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High Throughput SNP Genotyping with Two Mini-sequencing Assays

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Acta Biochimica et Biophysica Sinica 2004年第6期36卷 379-384页

作者： Chunqing LUO1,2., Libin DENG1#, and Changqing ZENG1,2. The institute of Genetic and Developmental Biology, the chinese {2. of sciences, Beijing 100101, china 2.eijing genomics institute, the chinese {2. of sciences, Beijing 1013.0, china The institute of Genetic and Developmental Biology the Chinese Academy of SciencesBeijing 100101 China Beijing genomics institute the Chinese Academy of SciencesBeijing 101300 China

Two mini-sequencing methods, FP-TDI (template-directed dye-terminator incorporation with fluorescence-polarization) and MassArray (matrix assisted laser desorption ionization time of flight detection mass spectrometry), were optimized. A numeric standard was introduced to evaluate the SNP scoring quality of FP-TDI assay, thus made the optimization work easier. At the same time, using multi-PCR technology, 8-plex genotyping of MassArray assay was successfully carried out, some softwares were developed and the data process of MassArray was highly automated. Then these two methods were applied to high throughput SNP genotyping, the accuracy, efficiency and robustness were compared. The result shows FP-TDI is more sensitive to the concentration of SNPprimer and PCR product, as well as extension cycles, the SNPprimer length of FP-TDI should be 2.–30 bp long, whereas MassArray assay prefers to be as short as only 16 *** 6440 SNP sites of human chromosome 3 were genotyped in a sample of 90 individuals, 4792.sites by FP-TDI assay and 1648 sites by MassArray assay, the success rates of FP-TDI and MassArray were 67.7% and 93.6% respectively. The throughput of MassArray was higher than FP-TDI, and the cost of MassArray was lower, MassArray was more suitable for high throughput SNP genotyping.

关键词： SNP genotyping FP-TDI MassArray high throughput

Cloning and expression of putative ethylene receptor genes in soybean plant

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Progress in Natural Science:Materials International 2007年第10期17卷 1152-1160页

作者： Xie Zongming1,2.3, Lei Gang1,2. Wuriyanghan Hada1,2. Tian Aiguo1, Zhang Jinsong1 and Chen Shouyi1(1. National Key Laboratory of Plant genomics, institute of Genetics and Developmental Biology, {2. academy of sciences, Beijing 100101, china 2. Graduate School of {2. academy of sciences, Beijing 100039, china 3. Xinjiang academy of Agricultural and Reclamation Science, Shihezi 832.00, china) National Key Laboratory of Plant genomics Institute of Genetics and Developmental Biology Chinese Acad. of Sci. Beijing 100101 China Graduate School Chinese Acad. of Sci. Beijing 100039 China Xinjiang academy of Agricultural and Reclamation Science Shihezi 832000 China

Ethylene plays important roles in plant growth, development, and stress responses, and ethylene receptors have been identified and studied extensively in various plant species. Here we report the cloning of four ethylene receptor genes from soybean, i.e. GmETR1, GmERS1, GmETR2.and GmEIN4. Construction of the phylogenic tree showed that GmETR1 and GmERS1 belong to subfamily I whereas GmETR2.and GmEIN4 belong to subfamily II. The four ethylene receptor genes showed different tissue-specific expression patterns in roots, stems, leaves, cotyledons, flowers, pods and seeds of soybean. These genes were differentially regulated by various abiotic stresses and plant hormones. The possible roles of the four genes in soybean plant were also discussed.

关键词： abiotic stress expression analysis ethylene receptor plant hormone soybean.

