基因组印记

根据孟德尔遗传定律,当一种性状从亲代传到子代,涉及这种性状的基因和染色体无论是来自父方或母方,传递所产生的表型效应都应该是完全相同的。但是这一普遍规律现已发现在哺乳动物某些组织和细胞中会出现例外．即控制某一表型的一对等位基因由于亲源不同而差异性表达,也就是说,机体只表达来自亲本一方的等位基因．而与其自身性别无关。这就称为基因组印记（genomic imprinting）。其中父（母）系等位基因不表达者,就称为父（母）系印记。它不遵循孟德尔遗传规律。     

环江香猪FSHβ、ESR及ZAR1基因多态性分析

环江香猪是广西著名的地方品种,本研究采用PCR-RFLP、HRM结合测序的方法分析了广西环江香猪繁殖状相关基因FSHβ、ESR和ZAR1基因的多态性。结果显示,首次成功克隆测序了环江香猪ZAR1基因的外显子3,与Gen Bank中的ZAR1基因(DQ231443,gi:83727928)外显子3序列100%同源。针对134头环江香猪检测样本中均未发现FSHβ和ESR基因的多态突变位点。对ZAR1基因的外显子3和部分内含子3测序后发现5个新突变SNP位点,未见有已报道的对猪产仔数产生显著影响的外显子3(C54T)突变。论文显示不同品种猪基因突变多态性亦呈现为不同的模式,研究结果对建立环江香猪基因标记辅助选择方法具有重要意义。     

藏猪和大约克猪MYL1基因多态性及差异表达分析

为研究猪不同品种的MYL1基因的多态性及mRNA表达差异.本试验以藏猪和大约克猪作为实验动物,采用一代测序技术对藏猪(59头)和大约克猪(58头)MYL1基因起始密码子上游3 kb区域进行多态性检测;利用实时荧光定量PCR技术检测MYL1基因在背最长肌、背脂、肝脏组织差异表达情况.结果表明:藏猪和大约克猪在起始密码子上...     

牛MA GEL2的基因结构及在组织和胎盘中印记状态分析

本研究旨在揭示牛PWS/AS (Prader-willi syndrome/angelman syndrome)印记区域中的MAGEL2 (Melanoma antigen-like gene 2)基因的结构特点及其在成年牛组织及胎盘中的印记状态。生物信息学软件分析发现,牛MAGEL2基因位于21号染色体,为单外显子蛋白编码基因,编码长为1 183个氨基酸的多肽序列,其产物为MAGE家族成员之一。构建MAGEL2基因的进化树,发现牛MAGEL2基因与羊相似性最高。MAGEL2在不同物种之间具有较高的保守性。应用基于单核苷酸多态性(single nucleotide polymorphisms, SNP)的RTPCR产物直接测序的方法分析MAGEL2在牛8个组织和器官(心,肝,脾,肺,肾,肌肉,脂肪和大脑)及胎盘的印记状态。比较杂合牛基因组PCR产物和RT-PCR产物在SNP位点的测序结果发现,MAGEL2基因在成年牛组织及胎盘中均为单等位表达,提示MAGEL2基因在牛中是印记的。     

Ascl2、Nap1l4和Osbpl5基因在牛胎盘中的表达分析

基因组印记是一个复杂的表观遗传修饰过程,从而使印记基因呈单基因表达的模式。大部分的印记基因在动物的胎盘中表达,通过调节胎盘的生长和转运能力而促进妊娠和胎儿发育。CDKN1C/KCNQ1OT1印记区域与人类的BWS综合症以及肿瘤的发生有关。Ascl2、Nap1l4和Osbpl5是位于CDKN1C/KCNQ1OT1印记区域的3个基因,其在小鼠和人胎盘中的表达存在差异,在小鼠的胎盘中3个基因是印记的,而在人中为非印记。本研究利用基于单核苷酸多态性(single-nucleotide polymorphism, SNP)的RT-PCR产物直接测序法分析了Ascl2、Nap1l4和Osbpl5在牛的胎盘中表达状态,结果发现Ascl2、Nap1l4和Osbpl5基因在牛的胎盘中的表达与人相似,是双等位基因表达,说明Ascl2、Nap1l4和Osbpl5在胎盘中印记表达模式具有物种特异性。     

