黄瓜cDNA芯片的构建及其在黄瓜缺镁胁迫下基因差异表达研究中的应用

 基因芯片技术在研究植物基因功能中发挥着越来越重要的作用, 本试验通过GenBank搜索已发布的生物代谢途径中的关键酶基因序列, 设计特异性引物, 采用RT-PCR法扩增这些酶的相应基因片段,在完成432个基因片段的克隆分离、测序和生物信息学分析工作的基础上研制出国内首张黄瓜cDNA芯片。该芯片含有9个质控cDNA片段和423个cDNA 探针, 涉及光反应、卡尔文循环、碳水化合物代谢、水-水循环、信号传导、激素代谢、光呼吸、防御、蛋白与氨基酸代谢等多个代谢途径。利用该芯片对黄瓜缺镁胁迫下基因表达谱进行了初步研究, 并发现在缺镁胁迫下差异表达的22个基因, 其中10个基因的表达受缺镁处理抑制, 12个基因的表达受缺镁处理诱导。该芯片的研制为进一步研究黄瓜功能基因的高通量时空变化提供了有效的技术支撑。     

DNA微阵列技术在柑橘研究中的应用

DNA微阵列技术是一项能够分析基因组、基因表达特征性图谱的新技术,使研究人员能同时对成千上万的相互作用的基因展开研究。随着Affymetrix公司生产的世界上第一个商品化的柑橘基因组阵列(Citrus Genome Array)的诞生,该技术在柑橘基因组学和相关科学研究中扮演着越来越重要的角色。综述了运用DNA微阵列技术在柑橘研究中的现状,重点说明了该技术在柑橘重要功能基因的检测及基因差异表达研究中的应用,介绍了柑桔研究中DNA微阵列的种类,及其在代谢途径分析、突变检测和突变机理分析等其他领域的研究现状,并对其在病原检测方面的可能应用也进行了探讨。     

Genomic evaluation using SNP‐ and haplotype‐based genomic relationship matrices in a closed line of Duroc pigs

A simulation analysis and real phenotype analysis were performed to evaluate the impact of three different relationship matrices on heritability estimation and prediction accuracy in a closed‐line breeding of Duroc pigs. The numerator relationship matrix (NRM), single nucleotide polymorphism (SNP)‐based genomic relationship matrix (GRM) (G_S), and haplotype‐based GRM (G_H) were applied in this study. We used PorcineSNP60 genotype array data (38 114 SNPs) of 831 Duroc pigs with four selection traits. In both heritability estimation and prediction accuracy, the accuracy depended on the number of animals with records. For heritability estimation, a large difference in the results among three relationship matrices was not shown, but the trend of the estimated heritabilities between GRMs, that is G_S < G_H, was shown in this population. For the accuracy of prediction values in test animals, the accuracies of prediction values obtained by two GRMs were higher than that by the NRM in this population. The accuracies obtained by GRMs using animals with no records were lower than that by the NRM using animals with their performance records, but were close to that by the NRM using animals with full‐sib testing records.     

利用棉花寡核苷酸芯片研究红麻花药表达谱的可行性分析

利用Agilent棉花寡核苷酸表达谱芯片,以2个同核异质的红麻材料P3A和P3B为试材,分析了棉花表达谱芯片应用于红麻表达谱研究的可行性。结果表明:芯片杂交扫描图像信号清晰,信噪比较高,整体背景较低,各阳性质控点信号均匀一致,空白点和阴性质控点信号低。P3A在棉花的寡核苷酸芯片上共检测到37 640个表达位点,占芯片总位点的85.93%;P3B在棉花的寡核苷酸芯片上共检测到37 629个表达位点,占芯片总位点的85.90%。34 290个探针在不育系和保持系中均具有显著的杂交信号,占总探针数43 803的78.28%。表明红麻花药cRNA与棉花表达谱芯片上寡核苷酸探针之间的远缘杂交非常有效,用棉花寡核苷酸芯片研究红麻的基因表达是可行的。     

