37 resultados para Molecular Marker
em Chinese Academy of Sciences Institutional Repositories Grid Portal
Resumo:
Zhikong scallop Chlamys farreri(Jones et Preston) is an economically important species in China. Understanding its immune system would be of great help in controlling diseases. In the present study, an important immunity-related gene, the Lipopolysaccharide and Beta-1,3-glucan Binding Protein (LGBP) gene, was located on C. farreri chromosomes by mapping several lgbp-containing BAC clones through fluorescence in situ hybridization (FISH). Through the localization of various BAC clones, it was shown that only one locus of this gene existed in the genome of C. farreri, and that this was located on the long arm of a pair of homologous chromosomes. Molecular markers, consisting of eight single nucleotide polymorphism (SNPs) markers and one insertion-deletion (indel), were developed from the LGBP gene. Indel marker testing in an F1 family revealed slightly distorted segregation (p = 0.0472). These markers can be used to map the LGBP gene to the linkage map and assign the linkage group to the corresponding chromosome. Segregation distortion of the indel marker indicated genes with deleterious alleles might exist in the surrounding region of the LGBP gene.
Resumo:
"Da-Huang" (Radix et Rhizoma Rhei, medicinal rhubarb), a famous and important Traditional Chinese Medicine, has often been confused with the adulterant species in the same genus, Rheum. Through sequencing the trnL (UAA)/trnF (GAA) regions of chloroplast DNA of thirteen species of Rheum (three medicinal rhubarb species and ten adulterant ones), a molecular marker of the medicinal species was found. A pair of PCR primers based on the sequences, was thus designed, which amplified a highly specific DNA fragment in medicinal rhubarb exclusively, and absent in the adulterants at all under an optimized PCR condition.
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An out-of-Africa dispersal route has been proposed for many organisms, including modern man. However, counter examples of in-to-Africa dispersal routes are less common. In the present article, the phylogenetic relationships within the Labeoninae, a subfamily of cyprinid fishes distributed in Asia and Africa, were analyzed to investigate the biogeographic processes governing the modern distribution of these Asian and African cyprinids. The mitochondrial DNA cytochrome b gene was used as a molecular marker. The phylogenetic analysis indicated that the subfamily Labeoninae is a monophyletic group, with some Asian labeonins located at the basal position. Two subclades were found that contained both African and Asian species, which highlighted a need for further biogeographic analysis. Based on this analysis, it is proposed that the centre of origin of the Labeoninae was in East Asia. Molecular clock estimation suggests that the Labeoninae arose by the Early Miocene (similar to 23 MYA) during the period of the second Tibetan uplift. Subsequently, two dispersal events of labeonins from Asia into Africa occured in the Early Miocene (similar to 20 MYA) and Late Miocene (similar to 9 MYA) and serve as examples counter to out-of-Africa dispersal.
Resumo:
Microsatellites have become the preferred molecular markers for strain selection and genetic breeding in fish. In this study a total of 105 microsatellites were isolated and identified in gibel carp (Carassius auratus gibelio) by microsatellite sequence searches in GenBank and other databases and by screening and sequencing of positive clones from the genomic library enriched for AG and GATA repeats. Moreover, nineteen microsatellites were randomly selected to design locus-specific primer pairs, and these were successfully used to identify and discriminate different cultured strains of gibel carp including strains A, D, L, and F. Three different types of microsatellite pattern were distinguished by the number and length of fragments amplified from the 19 primer pairs, and some microsatellite primer pairs were found to produce different microsatellite patterns among strains and strain-specific fragments. In addition, some duplicated alleles were also detected in two microsatellite patterns. Therefore, the current study provides direct molecular markers to discriminate among different cultured strains for selective breeding and aquaculture practice of gibel carp.
Resumo:
Twenty-seven Porphyra lines from 5 classes, including lines widely used in China, wild lines, and lines introduced to China from abroad in recent years, were screened by means of amplified fragment length polymorphism (AFLP) with 24 primer pairs. From the generated AFLP products, 13 bands that showed stable and repeatable AFLP patterns amplified by primer pairs M-CGA/E-AA and M-CGA/E-TA were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with digitals 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band. On the basis of these results, computerized AFLP DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique AFLP,fingerprinting pattern and can be easily distinguished from others. Software called PGI-AFLP (Porphyra germplasm identification-AFLP) was designed for identification of the 27 Porphyra lines. In addition, 21 specific AFLP markers from 15 Porphyra lines were identified; 6 AFLP markers from 4 Porphyra lines were sequenced, and 2 of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed AFLP DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification, and resource protection of the Porphyra lines.
