Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.     

Differential Gene Expression Analysis by DNA Microarray Technology and Its Application in Molecular Oncology

Accumulation of genetic and epigenetic aberrations leads to malignant transformation of normal cells. Functional studies of cancer using genomic and proteomic tools aim to reveal the true complexity of the processes leading to cancer development in humans. Until recently, diagnosis and prognosis of cancer was based on conventional pathologic criteria and epidemiological evidence. Certain tumors were divided only into relatively broad histological and morphological subcategories. Rapidly developing methods of differential gene expression analysis promote the search for clinically relevant genes changing their expression levels during malignant transformation. DNA microarrays offer a unique possibility to rapidly assess the global expression picture of thousands genes in any given time point and compare the results of detailed combinatory analysis of global expression profiles for normal and malignant cells at various functional stages or separate experimental conditions. Acquisition of such genetic portraits allows searching for regularity and difference in expression patterns of certain genes, understanding their function and pathological importance, and ultimately developing the molecular nosology of cancer. This review describes the basis of DNA microarray technology and methodology, and focuses on their application in molecular classification of tumors, drug sensitivity and resistance studies, and identification of biological markers of cancer.     

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the “gold standard” enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.     

新一代高通量RNA测序数据的处理与分析

随着新一代高通量DNA测序技术的快速发展,RNA测序(RNA-seq)已成为基因表达和转录组分析新的重要手段．RNA-seq技术产生的海量数据为生物信息学带来了新的机遇和挑战．有效地对测序数据进行针对性的生物信息学处理和分析,成为RNA-seq技术能否在科学探索中发挥重大作用的关键．以新一代Illumina/Solexa测序平台所产生的数据为例,在扼要介绍高通量RNA-seq测序流程的基础上,对RNA-seq数据处理和分析的方法和现有软件做一个较为全面的综述,并对其中有待进一步研究的问题进行展望．     

A Microarray-Based Comparative Analysis of Gene Expression Profiles During Grain Development in Transgenic and Wild Type Wheat

Global, comparative gene expression analysis is potentially a very powerful tool in the safety assessment of transgenic plants since it allows for the detection of differences in gene expression patterns between a transgenic line and the mother variety. In the present study, we compared the gene expression profile in developing seeds of wild type wheat and wheat transformed for endosperm-specific expression of an Aspergillus fumigatus phytase. High-level expression of the phytase gene was ensured by codon modification towards the prevalent codon usage of wheat genes and by using the wheat 1DX5HMW glutenin promoter for driving transgene expression. A 9K wheat unigene cDNA microarray was produced from cDNA libraries prepared mainly from developing wheat seed. The arrays were hybridised to flourescently labelled cDNA prepared from developing seeds of the transgenic wheat line and the mother variety, Bobwhite, at three developmental stages. Comparisons and statistical analyses of the gene expression profiles of the transgenic line vs. that of the mother line revealed only slight differences at the three developmental stages. In the few cases where differential expression was indicated by the statistical analysis it was primarily genes that were strongly expressed over a shorter interval of seed development such as genes encoding storage proteins. Accordingly, we interpret these differences in gene expression levels to result from minor asynchrony in seed development between the transgenic line and the mother line. In support of this, real time PCR validation of results from selected genes at the late developmental stage could not confirm differential expression of these genes. We conclude that the expression of the codon-modified A.␣fumigatus phytase gene in the wheat seed had no significant effects on the overall gene expression patterns in the developing seed.     

荔枝VQ基因家族鉴定及参与非生物胁迫应答的功能分析

【目的】鉴定荔枝（Litchi chinensis）VQ基因家族,并分析其在不同组织的表达模式及在低温、高温、干旱和盐胁迫下的应答。【方法】利用生物信息学方法在荔枝全基因组中鉴定LcVQ基因,并对其理化性质、亚细胞定位、基因结构和保守基序等进行分析;利用MEGA 6.0软件构建系统发育树,分析荔枝、拟南芥和水稻VQ蛋白的系统发育关系;使用实时荧光定量PCR（quantitative real-time PCR,qRT-PCR）技术验证LcVQs对多种非生物胁迫的响应情况。【结果】荔枝中共鉴定获得可聚类为9个亚族的18个VQ基因（LcVQ1-LcVQ18）,依次分布在荔枝的11条染色体上,其编码蛋白的氨基酸数介于111~427 aa之间,分子质量为12.48~45.49 kD;除LcVQ15和LcVQ17定位于细胞质之外,其余LcVQ蛋白均定位于细胞核。顺式作用元件预测分析结果表明,LcVQs启动子区域包含大量植物生长发育响应元件、激素响应元件及逆境响应元件。转录组数据分析结果显示,不同组织中LcVQs的表达量具有明显差异,总体上分为普遍性表达和特异性表达。qRT-PCR结果显示,在低温、高温、干旱和盐胁迫处理下的3 h内,与对照相比分别有4,3,3,4个LcVQs明显上调表达,可快速响应相应胁迫。【结论】荔枝全基因组中有18个VQ家族成员,具有典型VQ保守结构域,能差异化响应多种非生物胁迫,为研究其抗逆机制奠定了基础。     

