Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

A BAC-based physical map of the apple genome

Genome-wide physical mapping is an essential step toward investigating the genetic basis of complex traits as well as pursuing genomics research of virtually all plant and animal species. We have constructed a physical map of the apple genome from a total of 74,281 BAC clones representing approximately 10.5x haploid genome equivalents. The physical map consists of 2702 contigs, and it is estimated to span approximately 927 Mb in physical length. The reliability of contig assembly was evaluated by several methods, including assembling contigs using variable stringencies, assembling contigs using fingerprints from individual libraries, checking consensus maps of contigs, and using DNA markers. Altogether, the results demonstrated that the contigs were properly assembled. The apple genome-wide BAC-based physical map represents the first draft genome sequence not only for any member of the large Rosaceae family, but also for all tree species. This map will play a critical role in advanced genomics research for apple and other tree species, including marker development in targeted chromosome regions, fine-mapping and isolation of genes/QTL, conducting comparative genomics analyses of plant chromosomes, and large-scale genomics sequencing.     

Expression of an HBcAg-based antigen carrying angiotensin II in Chlamydomonas reinhardtii as a candidate hypertension vaccine

In contrast to traditional pharmacological treatments for hypertension, immunotherapies serve as promising alternatives as they are low-cost and afford better patient compliance. In this study, a chimeric protein targeting Angiotensin II via genetic fusion to a nucleocapsid antigen from Hepatitis B virus (HBcAg), serving as a carrier, is designed. This candidate immunogen designated as HBcAgII has been expressed in the alga specie Chlamydomonas reinhardtii, serving as an attractive vaccine expression system and delivery host. This alga can be grown on minimal media under controlled environmental conditions, and can serve as a safe oral delivery vehicle. Transgenic C. reinhardtii lines have been developed, and the expected recombinant protein has been detected by Western blot and ELISA analyses. Levels of expression of this recombinant protein in some transgenic lines have reached 0.05 % of total soluble protein. The immunogenic properties of the HBcAgII algae-derived antigen will be assessed.     

DNA methylation profiles differ between field- andin vitro-grown leaves of apple

A reliable method has been previously developed to detect cytosine methylation at the 5′-CCGG-3′ sequence using isoschizomers (Msp I and Hpa II) and a modified amplified fragment length polymorphism (AFLP) technique. With this method, DNA methylation profiles were investigated in leaf tissues of apple (Malus × domestica cv. Gala) grown under two different growth conditions, field and tissue culture. A total of 1,622 AFLP bands were detected using 32 pairs of primers, and these banding patterns were assembled into three groups. Type I AFLP bands were present in both EcoR I/Hpa II and EcoR I/Msp I lanes. Type II bands were present in the EcoR I/Msp I lanes, but not in EcoR I/Hpa II lanes. Type III bands were present in EcoR I/Hpa II lanes, but not in the EcoR I/Msp I lanes. For leaf tissues of field- and in vitro-grown apples, the ratios of types I, II, and III to the total number of amplified fragments were 70 %, 24 %, and 6 %, and 71 %, 23 %, and 6 %, respectively. Although the ratios of the three types of banding patterns were similar in both leaf tissues, a few bands specific to either field-grown trees or in vitro-grown shoots were observed. This study provided evidence that changes in methylation occurred in apple leaf tissues subjected to tissue culture growth conditions.     

Genome-wide analysis and characterization of molecular evolution of the <Emphasis Type="Italic">HCT</Emphasis> gene family in pear (<Emphasis Type="Italic">Pyrus bretschneideri</Emphasis>)

Lignin is a major component of stone cells in pear fruit, which significantly affects fruit quality. Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT), a recently discovered enzyme in plants, is an important gene that participates in the formation of lignin. Although HCT gene cloning and expression patterns have been studied in several species, including pear, there is still no extensive genome-wide bioinformatics analysis on the whole gene family, and the evolutionary history of HCT gene family is still unknown. A total of 82 HCT genes were identified in pear, most of which have one or two exons, and all with the conserved HXXXD motif and transferase domains. Based on the structural characteristics and phylogenetic analysis of these sequences, the HCT gene family genes could be classified into four main groups. Structural analysis also revealed that 25 % of HCT genes share a MYB binding site. Expansion of the HCT gene family mostly occurred before the divergence between Arabidopsis and Rosaceae, with whole-genome duplication or segmental duplication events playing the most important role in the expansion of the HCT gene family in pear. At the same time, purifying selection also played a critical role in the evolution of HCT genes. Five of the 82 HCT genes were verified by qRT-PCR to correspond to the pattern of stone cell formation during pear fruit development. The genome-wide identification, chromosome localization, gene structures, synteny, and expression analyses of pear HCT genes provide an overall insight into HCT gene family and their potential involvement in growth and development of stone cells.     

Iron-histidine resonance raman band of deoxyheme proteins: effects of anharmonic coupling and glass-liquid phase transition

Weak anharmonic coupling of two soft molecular vibrations is shown to cause pronounced temperature dependence of the corresponding resonance Raman bands. The developed theory is used to interpret the temperature dependence of the iron-histidine band of deoxyheme proteins and model compounds. It is shown that anharmonic coupling of the iron-histidine and heme doming vibrations must cause pronounced broadening of the band, its asymmetry, and shift of its maximum to the red upon heating. It also can lead to a structured shape of this band at room temperature. Proper consideration of the anharmonic coupling allows simulation of the temperature dependence of the iron-histidine band shape of horse heart myoglobin in the temperature interval of 10-300 K, using the minimum number of necessary parameters. Analysis of this temperature dependence clearly shows that the iron-histidine band of deoxyheme proteins is sensitive to the glass-liquid phase transition in the protein hydration shell, which takes place at 160-190 K.