Gene cloning and expression level of chalcone isomerase during florescence and content of flavonoids in Fagopyrum dibotrys / 中草药

Objective: To clone and analyze the full-length cDNA of chalcone isomerase (CHI) gene from Fagopyrum dibotrys (FdCHI). To analyze the expression of CHI and the total flavonoids content during florescence of F. dibotrys. Methods: The cDNA sequence of CHI was cloned by homology cloning from F. dibotrys. The expression of CHI was analyzed in the different tissues during florescence by semi-quantitative RT-PCR. The content of total flavonoids was measured by AlCl3 method. Results: The open reading frame (ORF) of FdCHI was 750 bp and encoded a protein with 249 amino acids. Bioinforamtion analysis suggested that the amino acid sequence of FdCHI had the high homology with those in other plants. Gene expression analysis showed that FdCHI was highly expressed in the flowers, followed by the roots and leaves, while lower in the stems. The content of total flavonoids was the highest in the flowers then the leaves and stems, and the lowest was in the roots. Conclusion: The cDNA sequence of FdCHI is firstly obtained from F. dibotrys and its coding protein has the typical characteristic of CHI homologous protein. The gene expression of FdCHI shows the same to the total flavonoids content in the stems, leaves, and flowers, but different in the roots of F. dibotrys.

Molecular cloning of squalene synthase gene form Paris polyphylla and its expression in Escherichia coli / 中国中药杂志

<p><b>OBJECTIVE</b>To clone the cDNA sequence of squalene synthase gene from Paris polyphylla, and characterize the biological features of the obtained SQS.</p><p><b>METHOD</b>Using homology cloning and RACE technique, a full-length cDNA sequence of PpSQS gene was isolated from P. polyphylla. The obtained sequence was analyzed by bioinformatics softwares. A plasmid [named pET-30b (+)-PpSQS] was constructed for prokaryotic expression the recombinant PpSQS.</p><p><b>RESULT</b>The full-length cDNA of PpSQS gene is 1 498 bp, which contains a 1 212 bp ORF. Sequence analysis indicated that PpSQS encoded 403 amino acids residues with a calculated molecular weight (MW) of 46.36 kDa and an isoelectric point (pI) of 6.83. SDS-PAGE results showed that the recombinant PpSQS was expressed in Escherichia coli BL21 (DE3) by inducing with 1 mmol x L(-1) IPTG.</p><p><b>CONCLUSION</b>The full-length cDNA sequence of PpSQS gene was obtained from P. polyphylla, and its molecular features were consisted with classic SQS in plant. The recombinant PpSQS was successfully expressed in E. coli.</p>