Abiotic stress poses a great threat to plant growth and can lead to huge losses in yield. Gene enolase2 （EN02） is important in resistance to abiotic stress in various organisms. ENO2 T-DNA insertion mutant （enoZ） ...Abiotic stress poses a great threat to plant growth and can lead to huge losses in yield. Gene enolase2 （EN02） is important in resistance to abiotic stress in various organisms. ENO2 T-DNA insertion mutant （enoZ） plants of Arabidopsis thaliana showed complete susceptibility to sodium chloride treatment when were analyzed either as whole plants or by measuring root growth during NaCl treatment. Quantitative real-time RT-PCR （RT-qPCR） was performed to investigate the expression profile of EN02 in response to NaCl stress in Arabidopsis. The transcript level of EN02 was rapidly elevated in 300 mmol L-1 NaCl treatment. ENO2 also responded to 300 mmol L 1 NaCl treatment at the protein level. To illuminate the mechanism underlying EN02 resistance to salt at the transcriptional level, we studied the wild-type and enoZ Arabidopsis lines that were treated with 300 mmol L 1 NaCl for 18 h using 454 GS FLX, which resulted in an expressed sequence tag （EST） dataset. A total of 961 up-regulated and 746 down-regulated differentially expressed genes （DEGs） were identified in the pairwise comparison w-r-18 h：eno2^-18 h. The DEGs were identified and functionally annotated using the databases of Gene Ontology （GO） and the Kyoto encyclopedia of genes and genomes （KEGG）. The identified unigenes were subjected to GO analysis to determine biological, molecular, and cellular functions. The biological process was enriched in a total of 20 GO terms, the cellular component was enriched in 13 GO terms, and the molecular function was enriched in 11 GO terms. Using KEGG mapping, DEGs with pathway annotations contributed to 115 pathways. The top 3 pathways based on a statistical analysis were biosynthesis of the secondary metabolites （KO01110）, plant-pathogen interactions （KO04626）, and plant hormone signal transduction （KO04075）. Based on these results, EN02 contributes to increased resistance to abiotic stress. In particular, EN02 is involved in some of the metabolic stress response pathways in Arabidopsis. Our work also demonstrates that this EST dataset will be a powerful resource for further studies of EN02, such as functional analyses, investigations of biological roles, and molecular breeding. Additionally, 3-phosphoglycerate kinase （PGK）, 3-phosphoglycerate kinase 1 （PGK1）, triosephosphate isomerase （TPI）, and pyruvate kinase （PK） in glycolysis interactions with ENO2 were verified using the yeast two-hybrid experiment, and EN02 may regulate the expression of PGK, PGK1, TPI, and PK. Taken together, the results from this study reflects that EN02 gene has an important role in the response to the high salt stress.展开更多
Microsatellites, or simple sequence repeats (SSRs), are highly polymorphic, co-dominant genetic markers commonly used for population genetics analyses although de novo development of species specific microsatellites i...Microsatellites, or simple sequence repeats (SSRs), are highly polymorphic, co-dominant genetic markers commonly used for population genetics analyses although de novo development of species specific microsatellites is cost-and time-intensive. Orchidaceae is one of the most species-rich families of angiosperms with more than 30,000 species estimated. Despite its high species-diversity, microsatellites are available only for a few species and all were developed by only using Sanger sequencing methods. For the first time in orchids, we used 454 GS-FLX sequencing to isolate microsatellites in two species (Cypripedium kentuckiense and Pogonia ophioglossoides), and report preliminary results of the study. From 1/16th plate that was subjected to sequencing, 32,665 reads were generated, from which 15,473 fragments contained at least one SSR. We selected 20,697 SSRs representing di-, tri-, and tetra-nucleotides. While 3,674 microsatellites had flanking regions on both sides, useable primer pairs could be designed for 255 SSRs. The mean numbers of reads, SSRs, and SSR-containing reads useful for primer design estimated for other 15 orchid species using Sanger sequencing method were 166, 78 and 31, respectively. Results demonstrate that the efficiency of microsatellite isolation in orchids is substantially higher with 454 GS-FLX sequencing technique in comparison to the Sanger sequencing methods.展开更多
