Gene expression and metabolite profiling of developing highbush blueberry fruit indicates transcriptional regulation of flavonoid metabolism and activation of abscisic acid metabolism.

Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative real-time reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3'-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3'5'-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation of blueberry flavonoid biosynthesis.

The use of Group 3 LEA proteins as fusion partners in facilitating recombinant expression of recalcitrant proteins in E. coli.

Late embryogenesis abundant (LEA) proteins are intrinsically disordered proteins that accumulate in organisms during the development of dehydration stress tolerance and cold acclimation. Group 3 LEA proteins have been implicated in the prevention of cellular protein denaturation and membrane damage during desiccation and anhydrobiosis. We tested the ability of LEA proteins to facilitate recombinant expression of recalcitrant and intrinsic membrane proteins. Two Brassica napus Group 3 LEA proteins, BN115m and a truncated fragment of BNECP63, were fused to two target proteins identified as recalcitrant to overexpression in soluble form or outside of inclusion bodies. Fusion of a truncated peptide of BNECP63 is sufficient to provide soluble and high levels of recombinant overexpression of BNPsbS (an intrinsic membrane chlorophyll-binding protein of photosystem II light harvesting complex) and a peptide of the Hepatitis C viral polyprotein. Furthermore, fusion of the recombinant target proteins to BNECP63 or BN115 prevented irreversible heat- and freeze-induced precipitation. These experiments not only underscore the exploitation of LEA-type peptides in facilitating protein overexpression and protection, but also provide insights into the mechanism of LEA proteins in cellular protection.

Optimization of in vitro transcription and full-length cDNA synthesis using the T4 bacteriophage gene 32 protein.

We evaluated the effect of the T4 bacteriophage gene 32 protein (T4gp32) on in vitro transcription and reverse transcription. T4gp32 doubled the yield of in vitro transcripts obtained with T7 RNA polymerase and increased the yield of cDNA synthesis when used in combination with an RNaseH-deficient Moloney murine leukemia virus [Au: ok] reverse transcriptase. The positive effect could be correlated with the RNA chaperone activity of T4gp32. T4gp32 stimulated the synthesis of long cDNAs, particularly species longer than 7 kb. By comparison, thermal activation of reverse transcriptase with trehalose only boosted the production of shorter cDNAs. For the construction of an Arabidopsis thaliana cDNA library, where the average cDNA size is 1.2 kbp, both the presence of T4gp32 under standard reaction conditions as well as thermal activation resulted in similarly high percentages of full-length cDNA. However, the inclusion of T4gp32 in a standard reverse transcription reaction resulted in the highest cDNA yield. We conclude that the addition of T4gp32 in standard reverse transcription reactions can increase the quality and yield of full-length cDNA libraries.

Characterization of a novel insect digestive DNase with a highly alkaline pH optimum.

A novel DNase from the digestive tract of the spruce budworm (Choristoneura fumiferana) has been isolated and characterized. This DNase has two features that distinguish it from other known DNases: (1) it has a pH optimum of 10.5 to 11; (2) it plays an important role in the conversion of the insecticidal crystal protein from Bacillus thuringiensis to the active DNA-free toxin in the larval gut. Only one digestive DNase with an apparent molecular mass of 23 kDa was found and no associated carbohydrate was detected. It has some similarities to pancreatic DNase I in that divalent alkaline metal ion is required for activity and it is inhibited by monovalent cations. In particular, Mg(2+) and Ca(2+) were the most effective activators. Transition metal ions also activated the enzyme but were less effective. The enzyme is an endonuclease that hydrolyzes single and double stranded DNA but shows a higher specificity for single stranded DNA. The purified enzyme acted synergistically with proteases on crystals from Bacillus thuringiensis to yield the DNA-free toxin. To our knowledge, this is the first characterization of DNase activity in insect larvae and provides strong evidence that a DNase is an integral component of the larval digestive system.

Control of seed germination in transgenic plants based on the segregation of a two-component genetic system.

We have developed a repressible seed-lethal (SL) system aimed at reducing the probability of transgene introgression into a population of sexually compatible plants. To evaluate the potential of this method, tobacco plants were transformed with an SL construct comprising gene 1 and gene 2 from Agrobacterium tumefaciens whereby gene 1 was controlled by the seed-specific phaseolin promoter modified to contain a binding site for the Escherichia coli TET repressor (R). The expression of this construct allows normal plant and seed development but inhibits seed germination. Plants containing the SL construct were crossed with plants containing the tet R gene to derive plant lines where the expression of the SL construct is repressed. Plant lines that contained both constructs allowed normal seed formation and germination, whereas seeds in which the SL construct was separated from the R gene through segregation did not germinate. The requirements of such a method to efficiently control the flow of novel traits among sexually compatible plants are discussed.