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人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析 总被引:6,自引:0,他引:6
应用抑制差减杂交技术,分别以源于4年和1年生人参根组织cDNA群体作为检测子(tester)与驱赶子(driver),成功构建了与人参植物皂苷生物合成相关的差减cDNA文库,并时从中筛选的阳性cDNA克隆进行DNA测序及其序列分析、PCR及Northern印迹杂交鉴定.结果显示,获得的13个克隆为新基因序列.其中6个差减克隆系人参植物根生长发育阶段差异表达基因.目前,6个差异表达新基因的结构与功能仍在进一步研究中. 相似文献
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抑制差减杂交(SSH)技术及其在植物基因分离上的应用 总被引:12,自引:0,他引:12
SSH是一种基于抑制PCR和差减杂交技术建立的 ,在转录水平上研究基因表达的技术 ,具有稳定、高效、可靠的特点 ,可对生物的生长、发育、衰老、死亡等生命过程及生物或非生物逆境胁迫对生物所造成的影响等进行全面、系统的分析。简单介绍了SSH的基本原理、技术要点 ,并对技术本身的改进和提高及在转录水平上与其它技术方法的比较及其在植物基因分离上的应用进行了概述。 相似文献
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基因表达研究方法的进展 总被引:1,自引:0,他引:1
信使RNA的发现,遗传密码的破解,人们对蛋白质合成的基因调节了更深入的理解,在此基础上提出了基因表达的现代概念。随之出现了全球性的基因表达研究的热点。在检测和定量分析基因表达水平的过程中,出现了很多种方法和技术。本文就Northern杂交法、S1核酶保护法、差减杂交技术、差异显示法、cNDA文库的序列测定、基因表达的系列分析、基因表达芯片等技术作一简要的综述。 相似文献
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差异表达基因分离技术的研究进展 总被引:1,自引:0,他引:1
分离并克隆差异表达基因是生命科学的研究热点.近年来,以差示筛选、扣除杂交等基本方法为基础,先后出现了抑制差减杂交,微阵列技术等多种分析差异表达基因的技术, 使差异表达基因分离方法不断完善.对这些方法的优缺点、发展趋势及应用前景进行了简要综述. 相似文献
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基因表达分析方法及其研究进展 总被引:1,自引:0,他引:1
近几年来,随着功能基因组学研究的兴起,基因表达研究的分析方法也在不断发展,主要有:差减杂交、差异显示、表达序列标签、基因表达的序列分析、微阵列杂交等。简要评述这五种方法的原理、优缺点等。 相似文献
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植物类Rho相关G蛋白(Rho-related GTPases from plants,ROP)属于小G蛋白超家族,是高等植物体内广泛存在的一类重要信号分子,在植物生长发育过程中起着关键的调控作用.本实验室从香蕉果实采后抑制差减杂交文库中获得一个香蕉ROP基因,命名为MaROP1.半定量RT-PCR表明该基因在香蕉的根、球茎、叶、花和果实中的表达存在差异,其中在球茎中的表达量最高且与其它器官的表达差异显著.为进一步研究该基因的功能,构建了以绿色荧光蛋白(green fluorescent protein,GFP)为报告基因的融合植物表达载体pCAMBIA1302-MaROP1,并利用基因枪转化法将重组载体转入洋葱表皮细胞瞬时表达,荧光显微镜检测表明该基因表达产物定位在细胞膜上. 相似文献
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本文结合胡杨盐诱导基因和盐抑制基因的筛选,介绍了差减杂交(subtractivehybridization)技术的试验原理,设计思想和应用方法。 相似文献
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Li Xu Yijun Yuan Lin Zhang Li Wan Yusheng Zheng Peng Zhou Dongdong Li 《Tree Genetics & Genomes》2011,7(5):999-1010
Suppression subtracted hybridization (SSH) and dot blotting were used to identify differential gene expression in the mesocarp
and kernel of oil palm nuts. The different types of nut tissue show differences in fatty acid anabolism and the synthesis
of other important compounds. In total, 302 clones from forward SSH libraries and 238 clones from reverse SSH libraries were
identified following differential screening, respectively. Among these, 120 clones from the forward SSH library and 81 clones
from the reverse SSH library, showed tenfold or more differential expression levels, and were sequenced. Sequence analysis
revealed that 76 clones (28 from the forward SSH library and 48 from the reverse SSH library) represent non-redundant cDNA
inserts. The differential expression of 39 subset genes in the two different tissues was further confirmed by RT-PCR analysis.
