Molecular cloning and characterization of a chlorophyll degradation regulatory gene (ZjSGR) from Zoysia japonica.

The stay-green gene (SGR) is a key regulatory factor for chlorophyll degradation and senescence. However, to date, little is known about SGR in Zoysia japonica. In this study, ZjSGR was cloned, using rapid amplification of cDNA ends-polymerase chain reaction (PCR). The target sequence is 831 bp in length, corresponding to 276 amino acids. Protein BLAST results showed that ZjSGR belongs to the stay-green superfamily. A phylogenetic analysis implied that ZjSGR is most closely related to ZmSGR1. The subcellular localization of ZjSGR was investigated, using an Agrobacterium-mediated transient expression assay in Nicotiana benthamiana. Our results demonstrated that ZjSGR protein is localized in the chloroplasts. Quantitative real time PCR was carried out to investigate the expression characteristics of ZjSGR. The expression level of ZjSGR was found to be highest in leaves, and could be strongly induced by natural senescence, darkness, abscisic acid (ABA), and methyl jasmonate treatment. Moreover, an in vivo function analysis indicated that transient overexpression of ZjSGR could accelerate chlorophyll degradation, up-regulate the expression of SAG113, and activate ABA biosynthesis. Taken together, these results provide evidence that ZjSGR could play an important regulatory role in leaf chlorophyll degradation and senescence in plants at the molecular level.

Paternal origins of Chinese cattle.

To determine the genetic diversity and paternal origin of Chinese cattle, 302 males from 16 Chinese native cattle breeds as well as 30 Holstein males and four Burma males as controls were analysed using four Y-SNPs and two Y-STRs. In Chinese bulls, the taurine Y1 and Y2 haplogroups and indicine Y3 haplogroup were detected in seven, 172 and 123 individuals respectively, and these frequencies varied among the Chinese cattle breeds examined. Y2 dominates in northern China (91.4%), and Y3 dominates in southern China (90.8%). Central China is an admixture zone, although Y2 predominates overall (72.0%). The geographical distributions of the Y2 and Y3 haplogroup frequencies revealed a pattern of male indicine introgression from south to north China. The three Y haplogroups were further classified into one Y1 haplotype, five Y2 haplotypes and one Y3 haplotype in Chinese native bulls. Due to the interplay between taurine and indicine types, Chinese cattle represent an extensive reservoir of genetic diversity. The Y haplotype distribution of Chinese cattle exhibited a clear geographical structure, which is consistent with mtDNA, historical and geographical information.

AGPAT6 polymorphism and its association with milk traits of dairy goats.

As one of the eight members in the 1-acylglycerol-3-phosphate-O-acyltransferase (AGPATs) family, AGPAT6 is a crucial enzyme for the biosynthesis of glycerolipids and triacylglycerol in eukaryotes, as well as catalyzing the conversion from lysophosphatidic acid to phosphatidic acid. AGPAT6 can be considered as a candidate gene for regulating milk composition. DNA sequencing and PCR-RFLP methods were applied to detect genetic variation in the AGPAT6 gene in 549 Chinese dairy goats. Four polymorphisms (NC_007328.3:g.152G>C, 8124G>A, 9263C>G, 16436G>A) were detected in 5'UTR, intron 2, exon 4, and 3'UTR, respectively. For the KpnΙ locus, the frequencies of the AGPAT6-G allele were 0.955 and 0.936 for SN (Xinong Sannen) and GZ (Guanzhong) dairy goat breeds, respectively. In the PCR-RFLP analysis for KpnΙ, EcoRII, NcoΙ, and BglΙ, the frequencies of the G allele of AGPAT6 were 0.955 and 0.936, 0.694 and 0.819, 0.206 and 0.254, 0.729 and 0.623 for SN and GZ dairy goat breeds, respectively. The 9263C>G mutation revealed a synonymous genetic code of Thr (threonine). Associations between the four mutations and milk traits were analyzed in two dairy goat breeds. At the 9263C>G locus, genotype GG and CG individuals showed significantly better milk performance than genotype CC individuals (P < 0.05). Therefore, the G allele is suggested to be a molecular marker for milk production in dairy goats.

A novel polymorphism of the lactoferrin gene and its association with milk composition and body traits in dairy goats.