Resumo:
Sequence-related amplified polymorphism (SRAP) is a novel molecular marker technique designed to amplify open reading frames (ORFs). The SRAP analytic system was set up and applied to Porphyra germplasm identification in this study for the first time. Sixteen Porphyra lines were screened by SRAP technique with 30 primer combinations. In the analysis, 14 primer combinations produced stable and reproducible amplification patterns in three repetitive experiments. Among the total 533 amplified fragments, 522 (98%) were polymorphic, with an average of 38 fragments for each primer combination, ranging in size from 50 to 500 bp. The 533 fragments were visually scored one by one and then used to develop a dendrogram with Unweighted Pair-Group Method Arithmetic Average (UPGMA), and the 16 Porphyra lines were divided into two major groups at the 0.68 similarity level. From the total 533 fragments, I I amplified by two primer combinations, ME1/EM1 and ME4/EM6, were used to develop the DNA fingerprints of the 16 Porphyra lines. The DNA fingerprints were then converted into binary codes, with I and 0 representing presence and absence of the corresponding amplified fragment, respectively. In the DNA fingerprints, each of the 16 Porphyra lines has its unique binary code and can be easily distinguished from the others. This is the first report on the development of SRAP technique and its utilization in germplasm identification of seaweeds. The results demonstrated that SRAP is a simple, stable, polymorphic and reproducible molecular marker technique for the classification and identification of Porphyra lines. (c) 2007 Elsevier B.V. All rights reserved.
Resumo:
Swertia mussotii is an important species in Tibetan folk medicine. However, it is quite expensive and frequently adulterated, so reliable methods for authentication of putative specimens and preparations of the species are needed to protect consumers and to support conservation measures. We show here that the chloroplast (cp) DNA rpl16 intron has limited utility for differentiating S. mussotii from closely related species, since the cpDNA rpl16 sequences are identical in