REAL-TIME PCR方法测定转基因小麦中外源基因拷贝数

采用SYBR GreenⅠ real-time PCR方法检测7株转基因小麦中外源半夏凝集素基因的拷贝数。以小麦蜡质基因（wx012）作为内参基因,以未转基因小麦基因组DNA为内参基因标准品进行5倍梯度稀释得到内参基因CT值与起始模板量的相关性标准曲线：y=-0.2667x+6.98;以含半夏凝集素基因（pta）的质粒DNA为目的基的因标准品同样进行5倍梯度稀释,建立目的基因CT值与起始模板量的相关性标准曲线：y=-0.2118x+4.53。通过SYBR GreenⅠ real-time PCR分别获得每一样本中目的基因和内参基因的CT值,将CT值分别代入标准曲线计算该样本中内参基因和目的基因起始模板量,目的基因与内参基因起始模板量比值即是目的基因在该转基因植株中的拷贝数。计算结果为：单拷贝的有1株,2个拷贝1株,3拷贝和4拷贝的各有2株,其中有1株为假阳性植株。     

High-Throughput Multiplex Sequencing to Discover Copy Number Variants in Drosophila

Copy number variation (CNV) contributes in phenotypically relevant ways to the genetic variability of many organisms. Cost-effective genomewide methods for identifying copy number variation are necessary to elucidate the contribution that these structural variants make to the genomes of model organisms. We have developed a novel approach for the identification of copy number variation by next generation sequencing. As a proof of concept our method has been applied to map the deletions of three Drosophila deficiency strains. We demonstrate that low sequence coverage is sufficient for identifying and mapping large deletions at kilobase resolution, suggesting that data generated from high-throughput sequencing experiments are sufficient for simultaneously analyzing many strains. Genomic DNA from two Drosophila deficiency stocks was barcoded and sequenced in multiplex, and the breakpoints associated with each deletion were successfully identified. The approach we describe is immediately applicable to the systematic exploration of copy number variation in model organisms and humans.STRUCTURAL variation is known to contribute extensively to the genetic variability of humans, mammals, and many model organisms. One class of structural variant, termed copy number variation (CNV), includes deletions, duplications, insertions, and genomic rearrangements which affect the number of occurrences of a specific DNA sequence present in the genome (Redon et al. 2006). CNV is known to occur extensively in the Drosophila genome with functionally significant consequences (Bridges 1936; Dopman and Hartl 2007; Tibshirani and Wang 2008; Zhou et al. 2008). In one study of 15 Drosophila strains, as many as 10% of genes were observed to harbor CNVs (Emerson et al. 2008). Cryptic CNVs that affect the phenotype observed in a model organism have the potential to confound research on multiple levels. For example, a recent report indicates that terminal deletions on chromosome (chr) 2L are frequent among deficiency kit stocks with mutations on the second chromosome and that the associated deletion of lgl has distorted the results of several previous studies (Roegiers et al. 2009). Despite widespread existence of CNV, the biological consequences of this phenomenon remain largely unexplored due to the lack of efficient tools for detection and characterization.Until recently, comparative genomic hybridization with whole-genome tiling arrays (array-CGH) was the primary method for characterizing CNVs (Carter 2007); however, several limitations for this platform reduce its efficacy and efficiency. First, cross-hybridization and reliance on intensity scores lead to data that are difficult to interpret. Second, custom array design and optimization is labor intensive and costly. Third, array-CGH methods can only detect CNV, not other complex rearrangements such as balanced translocations and inversions. Finally, the overall cost of array-CGH methods is relatively high, particularly when high-resolution, whole-genome tiling arrays are employed.Direct sequencing using next-generation technology has several advantages that make it a potentially powerful alternative to array-CGH for identifying genomic structural variations, including deletions, duplications, and rearrangements (Campbell et al. 2008; Chiang et al. 2009). First, high-throughput sequencing methods overcome the inherent limitations of cross-hybridization and provide a digital count of sequence representation. Second, no prior knowledge or design work is necessary. Third, using paired-end sequencing it is possible to identify complex structural variations. Finally, the current cost of CNV discovery by sequencing is comparable or lower than that of array-CGH and is continuing to decline.In this report, we describe a sequencing-based strategy for high-throughput, cost-effective, genomewide characterization of structural variation at fine resolution by employing the Illumina sequencing platform. Deletions in three deficiency fly stocks were successfully characterized and the associated breakpoints were accurately determined. As we demonstrate, high-throughput sequencing provides an ideal and cost-effective platform for CNV characterization.     

