Isolation of the Flanking Sequences Adjacent to Transgenic T-DNA in Brassica napus Genome by an Improved Inverse PCR Method

[Objective] The research aimed to isolate flanking sequences adjacent to the transgenic T-DNA in Brassica napus by an improved inverse PCR method.[Method] Using single clone of transgenic FS4 in Brassica napus as the research materials,total DNA was extracted from transgenic Brassica napus by using modified CTAB method.After enzyme digestion and purification,self-joining was made.Two circles of nested PCR and the sequence alignment were carried out.[Result] A fragement with the size of 4.0 kb was amplified ...

Ac/Ds Transposition Activity in Transgenic Rice Population and DNA Flanking Sequence of Ds Insertion Sites

The Agrobacterium mediated transgenic rice ( Oryza saliva L.) population with inserts of maize transposon Activator/Dissociation (Ac/Ds) was investigated. DNA sequences flanking the T-DNA were analyzed with inverse PCR. Results showed that 65.4% of the T-DNA was integrated in different locations of rice genome, and some T-DNA flanking sequences were located on certain chromosomes. A number of T-DNA was found to have inserted into protein coding regions. In order to induce transposition of the inserted Ds elements, 354 crosses of Ac x Ds and Ds x Ac were constructed. The excision frequency of Ds element trans-activated by Ac transposase was 22.7% in the F-2 populations, and the transposition was confirmed with analyses of DNA sequences flanking the Ds elements. In addition to the transposition due to 'cut-paste' mechanism, Ds can replicate itself and integrate into a new locus, and inaccurate excisions were also found. A proportion of DNA segments flanking the Ds elements showed no homologies to sequences published in GenBank, of which two were registered under the accession numbers AF355153 and AF355770. The strategy of using transposon tagging for rice genomics study was discussed.

Analysis of the Flanking Sequence and EventSpecific Detection of Transgenic Line W-4 of Brassica napus

The genetically modified high-oleic rapeseed （Brassica napus L.） line W-4 was obtained by transforming a binary vector which harbored an inverted repeat expression cassette of fad2 gene into the rapeseed cultivar Westar.The transformation was mediated by Agrobacterium.The flanking sequences to both the left and right borders of T-DNA insertion site were amplified by thermal asymmetric interlaced PCR （TAIL-PCR） from the genomic DNA of the transgenic rapeseed line W-4.The flanking sequences to the right border was 290 bp in length and the nucleotide composition was 31.27％ for G＋C content while 68.73％ for A＋T content.The flanking sequence to the left border was 365 bp in length and the G＋C content was 32.6％ and the A＋T content was 67.4％,indicating that the T-DNA was integrated in the A/T-rich region.Further more,sequence alignment analysis showed a deletion of 62 bp including the right border of pCNFIRnos and the integration of the whole left border except a change of G to A.That was to say,the integration of the T-DNA in the transgenic line W-4 not involved in the vector sequences.Based on both flanking sequences as well as the left and right borders of the T-DNA sequences,two pairs of specific primers TLF/TLR and TRF/TRR were designed.Using the primers the event-specific PCR detection method for transgenic rapeseed line W-4 was established.By the PCR,two fragments of 485 and 405 bp were amplified from the W-4 genomic DNA as expected,while no products were amplified from the genomic DNA of other transgenic rapeseed lines and non-transgenic rapeseed line.And by the PCR it is possible to detect the W-4 genomic DNA from a mixed sample of genomic DNA.The limit of the detection for the qualitative PCR assay was 0.1％.The method developed in this work is highly specific,sensitive and suitable for event-specific detection of the transgenic rapeseed line W-4.

