| Summary: | 碩士 === 嘉南藥理科技大學 === 生物科技系暨研究所 === 97 === Eucalyptus camaldulensis (Murray red gum), Myrtaceae, are trees with remarkable growth and wildly distributed in arid and semi-arid climates. It was first introduced into Taiwan in 1896 by TFRI and found with excellent adaptability over 20-year tests. E. camaldulensis is known as a tree offering promise as a potential material for use in paper production, due to the superior quality of its pulp fibers and high pulp yield. To improve the wood quality for pulpwood E. camaldulensis, genetic engineering would be commercially used in the near future.
The superfamily of cellulose synthase genes (CesA) involves the production of cellulose and has been cloned in many species. However, little is currently known about the role of CesA genes in E. camaldulensis. Therefore, total RNA was isolated from young leaves of E. camaldulensis and subjected to a two-step RT-PCR using degenerated primers that are specific for the amino acid motifis ’CYVQFPQ’ forward primer and ’GWIYGS’ reverse prime, which are highly conserved in all known plant CesAs and contain the CSRII region. The clones were then sequenced and analyzed with similarity searches against the nucleotide database in GenBank by BLASTN. Interestingly, one 1079 bp putative clone, EucCesA7, was sequenced at both ends and shown 90% nucleotide identity to PttCesA2. To obtain the full-length cDNA of EucCesA7, a RACE PCR technique was adopted by using SMARTTM RACE cDNA amplification kit (Clontech). To further confirm that our new cDNA fragments (CSRII fragments, 5′ and 3′ RACE products) were originated from a single, full-length cDNA, PCR primers were designed based on the 5′ and 3′ untranslated region (UTR) sequences. These primers were used to amplify full-length of EucCesA7 cDNAs with 3485 bp in size by RT-PCR. One clone, pCR2.1TOPO-EucCesA7-FL.18, was obtained and showed highly 80% nucleotide identity. PttCesA2 plays a role in the primary cell wall synthesis, and may involve the gene regulation in the formation of tension wood. Phylogenetic analysis of the CesA sequences was also performed by aligning the sequences with 57 plant CesAs and showed that EuCesA7 belongs to the group that relate to the biosynthesis of primary cell wall.
Moreover, an in situ hybridization system has been performed to characterize the localization of EucCesA7 in Eucalyptus. To generate DIG-labeling probes, cDNAs of CSRII region were firstly subcloned into the pGEM-T Easy vector. DNA fragments between the SP6 and T7 promoter sequences in constructs were then amplified by PCR. DIG-labelled sense and antisense RNA probes were prepared using an in vitro transcription kit. This cRNA can be combined with the anti-digoxigenin AP, via of NBT and BCIP has pigment blue-violet color. Signals representing the expression of EucCesA7 could be detected in the cambial zone of leaves and stems during different developmental stages. Among all tested tissues, the eighth internode with the strongest signal. Moreover, semi-quantitative polymerase chain reaction, analysis for EucCesA7 gene expression was also conducted. In our preliminary results showed high expression level in xylem and low expression level in leaves when analyzed by PhotoCap gel quantitative software. In comparison with other EucCesA members, EucCesA7 had the highest expression level in xylem followed by EucCesA4 and EucCesA2. In leaf tissue, EucCesA7 has lower expression level than that of EucCesA2 and EucCesA4.
A binary construct harboring EucCesA7 with pBI121 backbone has been obtained in this study. In the near future, transgenic tobacco plants carrying EucCesA7 would be obtained to investigate the role of EucCesA7 in cellulose biosynthesis.
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