西藏绒山羊KA P9.2基因CDS克隆、外显子区多态性及表达分析

KAP9.2基因是角蛋白关联蛋白(keratin associated protein, KAP)中的一员,在毛发的形成过程中有重要的调控作用。本研究对西藏绒山KAP9.2基因CDS进行了克隆;采用直接测序法对绒山羊200个个体KAP9.2基因外显子区的遗传变异情况进行分析;并利用Real-time PCR分析了KAP9.2基因在不同海拔山羊中的表达。结果显示,西藏绒山羊KAP9.2基因CDS序列为576 bp,编码191个氨基酸;KAP9.2基因外显子存在25处SNP位点及一处30 bp的缺失突变,其中12处SNP为错义突变,其他13处为同义突变。遗传多态性分析表明KAP9.2基因多态性丰富,遗传变异大;连锁分析发现12与388位点、54与93位点、153与159位点、273与279位点完全连锁,H1为优势单倍型;Real-time PCR显示西藏绒山羊KAP9.2基因在高海拔地区m RNA表达水平显著高于特高海拔地区,推测该基因可能促进绒毛的生长。本研究结果揭示了西藏绒山羊KAP9.2基因的遗传多态性及其在不同海拔的表达,为进一步研究KAP9.2基因潜在的功能位点提供一定的理论依据。     

难免流产蜕膜组织遗传印记基因PEG10的表达

采用半定量逆转录聚合酶反应(RT-PCR)、原位杂交、免疫印记(Western blot)及免疫组织化学技术检测了36例难免流产患者蜕膜组织PEG10 (Paternally expressed gene 10) mRNA及蛋白的表达与分布, 并以36例同期正常早孕妇女为对照, 研究遗传印记基因在难免流产蜕膜组织中的表达, 探讨其在自然流产中的作用。RT-PCR结果显示, PEG10在两组蜕膜组织中均有表达, 正常妊娠组平均表达水平为0.5994±0.049, 难免流产组为0.1783±0.037, 两组比较具有显著性差异(P<0.05)。原位杂交、免疫组化及Western blot分析也显示PEG10的表达规律与RT-PCR结果相吻合。研究结果表明, 遗传印记基因PEG10维持一定水平的表达对早期胚胎发育和正常妊娠的维持有重要意义, 而其表达下调可能是导致难免流产的原因之一。     

豚鹿MHC-DOA基因克隆及其外显子2遗传多样性研究

主要组织相容性复合物(MHC)在脊椎动物的免疫系统中起着重要的作用,MHC-DOA基因是MHCⅡ类的非经典基因,其外显子2多态性丰富。豚鹿Axis porcinus是我国极度濒危的动物。本研究以成都动物园圈养的38头豚鹿为研究对象,提取豚鹿血液总RNA,反转录合成cDNA为模板,利用PCR方法克隆到豚鹿MHC-DOA序列;同时利用PCR方法扩增38头豚鹿的MHC-DOA基因外显子2基因并测序,再进行多态性分析。结果显示:豚鹿MHC-DOA基因开放阅读框长753 bp,编码250个氨基酸残基;蛋白质同源性分析表明,豚鹿MHC-DOA基因与东欧马鹿Cervus elaphus hippelaphus的同源性最高(98.4%);38头豚鹿的MHC-DOA外显子2共有10种单倍型,整体遗传多样性水平中等;MHC-DOA外显子2部分序列中发生了核苷酸的缺失和插入,进而引起DOA蛋白序列发生改变。豚鹿MHC-DOA多态性的研究对豚鹿种群遗传结构调查、遗传资源的保护具有重要意义。     

合作猪SLA-DQA基因外显子2多态性分析

旨在研究合作猪DQA基因外显子2多态性,确定其等位基因数、核苷酸多态位点、氨基酸多态位点及各个等位基因之间的遗传关系,分析其进化意义。选用PCR-SSCP对439只合作猪SLA-DQA基因外显子2的多态性进行检测;测序群体内因变异而产生的各等位基因序列,并分析序列数据。结果显示,在合作猪SLA-DQA外显子2中发现了7个新等位基因,共18个核苷酸多态位点,10个氨基酸多态位点。合作猪SLA-DQA外显子2具有较丰富的多态性,群体内可能蕴藏着更加丰富的遗传资源;合作猪SLA-DQA外显子2基因最初可能由一个等位基因突变分化成一大类基因;合作猪SLA-DQA外显子2序列与各个猪种的SLA-DQA外显子2序列具有较高的同源性,预示着这些猪种的SLA-DQA外显子2基因最早可能来源于其分歧之前的共同祖先原始序列;新发现的7个SLA-DQA外显子2等位基因,可能由遗传关系较近的两个等位基因突变产生。     

SIM1基因第8外显子的SNP对猪背膘厚的影响

采用PCR-SSCP方法检测了约克夏、杜洛克、皮特兰、长白猪、嘉兴黑猪和金华猪6个品种共169头猪的SIM1基因外显子8的SNP及其基因型频率。结果共发现CC、CT、TT 3种基因型, 其基因型频率在国内外猪品种之间具有较大差异。其中, 国内猪种嘉兴黑猪和金华猪只存在TT基因型, 而国外猪种约克夏、杜洛克、皮特兰、长白猪则都存在3种基因型。用最小二乘法分析SNP对长白猪、约克夏猪和杜洛克猪的背膘厚的效应的结果表明, 纯合基因型个体的背膘厚大于杂合基因型个体。SIM1基因型对国外猪种背膘厚有显著效应(P< 0.05), 并且不同部位效应不同。     