耐盐转基因大豆事件AtARA6-A001外源插入片段侧翼序列及其应用

转基因大豆AtARA6-A001事件采用农杆菌介导大豆遗传转化法获得,其受体为沈农9号,具有耐盐特性,目前已经进入环境释放阶段。为明确该转基因大豆材料的分子特征及其转基因检测方法,推进该转基因事件生物安全评价工作。本研究以转基因大豆AtARA6-A001为研究对象,利用Southern杂交方法及基因组重测序技术鉴定了外源基因的拷贝数及插入位点的位置和方向。同时利用PCR扩增技术获得了外源T-DNA的左右侧翼序列,并基于左右旁侧序列,建立了转AtARA6基因耐盐大豆A001事件的特异性定性PCR检测方法。此方法能特异性检测转基因大豆植株AtARA6-A001根、茎、叶、花和种子样品,并且能够特异性识别转基因大豆事件的身份,这为后续转基因大豆及其产品的检测和监管提供技术支持。

基于重测序鉴定GbTMEM214基因在陆地棉基因组的插入位点

【目的】利用农杆菌介导法获得了转GbTMEM214基因陆地棉品系,旨在明确转基因棉花株系中T-DNA插入位点的序列特征及检测方法,为其生物安全评价提供依据。【方法】基于基因组重测序技术,利用BLASTn将测序数据与陆地棉TM-1基因组序列进行比对分析,设计特异性引物通过聚合酶链式反应(polymerase chain reaction,PCR)验证插入位点。【结果】携带目标基因GbTMEM214的T-DNA整合在陆地棉基因组D13号染色体57019068~57019106 bp,T-DNA插入导致受体基因组37 bp的片段缺失;通过PCR扩增获得携带GbTMEM214基因的T-DNA插入位点的侧翼序列,由此建立了转GbTMEM214基因棉花的特异性检测方法。【结论】基于基因组重测序技术获得了转GbTMEM214基因棉花的T-DNA插入位点及侧翼序列信息,可为转基因棉花的生物安全评价提供技术参考。

获得构建miRNA高表达载体的pre-miRNA及其侧翼DNA序列的生物信息学方法

近年研究发现pre-miRNA发夹结构两侧的序列对miRNA的成熟至关重要。文章主要介绍了利用"miRBase"和"Ensembl"数据库获得构建microRNA高表达载体的pre-miRNA及其侧翼DNA序列的生物信息学方法:即先通过"miRBase"数据库获得pre-miRNA在"Ensembl"数据库的位置链接;然后在"Ensembl"数据库内获得pre-miRNA在基因组上的位置及碱基序列;最后在pre-miRNA序列导出窗口5'和3'侧翼序列输入框内填写所需侧翼序列的长度,进而获得pre-miRNA及其侧翼的DNA序列。

Analysis and validation of genome-specific DNA variations in 5′flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

The thirty-three 5′ flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment,; a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety ‘Chinese Spring’ was employed to carry out chromosome assignment. The subsequent cloning; DNA sequencing of PCR fragments validated the sequences specificity of the 5′ flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding; 5′ flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively,; therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background; the efficient selections of quality-related components in breeding programs.

Roles of flanking sequences in the binding between unimolecular parallel-stranded G-quadruplexes and ligands

G-quadruplexes attract more and more attention in recent years.Numerous small molecules which can induce or stabilize the formation of G-quadruplexes have been investigated on the purpose of anticancer drug development.As a motif existed in physiological condition,flanking sequences are an important part of G-quadruplexes but the study on the impact of flanking sequences on (G-quadruplex)-ligand binding is rarely reported.In this paper,the effects of flanking sequences on binding affinity between a series of unimolecular parallel-stranded G-quadruplex sequences derived from c-myc oncogene promoter (termed as c-myc G-quadruplexes) and their ligands are discussed in detail.The results showed that the flanking sequences on c-myc G-quadruplexes play key roles in (G-quadruplex)-ligand interaction.When a c-myc G-quadruplex is bound to its ligands,the flanking sequences might form a binding cavity above the terminal G-quartet,which could provide a suitable site for ligands to dock in.Moreover,the bases on flanking sequences could interact with ligand through π-π stacking,and finally form a sandwich-stacking mode (terminal G-quartet,ligand and bases on the flanking sequence).This mode could stabilize the (G-quadruplex)-ligand complex effectively and enhance the binding affinity dramatically.However,flanking sequences are also found to exhibit steric hindrance effect which could impede the (G-quadruplex)-ligand binding.