APOBEC3基因拷贝数变异与乳腺癌易感性的关联

为了探讨APOBEC3基因缺失拷贝数变异的多态性与汉族女性乳腺癌易感性的关联性,本研究应用聚合酶链式反应-限制性多态性内切酶技术检测281例乳腺癌和292例正常对照组。我们发现APOBEC3基因拷贝数变异位点的等位基因、基因型频率分布在乳腺癌和对照组之间比较有显著性差异(p<0.05),校正混杂因素后基因型Del/Del,Del/Ins,显性模型和加性模型下对乳腺癌的风险预测值分别为2.753 (95%Cl:1.363～3.560; p=0.005)、1.462(95%Cl:1.021～2.094; p=0.038)、2.282(95%Cl:1.155～3.508; p=0.018)和1.596(95%Cl:1.129～2.256; p=0.008)。研究表明APOBEC3基因的缺失变异位点多态性与乳腺癌的发病风险存在关联,等位基因del是乳腺癌发病的危险因素。本研究的结果可以为本地区群体的乳腺癌易感基因的筛选以及评估易感基因对患病的风险程度提供参考数据,为乳腺癌的防治、诊断和个体化治疗研究提供了帮助。     

Copy Number Variants in the Kallikrein Gene Cluster

The kallikrein gene family (KLK1-KLK15) is the largest contiguous group of protease genes within the human genome and is associated with both risk and outcome of cancer and other diseases. We searched for copy number variants in all KLK genes using quantitative PCR analysis and analysis of inheritance patterns of single nucleotide polymorphisms. Two deletions were identified: one 2235-bp deletion in KLK9 present in 1.2% of alleles, and one 3394-bp deletion in KLK15 present in 4.0% of alleles. Each deletion eliminated one complete exon and created out-of-frame coding that eliminated the catalytic triad of the resulting truncated gene product, which therefore likely is a non-functional protein. Deletion breakpoints identified by DNA sequencing located the KLK9 deletion breakpoint to a long interspersed element (LINE) repeated sequence, while the deletion in KLK15 is located in a single copy sequence. To search for an association between each deletion and risk of prostate cancer (PC), we analyzed a cohort of 667 biopsied men (266 PC cases and 401 men with no evidence of PC at biopsy) using short deletion-specific PCR assays. There was no association between evidence of PC in this cohort and the presence of either gene deletion. Haplotyping revealed a single origin of each deletion, with most recent common ancestor estimates of 3000-8000 and 6000-14 000 years for the deletions in KLK9 and KLK15, respectively. The presence of the deletions on the same haplotypes in 1000 Genomes data of both European and African populations indicate an early origin of both deletions. The old age in combination with homozygous presence of loss-of-function variants suggests that some kallikrein-related peptidases have non-essential functions.     

Heterochromatin Protein 1 Deleted Chromo Domain Decreases Gene Silencing of Transgene in Mouse

The heterochromatin protein 1 (HP1) regulates epigenetic gene silencing by promoting and maintaining chromatin condensation. To decrease gene silencing, the chromo domain (CD) in the M31 (the main HP1 in mouse) was deleted by site-directed mutagenesis. Vector pcDNA3.1(+)/M31-DeltaCD, in which the M31-DeltaCD is driven by the CMV promoter, and vector pcDNA3.1(+)/P1A3-M31-DeltaCD, in which the M31-DeltaCD is driven by a goat ss-casein promoter were constructed. The former vector was transfected into a murine fibroblast cell line, which can express enhanced green fluorescent protein (EGFP). EGFP expression, which was determined by flow cytometric analysis, increased approximately 80% in the transfected cells. After injection of the latter vector into transgenic mouse mammary glands, which can express human clotting factor IX (hFIX), the hFIX expression level in the mouse milk increased approximately 40-60% and hFIX in one mouse milk was maintained at a high concentration for over 10 days.     

Genome-Wide Analyses of Exonic Copy Number Variants in a Family-Based Study Point to Novel Autism Susceptibility Genes

The genetics underlying the autism spectrum disorders (ASDs) is complex and remains poorly understood. Previous work has demonstrated an important role for structural variation in a subset of cases, but has lacked the resolution necessary to move beyond detection of large regions of potential interest to identification of individual genes. To pinpoint genes likely to contribute to ASD etiology, we performed high density genotyping in 912 multiplex families from the Autism Genetics Resource Exchange (AGRE) collection and contrasted results to those obtained for 1,488 healthy controls. Through prioritization of exonic deletions (eDels), exonic duplications (eDups), and whole gene duplication events (gDups), we identified more than 150 loci harboring rare variants in multiple unrelated probands, but no controls. Importantly, 27 of these were confirmed on examination of an independent replication cohort comprised of 859 cases and an additional 1,051 controls. Rare variants at known loci, including exonic deletions at NRXN1 and whole gene duplications encompassing UBE3A and several other genes in the 15q11–q13 region, were observed in the course of these analyses. Strong support was likewise observed for previously unreported genes such as BZRAP1, an adaptor molecule known to regulate synaptic transmission, with eDels or eDups observed in twelve unrelated cases but no controls (p=2.3×10⁻⁵). Less is known about MDGA2, likewise observed to be case-specific (p=1.3×10⁻⁴). But, it is notable that the encoded protein shows an unexpectedly high similarity to Contactin 4 (BLAST E-value=3×10⁻³⁹), which has also been linked to disease. That hundreds of distinct rare variants were each seen only once further highlights complexity in the ASDs and points to the continued need for larger cohorts.     

