Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appr...Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient sub-stratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1×106 ml?1, the percentages of conidium germination and appressorium formation were (97.98±0.67)% and (97.88±0.45)%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 μg total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA (complementary DNA) library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.展开更多
Extraction of quality RNA for molecular biology applications from perennial woody plants like mulberry is complicated due to the presence of high polysaccharides, polyphenols and other secondary metabolites. Since the...Extraction of quality RNA for molecular biology applications from perennial woody plants like mulberry is complicated due to the presence of high polysaccharides, polyphenols and other secondary metabolites. Since the existing methods failed to yield quality RNA in sufficient quantity from leaf and root tissues of mulberry, in this study, we modified the CTAB-based protocol. The standardised protocol yielded high quantity (520.00 μg/g fresh weight of leaf tissue) of quality RNA and the RNA extracted was suitable for all downstream applications such as cDNA synthesis, PCR and whole transcriptome analysis. The method developed was also found to be useful for isolating good quality and quantity total RNA from desiccated and salinity stressed leaf tissues of mulberry. The protocol was also applied successfully to isolate total RNA from leaf tissues of other species such as cardamom, papaya and rice.展开更多
基金Project (Nos. 30671351 and 30870101) supported by the National Natural Science Foundation of China
文摘Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient sub-stratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1×106 ml?1, the percentages of conidium germination and appressorium formation were (97.98±0.67)% and (97.88±0.45)%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 μg total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA (complementary DNA) library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.
文摘Extraction of quality RNA for molecular biology applications from perennial woody plants like mulberry is complicated due to the presence of high polysaccharides, polyphenols and other secondary metabolites. Since the existing methods failed to yield quality RNA in sufficient quantity from leaf and root tissues of mulberry, in this study, we modified the CTAB-based protocol. The standardised protocol yielded high quantity (520.00 μg/g fresh weight of leaf tissue) of quality RNA and the RNA extracted was suitable for all downstream applications such as cDNA synthesis, PCR and whole transcriptome analysis. The method developed was also found to be useful for isolating good quality and quantity total RNA from desiccated and salinity stressed leaf tissues of mulberry. The protocol was also applied successfully to isolate total RNA from leaf tissues of other species such as cardamom, papaya and rice.