谷子CPP基因家族鉴定及外源硒响应分析

【目的】探究谷子CPP家族成员基本特征及其在外源硒处理下的响应模式,为谷子富硒高叶酸新品种选育提供新的遗传材料。【方法】借助生物信息学工具鉴定谷子CPP家族成员,采用qRT-PCR技术对CPP基因在不同组织及外源硒处理下的表达水平进行检测,为揭示谷子CPP家族生物学功能及外源硒响应机制奠定基础。【结果】(1)谷子基因组中包含9个CPP基因,定位于6条染色体上,依照基因在染色体的排布命名为SiCPP1～SiCPP9,氨基酸数目在220～899之间,相对分子量为23624.32～97253.16Da,等电点介于6.64～9.17之间,内含子数目为3～11个不等,亚细胞预测显示该家族所有成员均能在细胞核定位到,蛋白质二级结构预测表明该家族中占比最大的是α-螺旋其次是无规则卷曲。(2)系统进化分析表明,谷子CPP蛋白可分为4个亚家族,相同亚家族之间保守基序和结构域的数目及分布相似。(3)启动子分析发现谷子CPP家族中存在大量光、生长发育、激素以及胁迫响应元件。(4)荧光定量结果显示,谷子CPP家族成员在根、茎、叶和穗中差异表达。谷子CPP家族9个成员均对外源硒存在响应,其中SiCPP5、SiCPP6、SiCPP7和SiCPP8响应程度最强。【结论】谷子CPP家族成员具有组织表达特异性且对外源硒存在不同程度响应。     

陆地棉XTH基因家族全基因组鉴定及在纤维发育过程的表达分析

本研究从陆地棉TM-1基因组中鉴定出72个XTH家族基因,编码木葡聚糖内转糖苷酶/水解酶(XTH,xyloglucan endotransglycosylase/hydrolase),分别命名为Gh XTH01~Gh XTH72,分析了其基因结构、保守基序、系统进化、理化性质、亚细胞定位,并探究其在棉纤维发育不同时期的表达规律。结果表明,XTH家族基因分布在除At 07、Dt 07以外的24条棉花染色体上,根据系统发育树,将XTH家族基因分为3个亚组;XTH氨基酸序列有3个保守基序,保守性较强;多数XTH蛋白定位在细胞外。根据XTHs在纤维发育不同时期的表达量变化,将其分为4类。通过构建陆地棉与拟南芥XTH氨基酸序列进化树,推测Gh XTH15、Gh XTH28、Gh XTH36、Gh XTH49、Gh XTH59、Gh XTH62、Gh XTH63等基因在棉纤维发育过程中发挥重要作用。通过比较XTH家族基因在不同纤维品质陆地棉品种中的表达差异,推测在优质棉花品种中优势表达基因Gh XTH03、Gh XTH12、Gh XTH17、Gh XTH22、Gh XTH23、Gh XTH28、Gh XTH33、Gh XTH44、Gh XTH46、Gh XTH59等在纤维发育伸长过程中可能发挥着重要作用。上述结果为研究陆地棉XTH基因家族在棉纤维发育中的功能提供了参考依据。     

西葫芦CCD基因家族鉴定及其在果实发育中的表达北大核心CSCD

类胡萝卜素裂解双加氧酶(carotenoid cleavage dioxygenase,CCD)是类胡萝卜素氧化裂解途径中的关键酶,在植物生长发育、香气形成及胁迫响应等过程中均发挥着重要作用。该研究运用生物信息学方法从西葫芦全基因组中鉴定出13条具有完整RPE65保守结构域的CCD基因,为进一步解析CCD基因家族在西葫芦中的功能奠定基础。结果表明:(1)聚类分析显示,13个西葫芦CCD基因编码的蛋白可分为CCD1、CCD4、CCD7、CCD8、NCED、CCD1-like共6个亚组,且CCD8和CCD7亚组与其他家族成员的遗传距离较远。(2)顺式作用元件预测分析发现,CCD基因启动子中含有光信号、激素、环境胁迫和生长发育响应元件。(3)转录组数据分析显示,CCD家族基因具有组织表达特异性,其中3个CCD基因在组织中不表达,CpCCD1基因在叶和果实中显著高表达。(4)在果实发育过程中,8个CCD基因呈现上调表达,2个CCD基因呈现下调表达,其中CpCCD1、CpCCD4a、CpCCD4b、CpCCD8a这4个CCD基因在果实膨大生长期或成熟期出现显著高表达,推测它们可能在西葫芦果实发育过程中具有重要的调控作用。     

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression^1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo^4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy⁶. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- β - like ligand DBL-1, so this assay is appropriate for identification of TGF-β signaling components⁷. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.     

