基因芯片技术筛选转SlNAC1基因拟南芥差异表达基因

研究已表明植物特有的一些NAC(NAM,ATAF1/2,CUC2)转录因子可提高植物抗逆性,利用基因芯片技术筛选转SlNAC1基因拟南芥与野生型拟南芥间差异表达基因,能够为研究转基因拟南芥非生物胁迫抗性相关基因提供依据。结果显示,在转SlNAC1基因拟南芥43 604个基因中有3 046个差异表达2倍以上的基因。对差异表达5倍以上基因经过GO富集度统计学分析表明,细胞组分相关基因占33.05%;分子功能相关基因占33.95%;生物学过程相关基因占33.00%。对差异表达2倍以上基因进行KEGG信号通路分析,结果表明有2 431个基因涉及到88个不同的信号通路。通过筛选获得转基因拟南芥非生物胁迫抗性相关候选基因,为后续研究NAC转录因子的下游基因及其调控网络的构建提供方向和理论支撑。     

番茄NAC转录因子家族的鉴定及生物信息学分析

NAC转录因子家族是植物特有的一类转录因子,在植物的生长发育、器官建成及逆境胁迫和激素信号应答中均发挥重要作用。本研究在基因组范围内,利用生物信息学方法对番茄的NAC转录因子家族成员、分布及结构和功能等进行分析。预测结果显示番茄NAC家族包含102个蛋白质,分为12亚族,其中茄属特有的TNAC亚族中成员最多,具有25个,其他NAC转录因子与拟南芥NAc家族具有相似分类。保守基序分析,在番茄NAC结构域中包含7个保守的NAM基序,主要分布在序列的N端,表明这些基序的存在对NAC蛋白质功能的执行是必需的。理化性质和结构分析表明,番茄NAC蛋白质绝大多数是亲水蛋白质,主要以无规则卷曲构成,而α-螺旋、β-折叠和β-转角则散布于整个蛋白质中,在各亚族中没有规律。     

基因芯片技术分析转SlNAC10基因拟南芥非生物胁迫相关差异表达基因

NAC(NAM、ATAF1/2和CUC2)转录因子是植物特有的转录调控因子,在植物的器官建成、生长发育以及抵御非生物胁迫等方面发挥着至关重要的作用。该文利用基因芯片技术筛选转Sl NAC10基因拟南芥和野生型拟南芥非生物胁迫抗性相关差异表达基因,并通过实时荧光定量PCR对部分差异表达基因进行验证。芯片结果显示,差异表达2倍以上的基因有4 054个,其中与非生物胁迫相关基因有15个,与非生物胁迫相关的转录因子基因有14个,这些基因参与应答渗透胁迫、响应高盐、冷、热、高光强等胁迫。对差异表达2倍以上的基因进行GO(Gene Ontology)分析和KEGG(Kyoto Encyclopedia of Genes and Genomes)分析,发现这些基因在非生物胁迫相关的13个注释中富集,涉及相关代谢途径96个,其中包括植物激素信号转导、精氨酸和脯氨酸代谢、吲哚生物碱合成、谷胱甘肽代谢等。以上结果表明,SlNAC10可直接或间接调控多种下游基因的表达,提高植物抵御非生物胁迫的能力。     

苦荞NAC转录因子FtNAC17的鉴定及表达分析

植物特有的NAC转录因子参与植物生长发育和逆境响应。研究苦荞中NAC转录因子在非生物胁迫中的应答,为阐明基因功能提供理论依据。以苦荞为试验材料,克隆一个NAC家族基因,对其进行生物信息学分析。通过荧光定量PCR技术检测该基因在干旱、低温、盐、茉莉酸甲酯、脱落酸和赤霉素胁迫时的响应情况。该基因编码272个氨基酸,将其命名为FtNAC17,GenBank登录号为MT641452。基因结构分析表明,FtNAC17由2个外显子和1个内含子组成。氨基酸序列多重比对和进化关系分析表明,FtNAC17蛋白与拟南芥ANAC002亲缘关系最近,属于NAC转录因子家族中同一亚组。FtNAC17对干旱、低温、盐等非生物胁迫均有不同程度的响应。     

番茄低温响应转录因子SlNAC41克隆及表达分析

NAC转录因子在调控植物生长发育、生物及非生物逆境应答中发挥着重要作用。前期,我们通过对番茄幼苗在低温胁迫下的基因表达谱进行分析,发现Unigene SGN-U212711受低温诱导表达强烈。本研究从番茄中克隆了该基因,命名为Sl NAC41,其开放阅读框（ORF）1 173 bp,编码390个氨基酸,蛋白N端具有典型的NAM结构域,属于NAC转录因子家族成员。预测Sl NAC41蛋白分子量为43.5 k Da,等电点为5.2。实时荧光定量PCR分析表明,Sl NAC41在番茄各组织均有表达,在花中的表达量最高,在红熟果中的表达量最低。低温、干旱、高盐、甲基紫精（MV）、脱落酸（ABA）及乙烯利（ETH）处理均能诱导该基因的表达,其中,以低温和干旱诱导表达最为强烈。利用PLACE和Plant CARE对启动子序列进行预测分析发现,Sl NAC41启动子区含有大量响应光、病原菌侵染、激素、低温、脱水及盐胁迫的顺式作用元件。这些结果表明,Sl NAC41可能在番茄生物及非生物胁迫应答中发挥重要调控作用。     

