RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust an...RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.展开更多
Salinity is a serious threat to agricultural production. Potato （Solanum tuberosum） is an important food crop characterised for having low to moderate salinity tolerance. Tissue cultures may be relevant to improve s...Salinity is a serious threat to agricultural production. Potato （Solanum tuberosum） is an important food crop characterised for having low to moderate salinity tolerance. Tissue cultures may be relevant to improve salt tolerance in potato through selection of salt-tolerant cell lines and subsequent regeneration of plants. In this work, the authors used the random amplified polymorphic DNA （RAPD） markers to investigate the occurrence of genetic polymorphism in a potato calli line tolerant to 150 mM NaCI. Out of 40 primers screened, eight generated polymorphic patterns that distinguished salt-tolerant line from the control. Although the macroscopic appearance was similar in both lines, ultrastructural study revealed alterations in salt-grown cells. These showed that plastids less differentiated with a lower number of grana had more and larger starch grains than control cells. In conclusion, RAPD analysis revealed that NaCl-adapted line is a somaclonal variant and the ultrastructural study showed changes essentially at the plastids.展开更多
Elicitor-induced phosphorylation of tyrosine residues in proteins of potato was studied. Proteins of crude extract of suspension culture of potato were analyzed by one- and two-dimensional electrophoresis followed by ...Elicitor-induced phosphorylation of tyrosine residues in proteins of potato was studied. Proteins of crude extract of suspension culture of potato were analyzed by one- and two-dimensional electrophoresis followed by Western blotting with monoclonal antibodies PY20 to phosphotyrosine proteins. One- and two-dimensional electrophoresis revealed l l and 25 tyrosine-phosphorylated proteins, respectively. Glycoprotein increased the phosphorylation level of most of these proteins.展开更多
文摘RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.
文摘Salinity is a serious threat to agricultural production. Potato （Solanum tuberosum） is an important food crop characterised for having low to moderate salinity tolerance. Tissue cultures may be relevant to improve salt tolerance in potato through selection of salt-tolerant cell lines and subsequent regeneration of plants. In this work, the authors used the random amplified polymorphic DNA （RAPD） markers to investigate the occurrence of genetic polymorphism in a potato calli line tolerant to 150 mM NaCI. Out of 40 primers screened, eight generated polymorphic patterns that distinguished salt-tolerant line from the control. Although the macroscopic appearance was similar in both lines, ultrastructural study revealed alterations in salt-grown cells. These showed that plastids less differentiated with a lower number of grana had more and larger starch grains than control cells. In conclusion, RAPD analysis revealed that NaCl-adapted line is a somaclonal variant and the ultrastructural study showed changes essentially at the plastids.
文摘Elicitor-induced phosphorylation of tyrosine residues in proteins of potato was studied. Proteins of crude extract of suspension culture of potato were analyzed by one- and two-dimensional electrophoresis followed by Western blotting with monoclonal antibodies PY20 to phosphotyrosine proteins. One- and two-dimensional electrophoresis revealed l l and 25 tyrosine-phosphorylated proteins, respectively. Glycoprotein increased the phosphorylation level of most of these proteins.