S. mussotii and two other species of Swertia. However, the rDNA internal transcribed spacer (ITS) sequences differ significantly between S. mussotii and all of 13 tested potential adulterants. Thus, the ITS region provides a robust molecular marker for differentiating the medicinal S. mussotii from related adulterants. Therefore, a pair of allele-specific diagnostic primers based on the divergent ITS region was designed to distinguish S. mussotii from the other species. Authentication by allele-specific diagnostic PCR using these primers is convenient, effective and both simpler and less time-consuming than sequencing the ITS region.
Resumo:
葡萄属(Vitis.L.)植物隶属于葡萄科(Vitaceae),主要分布于北温带,最南可以分布到南美洲的委内瑞拉和亚洲的越南以及印度北部。本文通过对该属分类研究历史的回顾,认为该属存在的问题主要表现在如下几个方面: 1)葡萄属自1753 年由Carl Linne创立以来,虽经planchon于1887年做了修订,但属的范围仍需进一步界定;2)在Planchon之后的100多年中未见有一全面的分类学修订工作,出现在该属的800多个名称需要考证;3)对一些广布种的变异认识不足,导致了大量可疑种。针对这些问题本文进行了如下几个方面的工作: l、形态学:通过大量的野外工作和标本观察,对该属植物的主要性状做了分析,讨论了这些性状状态在葡萄属中的变异规律及演化趋势,将灌木状习性、退化的卷须以及不裂的叶片视为进化的性状。 2、细胞学:利用前人对葡萄属(Vitis.L.)染色体数目的统计及一些杂交实验分析的结果,结合形态学等方面的特征分析,认为在葡萄科,染色体基数X=10为原始的,而x=19则为衍生的。葡萄属的染色体基数xl9(2n=38),多倍体较少见;麝香葡萄属[Muscadinia (Planch.) Sma11]的染色体基数为x=10 (2n=20).与蛇葡萄属、酸蔹藤属和爬山虎属的一致。葡萄属和麝香葡萄属间的杂种是不育的。 3、孢粉学:对葡萄属32种5变种及麝香葡萄属[Muscadinia (Planch.) Sma11]1种的花粉外壁做扫描电镜观察,结果发现花粉外壁雕纹在这两属间和葡萄属内变异较小,对区分属以及属下种上类群意义不大,但对种的鉴别有重要的价值。 4、植物化学:前人对植物化学的工作表明,植物的一些次生代谢产物如类黄酮化合物在葡萄科各类群中的分布规律较好地反映了各类群间的关系。这些结果较好地支持了Planchon对葡萄属范围的界定。 5、山葡萄复合体(V.amurensis complex)包括山葡萄(V.amurensis Rupr.)、燕山葡萄(V.amuresis Rupr. var. dissecta Skvorts.=var.yanshanensisD.Z.Lu et H.P.Liang)、百花山葡萄(V.baihuashanensis M.S.Kang et D.Z.Lu)、复叶葡萄(V.piasezkii Maxim.)、少毛复叶葡萄[V. piasezkii Maxux1.var. pagnuccii (Planch.) Rehd.]共3个种和2个变种,广泛分布于中国北方,形态变异较大。本文对该复合体做了形态分析,并用RAPD (Random Amplified Polymorphic DNAs)分子标记方法分析了这几个类群的关系。综合这些结果,归并了燕山葡萄和百花山葡萄。 在上述工作的基础上,我们得出了如下的结论: l、葡萄属在葡萄科中是一个进化的类群。整理后的葡萄属包括8系62种、l亚种和15变种,其中有2个新系、1个新组合系、2个新变种、1个新组合种和l3个新异名。 2、本文赞同SmaU在1903年作出的分类学处理,把麝香葡萄作为一个独立的属,比葡萄属原始但与葡萄属有着最近的亲缘关系。 3、依据形态特征和APD分析结果把山葡萄复合体的3个种2变种归并为2种l变种,即山葡萄、复叶葡萄和少毛复叶葡萄,认为分子标记技术在分析属内近缘种闻关系上很有价值。 4、葡萄属具有东亚和北美2个现代分布中心,该属可能起源于北美的东部,在晚白垩纪经白令陆桥散布至欧亚大陆。
Resumo:
