魁蚶核糖体DNA基因转录间隔区的序列特征

Sequence analysis of ribosomal DNA internal transcribed spacers of bloody clam,Scapharca broughtonii

以相应引物PCR扩增魁蚶(Scapharcabroughtonii)的核糖体转录间隔子ITS 1和ITS 2片段,测序后得到长度分别为461bp和533bp的核苷酸序列(含引物)。其中A、T、G、C4种碱基的含量分别为21 91%、24 73%、28 20%和25 16%(ITS 1),27 02%、25 52%、25 52%和21 95%(ITS 2)。将这2个片段与其他双壳贝类的相应片段进行比较,发现ITS 1和ITS 2引物在贝类中具有良好的通用性。比较几种贝类的ITS 1片段发现有1～100bp长度不等、弥散状态的插入/缺失;对5种蚶科贝类的ITS 2相应片段进行比较,发现中间有75bp的保守区域,与本研究在魁蚶ITS 2群体序列研究中观察到的情况相同。

Mainly distributed in coast waters of China,Korea,Japan and southeast Russia,bloody clam Scapharca broughtonii has been a very important commercial bivalve in China.In the past few years,however,the culture intensity in China increased significantly due to strong demand of bloody clam in both domestic and overseas markets,which led to a potential pressure on stock management for this species.Therefore,it is very necessary and important to investigate genetic diversity and genetic structure of bloody clam populations.By now,very little research has been done in this field in bloody clam at DNA level.Ribosomal DNA internal transcribed spacer (ITS-1 and ITS-2) fragments are very useful nuclear molecular markers and have been used in phylogenetic analysis and population diversity studies in many species.Because the coding regions flanking the two segments are conserved,it is very convenient to design primers and amplify the two segments. ITS-1 and ITS-2 segments of bloody clam were amplified via PCR[94℃ pre-denaturalization 2min,35 cycles of 94℃for 45s,50℃ (ITS-1) or 52℃ (ITS-2) for 1min and 72℃ for 1min].The PCR products were ligated into T-vectors,cloned and directly sequenced by following regular procedures.The nucleotide sequences of ITS-1 and ITS-2 at 461bp and 534bp (primers included) were obtained,respectively.The contents of A,T,G and C were 21.91%,24.73%,28.20% and 25.16% in ITS-1;27.02%,25.52%,25.52% and 21.95% in ITS-2,respectively.The content of G+C in ITS-1 was higher than that in ITS-2 and the content of G+C in the two fragments was at a moderate level among bivalves according to previous researches. These two fragments were compared with the corresponding fragments from several other bivalves,such as oyster,scallops and clam.The results showed that the primers of ITS-1 and ITS-2 were universal in a variety of mollusk species.It was found that the insertions/deletions (ranging from 1 to 100bp) were dispersed randomly in the whole ITS-1 fragment.Four to five bases or even single base insertion/deletion was very common and 50-100bp of long insertion/deletion was found too.A seventy-five-base conserved segment in ITS-2 sequence was found by comparison among five Arcidae species,which was very similar to the result in our population study with the ITS-2 fragment in S.broughtonii.This work could be a good step forward to the studies of genetic diversity in bloody clam using sequence data of ITS-1 and ITS-2.If combined with mtDNA sequence data,ITS sequences could be more informative and useful in studies of populations,evolution and phylogenetics.