转基因玉米ND207转化事件特异性定性PCR检测方法及其标准化

转基因玉米ND207是中国农业大学研发的转mCry1Ab和mCry2Ab基因的抗虫玉米,本研究的目的是建立ND207转化事件特异性定性PCR检测方法。根据研发者提供的引物进行PCR扩增,PCR产物测序分析,获得了5’端旁侧序列262 bp,包括109 bp的载体序列和153 bp的玉米基因组序列, 3’端旁侧序列316 bp,包括76 bp的载体序列和240bp的玉米基因组序列。针对两端旁侧序列设计14条引物,组成25对引物组合进行引物筛选,选中3’端一对最佳引物优化PCR反应体系及反应条件,建立ND207的转化事件特异性定性PCR检测方法, PCR产物片段大小为166 bp。经过测试,该方法的检出限为0.1%,相当于20个拷贝的ND207特异分子片段。国内8家转基因生物安全检测机构对本方法进行了特异性测试、检出限测试和再现性测试,循环验证报告显示:该方法符合国家标准方法的各项要求,可在检测行业推广应用。ND207转化事件特异性定性PCR检测方法的建立,为我国ND207及其衍生品系的安全监管提供技术支撑。

大豆转化体E8A7027外源T-DNA分析及特异性检测体系建立

为进一步分析转AgGlpF基因耐盐转基因大豆E8A7027转化体的分子特征信息并对其进行特异性检测,本研究分离该转化体的旁侧序列并建立其特异性PCR检测体系。基于基因组重测序技术并结合Sanger测序技术,将基因组重测序分析获得的序列信息与参考大豆基因组进行对比,确定E8A7027转化体的T-DNA区在大豆基因组上的插入位点及其5′端和3′端的旁侧序列。根据旁侧序列设计PCR检测引物,并对检测体系的特异性、灵敏度进行实验室内验证。结果显示:转基因大豆E8A7027的整合位点为Chr09染色体的33886331位点,整合方式为单拷贝插入,受体基因组DNA在插入位点缺失了1段58 bp序列,分别获得E8A7027大豆的5′端旁侧序列910 bp和3′端旁侧序列802 bp。根据这两个旁侧序列设计引物建立的PCR检测方法,能够特异性地将E8A7027大豆转化体与其他转基因作物和非转基因大豆区分开,方法的检测灵敏度为0.05%。研究结果可为E8A7027转化体的安全评价和监管检测提供技术支撑。