Cis-Effects of Heterochromatin on Heterochromatic and Euchromatic Gene Activity in Drosophila Melanogaster

Chromosomal rearrangements that juxtapose heterochromatin and euchromatin can result in mosaic inactivation of heterochromatic and euchromatic genes. This phenomenon, position effect variegation (PEV), suggests that heterochromatic and euchromatic genes differ in their regulatory requirements. This report describes a novel method for mapping regions required for heterochromatic genes, and those that induce PEV of a euchromatic gene. P transposase mutagenesis was used to generate derivatives of a translocation that variegated for the light(+) (lt(+)) gene and carried the euchromatic white(+) (w(+)) gene on a transposon near the heterochromatin-euchromatin junction. Cytogenetic and genetic analyses of the derivatives showed that P mutagenesis resulted in deletions of several megabases of heterochromatin. Genetic and molecular studies showed that the derivatives shared a euchromatic breakpoint but differed in their heterochromatic breakpoint and their effects on seven heterochromatic genes and the w(+) gene. Heterochromatic genes differed in their response to deletions. The lt(+) gene was sensitive to the amount of heterochromatin at the breakpoint but the heterochromatic 40Fa gene was not. The severity of variegated w(+) phenotype did not depend on the amount of heterochromatin in cis, but varied with local heterochromatic environment. These data are relevant for considering mechanisms of PEV of both heterochromatic and euchromatic genes.     

增加植酸酶基因appA-m的拷贝提高其在巴斯德毕赤酵母的表达量

为了进一步提高植酸酶的发酵效价,降低植酸酶生产成本,对毕赤酵母表达载体pGAPZα-A进行了改造。将表达载体pPIC9的AOX1启动子序列引入pGAPZα-A,使之成为甲醇可诱导型表达载体pAOXZα,插入植酸酶基因appA-m后得到重组载体pAOXZα-appA-m。以染色体上带有一个拷贝的appA-m基因、发酵效价可达到7.5×106IU/mL发酵液的重组酵母菌株74#为受体菌进行转化,在该重组菌株的染色体上的另一位点整合含有植酸酶基因的表达盒,经筛选到高表达植酸酶的重组子。通过PCR进行验证,植酸酶基因被整合到重组酵母的染色体上,且受体菌中原有的植酸酶基因结构未改变。重组菌在5L发酵罐经甲醇诱导120h植酸酶蛋白表达量达到4mg/mL发酵液,酶活性(发酵效价)达到1.2×107IU/mL发酵液以上,较含单拷贝植酸酶基因的受体菌株表达量有较大程度提高。PCR检测及表达量分析证明改良的菌株具有很好的遗传稳定性和表达稳定性。     

Hypervariability in Gene Copy Number for the Glucose Transporter Genes in Trypanosomes

The copy number of the gene encoding the carrier protein responsible for the uptake of glucose in bloodstream form Trypanosoma brucei (THT1) was investigated in the genome of 55 different members of the subgenus Trypanozoon. The gene is present in multiple copies in tandem arrays on two homologous chromosomes in these organisms, and copy number varies both intra- and interspecifically. Variability is also apparent in the number of genes encoding a second hexose transporter (THT2) which is the only isoform expressed in procyclic organisms. Multiple copies of THT2 are conserved in representatives of the non-tsetse transmitted species, Trypanosoma evansi and Trypanosoma equiperdum.     

The Role of Heterochromatin in the Expression of a Heterochromatic Gene, the Rolled Locus of Drosophila Melanogaster

