cDNA-AFLP

摘要： 桂花是我国重要的传统名花香花植物,花色与花香是桂花两个重要的观赏品质,受开花进程的影响.为了解其开花进程中色与香的合成过程及相关分子基础,该研究利用不同花期的桂花花瓣和幼叶建立了适用于多个样本的cDNA-AFLP体系,并分析开花进程中基因的表达差异.结果表明:共获得了283个在花瓣中特异且差异表达的转录衍生片段(transcript-derived fragments,TDFs).其中,120个TDFs在数据库中无同源序列;12个有同源序列,但功能未知;150个已知生物学功能的序列,其主要功能包括次级代谢、初级代谢和发育过程等.对其中已知功能的6个TDFs进行qRT-PCR验证,4个转录水平较高的TDFs表达模式与AFLP分析结果基本一致.这对了解开花进程中桂花色与香的合成过程相关基因的表达奠定了基础,也为进一步研究桂花色香形成的分子机制提供了参考.

摘要： [目的]筛选金边也门铁花叶性状相关差异表达基因,为深入探索也门铁叶色形成的分子调控机制打下基础.[方法]基于cDNA扩增片段长度多态性分析技术(cDNA-AFLP),利用64对EcoR I和Mse I选择性扩增引物组合分析金边也门铁叶片绿色与黄色部分组织cDNA,经筛选、回收、克隆、测序和BLAST比对分析获得差异表达基因.[结果]利用64对引物组合共扩增出1726条可分辨的条带,并获得58条差异表达的转录衍生片段(TDFs),其中12条TDFs为特异性表达.12条特异性TDFs中,5条TDFs具有同源序列,另外7条TDFs无同源序列,为未知功能基因.在叶片黄化部位特异表达同源性为96％的TDFs序列M8E3B-2和M8E3B-3与大丽花花叶病毒Holland包涵体蛋白基因、紫茎泽兰明脉病毒基因、洋丁香黄化卷叶病毒基因等花椰菜花叶病毒科病毒基因具有较高的同源性.[结论]M8E3B-2和M8E3B-3两条TDFs序列或来源于一种花椰菜花叶病毒科病毒基因,且有可能参与了金边也门铁花叶叶色性状的形成过程.

摘要： 【目的】筛选金边也门铁花叶性状相关差异表达基因,为深入探索也门铁叶色形成的分子调控机制打下基础。【方法】基于cDNA扩增片段长度多态性分析技术(cDNA-AFLP),利用64对EcoRI和MseI选择性扩增引物组合分析金边也门铁叶片绿色与黄色部分组织cDNA,经筛选、回收、克隆、测序和BLAST比对分析获得差异表达基因。【结果】利用64对引物组合共扩增出1726条可分辨的条带,并获得58条差异表达的转录衍生片段(TDFs),其中12条TDFs为特异性表达。12条特异性TDFs中,5条TDFs具有同源序列,另外7条TDFs无同源序列,为未知功能基因。在叶片黄化部位特异表达同源性为96%的TDFs序列M8E3B-2和M8E3B-3与大丽花花叶病毒Holland包涵体蛋白基因、紫茎泽兰明脉病毒基因、洋丁香黄化卷叶病毒基因等花椰菜花叶病毒科病毒基因具有较高的同源性。【结论】M8E3B-2和M8E3B-3两条TDFs序列或来源于一种花椰菜花叶病毒科病毒基因,且有可能参与了金边也门铁花叶叶色性状的形成过程。