甘蔗镰孢菌插入突变体库的构建、致病变异突变体筛选及突变基因定位

由镰孢菌复合种引起的甘蔗梢腐病(pokkah boeng disease,PBD)是甘蔗的主要病害之一。在广西,甘蔗镰孢菌(Fusarium sacchari)是PBD的优势病原菌。尽管PBD的研究已有较长的历史,但甘蔗镰孢菌的致病分子机理远未得到阐明。为研究甘蔗镰孢菌的致病分子机理,本研究采用农杆菌介导的遗传转化(Agrobacterium tumefaciens-mediated transformation,ATMT)技术构建甘蔗镰孢菌FF001菌株的T-DNA插入突变体库。采用携带潮霉素B磷酸转移酶基因pTHR1-AH双元载体的农杆菌菌株AGL-1介导转化F.sacchari分生孢子,获得3018个转化子;随机选取12个转化子进行PCR检测和Southern杂交分析,结果显示所有检测突变株均插入潮霉素B磷酸转移酶基因,多数为单拷贝插入。采用离体叶片接种评价转化子的致病力,获得致病力明显减弱21个和致病力增强9个,共30个突变体。通过TAIL-PCR扩增测序并与甘蔗镰孢菌基因组序列比对,确定了其中27个突变株的T-DNA插入位点。观察到多个插入位点伴随有基因组片段缺失,导致1个插入位点可以影响1个以上的功能基因。T-DNA插入位点多为编码区和启动子区,少数为终止子区和间隔区。受影响的基因分别编码α-甘露糖苷酶(alpha-mannosidase)、中性氨基酸渗透酶(neutral amino acid permease)、寡聚高尔基复合体成分4(oligomeric golgi complex component 4)、寡肽转运蛋白(oligopeptide transporter)、酰基辅酶A脱氢酶(acyl-CoA dehydrogenase)、乙酰乙酰-CoA合成酶(acetoacetyl-CoA synthetase)、碳酐酸酶(carbonic anhydrase)、Bud 10蛋白(Bud 10 protein)、类驱动蛋白bimC(kinesin-related protein bimC)、锌指蛋白ASD4(zinc finger protein ASD4)、转录起始因子TFIID(transcription initiation factor TFIID)、角质酶G-box结合蛋白(cutinase G-box binding protein)、吖啶黄素敏感性控制蛋白(acriflavine sensitivity control protein ACR-2)、核类VCP蛋白(nuclear VCP-like protein)、热激蛋白HSP30(heat shock protein 30)、硫氧还蛋白(thioredoxin)、2C型蛋白磷酸酶(type 2C protein phosphatase)、核糖核酸外切酶(exoribonuclease)、硒蛋白(selenoprotein)、DNA聚合酶η(DNA polymerase eta)、存活因子1(survival factor 1)和乙醛脱氢酶(aldehyde dehydrogenase)等22个已知功能蛋白和16个未知功能蛋白。部分突变基因,如锌指蛋白ASD4、角质酶G-box结合蛋白、寡肽转运蛋白和2C型蛋白磷酸酶等,已分别在稻瘟病菌、稻曲病菌、胶孢炭疽菌和尖孢镰孢菌等植物病原真菌中证实为致病相关基因。多种类型致病相关突变体的获得,为深入研究甘蔗镰孢菌致病分子机理提供了新的材料。

基于重测序鉴定耐盐转基因大豆事件外源T-DNA整合位点及特异性检测

[目的]转基因大豆事件L8014是本研究前期利用农杆菌介导的大豆子叶节高效遗传转化技术体系,将来源于山菠菜的基因BADH导入大豆(Glycine max)‘Williams 82’而获得具有良好耐盐(1.5%NaCl)性状的转基因大豆(该材料已经完成环境释放试验)。T-DNA插入位点侧翼序列的获得与分析对于转基因事件的安全评价及应用非常重要,本文旨在明确该转基因大豆材料的分子特征及其检测方法,以进一步推进安全评价。[方法]利用Southern杂交检测转基因大豆外源基因的拷贝数;基于基因组重测序技术,采用BWA(Burrows-Wheeler-Alignment)方法将重测序数据与T-DNA序列以及大豆基因组序列进行比对分析。[结果]T-DNA在大豆基因组10号染色体上的基因组区间3925017—3926022位点,插入方式为反向插入;结合PCR扩增技术获得外源T-DNA整合位点的左、右边界侧翼序列,基于该序列建立了转BADH基因大豆事件L8014的特异性PCR检测方法。[结论]本研究获得了转基因事件的整合位点及侧翼序列,方法快速、结果可靠,为转基因大豆及其衍生产品的检测提供了技术支撑。

Reading Time and DNA Sequence Preference of TET3 CXXC Domain Revealed by Single-Molecule Profiling

Recognition of CpG dinucleotide DNA in epigenetic information flow plays a pivotal role for cellular differentiation and development.The TET3 CXXC domain binds to CpG DNA,serving a basic epigenetic information reading mechanism.During the selective recognition of a CpG motif by a CXXC domain from crowded binding sites in a gene sequence,the protein-DNA interactions are beyond CpG dinu-cleotide.However,the selective binding dynamics of CpG within a long DNA context by epigenetic enzymes have been rarely exploit-ed,which is hard for ensemble methods to probe.Here,we used single-molecule magnetic tweezers to quantitatively examine the dynamics of TET3's CXXC domain on a Hoxa9 promoter DNA.Our single-molecule binding profile revealed that CXXC-DNA interactions involve both CpG motifs and their flanking sequences.The residence time of TET3 CXXC differs by about 1000 times in five distin-guished CpG clusters in the context of a CpG island.Moreover,we performed multi-state hidden Markov modeling analysis on the zip-ping/unzipping dynamics of a CpG hairpin,discovering TET3 CXXC's preference on CpG motifs regarding the-2 to+2 flanking bases.Our results shed light on the selective binding dynamics of a CXXC on a gene sequence,facilitating studies on epigenetic information reading mechanisms.

