an significant foodstuff and oil crop in our country, it contains full protein and fat, the valve of multiple utilization is high. The antireversion force and the yield of soybean decrease greatly because of the severe continuous cropping. For the past few years, we learned about the allelopathy of secondary metabolite is one important reason of continuous cropping obstacle from study and research. And the study of secondary metabolism have become one hot spot of molecular bioresearch in plants. Phenylpropanoid metabolic pathway is one of the most important secondary metabolism pathway in plants.trans-cinnamic acid-4-hydroxylase (C4H, EC 1.14.13.11), 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 reveal the construction features of soybean C4H in the molecular level and provide a theoretical basis of increasing the activity of its expression. Test materials is SN14 in this study, the soybean trans-cinnamic acid-4-hydroxylase mRNA(C4H mRNA)and genome DNA (C4H DNAA) was cloned by RT-PCR and PCR, respectively. In addition, 3'termination sequence of Soybean C4H was cloned by RACE technique. The main structure and function of protein which is translated by C4H mRNA was predicted by bioinformatics analysis. The results were as follows:The trans-cinnamic acid-4- hydroxylase mRNA (C4H mRNA)(Accession Number: FJ770468) was cloned successfully from soybean leaves by RT-PCR. The acquired sequence is 1766bp in length and the sequence in coding region of C4H is 1521bp. Compare with the coding region of C4H mRNA announced(Accession Number: X92437), the resemblance is 98.77%, just one basic group is different(situate 1237position ), the homology of this gene order exists in other plants, the protein translated by the sequencing result compares with the foregone protein (Accession Number: Q42797), the resemblance is 99.80%, just one amino acids is different(situate 396 position ), a polypeptide was encoded by 506 amino acid residues.The soybean trans-cinnamic acid-4- hydroxylase genome DNA (C4H DNA) (Accession Number: HM036117). The sequencing result shows that C4H DNA contain one extron and no intron, the sequence of extron and C4H mRNA acquired are the same, completely.3'termination sequence of Soybean C4H was cloned by RACE technique, and updata information of FJ770468, the new sequence is 1799bp in length.The physical chemistry characteristics of C4H were analyzed on-line by ExPasy and TOPPRED, it shows that theory value of C4H PI and Mw is 5.30 and 39092; respectively; C4H is a soluble, hydrophilic protein, while there are two significant hydrophobic peaks and two transmembrane peptide by TOPPRED, the phyletic evolution tree was constructed by DNAMAN6.0. The secondary structure of C4H various amino acid residues were predicted by HNN, respectively, Alpha helix: 251 is 49.60%, Extended strand: 52 is 10.28%, Random coil: 203 is 40.12%; 3D structure was predicted by Geno3d,it shows that 57 extended strands contained 24 alpha helices inβ-folding area and 14 random coils. According to the amino acid sequence and structural analysis, it shows that C4H protein contains one conserved domain, viz. P450 domain. The existing probability and the anchoring probability of signal peptide were both 0.498 which were analyzed by SignalP, the cleavage site was between 28 and 29 position amino acids. |
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