裸子植物5S rRNA基因序列变异及二级结构特征

在高等植物中，5SrRNA基因一级结构是高度保守的，二级结构也相当一致。通过比较18种裸子植物5SrRNA基因序列和二级结构变异，发现55％的核苷酸位点是可变的，这种变异有68％发生在干区（双链区），其中一些变异，如双链的互补性核苷酸替代，GU配对等能够维系5SrRNA二级结构的稳定性。环区相对保守，这与5SrRNA三级结构折叠或在转录翻译过程中蛋白质、RNA的结合相关。另外，首次报道了松属环E区核苷酸的变异性，这可能与其他区域的变异一样，是假基因造成的结果。5SrRNA基因信息可反映大分类群的系统进化关系，但由于基因长度短，信息量小，其在近缘种系统分类的应用受到限制。

Characterization of 5S rRNA Gene Sequence and Secondary Structure in Gymnosperms

In higher plants the primary and the secondary structures of 5S ribosomal RNA gene are considered highly conservative.Little is known about the 5S rRNA gene structure,organization and variation in gymnosperms.In this study we analyzed sequence and structure variation of 5S rRNA gene in Pinus through cloning and sequencing multiple copies of 5S rDNA repeats from individual trees of five pines, P.bungeana,P.tabulaeformis,P.yunnanensis,P.massoniana and P.densata.Pinus bungeana is from the subgenus Strobus while the other four are from the subgenus Pinus (diploxylon pines).Our results revealed variations in both primary and secondary structure among copies of 5S rDNA within individual genomes and between species. 5S rRNA gene in Pinus is 120 bp long in most of the 122 clones we sequenced except for one or two deletions in three clones.Among these clones 50 unique sequences were identified and they were shared by different pine species.Our sequences were compared to 13 sequences each representing a different gymnosperm species,and to six sequences representing both angiosperm monocots and dicots.Average sequence similarity was 97 1% among Pinus species and 94.3% between Pinus and other gymnosperms.Between gymnosperms and angiosperms the sequence similarity decreased to 88 1%.Similar to other molecular data,significant sequence divergence was found between the two Pinus subgenera.The 5S gene tree (neighbor-joining tree) grouped the four diploxylon pines together and separated them distinctly from P.bungeana. Comparison of sequence divergence within individuals and between species suggested that concerted evolution has been very weak especially after the divergence of the four diploxylon pines.The phylogenetic information contained in the 5S rRNA gene is limited due to its shorter length and the difficulties in identifying orthologous and paralogous copies of rDNA multigene family further complicate its phylogenetic application. Pinus densata is a diploid hybrid between P.tabulaeformis and P.yunnanensis. Its 5S rDNA composition is consistent with its hybrid origin. 5S rRNA of all gymnosperms published so far could be folded into a general secondary structure.Variation in this secondary structure was detected among species.About 55% of the 120 bp nucleotide positions was variable,in which 68% was on stem regions.Nevertheless,the positions at the end of the stems and those adjacent to loops are conserved.Their stability directly determines the size of the loops.Some mutations such as compensatory base-pair substitutions,and G-U pairing could be regarded as mechanisms for maintaining a stable secondary structure.The loops of the secondary structure are also relatively conserved.It seems that stable helices are necessary for the function of the gene.The conserved nucleotides in the loops are probably involved in the interaction with proteins and/or RNAs or with other nucleotide in the formation of the tertiary structure.However,unlike other reports,Loop E was found quite mutable among pines.These variations together with those on stems might be caused by the presence of pseudogenes among our clones.A preliminary evaluation indicates that only seven of 50 unique sequences are potentially functional genes.