Cloning And Analysis Of Complete Nuclear Ribosomal DNA (nrDNA) Cistron Sequence Of Pyropia Yezoensis(Bangiales, Rhodophyta)

Pyropia yezoensis is one of the most economically important seaweed crops inChina. Meanwhile, it has been used as a model system in marine algae for moleculargenetics and genomics. Pyropia yezoensis has a heteromorphic alternation ofgenerations. The thallus is the macroscopic haploid gametophyte phase, which is used inthis study, while the conchocelis is the microscopic diploid sporophyte phase. Nucleargenomic DNA of individual is extracted by CTAB method, and the complete nuclearribosomal DNA (nrDNA) cistron sequence of Pyropia yezoensis is cloned and analyzed.The ribosomal RNA gene (rDNA) that possesses abundant repeat units is amulti-gene family with transcription activity. In eukaryotes each unit of the rRNA geneconsists of the internal spacer (IGS), the small-subunit (SSU) rRNA gene, the internaltranscribed spacer1(ITS1), the5.8S rRNA gene, the internal transcribed spacer2(ITS2), and the large-subunit (LSU) rRNA gene in order. The gene cloning andsequencing methods were used to study the ribosomal RNA gene and the internal spacerin Pyropia yezoensis. Through analyzing sequence data, we detect that introns thenumber of which range from zero to two are present in the small-subunit rRNA genes ofwhich the exon sequences (1834bp) are almost identical. The location, number andlength of introns are discriminatory between individuals. GenBank accession numbersof the18S rDNA are KJ578745, KJ578746, KJ578747, KJ643310and KJ643311,respectively. However, the LSU rDNA (GenBank No. KJ578748) of which the fulllength is4770bp has no introns，and the5.8S rRNA gene is159bp. Moreover, the5’ and3’ terminal sequences of the genic regions of the ribosomal cistron are extremelyconservative even among various species in red alga. In addition, the ITS1is371bp-372bp and the ITS2is532bp-535bp. GenBank accession number of the ITSregion is KJ608639. We sequenced the complete nucleotide of IGS region of Pyropia yezoensis for the first time. GenBank accession numbers of the IGS region is KJ608640.The IGS that is5984bp in length possesses abundant sequence repeats upstream andsome complex structures in the center region, which make sequencing untoward. It isdefinite that the shortest sequence length is13654bp，and the longest sequence length is15130bp after sequence analysis.Total RNA of individuals are extracted and transcribed to cDNA which are used asPCR templates of the SSU rRNA and LSU rRNA amplification products. The resultindicates that two introns in transcript of SSU rDNA are not excised at the same timeand there are no introns in mature SSU rRNA. The introns are absent in transcript ofLSU rDNA, therefore, there are also no introns in mature LSU rRNA.The entire SSU rDNA, LSU rDNA, ITS, and partial IGS sequence of four differentbiogeographic Pyropia yezoensis from Rudong, Haimen in Jiangsu Province, andHongdao, Qingdao in Shangdong Province were sequenced and analyzed. The resultsindicate that the sequence of Pyropia yezoensis from Rudong, Haimen and Qingdao arebasically the same, which are considered to be one species. The sequences of Pyropiayezoensis from Hongdao have great differences with the above. Therefore, it ispresumed that the two kind Pyropia yezoensis are the distant relative subspecies, oreven different Pyropia species. The revelation of the full length of rDNA cistron willoffer groundbreaking information to be widely utilized for classification system, geneticrelationship, and phyletic evolution in red alga.