DNA barcoding of Gaultheria L. in china(Ericaceae:Vaccinioideae)

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Journal of Systematics and Evolution 2011年第5期49卷 411-424页

作者： 1~He REN 1,2.Lu LU 1,2.Hong WANG 1,2.De-Zhu LI 1(Laboratory of Biodiversity and Biogeography,Kunming institute of Botany,chinese {2. of sciences,Kunming 6502.4,china) 2.Plant Germplasm and genomics Center,Germplasm Bank of Wild Species,Kunming institute of Botany,chinese {2. of sciences,Kunming 6502.4,china) (Laboratory of Biodiversity and Biogeography Kunming Institute of Botany Chinese Academy of Sciences Kunming 650204 China) (Plant Germplasm and genomics Center Germplasm Bank of Wild Species Kunming Institute of Botany Chinese Academy of Sciences Kunming 650204 China)

Four DNA barcoding loci,chloroplast loci rbcL,matK,trnH-psbA,and nuclear locus internal transcribed spacer(ITS),were tested for the accurate discrimination of the chinese species of Gaultheria by using intraspecific and interspecific pairwise P-distance,Wilcoxon signed rank test,and tree-based *** study included 186 individuals from 89 populations representing 30 *** all individuals,single locus markers showed high levels of sequencing universality but were ineffective for species *** chain reaction amplification and sequencing were successful for all four *** ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH-psbA.A combination of matK and ITS was the most efficient DNA barcode among all studied regions,however,they do not represent an appropriate candidate barcode for chinese Gaultheria,by which only 11 out of 30 species can be *** rbcL,matK,and trnH-psbA,which were recently proposed as universal plant barcodes,have a very poor capacity for species separation for chinese *** barcodes may be reliable tools to identify the evolutionary units of this group,so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.

关键词： DNA barcoding Ericaceae Gaultheria species discrimination taxonomy Vaccinioideae.

Cloning,expression and characterization of a nucleoside diphosphate kinase (NDPK) gene from tobacco

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Progress in Natural Science:Materials International 2007年第8期17卷 906-912页

作者： Zhou Qiyun~(1,2.*) Xie Zongming~(1**) Zhang Zhigang~1 Cao Yangrong~1 Zhang jinsong~(1***) Chen Shouyi~(1***) (1.National Key Laboratory of Plant genomics,institute of Genetics and Developmental Biology,{2. academy of sciences,Beijing100101,china 2.Graduate University of {2. academy of sciences,Beijing 100039,china) National Key Laboratory of Plant genomics Institute of Genetics and Developmental Biology Chinese Acad. of Sci. Beijing 100101 China Graduate University Chinese Acad. of Sci. Beijing 100039 China

Nucleoside diphosphate kinase (NDPK) is a housekeeping enzyme that maintains the intracellular levels of all (d)NTPs used in biosynthesis except *** we report that a full-length cDNA encoding nucleoside diphosphate kinase (NDPK) was cloned us- ing yeast two-hybrid approach.A tobacco NDPK gene was obtained and designated as ***1 is 704 bp in length and en- codes a putative 16.2.kD protein of 148 amino *** analysis showed that NtNDPK1 is highly homologous to other plant NDPK genes and identified as type I (NDPK1).RNA-gel blot analysis showed that there was no significant difference of NtNDPK1 ex- pression in roots,stems,leaves and *** expression of NtNDPK1 was induced by ABA and PEG and repressed by NaCl,but not significantly affected by Paraquat,wounding and low temperature (4℃) treatments,indicating that NtNDPK1 may play a certain role in response to abiotic *** vitro phosphorylation assay demonstrated that NtNDPK1 had autophosphoryLation activity.

关键词： abiotic stress autophosphorylation activity NtNDPK1 tobacco yeast two-hybrid

Overexpression of a tobacco small G protein gene NtRop1 causes salt sensitivity and hydrogen peroxide production in transgenic plants

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Science china(Life sciences) 2008年第5期51卷 383-390页

作者： CAO YangRong1,2. LI ZhiGang1,2. CHEN Tao1,2. ZHANG ZhiGang1, ZHANG JinSong1 & CHEN ShouYi1 1 National Key Lab of Plant genomics, institute of Genetics and Developmental Biology, {2. academy of sciences, Beijing 100101, china 2.Graduate School of {2. academy of sciences, Beijing 100039, china National Key Lab of Plant genomics Institute of Genetics and Developmental Biology Chinese Academy of Sciences Beijing China Graduate School of chinese academy of {2. Beijing China