大丽轮枝菌(Verticillim dahliae)突变体库的构建与分析

为了更加快捷地筛选大丽轮枝菌致病关键基因,利用聚乙二醇介导的原生质体转化法构建病原菌随机插入突变体库,对部分阳性转化子的生长表型指标和致病力进行分析,筛选生长发育与致病缺陷突变体并进行靶标基因定位。结果表明,本研究共获得13030多个阳性转化子,随机挑选5个转化子与V991野生型菌株进行比较分析,发现其中一个突变体在PDA和不同碳源培养基上的菌落直径、产孢量以及致病力均显著降低。侧翼序列测序结果结合Blast比对分析表明,该缺陷突变体中的潮霉素抗性基因表达盒定位于大丽轮枝菌3号染色体1528782 bp处,属于内切葡聚糖酶1基因(VDAG_04017)。综上所述,本研究通过大丽轮枝菌插入突变体库构建、突变体生长表型指标和致病力鉴定及靶标基因定位等一系列分子生物学手段,可初步鉴定病原菌致病相关基因,为从全基因组层面深入研究大丽轮枝菌致病机制奠定了一定基础。

转基因抗虫棉Bt基因插入区碱基组成分析

利用TAIL PCR的方法克隆不同来源的转基因抗虫棉中外源基因插入区的侧翼序列并对其进行序列和结构分析。结果表明 :同一个转基因的单株自交得到的不同株系中外源基因插入区的两侧DNA序列完全相同 ,不同的转基因抗虫棉中的外源基因插入位置各不相同。不同来源的转基因品种外源基因插入的上游侧翼片段含有一段残留质粒片段 ,外源基因插入的下游侧翼片段为富含AT碱基结构 ,其中泗棉 3号转基因抗虫品系中下游侧翼片段的AT碱基高达 92 %。Southern杂交结果显示这些侧翼序列为高AT含量的多拷贝序列 。

山羊FSHR基因5'端侧翼序列的克隆与分析

为找寻山羊FSH受体基因的SNP突变位点,进行FSH受体基因的多态性分析和探索该受体基因在山羊多胎中的可能作用,利用比较基因组学的方法,根据绵羊FSH受体基因序列设计引物,以湖南湘东黑山羊基因组为模板,PCR扩增了FSH受体基因5' 端侧翼调控区,通过克隆进行了序列测定及分析,并与牛、绵羊该区段序列进行了比较.结果表明,PCR扩增获得的片段为844 bp,与中国西门塔尔牛该段序列相比,同源性为94.08%,在-364 bp处的左翼发生了多碱基插入,湘东黑山羊该序列有5个碱基的插入,另在-625^-619 bp处的7个碱基缺失;与澳洲绵羊的FSHR基因比较,同源性为93.60%,在-367 bp处的左翼,湘东黑山羊该段序列有2个碱基的插入,在-630,-145,-97 bp 处各有1个碱基缺失.