分子系统发育分析的主要任务包括:(1)帮助建立生命之树(tree of life);(2)追踪基因和基因家族(gene family)的起源和进化, 以获知基因在进化过程中的功能分化和伴随发生的重要分子事件(key molecular events)和形态性状的关键创新(key innovation)。这两个方面在本研究中都有所涉及。对于前者,选用植物线粒体matR基因重建被子植物蔷薇类群的系统发育关系;对于后者,则以SET基因超家族为例,探讨其在真核生物中的进化分类以及与功能多样性的关系。 I 蔷薇类的分子系统学 蔷薇类(rosids)是基于分子数据建立的被子植物的主要分支之一,包含13个目,大约三分之一的被子植物物种。两个主要蔷薇类内部分支是豆类fabids(包含7个目)和锦葵类malvids(包含3个目)。然而,这两个分支内部,以及这两个分支与蔷薇类基部类群,包括牻牛儿苗目(Geraniales)、桃金娘目(Myrtales)和流苏子目(Crossosomatales)之间的关系大多是不清楚的。本研究中,我们选取174个物种来代表72个蔷薇类(rosids)的科,利用两个数据集,即线粒体matR单基因数据集和包括线粒体matR基因、两个质体基因(rbcL、 atpB)和一个核基因(18S rDNA) 的4基因数据集,重建蔷薇类在科以上分类阶元水平的系统发育关系。同时,还对线粒体matR基因的进化特征和用于大尺度系统发育分析的适合度和潜力进行了评价。 线粒体matR单基因数据支持malvids和大多数蔷薇类目的单系性质,然而,豆类(fabids)成员没有形成一个分支,其COM亚支,包括卫矛目(Celastrales)、酢浆草目(Oxalidales)、金虎尾目(Malpighiales)和蒜树科(Huaceae),分辨为锦葵类(malvids)的姐妹群。这个关系在最近根据花结构特征曾被提出过,但从未在之前的分子系统发育分析中得到分辨。4基因数据集支持首先是牻牛儿苗目(Geraniales),接着是桃金娘目(Myrtales)作为蔷薇类(rosids)的最基部的分支;流苏子目(Crossosomatales)是锦葵类(malvids)姐妹群,以及蔷薇类(rosids)的核心部分包括豆类(fabids),锦葵类(malvids)和流苏子目(Crossosomatales)。线粒体matR基因的进化特征分析显示,与两个叶绿体基因(rbcL 和atpB)比较,同义替代速率约是它们的1/4,而非同义替代速率接近于自身的同义替代速率,表明matR 基因具有松弛的选择压力。线粒体matR基因相对慢速的进化使非同源相似(homoplasious)突变减少,提高了系统发育信息的质量,同时,松弛的选择压力使非同义替代数量增加,弥补了慢速进化导致的系统发育信息数量不足的缺陷,这两个方面的结合使线粒体matR基因非常适用于被子植物在科以上水平的系统发育研究。 II SET基因超家族的系统发育基因组学分析 SET基因超家族基因编码含有SET结构域的蛋白,在真核生物中,SET-domain蛋白一般是多结构域(multi-domain)的。SET-domain蛋白具有对组蛋白H3和H4的N末端尾部进行赖氨酸残基甲基化修饰的酶活性;从异染色质形成到基因转录,甲基化的组蛋白广泛影响染色质水平的基因调控。依据SET结构域一级序列的相似性和结构域组织(domain architecture)特征,目前,SET-domain基因超家族被划分为4-7个家族。由于这些划分或者使用动物或者使用植物SET基因,只有少数其它类群的物种加入分析,因此这样的划分可能是不完整的。本研究采用系统发育基 因组学方法(phylogenomic approach),在真核生物范围内广泛取样,期望获得相对完整的SET-domain基因家族的 进化分类方案,在此基础上加深理解SET-domain基因的进化机制和功能多样性。 在提取了17个物种,代表5个真核超群的SET蛋白序列基础上,系统发育分析结合“结构域组织特征”鉴别了9个SET基因家族,其中一个是新的SET基因家族。以前的SET8和Class VI家族,及SMYD和SUV4-20家族分别合并为一个家族。大部分家族在进化过程中发生了2次以上的基因重复事件,通过获得不同的结构域产生具有不同功能的新基因。一个SET基因家族在进化过程中推测发生了从脊椎动物祖先向盘基网柄菌(Dictyostelium discoideum)的水平基因转移。
Resumo:
本文应用RAPD分标记技术对我国重要的油料作物“杂油59”杂种种子的Fl代杂种的纯度进了技术鉴定,并完善了这一技术,摸索出这一适合于目前生产应用的实用方法,填补了这一技术在油菜作物应用上的空白。用RFLP技术对我国重要的雄性不育材料“陕2A”细胞质进行了分子水平的鉴定,为证明“陕2A”是一类新型的雄性不育材料提供了重要的实验证据。 对甘蓝型油菜采用DNA快速提取法、酚仿法和CTAB法应用于不同的分子标记分析,实验结果显示: CTAB法适用于样品量大,纯度要术高的RFLP技术,酚仿法适用于引物筛选、DNA模板用量大的PCR反应,而快速提取法特别适合于生产上对种子纯度检测,是生产上推广前景很好的实用技术。 对甘蓝型油菜RAPD技术应用当中PCR体系的建立进行了探讨。实验结果显示:热启动对PCR结果的影响至关重要。而Mg++浓度、dNTP浓度、模板浓度、Tag酶用量对反应结果有不同程度的影响。经过反复实验:当PCR各组分按Mg++,2mM;dNTP,200uM;模板浓度,50ng - lOOng时,PCR的结果最好。PCR反应条件经反复实验后确定为:第一个循环:(热启动)94℃,Imin20sec.OoC 2min循环一次;第二个循环:(解链)94℃ 50sec,(退火)40℃ Imin30sec;(延伸)72℃lmin;循环40次。第三个循环:72℃lOmin,循环1次。反应总体积为20ul时最为适用。 用40个lOmer的RAPD随机引物对“杂油59”的2个亲本“垦C8”和“陕3A” 进行RAPD分析,共出现290条带,分布于3530-220bp之间。引物opA-06、opK-03、opK-13、opj-12出现阳性扩增。经重复实验后确定: opK-03的PCR结果重复性最好,该引物序列为:CCAGCTTAGG。用它对两个亲本进行RAPD分析,PCR结果共出现9条带,其中510bp、260bp为二条特征带。在Fl代中这两条特征带重现性很好。用50个商品用种萌发的F1单株进行验证,检测结果为3个个体没有出现510bp的特征带,4个个体没有出现260bp的特征带,有5个个体出现了其它带,纯度为78%,与生产用种的纯度相符。 通过对“杂优59”不同生育时期及不同取样部位作酯酶同工酶电泳方法与RAPD方法相比较,结果显示:RAPD方法可以弥补同工酶方法的缺限。由于它是基于基因水平的分析技术,可以不受环境条件、发育时期、取材部位等客观条件的限制,并具有取样量小、易操作、费用低、灵敏度高、可以检测出亲缘关系相当近的种闾或种内的材料,具有独到的优点。是值得今后在生产上推广的新技术。 用6个雄性不育材料线粒体的特异探针:ALXR 18(线粒体ATPaseα亚基);COB 640(脱辅基细胞色素-b);COX -I(细胞色素氧化酶亚基-I);COX -Ⅱ(细胞色素氧化酶亚基-II;PDC - 12(胡萝卜线粒体随机片断);C2(玉米线粒随机片断),对“陕2A”,Hybrides Polima,Ogura NSL 94/96, Ogura MLCH036, Ogura NSL, Polima, Fu27,Fu38, Anand等9个材料进行RFLP分析,结果显示:用限制性内切酶EcoR I消化后的DNA与探针COB 640杂交,“陕2A”材料在4.5 kb处缺失,与ALXR 18探针杂交,在4.4 kb、4.2 kb处也明显缺失,证明“陕2A”显然不同与其它不育材料。用ALXR l8为探针,与用内切酶Nc01的酶切片断作Sourthern杂交,在RFLP谱带上6.1 kb、2.4 kb、2.5 kb处明显缺带,进一步为“陕2A”是一种新型的甘蓝型油莱雄性不育系提供了证据。
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野大豆群体3和群体4属盐渍群体,其个体有的是抗盐的,有的是敏感的,有的是中等抗盐的,本文通过随机扩增多态性DNA (RAPD)和DNA扩增指纹(DAF)分析野大豆群体抗盐性与分子标记之间的关系,从而更好地研究野大豆群体的盐适应机理。通过12个RAPD引物和3个DAF引物扩增发现:引物OPF05,OPF19和OPH02的扩增产物中有与抗盐性可能相关的特异标记,分别是OPF05_(213);OPF19_(4361);OPF19_(1727);OPF19_(1400);OPF19_(700);OPH02_(1350)。这些特异标记在所研究的抗盐植株中都存在;在敏感型植株中都不存在;在中等抗盐植株中有的存在,有的不存在。以上表明野大豆群体的抗盐性与RAPD分子标记有一定的相关性。为进一步研究抗盐性的特异标记,本文对栽培大豆抗盐品种Morgan和文丰七号的特异DAF标记片断8-27_(240) (Zhong et al., 1997)进行了克隆测序,测序结果通过BLASTn程序与基因库中的基因序列进行同源比较,发现上的DNA序列中的19组(每组大约二十到三十个碱基)序列与基因库中的其它基因相应序列有很高的同源性,几乎全部100%同源。尤其目的序列第15个碱基到第33个碱基(共19个碱基)之间的序列与基因库中的25个基因的相应序列的同源性全部是100%,并且与之相比的基因大多来自动物和人。因而推测其有可能是保守区,而不是编码区。进一步用DNASIS软件分析其碱基组成(A/T含量是64.9%,G/C含量是35.1%)并进行翻译,结果同样表明此序列可能是一调控序列并非编码区。至于这段序列是否与抗盐紧密相关,这有待于以后把此序列转到敏感型植株中然后检测其抗性来验证。 本文还通过RAPD分析野大豆群体3和群体4的多态性,发现群体3的多态性明显高于群体4。野大豆群体3的抗盐性大于群体4早已通过生理指标的鉴定,至于多态性与抗盐性之间是否有必然联系,还需进一步研究讨论。利用RAPD数据,通过MEGA软件中的NJ计算遗传距离的方法对群体3和4进行聚类分析,研究野大豆群体间及群体内个体间的亲缘及进化关系,探讨野大豆群体盐适应机理的分子起源。