Jasmonates modulate the promotion effects induced by SNP on root development of wheat under osmotic stress through lipoxygenase activation

We investigated promotion effects of exogenous sodium nitroprusside (SNP) on wheat seedling (Triticum aestivum L.) lateral root (LR) and root hair development, and the relationship between endogenous jasmonate (JA) production and activity changes of lipoxygenase (LOX) isoenzymes under osmotic stress generated by 15 % PEG-6000. Our results showed that 25 or 50 μM SNP could significantly increase LR length and number whether or not the seedlings were under PEG stress. When 50 μM cPTIO, 50 μM SHAM or 50 μM NDGA was supplemented, the promotion effects of SNP were blocked. SNP could also induce the production of endogenous JAs in roots, and 25 μM SNP induced the maximum JA content. The effect of SNP on JA production could also be blocked by adding cPTIO, SHAM or NDGA. Furthermore, the activity of lipoxygenase (LOX) in roots was affected by SNP; the maximal activity of LOX also occurred in the roots treated by 25 μM SNP under PEG stress, or 50 μM SNP without PEG stress. LOX isoenzymes in roots were detected by electrophoresis; the results showed that 25 μM SNP could noticeably increase the activities of LOXII and LOXIII under PEG stress. Our results suggest that, under osmotic stress generated by PEG, the promotion effects of exogenous SNP on wheat LR and root hair development could be mediated by endogenous JAs through LOX activation.     

Interaction of sodium nitroprusside/liposome system with bovine serum albumin: a short-cut determination

A rapid determination of protein-liposome binding was developed to predict the circulation time of the system within an animal, which is a function of the amount and type of protein bound. The binding pattern of albumin to liposomes, with and without sodium nitroprusside (SNP), was analyzed by SDS-PAGE. Liposomes were made of egg yolk lecithin, soybean lecithin and dimyristoyl lecithin, and contained SNP. They bound 58%, 26% and 100% bovine serum albumin, respectively, when compared to their corresponding controls lacking SNP. The method applied is simpler and significantly faster than ordinary chemical determinations.     

Mosaicism of Solid Gold supports the causality of a noncoding A-to-G transition in the determinism of the callipyge phenotype

To identify the callipyge mutation, we have resequenced 184 kb spanning the DLK1-, GTL2-, PEG11-, and MEG8-imprinted domain and have identified an A-to-G transition in a highly conserved dodecamer motif between DLK1 and GTL2. This was the only difference found between the callipyge (CLPG) allele and a phylogenetically closely related wild-type allele. We report that this SNP is in perfect association with the callipyge genotype. The demonstration that Solid Gold-the alleged founder ram of the callipyge flock-is mosaic for this SNP virtually proves the causality of this SNP in the determinism of the callipyge phenotype.     

Nitric oxide treatment alleviates drought stress in wheat seedlings

The effects of sodium nitroprusside (SNP; nitric oxide donor) treatment on drought stress induced by PEG for different periods of time in wheat seedlings were investigated. Our results suggested that treatment for 2, 4 and 6 d with 15 % PEG could be termed as mild, moderate and severe stress, respectively. Drought stress induced accumulation of hydrogen peroxide and resulted in lipid peroxidation. On the other hand, activities of SOD, CAT and PAL increased under mild stress to counteract the oxidative injury and then decreased when the stress became severe (6 d). As the effect of SNP treatment, 0.2 mM enhanced wheat seedlings growth and kept high relative water content and alleviated the oxidative damage. However, 2 mM SNP aggravated the stress as a result of uncontrolled generation of reactive oxygen species and ineffectiveness of antioxidant systems.     

A missense mutation in AGTPBP1 was identified in sheep with a lower motor neuron disease