The small GTPases of Rop/Rho family is central regulators of important cellular processes in plants. Tobacco small G protein gene NtRop1 has been isolated; however, its roles in stress responses were unknown. In the present study, the genomic sequence of NtRop1 was cloned, which has seven exons and six introns, similar to the Rop gene structure from Arabidopsis. The NtRop1 gene was constitutively expressed in the different organs whereas the other six Rop genes from tobacco had differential expression patterns. The expression of the NtRop1 gene was moderately induced by methyl viologen, NaCl, and ACC treatments, but slightly inhibited by ABA treatment, with no significant induction by NAA treatment. The transgenic Arabidopsis plants overexpressing the NtRop1 showed increased salt sensitivity as can be seen from the reduced root growth and elevated relative electrolyte leakage. The hydrogen peroxide production was also promoted in the NtRop1-trangenic plants in comparison with wild type plants. These results imply that the NtRop1 may confer salt sensitivity through activation of H2.2.production during plant response to salt stress.

关键词： small G protein NtRop1 salt sensitive hydrogen peroxide

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Next-generation sequencing technology:A technology review and future perspective

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Science china(Life sciences) 2010年第1期53卷 44-57页

作者： ZHOU XiaoGuang1,REN LuFeng1,LI YunTao2.ZHANG Meng1,YU YuDe2.& YU Jun1 1 Key Laboratory of Genome sciences and Information,Beijing institute of {2.,chinese academy of sciences,Beijing 10002.,china 2.institute of Semiconductors,chinese academy of sciences,Beijing 100083,china Key Laboratory of Genome sciences and Information Beijing Institute of Genomics Chinese Academy of Sciences Beijing China institute of Semiconductor Chinese Academy of Sciences Beijing China

As one of the most powerful tools in biomedical research,DNA sequencing not only has been improving its productivity at an exponential growth rate but has also been evolving into a new layout of technological territories toward engineering and physical disciplines over the past three *** this technical review,we look into technical characteristics of the next-generation sequencers and provide insights into their future development and *** envisage that some of the emerging platforms are capable of supporting the USD1000 genome and USD100 genome goals if given a few years for technical *** also suggest that scientists from china should play an active role in this campaign that will have a profound impact on both scientific research and societal healthcare systems.

关键词： genomics DNA sequencing next generation sequencing technologies sequencer

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BUD2.encoding an S-adenosylmethionine decarboxylase,is required for Arabidopsis growth and development

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Cell Research 2006年第5期16卷 446-456页

作者： Chunmin Ge~(1,2.*) Xia Cui~(1,2.*) Yonghong Wang~1 Yuxin Hu~3 Zhiming Fu~1 Dongfen Zhang~(1,2. Zhukuan Cheng~1 Jiayang Li~1 ~1State Key Laboratory of Plant genomics and National Center for Plant Gene Research,institute of Genetics and Developmental Biology,{2. academy of sciences,Beijing 100101,china ~2.raduate School,{2. academy of sciences,Beijing 100101,china ~3institute of Botany,{2. academy of sciences,Beijing 100093,china State Key Laboratory of Plant genomics and National Center for Plant Gene Research Institute of Genetics and Developmental Biology Chinese Academy of Sciences Beijing China Graduate School Chinese Academy of Sciences Beijing China institute of Botany Chinese Academy of Sciences Beijing China

Polyamines are implicated in regulating various developmental processes in plants,but their exact roles and how theygovern these processes still remain *** report here an Arabidopsis bushy and dwarf mutant,bud2.which resultsfrom the complete deletion of one member of the small gene family that encodes S-adenosylmethionine decarboxylases(SAMDCs) necessary for the formation of the indispensable intermediate in the polyamine biosynthetic ***2.plant has enlarged vascular systems in inflorescences,roots,and petioles,and an altered homeostasis *** double mutant of bud2.and samdcl,a knockdown mutant of another SAMDC member,is embryo lethal,demonstrat-ing that SAMDCs are essential for plant *** results suggest that polyamines are required for the normalgrowth and development of higher plants.