Functionally annotated blasting against the GenBank non-redundant protein database classified all 76 candidate genes into
six categories, according to their putative functions. Interestingly, our results show that a group of significantly differentially
expressed genes are involved in processes associated with oil palm nut maturation, such as the synthesis of medium-chain saturated
fatty acids and phytic acid, nut development, and stress/defense responses. This study describes some relationships between
gene expression and metabolic pathways in mature oil palm nuts, and contributes to our understanding of oil palm nut ESTs. 相似文献
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Voiblet C Duplessis S Encelot N Martin F 《The Plant journal : for cell and molecular biology》2001,25(2):181-191
Ectomycorrhiza development alters gene expression in the fungal and plant symbionts. The identification of a large number of genes expressed exclusively or predominantly in the symbiosis will contribute greatly to the understanding of the development of the ectomycorrhizal symbiosis. We have constructed a cDNA library of 4-day-old Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza and sequenced 850 cDNAs cloned randomly or obtained through suppression subtractive hybridization (SSH). Based on the absence of a database match, 43% of the ectomycorrhiza ESTs are coding for novel genes. At the developmental stage analysed (fungal sheath formation), the majority of the identified sequences represented 'housekeeping' proteins, i.e. proteins involved in gene/protein expression, cell-wall proteins, metabolic enzymes, and components of signalling systems. We screened arrayed cDNAs to identify symbiosis-regulated genes by using differential hybridization. Comparisons of signals from free-living partners and symbiotic tissues revealed significant differences in expression levels (differential expression ratio >2.5) for 17% of the genes analysed. No ectomycorrhiza-specific gene was detected. The results successfully demonstrate the use of the cDNA array and SSH systems as general approaches for dissecting symbiosis development, and provide the first global picture of the cellular functions operating in ectomycorrhiza. 相似文献
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Glandular trichomes are special organs involved in plant defense response and synthesis of volatile secondary metabolites, analyzing trichome specific expressed sequence tags will help us further understand the specific function of plant trichomes. In this paper, suppression subtractive hybridization(SSH) based on magnetic beads technology was used to isolate differential expressed genes of the glandular trichomes in Lycopersicon esculentum. The differential expressing cDNA library was constructed using the glandular trichomes cDNA as tester and the cDNA from the stem without glandular trichomes as driver. After randomly sequencing 108 differential ESTs, Blast2go program was used to do blastx, functional annotation and metabolism analysis. The results show that most ESTs are related to substance metabolism, response to stress, biotic or abiotic stimulus, and have binding and catalytic function. These differential genes lay the foundation for further research on defense mechanism of the tomato trichomes. 相似文献