普通小麦祖先种类TaNAC2a基因的生物信息和表达分析

采用生物信息学方法,利用核酸、蛋白数据库对普通小麦祖先种乌拉尔图小麦(Triticum urartu L.)和粗山羊草(Aegilops tauschii L.)NAC转录因子基因家族进行分析,分别鉴定出107、126个NAC蛋白家族成员。根据拟南芥、水稻NAC基因家族分类系统,将其分为15个亚族。通过与抗逆相关基因TaNAC2a进行同源进化树分析,发现5个TuNAC、6个AetNAC基因与其高度同源,对这些基因的蛋白结构域、基因结构、启动子顺式作用元件及组织表达特性进行分析。结果表明,11个NAC蛋白具有典型的NAC结构域。进化关系较近的基因具有相似基因结构;启动子区域预测发现其均含有逆境胁迫响应作用元件。实时荧光定量PCR结果显示,TuNAC、AetNAC基因分别在乌拉尔图小麦和粗山羊草根、胚芽鞘、叶组织中均有表达,并呈现出明显的组织表达特异性。通过芯片表达数据和逆境胁迫基因表达试验,推测AetNAC2c基因可能参与植物干旱胁迫响应,AetNAC2b可能参与调控植物的耐旱、耐低温胁迫反应。上述分析结果为普通小麦祖先种基因家族的系统研究,优异候选功能基因的预测、筛选提供了试验依据。     

乌拉尔图小麦NAC转录因子的筛选与分析

NAC是植物特有的具有多种功能的一类转录因子,广泛参与植物的生长发育,器官建成及抗逆境胁迫等反应.目前有关NAC转录因子的研究主要针对模式植物(如拟南芥和水稻),而在小麦中的研究相对较少.本文利用生物信息学方法,获得乌拉尔图小麦(Triticum urartu)NAC转录因子家族基因的全长序列,并对其进化关系,生物学功能,染色体定位以及基因复制等进行预测与分析,同时利用荧光定量PCR验证相关转录因子在非生物胁迫下的表达模式.结果显示,共筛选得到87个乌拉尔图小麦全长NAC转录因子,通过进化树分析将其分为7个亚族,其中39个NAC 转录因子被定位在7条染色体上.通过基因复制分析发现,有5对NAC转录因子基因发生了复制.进一步通过荧光定量验证4个NAC转录因子在非生物胁迫下的表达模式,发现4个转录因子均受不同胁迫而上调表达.     

基于基因芯片技术的转Slnac2基因拟南芥差异表达基因分析

NAC转录因子,对植物的生长发育及抵御逆境胁迫起着重要的作用。实验室前期克隆了辽宁碱蓬(Suaeda liaotungensis)Slnac2基因,转Slnac2拟南芥(Arabidopsis thaliana)提高了抵御盐胁迫的能力。本研究利用基因芯片技术筛选转SlNAC2拟南芥差异表达基因,共筛选出差异表达2倍以上的基因1 258个。GO富集度分析结果显示,分子功能相关基因占34.06%,细胞组分相关基因占33.13%,生物学过程相关基因占32.81%。KEGG分析表明,差异表达基因涉及63个信号通路,主要有植物激素信号转导、核糖体、植物病原物相互作用和抗坏血酸代谢等。通过实时荧光定量PCR对部分差异基因进行验证,所得结果与基因芯片结果一致。本研究揭示,SlNAC2可调控多个下游基因的表达,提高植物在逆境胁迫下的生存能力。     

紫花苜蓿NAC转录因子MsNAC1基因的克隆、生物信息学分析及非生物逆境胁迫下的表达分析

NAC转录因子是高等植物所特有的具有多种生物功能的转录因子,在植物生长发育、抵抗逆境和激素调节等过程中发挥着重要作用。本研究利用RACE-PCR技术,克隆获得了紫花苜蓿NAC转录因子Ms NAC1(Gen Bank登录号为JN099384.1)基因的c DNA序列。生物信息学分析显示,Ms NAC1基因的开放阅读框(ORF)为993 bp,编码一个由330个氨基酸残基组成的亲水性蛋白,N-端具有保守的NAM结构域,C-端高度变异,具备NAC转录因子的基本特征;Ms NAC1蛋白被定位在细胞核中,含有2条核定位信号序列,具有9个糖基化位点和23个磷酸化位点,三级结构为对称的同型二聚体。多重比对发现,Ms NAC1蛋白与拟南芥ATAF1和水稻Os NAC6蛋白的同源性较高;系统进化分析表明,Ms NAC1蛋白属于NAC转录因子家族中的ATAF亚族,与ATAF1的亲缘关系最近。非生物逆境胁迫下的表达分析显示,Ms NAC1基因在高盐、干旱和低温胁迫下表达量均呈现先上调后下调的趋势,不同处理时间的差异达到极显著水平,并且根中的表达量上调幅度高于叶片,说明该基因可能参与调控非生物逆境胁迫的生理响应。     