新疆甜瓜Fom-2基因同源序列的克隆及分析

近年来甜瓜枯萎病蔓延迅速,危害严重,分离克隆甜瓜抗病基因,利用转基因技术改良甜瓜抗病品性是一种从根本上解决甜瓜枯萎病危害的新途径。根据已知Fom-2基因序列设计特异引物,采用RT-PCR技术从新疆甜瓜抗病性品种黄旦子中扩增出580bp的cDNA片段,序列分析表明它与已发表Fom-2基因具有高度同源性基因片段,命名为X-Fom-2。以X-Fom-2为探针对新疆甜瓜黄旦子、伽什瓜、红心脆、金丽-2号进行Southem blot和Northem blot,结果表明,X-Fom-2以多拷贝形式存在。X-Fom-2在抗病品种黄旦子、金丽2号有表达且在病原菌诱导后增强,而在感病品种伽什瓜、红心脆中没有表达。     

Gene Regulation in Continuous Cultures: A Unified Theory for Bacteria and Yeasts

During batch growth on mixtures of two growth-limiting substrates, microbes consume the substrates either sequentially (diauxie) or simultaneously. The ubiquity of these growth patterns suggests that they may be driven by a universal mechanism common to all microbial species. Recently, we showed that a minimal model accounting only for enzyme induction and dilution, the two processes that occur in all microbes, explains the phenotypes observed in batch cultures of various wild-type and mutant/recombinant cells (Narang and Pilyugin in J. Theor. Biol. 244:326–348, 2007). Here, we examine the extension of the minimal model to continuous cultures. We show that: (1) Several enzymatic trends, attributed entirely to cross-regulatory mechanisms, such as catabolite repression and inducer exclusion, can be quantitatively explained by enzyme dilution. (2) The bifurcation diagram of the minimal model for continuous cultures, which classifies the substrate consumption pattern at any given dilution rate and feed concentrations, provides a precise explanation for the empirically observed correlations between the growth patterns in batch and continuous cultures. (3) Numerical simulations of the model are in excellent agreement with the data. The model captures the variation of the steady state substrate concentrations, cell densities, and enzyme levels during the single- and mixed-substrate growth of bacteria and yeasts at various dilution rates and feed concentrations. This variation is well approximated by simple analytical expressions that furnish deep physical insights. (4) Since the minimal model describes the behavior of the cells in the absence of cross-regulatory mechanisms, it provides a rigorous framework for quantifying the effect of these mechanisms. We illustrate this by analyzing several data sets from the literature.     

从Myriad案谈基因专利的正当性及美国对基因专利授权实质性要件分析

按照专利制度构建的本质,基因专利的作用在于激励产业创新,促进基因研究的发展。但基因专利从产生以来就一直存在着争议。2011年美国Myriad案对分离DNA序列的可专利性具有不同的观点,从Myriad I案认为分离的DNA是不可专利的客体,到Myriad II上诉案中联邦巡回上诉法院推翻地方法院的观点,认为分离的DNA具有不同的化学结构,满足专利客体的适格性,但同时也反射出了对DNA序列可专利性的怀疑。Myriad案引起了美国、欧洲和澳大利亚司法审判中就基因专利适格性问题的较大争议。本文结合美国Myriad案来分析DNA序列作为专利客体的适格性以及目前美国对基因专利授权的实质性条件。     

Combining Rapid Data Independent Acquisition and CRISPR Gene Deletion for Studying Potential Protein Functions: A Case of HMGN1

CRISPR‐Cas gene editing holds substantial promise in many biomedical disciplines and basic research. Due to the important functional implications of non‐histone chromosomal protein HMG‐14 (HMGN1) in regulating chromatin structure and tumor immunity, gene knockout of HMGN1 is performed by CRISPR in cancer cells and the following proteomic regulation events are studied. In particular, DIA mass spectrometry (DIA‐MS) is utilized, and more than 6200 proteins (protein‐ FDR 1%) and more than 82 000 peptide precursors are reproducibly measured in the single MS shots of 2 h. HMGN1 protein deletion is confidently verified by DIA‐MS in all of the clone‐ and dish‐ replicates following CRISPR. Statistical analysis reveals 147 proteins change their expressions significantly after HMGN1 knockout. Functional annotation and enrichment analysis indicate the deletion of HMGN1 induces histone inactivation, various stress pathways, remodeling of extracellular proteomes, cell proliferation, as well as immune regulation processes such as complement and coagulation cascade and interferon alpha/ gamma response in cancer cells. These results shed new lights on the cellular functions of HMGN1. It is suggested that DIA‐MS can be reliably used as a rapid, robust, and cost‐effective proteomic‐screening tool to assess the outcome of the CRISPR experiments.     

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day ^1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.