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Cloning and Identification of Coumaric Acid3-hydroxylase Gene

Author: NieHuiZhong
Tutor: XueYongChang
School: Dalian University of Technology
Course: Biochemical Engineering
Keywords: lignin c3h RT-PCR bioinformatics
CLC: Q943.2
Type: Master's thesis
Year: 2008
Downloads: 5
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Abstract


Lignin is an important biopolymer in plant with important biological function in theplant growth and development. The biosynthesis of lignin is catalyzed by multiple enzymesystem，Coumaric Acid3-Hydroxylase（C3H） is one of the key lignin biosynthetic enzymescontrolling the production of different monolignols. In this paper c3h gene was cloned frompoplar（Populus×euramericana cv.“74/76”）, and its bioinformational function was alsostudied. The Main results are as follows:1. Genomic DNA was extracted from poplar（Populus×euramericana cv.“74/76”）byCTAB Method. Three pairs of primers were designed by Primer Premier5.0software and beused to amplify the c3h gene from poplar genomic DNA. A pair of suitable primers wasselected and PCR amplification system was optimized.2. The c3h gene was amplified by RT-PCR from poplar developing xylem total RNA.The amplified fragment is about1000bp as we expected, it was cloned into pMD20-T vectorand transformed into JM109competent cell, and the recombinant plasmid was obtained, andidentified by restriction enzyme cutting, PCR amplification and sequencing.3. The sequencing results indicated that the amplified DNA fragment is994bp andcontained a495bp ORF（open reading frame）.Then amplified fragment was cloned intopMD20-T vector with the forward direction. Compared with mRNA sequence of C3H in Pinustaeda（accession number: AY064170）,the homology was70%.Alignment with amino acid ofC3H in Pinus taeda（accession number: AAL47685.1）showed that their homology was91%.So the cloned DNA fragment could primarily classified as a member of the c3h family. Itwas submitted to GenBank, the accession number was AM920690. We named therecombinant plasmid pMD20-T-c3h.

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CLC: > Biological Sciences > Botany > Plant Cell Genetics > Plant Genetic Engineering
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