基金funded by the National Natural Science Foundation of China (31470399 and 31270365)
文摘Abiotic stress poses a great threat to plant growth and can lead to huge losses in yield. Gene enolase2 （EN02） is important in resistance to abiotic stress in various organisms. ENO2 T-DNA insertion mutant （enoZ） plants of Arabidopsis thaliana showed complete susceptibility to sodium chloride treatment when were analyzed either as whole plants or by measuring root growth during NaCl treatment. Quantitative real-time RT-PCR （RT-qPCR） was performed to investigate the expression profile of EN02 in response to NaCl stress in Arabidopsis. The transcript level of EN02 was rapidly elevated in 300 mmol L-1 NaCl treatment. ENO2 also responded to 300 mmol L 1 NaCl treatment at the protein level. To illuminate the mechanism underlying EN02 resistance to salt at the transcriptional level, we studied the wild-type and enoZ Arabidopsis lines that were treated with 300 mmol L 1 NaCl for 18 h using 454 GS FLX, which resulted in an expressed sequence tag （EST） dataset. A total of 961 up-regulated and 746 down-regulated differentially expressed genes （DEGs） were identified in the pairwise comparison w-r-18 h：eno2^-18 h. The DEGs were identified and functionally annotated using the databases of Gene Ontology （GO） and the Kyoto encyclopedia of genes and genomes （KEGG）. The identified unigenes were subjected to GO analysis to determine biological, molecular, and cellular functions. The biological process was enriched in a total of 20 GO terms, the cellular component was enriched in 13 GO terms, and the molecular function was enriched in 11 GO terms. Using KEGG mapping, DEGs with pathway annotations contributed to 115 pathways. The top 3 pathways based on a statistical analysis were biosynthesis of the secondary metabolites （KO01110）, plant-pathogen interactions （KO04626）, and plant hormone signal transduction （KO04075）. Based on these results, EN02 contributes to increased resistance to abiotic stress. In particular, EN02 is involved in some of the metabolic stress response pathways in Arabidopsis. Our work also demonstrates that this EST dataset will be a powerful resource for further studies of EN02, such as functional analyses, investigations of biological roles, and molecular breeding. Additionally, 3-phosphoglycerate kinase （PGK）, 3-phosphoglycerate kinase 1 （PGK1）, triosephosphate isomerase （TPI）, and pyruvate kinase （PK） in glycolysis interactions with ENO2 were verified using the yeast two-hybrid experiment, and EN02 may regulate the expression of PGK, PGK1, TPI, and PK. Taken together, the results from this study reflects that EN02 gene has an important role in the response to the high salt stress.
文摘Microsatellites, or simple sequence repeats (SSRs), are highly polymorphic, co-dominant genetic markers commonly used for population genetics analyses although de novo development of species specific microsatellites is cost-and time-intensive. Orchidaceae is one of the most species-rich families of angiosperms with more than 30,000 species estimated. Despite its high species-diversity, microsatellites are available only for a few species and all were developed by only using Sanger sequencing methods. For the first time in orchids, we used 454 GS-FLX sequencing to isolate microsatellites in two species (Cypripedium kentuckiense and Pogonia ophioglossoides), and report preliminary results of the study. From 1/16th plate that was subjected to sequencing, 32,665 reads were generated, from which 15,473 fragments contained at least one SSR. We selected 20,697 SSRs representing di-, tri-, and tetra-nucleotides. While 3,674 microsatellites had flanking regions on both sides, useable primer pairs could be designed for 255 SSRs. The mean numbers of reads, SSRs, and SSR-containing reads useful for primer design estimated for other 15 orchid species using Sanger sequencing method were 166, 78 and 31, respectively. Results demonstrate that the efficiency of microsatellite isolation in orchids is substantially higher with 454 GS-FLX sequencing technique in comparison to the Sanger sequencing methods.
