赤桉CCoAOMT亚家族的基因克隆及可变剪接分析

Gene Cloning and Alternative Splicing of CCoAOMT Subfamily Genes from Eucalyptus camaldulensis

通过同源克隆策略和RACE技术获得赤桉CCoAOMT亚家族2个成员的全长cDNA序列。CCoAOMT1基因的全长cDNA序列为1 020 bp,其中可读框ORF长度为738 bp,5’-UTR为106 bp,3’-UTR为176 bp;CCoAOMT2基因的全长cDNA序列为1 047 bp,其中可读框ORF长度为741 bp,5’-UTR为125 bp,3’-UTR为158bp。CCoAOMT1和CCoAOMT2基因都由5个外显子组成,主要的区别在于前者第1个内含子存在5’端可变剪接位点,形成2条不同长度的mRNA,一条的ORF长度为738 bp,而另一条比前者缺失了42 bp,但不影响其余氨基酸的读码框顺序。赤桉CCoAOMT1基因在茎段中可变剪接发生在5—9月间,是桉树一年中树高和直径增长最快的阶段,这个可变剪接可能与组织器官生长发育有关。蛋白质三维结构分析表明,CCoAOMT能形成正确的酶活性中心与底物结合位点。抑制CCoAOMT基因表达,降低桉树木质素对于减少制浆造纸工业污染具有重要意义。

In this study,the homology-based RT-PCR method and the rapid amplification of cDNA ends （RACE） method were used to clone full-length cDNAs of two CCoAOMT subfamily genes in Eucalyptus camaldulensis.The full-length cDNA of CCoAOMT1 gene is 1 020 bp and contains 738 bp ORF,106 bp 5 ＇-UTR and 176 bp 3 ＇-UTR,and the full-length cDNA of CCoAOMT2 gene is 1 047 bp and contains 741 bp ORF,125 bp 5 ＇-UTR and 158 bp 3 ＇-UTR.The alignment result showed that CCoAOMT1 and CCoAOMT2 genomic DNA both contain five exons.Their main difference lay in that there was a alternative 5 ＇ splice site at the CCoAOMT1 ＇ s first intron,which could result in formation of two mRNA with different lengths.One mRNA contains 738 bp ORF,and the other has a 42 bp deletion,however this splicing doesn＇ t change the reading frame sequence of posterior amino acids.The alternative splicing of CCoAOMT1 gene expresses from May to September,at the most vigorous growing stages of Eucalyptus stem,suggesting that this alternative splicing may involve in the development or growth of tissues and organs.Analysis result of protein＇s three-dimensional structure showed that the CCoAOMT can form the correct active center of enzyme and the substrate binding site.Limitation of the CCoAOMT genes＇ expression by genetic manipulation may serve as a way to reduce the lignin content in Eucalyptus,which presents a significant prospect in reducing pollutants generated from pulping industry.