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Cloning, Identification and Sequence Analysis of Cellulose Synthases Gene in Plant

Author: LiuChangBin
Tutor: XueYongChang
School: Dalian University of Technology
Course: Biochemical Engineering
Keywords: Cellulose Cellulose synthase (CesA) RT-PCR Sequence Analysis
CLC: Q943.2
Type: Master's thesis
Year: 2008
Downloads: 6
Quote: 0
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Abstract


Cellulose is one kind of natural polysaccharide catalysised by cellulose synthase. It’sthe most redundant biomacromolecule, important renewable resources, and also the maincomponent of plant cell wall. Cellulose, as one kind of potential bioenergy, is also a veryimportant natural material for pulp and paper industry. There are a number of genes whichcompose a superfamily encoding the cellulose synthases. All of the cellulose in the livingbody is catalysised by cellulose synthase.Genomic DNA was extracted from Populus×euramericana cv.“74/76” by CTABmethod. Two pairs of primers were designed by Primer Premier5.0software for PCRamplification. Through optimizing the influence factors （annealing temperature, primer,dNTP and DNA template concentration） of PCR amplification, we got the optimal primersand amplification condition for cellulose synthases gene （CesA） amplification.The total RNA was extracted from second xylem of Populus×euramericana cv.74/76”stem. Three DNA fragments were amplified by RT-PCR at55°C from the total RNA. Theywere recycled and cloned into pMD20-T vector and transferred into the JM109componentcell. After cultured16hours on LB solid medium containing Amp, X-Gal and IPTG, thewhite colony was selected, and identified by PCR, restriction enzyme cutting and sequencing.The result of sequencing shows that the amplified DNA fragment was883bp, andcloned into pMD20-T vector with forward direction. The identity of the fragment with Populustremula×Populus tremuloides CesA1is99%. The predicted amino acid sequence analysisof the fragment shows that it code a zinc finger and two transmembrane domains which areconserved structure in cellulose synthases （CesA）. So, the fragment we cloned is a fragmentof CesA and we registed it to GenBank, the accession numbers is AM921697; moreover, theplasmid containing the target fragment was named as pMD20-T-CesA and kept in our lab forthe following studies.

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