摘要： [目的]探明高丹草杂种及其亲本叶片基因差异的多样性.[方法]以4份高粱不育系和5种类型苏丹草为亲本,按照NCⅡ设计组配成20个杂交组合,测量各杂交组合及亲本的田间形态指标和生理生化指标,采用cDNA-AFLP差异显示技术分析其基因差异多样性,计算29份材料的遗传相似系数并进行UPGMA聚类分析.[结果]20份高丹草杂种及其9份亲本材料的cDNA-AFLP分析结果显示,12对引物组合共扩增出336个位点,其中特异性位点276个,占总数的82.14％,平均每对引物组合扩增出特异性位点23个.29份材料的观测等位基因数(No)为1.870 1,有效等位基因数(Ne)为1.736 5,Nei's基因多样性指数(H)为0.389 8,Shannon信息指数(I)为0.556 9,显示了高丹草杂种及其亲本苗期叶片基因表达的复杂性.聚类分析结果显示,29份材料间的遗传相似系数为0.508～0.876,以遗传相似系数0.54为基准,可将29份材料分为4类,其中亲本材料主要在第Ⅰ类和第Ⅳ类,杂种主要在第Ⅱ类和第Ⅲ类.[结论]构建了高丹草cDNA-AFLP差异显示技术反应体系,表明高丹草及其亲本苗期叶片基因具有差异多样性.%[Objective] This study investigated the genetic diversity differences in leaves of hybrids and parents of Sorghum-sudangrass.[Method] Four sorghum sterile lines and five Sudan grass were used as parents to formulate 20 hybrid combinations based on the NC Ⅱ design.Field shape indexes and physiological and biochemical indexes of the hybrids and parents were measured.Genetic diversity differences were analyzed using cDNA-AFLP differential display technique,genetic similarity coefficients of the 29 materials were calculated,and cluster analysis was conducted.[Result] The cDNA-AFLP analysis of 29 sorghum hybrids and their parents showed that 12 pairs of primer combinations amplified 336 sites,including 276 specific sites with the ratio of 82.14％.The average specific sites of each primer combination was 23.The number of alleles (No) was 1.870 1,the effective number of alleles (Ne) was 1.736 5,the Nei's gene diversity index (H) was 0.389 8,and the Shannon (I) was 0.556 9,indicating the complexity in gene expression of the hybrids and parents.Cluster analysis showed that the genetic similarity coefficients of 29 Sorghum-sudangrass materials were 0.508-0.876.Using 0.54 as standard,the 29 materials can be divided into 4 categories.The parental materials were mainly in types Ⅰ and Ⅳ,and the hybrids were mainly in types Ⅱ and Ⅲ.[Conclusion] The reactive system of cDNA-AFLP differential display technique was established,and it showed that the leaves of Sorghum-sudangrass hybrids and their parents had genetic diversity at seedling stage.

摘要： [目的]为了探究矮化突变体AS98发生变异的分子生物学机制,[方法]本研究利用正常陆地棉品种LHF10W99(简称LHF)与AS98杂交后代分离出的正常表现型植株和极端矮化表现型植株,在现蕾期提取2种株型的茎尖、茎和根的RNA,利用cD-NA-AFLP技术分析2种植株茎尖的表达差异.[结果]筛选到差异表达的片段32条,对这32条片段进行克隆测序,利用生物信息学进行分析发现其中一条(HD61)与拟南芥的阿拉伯半乳聚糖蛋白的同源性较高.用棉花的阿拉伯半乳聚糖蛋白(GhAGP)的序列设计引物,用RT-PCR方法检验阿拉伯半乳聚糖蛋白在2类植株的茎尖、茎和根等组织上的表达量,结果表明,GhAGP基因在突变型和正常型材料的茎、根上的表达量基本相同,但是在茎尖上却表现出明显的差异,突变型茎尖上的表达量显著低于正常型茎尖的表达量,说明GhAGP可能参与棉花茎尖细胞的伸长生长过程.[结论]AS98的半乳糖代谢的改变可能与赤霉素(GA)合成中基因发生突变有关,不能合成正常具有生物活性的GA,使得阿拉伯半乳聚糖蛋白等基因在AS98茎尖的表达减少甚至缺乏.%[Objective] The present paper aims to get the molecular biology mutation mechanism of dwarf mutant AS98.[Method] The normal upland cotton LHF10W99 was crossed with AS98,and the normal plant and extreme dwarf plant from the hybrid offspring plants were isolated.The RNA of stem tip,stem and root from these two kind plants in the budding period were extracted,and the expressing difference in the stem tip of two type plants was analyzed by cDNA-AFLP.[Result] 32 fragments were amplified and sequenced,and one of fragment HD61 exhibited high homology with Arabidopsis Arabinogalactan protein using bioinformatics analysis.Then the special primers were designed according to the cotton Arabinogalactan Protein (GhAGP) sequences,the RT-PCR was done in stem tip,stem and root to verify the expressing difference between the two type plants.The GhAGP expression were the same in stem and root of normal and dwarf mutant plants,while there were obvious difference in the stem tip:the expression of mutant stem tip was significantly lower than normal stem tip,indicating that the GhAGP might participate in the process of cell elongation growth of cotton stem tip.[Conclusion] The change of AS98 galactose metabolism maybe has relationship with the gibberellin (GA) synthesis gene mutation;The AS98 cannot synthesize bioactive GA normally and reduce or lack the gene expression of GhAGP in AS98 stem tip.