关键词： BUD2 polyamines embryogenesis plant architecture Arabidopsis

Preparing a re-sequencing DNA library of 2.cancer candidate genes using the ligation-by-amplification protocol by two PCR reactions

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Science china(Life sciences) 2009年第5期52卷 483-491页

作者： SU YeYang1,2.3, LIN Lin1,2.3, TIAN Geng1,2.3, CHEN Chen2.3, LIU Tao2.3, XU Xingya3, QI XinPeng1,2.3, ZHANG XiuQing2.3& YANG HuanMing2.3 1 Graduate School of chinese academy of {2., Beijing 100049, china 2.Beijing institute of genomics, chinese academy of {2., Beijing 10002., china 3 Beijing genomics institute, Beijing {12., china Graduate School of chinese academy of {2. Beijing China Beijing institute of genomics Chinese Academy of Sciences Beijing China Beijing genomics institute Beijing China

To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing service, with subsequent bioinformatic software and laboratory methods developed to expand their applications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven't benefited much from those next-generation sequencing technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies. Although several methods have been proposed to solve this problem, including microarray-based genomic selections and selector-based technologies, they require advanced equipment and procedures which limit their applications in many laboratories. By contrast, we overcame such potential drawbacks by utilizing a ligation by amplification (LBA) protocol, a method using a pair of Universal Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences of two human cancer genes: BRCA1 and BRCA2.as target regions, specifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both a conventional Sanger sequencer ABI 3730xl DNA Analyzer and the next-generation 'synthesis by sequencing technology' Illumina/Solexa Genome Analyzer. Bioinformatic analysis demonstrated the utility and efficiency (including the coverage and depth of each target sequence and the SNPs detection effectiveness) o

关键词： health genomics the next-generation sequencing techonology sequencing technology large-scale genomics PCR

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Identification of human dopamine receptors agonists from chinese herbs

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Acta Pharmacologica Sinica 2007年第1期28卷 132-139页

作者： Yi-lin ZHANG~(1,2.3,4) Hai-qing ZHANG~(1,2.3,4) Xiao-yu LIU~3 Shi-neng HUA~3 Lu-bing ZHOU~3 Jun YU~2.Xue-hai TAN~(3,5)1 Graduate School of the chinese academy of {2.,Beijing 100039,china 2.Beijing genomics institute,chinese academy of {2. Beijing 1013.0,china 3 HD Bio{2. Co Ltd,Shanghai 2.12.3,china Graduate School of the chinese academy of {2. Beijing China Beijing genomics institute Chinese Academy of Sciences Beijing China HD Biosciences Co Ltd Shanghai China

Aim:To find human dopamine receptors,especially Dl-like receptor specific ago-nists from chinese herbs as potential antihypertension drug ***:TwoD l-like receptor cell lines carrying a μ-lactamase reporter gene,and a D2.receptorcell line coexpressing a promiscuous G protein G15 were constructed using HEK2.3cells.A natural compound library made from fractionated samples of herbal ex-tracts was used for high-throughput screening（HTS）against one of the cell lines,HEK/D5R/*** interested hits were evaluated for their activities againstvarious dopamine ***:Fourteen hits were identified from primaryscreening,of which 2.of the better hit samples,HD052. and HD0059,were selectedfor further material and activity analysis,and to obtain 2.compounds that ap-peared as 2.single peaks in HPLC,HD052.H01 and ***0059H01could activate D 1,D2.and D5 receptors,with EC50values of 2.2. μg/mL,0.85 μg/mL,and 1.41 μg/mL,***052.H01 could only activate DlR and D5Rwith EC50values of 2.95 μg/mL and 8.38 μg/***:We established cell-based assays for 3 different human dopamine receptors and identified specificagonists HD052.H01 and HD0059H01 through *** specific agonist to D1-like receptors,HD052.H01,may become a new natural product-based drug leadfor antihypertension treatment.

关键词： dopamine receptors natural products high throughput screening hypertension