Plant Molecular Biology Reporter

We describe a modification of a protocol for the isolation of BAC DNA using a silica membrane-based kit designed for the isolation of plasmid DNA. The major advantages of this protocol are the expediency of the procedure, the high yield and purity, and the high quality of the BAC DNA that is suitable for direct sequencing.

Reverse transcription PCR (RT-PCR) has been proven to be a useful method in the analysis of gene expression, especially for detecting low abundance mRNA transcripts. However, quantitation by RT-PCR can be difficult due to different reverse transcription and PCR amplification efficiencies and sample-to-sample and tube-to-tube variations. We have used synthetic RNA that contains the same primer sequences as the target mRNA, as an internal standard, to improve the accuracy of RT-PCR quantitation. Products generated from the target gene by RT-PCR differ in size from those of the synthetic internal standard. Using the ratios of two species of PCR products as a quantitation index, sample-to-sample and tube-to-tube variations can be eliminated. The method we present here can be widely applied to analyzing gene expression in different tissues or cell types with minimal effort to produce standard curves.

The calmodulin antagonist N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) binds to calmodulzin and inhibits Ca2+/calmodulin-regulated enzyme activities. In plant cells, W7 inhibits the activity of calcium-dependent protein kinases (CDPKs)—the major calcium sensors in plants. In the present study, we examined the effect of W7 on increased resveratrol biosynthesis and expression of CDPK and stilbene synthase (STS) genes in a cell culture of Vitis amurensis Rupr. We used coumaric acid (CA), salicylic acid (SA), and phenylalanine (Phe) to increase the content of resveratrol in V. amurensis calli, since its content is low under standard conditions. W7 significantly decreased resveratrol production and expression of STS genes in CA-, SA-, and Phe-treated grape cells. Also, treatment of the V. amurensis calli with SA, Phe, or CA considerably increased expression of VaCDPK1a (with SA, Phe), VaCDPK1L (with SA, Phe), VaCDPK2a (with Phe) genes, and decreased expression of VaCDPK3a (with CA). Addition of W7 to CA-, SA-, and Phe-treated grape cells reversed this effect, resulting in increased VaCDPK3a expression and decreased VaCDPK1a, VaCDPK1L, and VaCDPK2a expression. The results obtained suggest that CDPK activities might play an important role in resveratrol biosynthesis.

Although the increasing number of expressed sequence tags (ESTs) from the public domain has facilitated the detection of single nucleotide polymorphisms (SNPs), further validation is needed before they can be used as markers. For SNP validation, we have compared 2 independent methods: (1) the primer extension method followed by capillary electrophoresis on an ABI PRISM 3100 Genetic Analyzer and (2) nested PCR followed by agarose-based visualization. We present an assessment of the efficiency and costs associated with these methods, based on a sample of barley cultivars.

A spikelet-specific cDNA clone, OsMSP (Oryza sativa meiosis serine protease), was isolated from a rice cDNA library constructed by suppression subtraction hybridization (SSH). Its expression level was significantly reduced in the thermo-sensitive genic male-sterile rice line at sterile temperature (30/26°C, day/night) compared with that at fertile temperature (23/21°C, day/night) conditions. In contrast, no significant differences were observed in the normal fertile line under the above two sets of temperature conditions. The cDNA is 2,867 bp in length with an open reading frame (ORF) encoding 837 amino acid residues. Northern blots revealed that OsMSP transcripts were specifically present in spikelets at meiosis, and reached the highest level at early microspore stage. mRNA in situ hybridization confirmed that OsMSP was a rice microsporocyte-specific gene and its expression was limited to early microspore developmental stage. BLAST and phylogenetic analyses revealed that OsMSP shared high similarities with subtilisin-like serine protease genes from other organisms at both nucleotide and amino acid levels.

A reliable and efficient protocol is given for the isolation of mRNA from the periderm of potato tubers and sweet potato storage roots. The method relies on a urea-based lysis buffer and lithium chloride to concentrate total RNA away from most of the cytoplasmic components and to prevent oxidation of phenolic complexes. To enhance the physical separation of the RNA from other macromolecular components, the RNA fraction was incubated in the presence of the cationic surfactant Catrimox-14. Poly(A)+ mRNA was separated from total RNA and other contaminants by using Promega's MagneSphere technology. The mRNA was suitable for cDNA library construction and RNA fingerprinting.