改进的反向PCR技术克隆转移基因的旁侧序列

对传统的反向ＰＣＲ 技术作了一些改进： 用巢式ＰＣＲ 扩增含量极少的靶序列； ＰＣＲ 反应体系中加入５ ％ 的甲酰胺以减少非特异性扩增． 结果表明， 改进的反向ＰＣＲ 体系是克隆人基因组已知片段旁侧序列的高度灵敏、高度特异的方法．

大豆二核苷酸SSR侧翼序列保守性分析

从NCBI下载了大豆的EST和GSS序列,以SSR两侧序列各100 nt的片段作为分析材料,统计SSR数目、计算侧翼序列碱基组成,对SSR侧翼序列进行了序列比对和数据库blast搜索。结果表明:大豆SSR分布频率为1/6.67kb,侧翼序列GC含量为37.83%,二核苷重复是SSR的主要类型(65.7%),同一种重复基元的SSR存在多类型侧翼序列,同一类型的侧翼序列高度保守,不同类型的侧翼序列之间相似性较小,尤其是非SSR区域可以存在与SSR侧翼序列相同的序列。由上述结果推断SSR引物可能扩增出不含SSR的产物,易位可以产生形成新类型SSR。这一分析结果有助于提高SSR引物设计效率和进一步阐明SSR的形成机理。

棉花黄萎病菌T-DNA插入突变体表型特征和侧翼序列分析

【目的】为鉴定棉花黄萎病菌随机插入突变体的表型特征,构建黄萎病菌遗传转化和功能基因研究的技术平台。【方法】采用农杆菌介导的T-DNA插入和高效TAIL-PCR方法。【结果】获得了2628个棉花黄萎病菌VD991的T-DNA随机插入的突变体和15个突变体插入位点的侧翼序列。部分突变体的表型特征和插入位点侧翼序列分析表明,①突变体的菌落形态分为菌丝型、菌核型和中间型3类,菌核型约占7.3%;②在摇瓶培养条件下,突变体产孢高峰在接种后第5—6天,菌核型突变体的产孢能力普遍高于菌丝型;③棉花黄萎病菌VD991可以产生胞外蛋白酶、淀粉酶、纤维素酶和果胶酶。筛选获得蛋白酶分泌缺失的突变体3个,淀粉酶分泌缺失的突变体1个,果胶酶分泌降低的突变体6个;④菌核型突变体的致病力普遍高于野生型。获得了6个致病力减弱的突变体;⑤突变体侧翼序列与同种的大丽轮枝菌菌株VDLs.17基因组的序列一致性在95%—100%之间,与不同种的黑白轮枝菌VaMs.102序列的一致性为87%—94%。【结论】农杆菌介导的T-DNA随机插入可用于棉花黄萎病菌突变体的构建,突变体微菌核的产生与其产孢能力、致病性存在相关关系。美国大丽轮枝菌菌株VDLs.17基因组可用作黄萎病菌VD991功能基因研究的参考序列。

鳜鱼(Siniperca chuatsi)β-肌动蛋白基因cDNA全序列与5′侧翼区的克隆与分析

采用RT-PCR及RACE法,分离、克隆鳜鱼β-肌动蛋白基因cDNA全序列,再用基因组步行法(Genome Walker)克隆鳜鱼β-肌动蛋白基因5′调控区。序列分析结果表明,鳜鱼β-肌动蛋白基因全长1897bp,其中5′-UTR长94bp,3′-UTR长675bp,编码区长1128bp,编码375个氨基酸。将所得序列与其它动物类群的β-肌动蛋白基因序列进行比较分析显示,鱼类、两栖类、鸟类、哺乳类等不同类群脊椎动物β-肌动蛋白氨基酸序列同源性均在96%以上,说明该基因在生物进化过程中高度保守。通过鳜鱼与其它脊椎动物β-肌动蛋白基因的核苷酸序列构建的进化树显示,脊椎动物β-肌动蛋白聚类成3个分支,鱼类β-肌动蛋白基因形成一个独立的分化群,说明鱼类β-肌动蛋白基因起源于一个共同祖先。克隆得到的鳜鱼β-肌动蛋白基因5'侧翼序列长1399bp,对其进行序列分析,在其起始密码字ATG上游200bp范围内发现含有CAATbox、CC(A/T)6GG(CArGbox)、TATA box对转录调控起重要作用的顺式元件,同时在侧翼区也发现含有GC box、MYOD、YY1、SP1、GATA等多个潜在调控元件。鳜鱼β-肌动蛋白基因5′侧翼序列的克隆成功,为今后转基因鳜鱼的研究工作奠定了基础。