| Lignin is polymerized by three basic monolignols, the content and monolignol composition are related to plant’s utlizaition. There are many different enzymes in lignin biosynthesis pathway; HCT (Shikimate/Quinate Hydroxycinnamoyltransferase) was the latest discovered enzymes for lignin biosynthesis, controlling the transformation of H-monomer and G/S-monomer. In this paper, hct gene fragment was amplified from RNA extracted from second xylem of Populus xeuramericana cv."74/76" stem, and then cloned into pMD20-T vector. After sequencing, the fragment was analysed. The Main results are as follows:1. Genomic DNA was extracted from Populus xeuramericana cv."74/76" by CTAB method. Two pairs of primers were designed by Primer Premier5.0for the amplification of hct. A pair of suitable primers was selected and PCR amplification condition was optimized.2. The hct gene was amplified by RT-PCR from poplar developing xylem total RNA. The amplified fragment is about700base pairs as we expected. It was recycled and cloned into pMD20-T vector and transferred into JM109component cell. After cultured for16hours on LB/Amp/X-gal/IPTG solid medium, the recombinanted plasmid was extracted from white colony, and identified by PCR, restriction enzyme cutting and sequencing.3. The sequencing results shows that the amplified DNA fragment was709bp containing a708bp ORF (open reading frame), and cloned into pMD20-T vector with forward direction. The identity of the fragment with Populus trichocarpa complete sequcece is98%, with HCT1of Populus trichocarpa is91%. The putative amino acid sequence belongs to transferase superfamily, containing a commom motif DFGWG of HCT. Alignment with amino acid of two HCTs in Populus trichocarpa showed that their homology was98%and91%respectively. So the cloned DNA fragment is hct and we registered it to GenBank, the accession number is FM202091. The recombinant plasmid was named as pMD20-T-hct and kept in our lab for further research. |
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