A type of lower motor neuron (LMN) disease inherited as autosomal recessive in Romney sheep was characterized with normal appearance at birth, but with progressive weakness and tetraparesis after the first week of life. Here, we carried out genome-wide homozygosity mapping using Illumina Ovine SNP50 BeadChips on lambs descended from one carrier ram, including 19 sheep diagnosed as affected and 11 of their parents that were therefore known carriers. A homozygous region of 136 consecutive single-nucleotide polymorphism (SNP) loci on chromosome 2 was common to all affected sheep and it was the basis for searching for the positional candidate genes. Other homozygous regions shared by all affected sheep spanned eight or fewer SNP loci. The 136-SNP region contained the sheep ATP/GTP-binding protein 1 (AGTPBP1) gene. Mutations in this gene have been shown to be related to Purkinje cell degeneration (pcd) phenotypes including ataxia in mice. One missense mutation c.2909G>C on exon 21 of AGTPBP1 was discovered, which induces an Arg to Pro substitution (p.Arg970Pro) at amino-acid 970, a conserved residue for the catalytic activity of AGTPBP1. Genotyping of this mutation showed 100% concordant rate with the recessive pattern of inheritance in affected, carrier, phenotypically normal and unrelated normal individuals. This is the first report showing a mutant AGTPBP1 is associated with a LMN disease in a large mammal animal model. Our finding raises the possibility of human patients with the same etiology caused by this gene or other genes in the same pathway of neuronal development.     

Fluorescence microscopic study of cells and polykaryons in the early periods following polyethylene glycol treatment

A fluorescence-microscopical study is made of cultured murine fibroblasts (L-cells) in early periods after the treatment with polyethylene glycol (PEG). Optimal conditions of fusion procedure were found under which the effectiveness of fusion was the highest and the toxical effect of PEG the lowest. The number of dead cells after the treatment with PEG did not exceed 10%. No significant changes in chromatin cytochemical properties (Acridine Orange and Olivomycin binding) were observed in the early periods of PEG treatment, that allows to use PEG for studying chromatin properties in hybrid cells obtained by PEG fusion. By means of PEG fusion, the hybrid cells with prematurely condensed chromosomes and also hybrids between animal and yeast cells have been obtained.     

A genome-wide association study of osteochondritis dissecans in the Thoroughbred

Osteochondrosis is a developmental orthopaedic disease that occurs in horses, other livestock species, companion animal species, and humans. The principal aim of this study was to identify quantitative trait loci (QTL) associated with osteochondritis dissecans (OCD) in the Thoroughbred using a genome-wide association study. A secondary objective was to test the effect of previously identified QTL in the current population. Over 300 horses, classified as cases or controls according to clinical findings, were genotyped for the Illumina Equine SNP50 BeadChip. An animal model was first implemented in order to adjust each horse's phenotypic status for average relatedness among horses and other potentially confounding factors which were present in the data. The genome-wide association test was then conducted on the residuals from the animal model. A single SNP on chromosome 3 was found to be associated with OCD at a genome-wide level of significance, as determined by permutation. According to the current sequence annotation, the SNP is located in an intergenic region of the genome. The effects of 24 SNPs, representing QTL previously identified in a sample of Hanoverian Warmblood horses, were tested directly in the animal model. When fitted alongside the significant SNP on ECA3, two of these SNPs were found to be associated with OCD. Confirmation of the putative QTL identified on ECA3 requires validation in an independent sample. The results of this study suggest that a significant challenge faced by equine researchers is the generation of sufficiently large data sets to effectively study complex diseases such as osteochondrosis.     

Responses of in vitro-cultured <Emphasis Type="Italic">Allium hirtifolium</Emphasis> to exogenous sodium nitroprusside under PEG-imposed drought stress

Drought stress is a major threat to plant production in semi-arid and arid areas of the world. This research was laid out to asses the effects of sodium nitroprusside (SNP) as a nitric oxide donor on growth, physiological and biochemical changes of in vitro-cultured Allium hirtifolium under polyethylene glycol (PEG) induced drought stress. Basal plate explants of A. hirtifolium were cultured on MS medium containing different levels of PEG (0, 2, 4, 8 and 16 mM) and SNP (0, 10, 40 and 70 µM). After prolonged drought, growth responses, oxidative stress indicators, and phytochemical variations of regenerated plantlets with or without PEG and/or SNP treatments were recorded. Water limitation reduced regeneration potential of explants and consequently number of shoots per explant. Relative water content, total chlorophyll and carotenoid contents of regenerated A. hirtifolium plantlets decreased, but accumulation of malondialdehyde, H₂O₂ and proline and the activities of superoxide dismutase, ascorbate peroxidase, catalase and peroxidase enzymes increased with decreasing water availability. Total phenol and allicin contents were also increased in response to drought stress. Exogenous SNP in 10 and particularly in 40 µM was effective in enhancing regeneration rate and relative water content as well as protecting photosynthetic pigments under different levels of water availability. SNP also inhibited the hydrogen peroxide (H₂O₂) accumulation and lipid peroxidation in cell membranes via increasing the activities of superoxide dismutase and ascorbate peroxidase enzymes and accumulating proline and allicin. In general, these results suggest that exogenous SNP at 40 µM not only could somewhat protect A. hirtifolium from drought stress, but also can help to improve the propagation and allicin production of that plant under in vitro condition.