Molecular Cloning And Variation Analysis Of CCR Gene In Spotted Gums And Its Relatives

The Cinnamoyl CoA reductase (CCR) gene is involved in determining lignin content and composition in plant secondary cell walls and has been associated with wood strength characteristics in Eucalyptus. The Corymbia (Bloodwoods and Ghost gums) were once considered a subgenus of Eucalyptus but are now recognised as a separate genus. Now the eucalypts is consisted of the genus Eucalyptus Corymbia and Angophora. The spotted gum complex (Genus Corymbia, Section Politaria) is considered the most important taxa for forestry. 20 samples of Corymbia in total, with representatives from 5 major sections, Politaria, Rufaria, Ocharia, Blakearia and Cadagaria were used as samples. E. gunnii of Eucalyptus or Angophora spp. was used as an outgroup fot the phylogenetic study.In this study, Qiagen DNeasy^TM 96 Plant Kit was used to extract Corymbia genomic DNA, and PVP was added to prevent the phenolic to inhibit the enzyme activity. It is a high efficiency method DNA extraction. E. gunnii CCR gene then the sample 7782 obtained from the experiment was used to design primers to amplify Corymbia CCR gene. Specific primer pairs CCR₄20F and CCR₁678R, CCR₁610F (self-designed) and CCR₃198R, CCR_AF (self-designed) and CCR₃198R, and CCR_BF (self-designed) and CCR_ER (self-designed) were successful in amplifying target fragments. Cut and purified the target fragments from agarose gel for direct sequencing or cloning and sequencing. The sequencing reaction was improved therefore the fragments long to be 1 kb could be successfully sequenced for single fragment. The modified sequencing cleanup was as follows: RNase and DNase free water 40μl, 3M Sodium acetate 5μl,AR Ethanol 125μl without EDTA.The 16 CCR genes were rectified and constructed contigs by solfware Sequencher 4．5 and aligned by ClustalW automatically and manually.After running BLAST with other plant CCR genes available in GenBank, the 16 CCR genes had high identities compared to 20 plant species, and parts of them reached 97%.Software MEGA 3．1 was applied to analyse the variation and distribution of the 20 species or individuals of Corymbia, focusing on spotted gums (n=8) (Genus Corymbia, Section Politaria) as well as 4 CCV cloned subsequently for the future association study. The sections Rufaria, Blakearia, Ochraria and Cadagaria were sequenced so as to analyse the CCR gene and reconstruct the phylogenetic tree.It is popular to use the gene to reconstruct the phylogenetic tree. The section levels of Corymbia were well resolved in the phylogenetic tree with or without the E. gunnii outgroup. Each section formed monophyletic clade, but it seemed more reasonable without the present of the E. gunnii outgroup. It is not well resolved in species level within sections, likely to be the rate of gene evolution, substitution, indels, SSR or intergenic recombination, etc. Although the affinity of Angophora was more closed to Corymbia compared to E. gunnii as an outgroup, 4 sections were not well resolved because of the present of polyphyletic clades.Sequence variation was detected in all introns and exons analysed and found 292 variation sites and 171 parsimonious information sites within Corymbia, 65 variation sites and 38 parsimonious information sites within Politaria, and 37 variation sites and 9 parsimonious information sites within Corymbia citriodora subsp. variegata (CCV). Introns were more variable than exons. These variation sites provided information for the wood properties of the spotted gums and it relatives, likely to be responsible for microfibril angle and lignin contents and composition, which could provide fundamental data for the future association study for spotted gums, especially CCV.Sample 7790 (CCV) CCR was certified to be a multi-copy gene after cloned and sequenced. The analysis results showed 7790 CCR was likely to be 3 copies. This is a new discovery for the first time to publish the multi-copy gene of Corymbia CCR in eucalypts. The CCR of Eucalyptus is single copy as well as the 2 species of Angophora in this study.For the first time, this study publishes Corymbia and Angophora CCR genes. 16 CCR sequences of Corymbia samples have been submitted to the GenBank, and the additional 12 sequences including Angophora CCR have got the GenBank accession number. These CCR genes, phylogenetic analysis and exon and intron variation analysis provide fundamental genetic data for the further CCR study, CCR transfergenic plant and cross breeding researches for Corymbia spp. especially the spotted gums.