摘要： Hypoxia is one of the major stresses in aquaculture animals.Bighead carp (Hypophthalmichthys nobilis) is one of the most important aquaculture fish in China,which is sensitive to hypoxia stress,and the related genetic research is rarely reported.In this study,six hypoxia-sensitive samples (S group) and six hoypoxia-tolerant samples (T group) from a hypoxia stress experiment of bighead carp were selected,and differentially expressed genes between the two groups were analyzed using cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique.A total of 10 571 transcript-derived fragments (TDFs) were obtained from 256 primer combinations of the EcoR Ⅰ and Mse Ⅰ AFLP system,and the number of TDFs produced by a single primer combination ranged from 21 to 63,with an average of 41.Four types of differential straps were screened in the hypoxia stress experiment of bighead carp,including A (up-regulated band),B (down-regulated band),C (the fragment only expressed in one sample) and D (the fragment expressed in all samples),and bands of types A and B were chosen as target bands.Among those 221 differentially expressed TDFs between two groups,137 (62％) were up-regulated,and 84 (38％) were down-regulated.70 TDFs were successfully cloned and sequenced,and the length of fragments ranged from 96 to 402 bp.The results of sequence blast against public databases revealed that 29 TDFs (TDF42～TDF70) had no hits to any known homologous sequences,9 TDFs.(TDF33～TDF41) were unknown functional sequences,and 32 TDFs (TDF1～TDF32) showed homology to genes of known functions.Among the 32 differentially expressed TDFs,TDF29 and TDF32 sequences were different but both encoded the same protein (S-adenosylmethionine synthase).These functional genes participated in such physiological pathways as transcriptional regulation (16％),stress and signal transduction (9％),energy metabolism (7％),protein synthesis (4％),immune defense (4％) and cell proliferation (6％).Four TDFs with the genes of known functions (ANXA6,TRIM25,Tnp and RBAT) were randomly selected for verification by qRT-PCR analysis.For ANXA6,TRIM25 and Tnp,the expression patterns from qRT-PCR were consistent with those from AFLP analysis,while the expression pattern of RBAT was different in two methods.The results of this study would facilitate revealing molecular mechanism of genetic regulation responsible to hypoxia stress in bighead carp,and they would also provide useful information for potential gene (marker)-assisted selection towards hypoxia-tolerant variety in aquaculture of this species.%低氧是水产养殖动物面临的主要胁迫之一.鳙(Hypophthalmichth ys nobilis)是我国最重要的淡水养殖鱼类之一,其对低氧胁迫敏感,但相关的遗传基础研究报道很少.本研究利用cDNA-扩增片段长度多态性(cDNA-amplified fragment length polymorphism,cDNA-AFLP)技术对低氧敏感和低氧耐受两组鱼进行比较分析,鉴定鳙与低氧反应相关的信号通路中的差异表达基因.从EcoR Ⅰ和Mse Ⅰ AFLP体系的256个引物组合共获得10 571条转录衍生片段(transcript-derived fragments,TDFs).其中,低氧胁迫后差异表达的TDFs数目为221个,包括上调表达137个(62％),下调表达84个(38％).对70个差异TDFs成功地进行了切胶回收、PCR产物克隆和测序,序列比对分析后发现:29个TDFs在数据库中未检索到同源序列;9个TDFs比对到未知功能序列;32个TDFs比对到已知生物学功能的序列,其功能包括转录调控、应激和信号转导、能量代谢、蛋白质合成、免疫防御以及细胞生长与增殖.随机选取4个己知功能的TDFs(ANXA6、TRIM25、Tnp和RBAT)进行qRT-PCR验证,结果显示:ANXA6、TRIM25和Tnp在低氧胁迫后的表达模式与AFLP分析结果一致,RBAT与AFLP结果不一致.本研究结果有助于揭示鳙应对低氧逆境的生理和遗传调控机制,同时对鳙抗逆品种的分子标记辅助选育也有一定的参考价值.