Microarray analysis of Longissimus thoracis muscle gene expressions in vitamin A‐restricted Japanese Black steers in middle fattening stage

Vitamin A (VA) restriction in beef cattle improves meat marbling; however, the underlying molecular mechanisms remain incompletely understood. We performed microarray analysis to clarify the effect of VA restriction on Longissimus thoracis gene expressions in Japanese Black steers. Six Japanese Black steers 13–14 months of age were divided into two groups: S group (n = 3), which received VA supplementation, and R group (n = 3), in which dietary VA intake was restricted. Steers were fattened for 7 months, following which tissue samples were obtained. Extracted RNA samples were analyzed by Affymetrix Genechip Bovine Genome Array. Lists of genes highly expressed in the R and S groups were obtained. The lists were functionally interpreted using functional annotation software, DAVID. In the R and S groups, 48 and 40 genes were significantly highly expressed, respectively. The gene list of the R group included CD36, LPL, GPAM, DGAT2, and SCD and additional genes annotated ‘PPAR signaling pathway,’ ‘lipid biosynthesis’ and ‘mitochondrion,’ whereas that of the S group included COL1A2, FN1 and DCN and additional genes annotated ‘extracellular matrix.’ Changes in the expression of these genes are possibly involved in marbling improvement in beef cattle by VA restriction.     

Gene expression profiles of CD4/CD8 double‐positive T cells in porcine peripheral blood

A characteristic subset of T cells, known as double positive T cells (DPTC) and expressing both cluster of differentiation 4 (CD4) and CD8, is observed in porcine peripheral blood. Previous studies suggested that DPTC might be memory cells. However, detailed phenotypes and functions of DPTC are yet to be fully elucidated and thus, the relatedness of DPTC with memory phenotypes remains unclear. In this study, DPTC gene expression profiles in peripheral blood were analyzed by DNA microarray in Experiment 1 and compared with those of CD4 single positive T cells (4SPTC) and CD8 single positive T cells (8SPTC). Expressions of IFNG, CCL5, NCK2, CCR2 and ITGB1 were higher than that of 4SPTC and 8SPTC. In contrast, expressions of CCR7 and SELL were lower than that of 4SPTC and 8SPTC. These results suggested that DPTC were either effector T cells or effector memory T cells (T_EM). Next, to determine whether DPTC were effector T cells or T_EM, differences in the response of DPTC and 8SPTC against immunized/primed antigens were compared (Experiment 2). While DPTC showed quick elevation of IL2 and CD25 gene expressions against in vitro stimulation of primed/immunized antigens, 8SPTC did not. These results suggest that at least some DPTC likely belong to T_EM.     

Comparing deregression methods for genomic prediction of test‐day traits in dairy cattle

We aimed to investigate the performance of three deregression methods (VanRaden, VR; Wiggans, WG; and Garrick, GR) of cows’ and bulls’ breeding values to be used as pseudophenotypes in the genomic evaluation of test‐day dairy production traits. Three scenarios were considered within each deregression method: (i) including only animals with reliability of estimated breeding value (REL_EBV ) higher than the average of parent reliability (REL_PA ) in the training and validation populations; (ii) including only animals with REL_EBV higher than 0.50 in the training and REL_EBV higher than REL_PA in the validation population; and (iii) including only animals with REL_EBV higher than 0.50 in both training and validation populations. Individual random regression coefficients of lactation curves were predicted using the genomic best linear unbiased prediction (GBLUP), considering either unweighted or weighted residual variances based on effective records contributions. In summary, VR and WG deregression methods seemed more appropriate for genomic prediction of test‐day traits without need for weighting in the genomic analysis, unless large differences in REL_EBV between training population animals exist.     