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体细胞无性系变异是植物组织培养过程中发生的一种较为普遍的现象,这种现象在遗传学理论和育种实践上都很值得研究。应用这种变异进行筛选人们已经获得了一大批生产上有广泛意义的细胞突变体。但对于无性系变异发生的机理,人们虽然也进行了详细研究,提出了不少假说,但一直未能给出一个较为全面的解释。有些解释仍然建立在推测的基础上,有待进一步的研究。在这些解释中,利用转座子活化进行解释是最使人感兴趣的一种,也是较有说服力的一种。我们的实验分为两个部分,一是对无性系变异发生与转座子的关系进行了初步的探索,一是利用体细胞无性系变异筛选烟草黑胫病细胞突变体的应用进行了研究。 对转座因子的转活性进行分析,通常采用标记基因的表型检测。本实验用农杆菌双元载体pSLJ721(Ac∷GUS)转化烟草(品种:红花大金元), 然后对转基因烟草的愈伤组织进行GUS酶活性的组织化学分析。通过GUS活性变化来分析转座活性。结果表明组织培养可以增强转座子的活性。间接证明转座子活化是体细胞无性系变异的原因之. 本论文实验方面的另一部分是体细胞无性系变异在烟草抗黑茎病研究上的应用。烟草黑茎病是由烟草致病疫霉引起的一种烟草主要病害。首先从云南地区黑胫病病区分离、纯化黑胫病(Phytophythora parasitica via. Nicotina)病原菌,同时利用红花大金元为实验对象,以组织培养中自然发生的元性系变异为基础,以50%以及80%的黑胫病病菌粗毒素为选择压力,筛选出抗黑胫病毒素的烟草株系,用离体叶片法和茎部接种法进一步鉴定其对黑胫病病菌的抗性。最后进行田间检测以期获得综合性状优良的抗黑胫病细胞突变体。另外我们还利用随机引物扩增多态DNA(RAPD)分析技术,对选择到的九个抗病株系和六株对照的DNA进行分析。在使用的70种随机引物中,共有57个引物产生可检测扩增产物,产生306条搁增带,其中有多态性的条带数为51条,突变体的平均RAPD条带变异率为1.85%,其中有两个RAPD条带(CYA-17-100和Sangon03-350)为突变体所特有,可初步判断为抗黑胫病的RAPD标记。对突变体和对照植株叶片水溶性蛋白SDS电泳观察到一些条带的变异,但没有找到突变体特异的共同条带。
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拟南芥ast (anthocyanin spotted testa) 突变体是由碳离子束诱导产生的与花青苷生物合成有关的突变体,受单隐性核基因控制。由于花青苷的异常积累,突变体未成熟种子的种皮呈现紫红色的斑点;野生型植株幼嫩的种皮没有花青苷的异常积累,呈淡绿色。初步作图分析表明,AST基因定位于拟南芥第I号染色体上,并且位于SSLP分子标记nga280和CAPS分子标记PAB5之间。 AST基因与SSLP分子标记nga280紧密连锁,遗传距离为3.2cM;与CAPS分子标记PAB5相距较远,遗传距离为21.1cM。 采用DDRT-PCR的策略,分析野生型与突变型植株未成熟角果中基因表达的差异。通过调整DDRT-PCR中总RNA、锚定引物、随机引物、cDNA和dNTP等关键试剂的用量,优化了适用于银染检测的DDRT-PCR方法。PCR扩增产物经6%变性聚丙烯酰胺凝胶垂直电泳分离后,银染能检测到多而清晰的条带。泳道中的条带数最少为40个,最多达80个,平均为60个,条带大小分布在100bp-900bp范围,银染的灵敏度为5pg/mm2。此方法操作简便快速,灵敏度高,重复性好。采用这个改良的的方法,分析了拟南芥野生型和ast突变型植株未成熟角果中16,000个cDNA扩增产物条带,从中筛选出28个差异条带。二次PCR扩增后,进一步筛选出10个差异表达的cDNA条带,其中6个是野生型特异表达的,4个是突变型特异表达的。对这10个差异片段进行测序。BLASTN分析表明,这10个差异表达的cDNA片段与数据库中花青苷生物合成途径中的结构基因和调节基因序列没有同源性,表明用DDRT-PCR的方法克隆特定的AST基因有一定的局限性。 利用图位克隆(map-based cloning)的策略,对拟南芥AST 基因进行克隆。根据拟南芥数据库中的SNPs (simple nucleotide polymophisms) 序列和插入/缺失多态性(insertion/deletion polymorphisms)序列,设计了一系列分子标记。利用这些分子标记,对600个F2代有突变表型的植株进行重组子筛选,完成了对拟南芥AST基因的精细作图,成功地将AST 基因定位到BAC克隆T13M11上。初步确定该BAC克隆中的基因T13M11.8 可能是AST基因。该基因的DNA序列长1432bp,含有6个外显子和5个内含子,编码的蛋白与花青苷生物合成途径中的二氢黄酮醇4-还原酶有较高的同源性。功能互补实验正在进行当中。