摘要： In order to investigate the molecular mechanism of the regulation of maize growth under partial plastic film-mulched cultivation,the differentially expressed genes were screened by 90 pairs of single primers using cDNA-AFLP technique,and bioinformatics analysis was carried out with Jundan 29 as the material under openfield-cultivation for the control and partial plastic film-mulched.The results showed that a total of 2 298 differential fragments were amplified by the 90 primer pairs,of which 1 429 were up-regulated (45％ of the total number of bands),869 were down-regulated (23.7％of the total number of bands).877 genes were expressed without difference (27.6％ of the total number of bands).Twenty different differential fragments were selected and sequenced.Used Blast database comparison and gene annotation analysis,fitteen fragments could be divided into 6 groups according to the functions,namely cytoskeleton genes (13.3％),energy metabolism gens (13.3％),cell rescue and defense genes (6.7％),transcription genes (40％),intracellular transport related genes (6.7％) and unknown function (20％).Some important genes were analyzed.For example,RPM1 protein plays a crucial role in adversity resistance,MYM zinc finger protein,BCAS2 protein and EIF3B are related to the regulation of transcription and translation.This study would provide basic data for revealing the molecular mechanism of partial plastic filmmulched affecting maize growth and development.%为了探讨半膜覆盖调控玉米生长的差异表达基因及分子机制,以‘浚单29’为材料,露地栽培作为对照,与半膜覆盖间隔种植,利用cDNA-AFLP技术,采用90对单引物筛选半膜覆盖下差异表达基因,并进行生物信息学分析.结果表明,利用90对单引物组合共扩增出2 298条差异片段,其中上调表达的有1 429条,占总条带数的45％;下调表达的有869条,占总条带数的23.7％;无差异表达的有877条,占总条带数的27.6％.选择重复扩增稳定的20条差异片段进行回收测序,经过Blast数据库对比和基因注释分析,再将所得的15条差异片段按功能分为细胞骨架相关基因(13.3％)、能量代谢相关基因(13.3％)、细胞挽救和防御相关基因(6.7％)、转录过程相关基因(40％)、胞内转运相关基因(6.7％)和未知功能基因(20％)6大类.分析其中一些重要的基因,如RPM1在逆境抗性中起作用,NYM锌指蛋白、BCAS2蛋白和EIF3B翻译起始因子都与调控转录翻译相关.通过cDNA-AFLP技术筛选了多个半膜覆盖下差异表达的基因,为揭示半膜覆盖影响玉米生长发育的分子机制提供了基础资料.

摘要： 在介绍cDNA-AFLP技术原理与操作步骤的基础上,分析了应用cDNA-AFLP分子标记技术分析蓖麻种子脂肪酸含量差异基因表达过程中的主要影响因素,以期为后续利用该标记技术分析不同发育时期蓖麻种子脂肪酸含量的差异表达提供技术支持.

摘要： 本研究采用cDNA-AFLP技术建立了水稻与稻瘟病菌互作的基因表达图谱,对差异表达基因功能进行分析.利用64对引物进行筛选,共获得了960个转录衍生片段(transcript-derived fragment TDF).回收测序200条片段,获得134条可读序列,通过BLAST比对分析,得到74个不重复的差异TDFs.对74个TDFs对应的聚丙烯酰胺凝胶上表达变化情况进行统计,接种稻瘟病菌后基因诱导表达上调多余下调表达,早期上调表达基因有6个,晚期上调表达基因有11个,其他形式上调表达基因32个,各种下调表达基因25个.

摘要： @@利用cDNA-AFLP结合QRT-PCR技术，分析高温胁迫早期茄子耐热品系叶片基因表达谱的变化情况，以期为深入研究茄子耐热性分子机理奠定基础。

摘要： @@利用cDNA-AFLP结合QRT-PCR技术，分析高温胁迫早期茄子耐热品系叶片基因表达谱的变化情况，以期为深入研究茄子耐热性分子机理奠定基础。

摘要： @@利用cDNA-AFLP结合QRT-PCR技术，分析高温胁迫早期茄子耐热品系叶片基因表达谱的变化情况，以期为深入研究茄子耐热性分子机理奠定基础。

摘要： @@利用cDNA-AFLP结合QRT-PCR技术，分析高温胁迫早期茄子耐热品系叶片基因表达谱的变化情况，以期为深入研究茄子耐热性分子机理奠定基础。

摘要： 以具有性早熟特征的济宁百日鸡为材料，选择同期饲养而开产时间差别较大的个体各5只，分别取其卵巢组织，利用cDNA-AFLP差异显示方法，分析在开产和未开产的鸡卵巢组织中差异表达的基因。用54对引物扩增出403个差异显示片段，经二次扩增，胶回收及克隆测序后，最终得到27个与GeneBank数据库高度同源的差异表达序列，其中有14个片段与鸡已知的功能基因序列相似，7个是鸡的假定蛋白，2个是鸡的EST，4个在数据库中未找到显著相似的序列。这些差异表达的基因或蛋白有的与miRNA有关，如M1E4差异片段同源基因，其编码蛋白是miRNA的靶蛋白，而M5E1差异片段同源基因与miRNA的合成有关;有的是跨膜蛋白或锌指蛋白等;有的蛋白功能未知，但却在开产的卵巢特异表达。