Differentially expressed genes associated with Staphylococcus aureus mastitis of Canadian Holstein cows

To study pathway specific gene expression within the immune-endocrine axis of dairy cows with Staphylococcus aureus mastitis, mRNA was collected from blood mononuclear cells (BMCs) and milk somatic cells (MSCs) of cows (n = 7) identified as culture positive for S. aureus and their matched negative control cows (n = 7) with no evidence of S. aureus mastitis. Labeled cDNA probes derived from BMCs and MSCs of infected and healthy cows were applied to a bovine immune-endocrine cDNA array containing 167 genes. Genes with a log₂ ratio ≥ 0.5 were considered to be up-regulated and genes with a log₂ ratio ≤ −0.5 to be down-regulated. In total, 22 genes were differentially displayed in BMCs and 16 genes in MSCs of case versus controls. Expression of selected genes in BMCs and MSCs were confirmed by real-time PCR. The RT-PCR results were highly correlated with microarray measurements. Some of these genes, such as interleukin (IL)-8 have been previously implicated in other bacterial diseases, and are known to regulate immune responses; whereas, others may reflect novel pathways or genes involved in progressive mammary gland disease. For example, IL-18 was up-regulated in BMCs but not MSCs of mastitic quarters, while IL-17 was more highly expressed in MSCs compared to BMCs. This study identified a number of differentially expressed genes associated with bovine S. aureus mastitis and demonstrates the intricacy of the patterns of gene expression that influence host response to a complex pathogen of significant relevance to both human and veterinary medicine.     

Dissection of additive genetic variability for quantitative traits in chickens using SNP markers

The aim of this study was to separate marked additive genetic variability for three quantitative traits in chickens into components associated with classes of minor allele frequency (MAF), individual chromosomes and marker density using the genomewide complex trait analysis (GCTA) approach. Data were from 1351 chickens measured for body weight (BW), ultrasound of breast muscle (BM) and hen house egg production (HHP), each bird with 354 364 SNP genotypes. Estimates of variance components show that SNPs on commercially available genotyping chips marked a large amount of genetic variability for all three traits. The estimated proportion of total variation tagged by all autosomal SNPs was 0.30 (SE 0.04) for BW, 0.33 (SE 0.04) for BM, and 0.19 (SE 0.05) for HHP. We found that a substantial proportion of this variation was explained by low frequency variants (MAF <0.20) for BW and BM, and variants with MAF 0.10–0.30 for HHP. The marked genetic variance explained by each chromosome was linearly related to its length (R² = 0.60) for BW and BM. However, for HHP, there was no linear relationship between estimates of variance and length of the chromosome (R² = 0.01). Our results suggest that the contribution of SNPs to marked additive genetic variability is dependent on the allele frequency spectrum. For the sample of birds analysed, it was found that increasing marker density beyond 100K SNPs did not capture additional additive genetic variance.     

Effects of a breeding scheme combined by genomic pre‐selection and progeny testing on annual genetic gain in a dairy cattle population

The effectiveness of the incorporation of genomic pre‐selection into dairy cattle progeny testing (GS‐PT) was compared with that of progeny testing (PT) where the fraction of dam to breed bull (DB) selected was 0.01. When the fraction of sires to breed bulls (SB) selected without being progeny tested to produce young bulls (YB) in the next generation was 0.2, the annual genetic gain from GS‐PT was 13% to 43% greater when h² = 0.3 and 16% to 53% greater when h² = 0.1 compared with that from PT. Given h² = 0.3, a selection accuracy of 0.8 for both YB and DB, and selected fractions of 0.117 for YB and 0.04 for DB, GS‐PT produced 40% to 43% greater annual genetic gain than PT. Given h² = 0.1, a selection accuracy of 0.6 for both YB and DB, and selected fractions of 0.117 for YB and 0.04 for DB, annual genetic gain from GS‐PT was 48% to 53% greater than that from PT. When h² = 0.3, progeny testing capacity had little effect on annual genetic gain from GS‐PT. However, when h² = 0.1, annual genetic gain from GS‐PT increased with increasing progeny testing capacity.