Milk composition and body measurement traits, influenced by genes and environmental factors, play important roles in value assessments of efficiency and productivity in dairy goats. Lactoferrin (LF), involved in the efficient expression of protein in milk, is also an anabolic factor in skeletal tissue and a potent osteoblast survival factor. Therefore, it is an important candidate gene for milk composition and body measurement trait selection in marker-assisted selection. We employed PCR-SSCP and DNA sequencing to screen the genetic variations of the LF gene in 549 Chinese dairy goats. A novel single-nucleotide polymorphism (SNP) (G198A in exon II) of the LF gene was detected. The frequencies of the AA genotype were 0.0285 and 0.0261 in GZ and SN populations, respectively. Both populations were found to have low levels of polymorphism and were in Hardy-Weinberg disequilibrium (P < 0.05). We found significant (P < 0.05) associations of the SNP marker with milk protein and acidity in the total population; animals with the AA genotype had higher mean values for milk protein than those with the GA genotype. Animals with genotype AA had higher mean values for withers height than those with genotype GG (P < 0.05). We concluded that this SNP of the LF gene has potential as a genetic marker for milk composition and body traits in dairy goat breeding.

Ultra-rapid freezing by spraying/plunging: pre-cooling in the cold gaseous layer.

A thermophysical model is established to analyse the influence of pre-cooling of a biological specimen in the cold gas layer associated with spray-freezing techniques. The basic principles governing the process of pre-cooling are provided. It is concluded that pre-cooling is one of the major limiting steps in attaining an overall ultra-rapid cooling rate. Pre-cooling has a substantial influence on the nature of the final frozen specimens. In order completely to avoid crystallization before entry into the liquid cryogen and maximize the overall cooling rate of the specimen, precautions should be taken to control the height of the gaseous layer and the size of the specimen. The probability of the specimen being frozen in the cold gaseous layer is reduced by increasing the entry speed. The effectiveness, however, becomes less marked at speeds greater than 10 m s. In order to minimize the risk of misinterpreting the measured cooling rate, it is necessary to specify the pre-cooling conditions. The pre-cooling effect is much more evident in liquid helium than in cryogens such as propane, ethane, Freon 12, 13 and 22.

Physical aging of glassy state: DSC study of vitrified glycerol systems.

After annealing during the glass transition temperature (Tg) range or at sub-Tg temperatures, a "Tg overshoot" has been observed by differential scanning calorimetry in vitrified 55 and 80% (W/W) glycerol solutions. The temperature dependence of the overshoot is most pronounced immediately below the Tg and always increases in amplitude with lengthened annealing periods, whether annealed during Tg range or below. Of particular note is the anomalous shift in devitrification temperature (Td) to lower temperatures following increased time of annealing at sub-Tg temperatures. The Tg obtained on warming also occurs at higher temperatures with increased annealing times. The results allow us to suggest the existence of the time and temperature dependency of the glassy state. Accordingly, it may be desirable to maintain vitrified biological systems at temperatures sufficiently below Tg so that the extent of relaxation in the glass system is avoided or minimized during cryopreservation. The relaxation effects must be taken into account prior to warming a biological system after a long-term cryopreservation. These effects should also be considered in studies of vitrification solutions.

The compensatory 'rebound' of reactive astrogliosis: glial fibrillary acidic protein immunohistochemical analysis of reactive astrogliosis after a puncture wound to the brain of rats with portocaval anastomosis.

This study was designed to compare the degree of reactive astrogliosis occurring around a puncture wound in the brain of normal rats and at different intervals after a similar puncture wound in rats with a portocaval anastomosis. The gliosis was evaluated by the number of astrocytes, the thickness of their processes and the intensity of the glial fibrillary acidic protein immunoreactivity. After the puncture wound in the brain of rats with a portocaval anastomosis, the gliosis varied at different intervals being: (1) decreased at 10 days, (2) markedly increased at 5 weeks and (3) significantly decreased at 8, 12, and 16 weeks. These findings suggest that 5 weeks after portocaval anastomosis, an active proliferation of the metabolically altered astrocytes occurs with heightened synthesis of glial fibrillary acidic protein in the period of adaptive compensation, the so-called compensatory 'rebound'. At 8 weeks or more after portocaval anastomosis, these altered astrocytes were considered to be in the phase of decompensation and incapable of maintaining the reactive response which occurred in normal rats. The compensatory rebound and decompensatory 'decline' illustrate the dynamic plasticity of the reactive astrogliosis.