肾形肾状线虫个体间rDNA内转录间隔区的变异

Interindividual variability of internal transcribed spacer region of ribosomal DNA in Rotylenchulus reniformis

利用DNA序列分析技术对我国浙江、福建和重庆3个地区的肾形肾状线虫（Rotylenchulus reniformis,RN）种内群体及群体内个体间核糖体RNA基因（rDNA）内转录间隔区（internal transcribed spacer,ITS）序列进行PCR扩增及序列变异分析。结果显示不同的群体间均获得2种PCR产物,标记为变异类型Ⅰ（RN_VAR1）和变异类型Ⅱ（RN_VAR2）。基于每个群体内2条雌虫分别挑取各变异类型的5个克隆测序,共得到60个克隆序列。经序列比对分析,发现肾形肾状线虫rDNA基因2种类型的ITS区变异很大,其中变异类型Ⅰ序列长度为705-712bp,鸟嘌呤和胞嘧啶（guanine and cytosine content,GC）含量为45.1%-46.7%;变异类型Ⅱ序列长度为854-860bp,GC含量为48.4%-50.0%。2种类型的ITS相似度（包括5.8SRNA基因）仅为62.1%-65.6%,而各rDNA变异类型内部各个克隆序列也存在差异,变异类型Ⅰ内个体间相似度为89.9%-100%;变异类型Ⅱ内个体间相似度为91.4%-99.8%。系统进化分析表明2种变异序列明显分成2支,同时通过ITS序列分析无法将3个地方群体区分开来。经实时荧光定量PCR（quantitative real-time PCR,qPCR）检测发现RN_VAR1含量稍大于RN_VAR2,分别占保守18S基因的rDNA重复单位含量的56%和40%。同时,还发现肾形肾状线虫的2种rDNA-ITS变异类型与已报道的2种18SRNA基因变异类型相对应,表明肾形肾状线虫存在2种rDNA变异类型。

Summary Ribosomal RNA gene（rDNA）is conservative and it is composed of numerous copies of tandemly repeated transcription units within the genome that has been recognized as an attractive marker for phylogenetic studies.It consists of the 18 S,5.8S,and 28 Sgenes as well as internal transcribed spacer（ITS）region and external transcribed spacer（ETS）region.As members of a sequence family,the multiple copies of the rDNA do not evolve independently.They tend to evolve in a concerted fashion,which means that in a species the repeats evolve together.Previous study shows that there are two major variants in the 18 SrRNA gene of the single Rotylenchulus reniformis.The variation within ITS region of rRNA gene among R.reniformis populations from Zhejiang,Fujian andChongqing of China was reported.Two bands of 18S-ITS sequence with lengths of 1 250 bp and 1 400 bp（include≈480bp 18 SrDNA sequence,the whole ITS sequence and ≈50bp 28 SrDNA sequence）were amplified from each nematode of three populations of R.reniformis,which named as variant 1（RN_VAR1）and variant 2（RN_VAR2）,respectively.The amount of variation was assessed by sequencing five clones from each variant from six female reniform nematodes of three populations, with a total of 60 sequences.These sequences were distinguished,based on multiple sequence alignment（MSN）,the percent identity and guanine and cytosine content（GC）content of the ITS and clustering in phylogenetic trees.The length and GC content of ITS region were characterized by RN_VAR1（705-712 bp,45.1%-46.7%）and RN_VAR2（854-860 bp,48.4%-50.0%）,respectively.The percent identity between RN_VAR1and RN_VAR2was relatively low which only ranged between62.1% and 65.6%.The percent identity between all clones in RN_VAR1 was 89.9%-100%,and that in RN_VAR2was 91.4%-99.8%.Phylogenetic tree analyses based on neighbor-joining method showed that both RN variants could be distinguished from other nematodes by ITS sequence alignment, while it was hard for differentiation of R.reniformis intrapopulation.The quantitative real-time polymerase chain reaction（qPCR）experiments showed that RN_VAR1made up 56% of rDNA units with relatively well-conserves in 18 SRNA gene,whereas RN_VAR2was 40%.The two ITS variants,RN_VAR1and RN_VAR2,were consistent to the reported two 18 SRNA gene variants,respectively.The results confirm that there are two rDNA variant types in reniform nematode.This variation may be as a result of ancestral interspecific hybridization or gene recombination between sister chromatids,leading to the coevolution of the rDNA sequences on these chromosomes.