摘要： 以具有性早熟特征的济宁百日鸡为材料，选择同期饲养而开产时间差别较大的个体各5只，分别取其卵巢组织，利用cDNA-AFLP差异显示方法，分析在开产和未开产的鸡卵巢组织中差异表达的基因。用54对引物扩增出403个差异显示片段，经二次扩增，胶回收及克隆测序后，最终得到27个与GeneBank数据库高度同源的差异表达序列，其中有14个片段与鸡已知的功能基因序列相似，7个是鸡的假定蛋白，2个是鸡的EST，4个在数据库中未找到显著相似的序列。这些差异表达的基因或蛋白有的与miRNA有关，如M1E4差异片段同源基因，其编码蛋白是miRNA的靶蛋白，而M5E1差异片段同源基因与miRNA的合成有关;有的是跨膜蛋白或锌指蛋白等;有的蛋白功能未知，但却在开产的卵巢特异表达。

摘要： 以具有性早熟特征的济宁百日鸡为材料，选择同期饲养而开产时间差别较大的个体各5只，分别取其卵巢组织，利用cDNA-AFLP差异显示方法，分析在开产和未开产的鸡卵巢组织中差异表达的基因。用54对引物扩增出403个差异显示片段，经二次扩增，胶回收及克隆测序后，最终得到27个与GeneBank数据库高度同源的差异表达序列，其中有14个片段与鸡已知的功能基因序列相似，7个是鸡的假定蛋白，2个是鸡的EST，4个在数据库中未找到显著相似的序列。这些差异表达的基因或蛋白有的与miRNA有关，如M1E4差异片段同源基因，其编码蛋白是miRNA的靶蛋白，而M5E1差异片段同源基因与miRNA的合成有关;有的是跨膜蛋白或锌指蛋白等;有的蛋白功能未知，但却在开产的卵巢特异表达。

摘要： 以具有性早熟特征的济宁百日鸡为材料，选择同期饲养而开产时间差别较大的个体各5只，分别取其卵巢组织，利用cDNA-AFLP差异显示方法，分析在开产和未开产的鸡卵巢组织中差异表达的基因。用54对引物扩增出403个差异显示片段，经二次扩增，胶回收及克隆测序后，最终得到27个与GeneBank数据库高度同源的差异表达序列，其中有14个片段与鸡已知的功能基因序列相似，7个是鸡的假定蛋白，2个是鸡的EST，4个在数据库中未找到显著相似的序列。这些差异表达的基因或蛋白有的与miRNA有关，如M1E4差异片段同源基因，其编码蛋白是miRNA的靶蛋白，而M5E1差异片段同源基因与miRNA的合成有关;有的是跨膜蛋白或锌指蛋白等;有的蛋白功能未知，但却在开产的卵巢特异表达。

摘要： 以具有性早熟特征的济宁百日鸡为材料，选择同期饲养而开产时间差别较大的个体各5只，分别取其卵巢组织，利用cDNA-AFLP差异显示方法，分析在开产和未开产的鸡卵巢组织中差异表达的基因。用54对引物扩增出403个差异显示片段，经二次扩增，胶回收及克隆测序后，最终得到27个与GeneBank数据库高度同源的差异表达序列，其中有14个片段与鸡已知的功能基因序列相似，7个是鸡的假定蛋白，2个是鸡的EST，4个在数据库中未找到显著相似的序列。这些差异表达的基因或蛋白有的与miRNA有关，如M1E4差异片段同源基因，其编码蛋白是miRNA的靶蛋白，而M5E1差异片段同源基因与miRNA的合成有关;有的是跨膜蛋白或锌指蛋白等;有的蛋白功能未知，但却在开产的卵巢特异表达。

摘要： 利用cDNA-AFLP技术分离到10个与辣椒抗疫病反应相关的TDFs(transcript-derived-fragments)。Blastn结果表明：4个TDFs与抗病信号传导途径中的一些酶类基因同源性较高，2个TDFs与抗病基因有同源性,1个TDF与马铃薯MYST型乙酰转移酶基因有较高同源性，3个TDFs无同源性。