Constitutive heterochromatic regions of chromosomes are those that remain condensed through most or all of the cell cycle. In Drosophila melanogaster, the constitutive heterochromatic regions, located around the centromere, contain a number of gene loci, but at a much lower density than euchromatin. In the autosomal heterochromatin, the gene loci appear to be unique sequence genes interspersed among blocks of highly repeated sequences. Euchromatic genes do not function well when brought into the vicinity of heterochromatin (position-effect variegation). We test the possibility that the blocks of centromeric heterochromatin provide an environment essential for heterochromatic gene function. To assay directly the functional requirement of autosomal heterochromatic genes to reside in heterochromatin, the rolled (rl) gene, which is normally located deep in chromosome 2R heterochromatin, was relocated within small blocks of heterochromatin to a variety of euchromatic positions by successive series of chromosomal rearrangements. The function of the rl gene is severely affected in rearrangements in which the rl gene is isolated in a small block of heterochromatin, and these position effects can be reverted by rearrangements which bring the rl gene closer to any large block of autosomal or X chromosome heterochromatin. There is some evidence that five other 2R heterochromatic genes are also affected among these rearrangements. These findings demonstrate that the heterochromatic genes, in contrast to euchromatic genes whose function is inhibited by relocation to heterochromatin, require proximity to heterochromatin to function properly, and they argue strongly that a major function of the highly repeated satellite DNA, which comprises most of the heterochromatin, is to provide this heterochromatic environment.     

基因拷贝数与染色体位置对酵母表达乙肝病毒融合表面抗原SA-28基因的影响

应用ＦＬＰ重组酶介导的染色体定点整合技术,将带有不同拷贝数的乙肝病毒融合表面抗原ＳＡ－２８基因表达单元的质粒整合在酵母不同的染色体位点,并测定了ＳＡ－２８基因的表达情况,初步研究了基因拷贝数与染色体位置对酵母表达外源基因的影响。结果表明ＳＡ－２８基因在ＨＩＳ３位点整 合时的表达水平随基因拷贝数的增加而提高,遵循基因剂量效应;在某些染色体位点整２合时,插入方向对其表达有不同程度的影响,呈现出明显的染     

利用实时荧光定量PCR法检测转基因水稻外源基因拷贝数的研究

转基因植物中外源基因拷贝数是影响目的基因表达水平和遗传稳定性的重要因素,因此外源基因拷贝数的检测成为转基因研究的关键．利用高通量、快速、灵敏的SYBR Green Ⅰ荧光定量实时PCR法,检测了转大麦烟酰胺合成酶基因（NASl）水稻中外源基因拷贝数．以蔗糖磷酸合成酶基因（SPS）作为水稻的内源参照基因．通过梯度稀释法．分别获得了NAS1和SPS基因的Ct值与起始模板数的相关性标准曲线,相关系数分别为0．99976和0．99571,相关性高．通过目的基因NAS1和水稻内源参照基因SPS起始模板数的比较,获得了目的基因在转基因水稻中的拷贝数。在8株转基因株系中,1株为假阳性,1株拷贝数为1,3株拷贝数为2,其余3株拷贝数分别为3、4和7．而阴性对照拷贝数为0．这种方法快速、简便、准确,可以满足转基因育种工作中对后代优良株系的选择．     

Dietary Variation and Evolution of Gene Copy Number among Dog Breeds

Prolonged human interactions and artificial selection have influenced the genotypic and phenotypic diversity among dog breeds. Because humans and dogs occupy diverse habitats, ecological contexts have likely contributed to breed-specific positive selection. Prior to the advent of modern dog-feeding practices, there was likely substantial variation in dietary landscapes among disparate dog breeds. As such, we investigated one type of genetic variant, copy number variation, in three metabolic genes: glucokinase regulatory protein (GCKR), phytanol-CoA 2-hydroxylase (PHYH), and pancreatic α-amylase 2B (AMY2B). These genes code for proteins that are responsible for metabolizing dietary products that originate from distinctly different food types: sugar, meat, and starch, respectively. After surveying copy number variation among dogs with diverse dietary histories, we found no correlation between diet and positive selection in either GCKR or PHYH. Although it has been previously demonstrated that dogs experienced a copy number increase in AMY2B relative to wolves during or after the dog domestication process, we demonstrate that positive selection continued to act on amylase copy number in dog breeds that consumed starch-rich diets in time periods after domestication. Furthermore, we found that introgression with wolves is not responsible for deterioration of positive selection on AMY2B among diverse dog breeds. Together, this supports the hypothesis that the amylase copy number expansion is found universally in dogs.     

Copies of a Stellate Gene Variant Are Located in the X Heterochromatin of Drosophila Melanogaster and Are Probably Expressed

Two variants of X chromosome Stellate genes responsible for crystal formation in XO male primary spermatocytes occupy different genome positions. The majority if not all of the 1250-bp Stellate genes are located at the 12E site where the Ste locus has been mapped and almost all of the 1150-bp Stellate repeats are concentrated in the distal X heterochromatin. Sequencing of Stellate genes derived from X heterochromatin reveals the preservation of their open reading frames and precise matching with some Stellate cDNAs reported earlier. At least some heterochromatic Stellate genes are suggested to be expressed and, therefore, involved in the interaction with the Y chromosome locus Su(Ste), as are the Stellate genes from 12E.