We reported our preliminary study on mapping of the expressed sequence tag (EST) using single-stranded DNA conformation polymorphism (SSCP) on polymerase chain reaction (PCR) product. Five ESTs primer pairs, developed...We reported our preliminary study on mapping of the expressed sequence tag (EST) using single-stranded DNA conformation polymorphism (SSCP) on polymerase chain reaction (PCR) product. Five ESTs primer pairs, developed from nuclear genes including CAD(cinnamyl-alcohol dehydrgenase), CHS(chalcone synthase), NIR(nitrite reductase), ARA554 (cDNA expressed in differentiating xylem in Pinus taedae) and GS(glutamine synthetase), were selected to optimize the experimental protocol and generate mapping markers in the megagemtophytes from a single tree of Pinus massoniana. Our ultimate goal was to use SSCP approach to construct a transcriptional map for comparative mapping studies in P. massoniana. The efficiency to construct a transcriptional map in P. massoniana based on the published EST primer pairs derived from other pine species critically depended on the successful amplification of EST fragments and the ratio of the heterozygous loci revealed in P.massoniana. In this study, 3 (60) out of 5 tested EST primer pairs were succeeded in amplification, however, only 1(20) gene was heterozygous in the tested tree. The ratio of heterozygous loci detected in this study was similar to that revealed by anonymous marker in P.taeda. Therefore, a low efficiency would be expected if the map would be constructed using single pedigree. The segregation ratio of loci revealed by primer pairs would be higher if multiple pedigrees would be used. We proposed that consensus mapping approach based on multiple-pedigree should be used for EST mapping and therefore to increase the efficiency of EST mapping.展开更多
The traditional culture-dependent plate counting and culture-independent small-subunit-ribosomal RNA gene-targeted molecular techniques, Single-Strand Conformation Polymorphism (SSCP) and ter-minal Restriction Fragmen...The traditional culture-dependent plate counting and culture-independent small-subunit-ribosomal RNA gene-targeted molecular techniques, Single-Strand Conformation Polymorphism (SSCP) and ter-minal Restriction Fragment Length Polymorphism (tRFLP) combined with 16S rDNA clone library were adopted to investigate the impacts of secretion from Camptotheca acuminata (abbreviated to Ca) roots on the quantities and structure of eukaryotic microbes and bacteria in the rhizosphere, and the possi-bility that Ca controls exotic invasive plant Eupatorium adenophorum (Ea). The counting results indi-cated that the number of bacteria increased in turn in rhizospheres of Ea, Ca-Ea mixed culture and Ca, while that of eukaryotic microbes decreased. PCR-SSCP profiles showed eukaryotic microbial bands (corresponding to biodiversity) in rhizosphere of Ea were more complex than those of Ca and CE. Meristolohmannia sp., Termitomyces sp. and Rhodophyllus sp. were the dominant populations in the rhizosphere of Ca. Bacterial terminal restriction fragments (TRFs) profiles showed no difference among three kinds of rhizospheres, and the sequences of the 16S rDNA clone library from Ca rhizospheres were distributed in 10 known phyla, in which phylum Proteobacteria were the absolute dominant group and accounted for 24.71% of the cloned sequences (δ-Proteobacteria accounted for up to 17.65%), and phyla Acidobacteria and Bacteroidetes accounted for 16.47% and 10.59% of the cloned sequences, respectively. In addition, high performance liquid chromatography detected a trace amount of camp-tothecin and hydroxycamptothecin in the rhizospheric soil of Ca and CE, but examined neither camp-tothecin nor hydroxycamptothecin in rhizospheric soil of Ea. Therefore, invasion and diffusion of Ea evidently depended on distinguishing the eukaryotic community structure, but not on that of the bac-terial pattern. Ca was able to alter the eukaryotic community structure of invasive Ea by secreting camptothecin and hydroxycamptothecin into rhizospheres, and may bene展开更多
文摘We reported our preliminary study on mapping of the expressed sequence tag (EST) using single-stranded DNA conformation polymorphism (SSCP) on polymerase chain reaction (PCR) product. Five ESTs primer pairs, developed from nuclear genes including CAD(cinnamyl-alcohol dehydrgenase), CHS(chalcone synthase), NIR(nitrite reductase), ARA554 (cDNA expressed in differentiating xylem in Pinus taedae) and GS(glutamine synthetase), were selected to optimize the experimental protocol and generate mapping markers in the megagemtophytes from a single tree of Pinus massoniana. Our ultimate goal was to use SSCP approach to construct a transcriptional map for comparative mapping studies in P. massoniana. The efficiency to construct a transcriptional map in P. massoniana based on the published EST primer pairs derived from other pine species critically depended on the successful amplification of EST fragments and the ratio of the heterozygous loci revealed in P.massoniana. In this study, 3 (60) out of 5 tested EST primer pairs were succeeded in amplification, however, only 1(20) gene was heterozygous in the tested tree. The ratio of heterozygous loci detected in this study was similar to that revealed by anonymous marker in P.taeda. Therefore, a low efficiency would be expected if the map would be constructed using single pedigree. The segregation ratio of loci revealed by primer pairs would be higher if multiple pedigrees would be used. We proposed that consensus mapping approach based on multiple-pedigree should be used for EST mapping and therefore to increase the efficiency of EST mapping.
基金the Excellent Young Teacher’s Innovation Foundation of Northeast Forestry University to Yang FengJian,the Key Research Fund of Ministry of Educa-tion of China (Grant No.104191) the Forestry Noxious Plant Investigation Fund of State Forestry Administration of China to Zu YuanGang
文摘The traditional culture-dependent plate counting and culture-independent small-subunit-ribosomal RNA gene-targeted molecular techniques, Single-Strand Conformation Polymorphism (SSCP) and ter-minal Restriction Fragment Length Polymorphism (tRFLP) combined with 16S rDNA clone library were adopted to investigate the impacts of secretion from Camptotheca acuminata (abbreviated to Ca) roots on the quantities and structure of eukaryotic microbes and bacteria in the rhizosphere, and the possi-bility that Ca controls exotic invasive plant Eupatorium adenophorum (Ea). The counting results indi-cated that the number of bacteria increased in turn in rhizospheres of Ea, Ca-Ea mixed culture and Ca, while that of eukaryotic microbes decreased. PCR-SSCP profiles showed eukaryotic microbial bands (corresponding to biodiversity) in rhizosphere of Ea were more complex than those of Ca and CE. Meristolohmannia sp., Termitomyces sp. and Rhodophyllus sp. were the dominant populations in the rhizosphere of Ca. Bacterial terminal restriction fragments (TRFs) profiles showed no difference among three kinds of rhizospheres, and the sequences of the 16S rDNA clone library from Ca rhizospheres were distributed in 10 known phyla, in which phylum Proteobacteria were the absolute dominant group and accounted for 24.71% of the cloned sequences (δ-Proteobacteria accounted for up to 17.65%), and phyla Acidobacteria and Bacteroidetes accounted for 16.47% and 10.59% of the cloned sequences, respectively. In addition, high performance liquid chromatography detected a trace amount of camp-tothecin and hydroxycamptothecin in the rhizospheric soil of Ca and CE, but examined neither camp-tothecin nor hydroxycamptothecin in rhizospheric soil of Ea. Therefore, invasion and diffusion of Ea evidently depended on distinguishing the eukaryotic community structure, but not on that of the bac-terial pattern. Ca was able to alter the eukaryotic community structure of invasive Ea by secreting camptothecin and hydroxycamptothecin into rhizospheres, and may bene