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黄瓜芽黄突变体抑制消减杂交文库的构建及初步分析 总被引:3,自引:0,他引:3
利用抑制消减杂交技术(suppression subtractive hybridization,SSH)分离了黄瓜芽黄突变体及其野生型之间差异表达的cDNA片段.以突变体和野生型分别作检测子和驱赶子,建立正向和反向两个消减杂交cDNA文库;经阳性克隆鉴定,在正向文库中获得特异表达的阳性克隆有133个,在反向文库中得到的阳性克隆有73个.测序后将所得到的159条非重复且非黄瓜的ESTs(登录号:GH270133~GH270291)进行序列同源性比对分析,发现这些ESTs分别与叶绿素合成、光合系统、信号转导、转录因子、氨基酸代谢、糖类代谢、脂类代谢等相关酶及蛋白基因高度同源. 相似文献
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Drought is one of the prime abiotic stresses responsible for limiting agricultural productivity. A number of drought responsive genes have been isolated and functionally characterized but these studies have been restricted to a few model plant systems. Very few drought responsive genes have been reported till date from non model drought tolerant plants. The present study aimed at identifying differentially expressed genes from a drought tolerant, non-model plant, Ziziphus nummularia (Burm.f.) Wight & Arn. One month old seedlings of Z. nummularia were subjected to drought stress by 30% Polyethylene glycol (PEG 6000) treatment for 6, 12, 24, 48 and 72 h. A significant reduction in RWC and increase in proline was observed at 24 h and 48 h of treatment. Suppression subtractive hybridization (SSH) library was constructed with drought stressed seedlings after 24 h and 48 h of PEG 6000 treatment. A total of 142 and 530 unigenes from 24 h and 48 h library were identified respectively. Gene ontology studies revealed that about 9.78% and 15.07% unigenes from 24 h and 48 h SSH libraries were expressed in “response to stress”. Fifteen putative drought responsive genes identified in SSH library were validated for drought responsive differential expression by RT-qPCR. Significant changes in fold expressions were observed with time in the treated samples compared to the control. A heat map revealing the expression profile of genes was constructed by hierarchical clustering. Various genes identified in SSH libraries can serve as a resource for marker discovery and selection of candidate genes to improve drought tolerance in other susceptible crops. 相似文献
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抑制性消减杂交技术(suppression subtractive hybridization,SSH)是目前被广泛用于寻找差异表达基因方面的一种技术,因其具有假阳性率低、灵敏度高、重复性好、特异性强等特点而被大多数研究者所采用。该技术的优势在于可以在转录水平对不同环境、不同生理条件下的组织或细胞进行基因差异表达方面的研究。随着近年来分子生物学的不断发展,对差异表达基因的筛选及克隆已逐渐成为研究的热点。本文主要对抑制性消减杂交技术在鹅、鸭和鸡这三种常见禽类的生产性能、抗病机理以及品种差异等方面研究中的应用进行综述,从而为采用抑制性消减杂交技术研究生命活动的分子作用机制提供更多的参考。 相似文献
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番茄茎腺毛差异表达序列分离与分析 总被引:1,自引:0,他引:1
腺毛是参与植物防御性反应与次生代谢挥发物合成的特异化器官,了解腺毛专一性表达序列标签将有助于我们进一步认识植物腺毛的特异性功能.本文应用磁珠介导的抑制消减杂交方法(SSH),以番茄茎腺毛为检测子(tester),去除腺毛的番茄茎为驱动子(driver),构建番茄茎腺毛差异表达cDNA文库,分离腺毛差异表达基因.随机挑选了108个差异ESTs进行测序,测序结果使用Blast2go程序进行blastx比对、功能注释和KEGG代谢路径分析.结果表明,绝大部分ESTs功能与胁迫响应、物质代谢、生物及非生物刺激反应相关,具有结合、催化功能.这些分离的差异ESTs为进一步研究番茄腺毛的植物防御性机制奠定了基础. 相似文献
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植物铝胁迫响应基因的研究进展 总被引:1,自引:0,他引:1
铝毒是酸性土壤中植物生长和作物生产的主要限制因子.近年来的很多研究应用差异显示PCR、抑制差减cDNA文库和DNA微正列等技术,在一些铝耐受型和敏感型植物中鉴定了很多铝胁迫响应基因.本研究通过参阅国内外有关报道和结合本实验室的研究成果,从铝诱导的通道蛋白、代谢相关、胁迫和细胞死亡以及信号转导相关基因4个方面的研究进展进行了综述. 相似文献