It has been reported that there was a linkage of 5S rRNA gene to 18S-5.8S-25S rRNA gene in a few of species in Ochrophyta.In regard to the usual two positions of linked 5S rDNA to the 3′end of 25S rDNA,two pairs of p...It has been reported that there was a linkage of 5S rRNA gene to 18S-5.8S-25S rRNA gene in a few of species in Ochrophyta.In regard to the usual two positions of linked 5S rDNA to the 3′end of 25S rDNA,two pairs of primers were designed for amplification to verify this linkage of two genes in a kelp cultivar of Saccharina japonica,one of species in Ochrophyta.This result supplemented the previous report that 5S rDNA was unlinked to 25S rDNA in this kelp.In order to simultaneously visualize this unlinkage of two genes,dual-color fluorescence in situ hybridization(FISH)technique was applied to the cytogenetics of S.japonica.Dual-color FISH images showed that two and four hybridization signals were present in the kelp gametophyte and sporophyte,respectively,metaphase nuclei hybridized simultaneously with the labeled probes of 18S rDNA and 5S rDNA.Both haploid and diploid karyotypes in decreasing length of chromosomes showed that 18S-5.8S-25S rDNA was localized at the interstitial region of Chromosome 23,whereas 5S rDNA resided at the sub-telomeric region of Chromosome 27.These karyotypes suggested that the kelp nuclear genome had only one locus of each rRNA gene,and their loci on different chromosomes indicated the physical unlinkage of 5S rDNA to 18S-5.8S-25S rDNA in this kelp.Therefore,dual-color FISH seems to be a powerful technique for the discrimination and pairing of chromosomes featured in both small size and nearly identical shape in S.japonica.展开更多
Objective:To develop the current single primer PCR-based genome-walking method with Scenedesmus sp.Methods:The unknown 5’and/or 3’flanking regions for a specific conserved sequence were optimized and the current sin...Objective:To develop the current single primer PCR-based genome-walking method with Scenedesmus sp.Methods:The unknown 5’and/or 3’flanking regions for a specific conserved sequence were optimized and the current single primer PCR-based genome-walking method were developed.Alignment was between the related species of microalga and Scenedesmus sp.For 18S rDNA,we selected the species Scenedesmus sp.,Chlorella sp.,and Chlamydomonas sp.For the rbcL gene from the chloroplast genome,alignment was done between Scenedesmus sp.,and Chlamydomonas sp.Results:Obtaining a small conserved sequence for any gene family is something that can be achieved quite easily.However,identifying the whole gene is often difficult.After investigating and testing,some of the current protocols using to get the unknown 5’and/or 3’flanking regions for a specific conserved sequence,we developed the current single primer PCR-based genome-walking method.We performed two consecutive PCR reactions;band extraction and the PCR product were sequenced.We got our results by testing the method on three genes from the total DNA of Scenedesmus sp.;two genes had a fully known sequence in gene bank(18S rDNA and rbcL),but the third one has not yet been identified(rbcS).We designed our primers based on the alignment between the related species and to each other.We also tested two different DNA polymerases Ex Taq and TLA polymerase.Conclusions:Results from our study suggest that Ex Taq is the most suitable polymerase for the current protocol.展开更多
基金Supported by the National Natural Science Foundation of China(Nos.41376136,31201992)the Double First-Class Discipline of Fisheries Science in China。
文摘It has been reported that there was a linkage of 5S rRNA gene to 18S-5.8S-25S rRNA gene in a few of species in Ochrophyta.In regard to the usual two positions of linked 5S rDNA to the 3′end of 25S rDNA,two pairs of primers were designed for amplification to verify this linkage of two genes in a kelp cultivar of Saccharina japonica,one of species in Ochrophyta.This result supplemented the previous report that 5S rDNA was unlinked to 25S rDNA in this kelp.In order to simultaneously visualize this unlinkage of two genes,dual-color fluorescence in situ hybridization(FISH)technique was applied to the cytogenetics of S.japonica.Dual-color FISH images showed that two and four hybridization signals were present in the kelp gametophyte and sporophyte,respectively,metaphase nuclei hybridized simultaneously with the labeled probes of 18S rDNA and 5S rDNA.Both haploid and diploid karyotypes in decreasing length of chromosomes showed that 18S-5.8S-25S rDNA was localized at the interstitial region of Chromosome 23,whereas 5S rDNA resided at the sub-telomeric region of Chromosome 27.These karyotypes suggested that the kelp nuclear genome had only one locus of each rRNA gene,and their loci on different chromosomes indicated the physical unlinkage of 5S rDNA to 18S-5.8S-25S rDNA in this kelp.Therefore,dual-color FISH seems to be a powerful technique for the discrimination and pairing of chromosomes featured in both small size and nearly identical shape in S.japonica.
基金Supported by Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education,Science and Technology(2010-0013600).
文摘Objective:To develop the current single primer PCR-based genome-walking method with Scenedesmus sp.Methods:The unknown 5’and/or 3’flanking regions for a specific conserved sequence were optimized and the current single primer PCR-based genome-walking method were developed.Alignment was between the related species of microalga and Scenedesmus sp.For 18S rDNA,we selected the species Scenedesmus sp.,Chlorella sp.,and Chlamydomonas sp.For the rbcL gene from the chloroplast genome,alignment was done between Scenedesmus sp.,and Chlamydomonas sp.Results:Obtaining a small conserved sequence for any gene family is something that can be achieved quite easily.However,identifying the whole gene is often difficult.After investigating and testing,some of the current protocols using to get the unknown 5’and/or 3’flanking regions for a specific conserved sequence,we developed the current single primer PCR-based genome-walking method.We performed two consecutive PCR reactions;band extraction and the PCR product were sequenced.We got our results by testing the method on three genes from the total DNA of Scenedesmus sp.;two genes had a fully known sequence in gene bank(18S rDNA and rbcL),but the third one has not yet been identified(rbcS).We designed our primers based on the alignment between the related species and to each other.We also tested two different DNA polymerases Ex Taq and TLA polymerase.Conclusions:Results from our study suggest that Ex Taq is the most suitable polymerase for the current protocol.