光皮桦miR164及其靶基因NAC1在低氮胁迫中的表达分析

氮是植物生长发育所必需的大量营养元素,植物缺氮后严重影响地上部分生物量的积累,因此,揭示植物如何抵抗或适应低氮胁迫的分子机制具有重要意义。杨树(Populus tremula × P. alba)NAC1(NAM, ATAF, CUC 1)基因位于调控网络上游,在低氮环境下调控下游关键基因的表达,进而调控根系生长以抵抗低氮胁迫。本文以光皮桦(Betula luminifera)G49-3无性系组培苗为材料,探讨了miR164及其靶基因NAC1对低氮胁迫的响应。通过RACE技术克隆了光皮桦NAC1基因(GenBank登录号：KT900889),全长1497 bp,编码358个氨基酸,N端具有高度保守的NAM结构域;运用5′-RACE验证了NAC1为miR164靶基因,切割位点在第10和11位碱基之间;采用qRT-PCR分析miR164与靶基因NAC1在低氮胁迫时的表达模式,发现miR164表达在根中的低氮处理前期(4 d)受到抑制,而后升高,而茎叶中表达模式与根不同;靶基因NAC1与miR164表达水平呈负相关,且在恢复实验组(重新添加全营养液)中,根中miR164表达上升,NAC1显示出相应的表达变化,暗示miR164及其靶基因NAC1可能在低氮胁迫响应中发挥调控功能。本研究结果有助于揭示miR164对NAC1在低氮胁迫响应中转录后水平的分子调控机制,为进一步研究miR164-NAC1在低氮胁迫响应中的功能提供有价值的信息。     

Genome-wide identification of Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families and their expression analyses in response to viral infection and abiotic stresses in Solanum lycopersicum

Dicer, Argonaute and RNA-dependent RNA polymerase form the core components to trigger RNA silencing. Although tomato (Solanum lycopersicum) is a dicotyledon model plant, no systematic analysis and expression profiling of these genes in tomato has been undertaken previously. In this study, seven Dicer-like (SlDCLs), 15 Argonaute (SlAGOs) and six RNA-dependent RNA polymerase (SlRDRs) genes were identified in tomato. These genes were categorized into four subgroups based on phylogenetic analyses. Comprehensive analyses of gene structure, genomic localization and similarity among these genes were performed. Their expression patterns were investigated by means of expression models in different tissues and organs using online data and semi-quantitative RT-PCR. Many of the candidate genes were up-regulated in response to Tomato yellow leaf curl virus infection and abiotic stresses. The expression models of tandem gene duplications among SlDCL2s indicated the DCL2 family plays an important role in the evolution of tomato.     

Evidence of cryptic introgression in tomato (Solanum lycopersicum L.) based on wild tomato species alleles

ABSTRACT: BACKGROUND: Many beneficial traits (e.g. disease or abiotic stress resistance) have been transferred into crops through crosses with their wild relatives. The 13 recognized species of tomato (Solanum section Lycopersicon) are closely related to each other and wild species genes have been extensively used for improvement of the crop, Solanum lycopersicum L. In addition, the lack of geographical barriers has permitted natural hybridization between S. lycopersicum and its closest wild relative Solanum pimpinellifolium in Ecuador, Peru and northern Chile. In order to better understand patterns of S. lycopersicum diversity, we sequenced 47 markers ranging in length from 130 to 1200 bp (total of 24 kb) in genotypes of S. lycopersicum and wild tomato species S. pimpinellifolium, Solanum arcanum, Solanum peruvianum, Solanum pennellii and Solanum habrochaites. Several of the markers had previously been hypothesized as carrying wild species alleles within S. lycopersicum, i.e., cryptic introgressions. RESULTS: Each marker was mapped with high confidence (e < 1 x 10-30) to a single genomic location using BLASTN against tomato whole genome shotgun chromosomes (SL2.40) database. Neighbor-joining trees showed high mean bootstrap support (86.8 plus or minus 2.34%) for distinguishing red-fruited from green-fruited taxa for 38 of the markers. Hybridization and parsimony splits networks, genomic map positions of markers relative to documented introgressions, and historical origins of accessions were used to interpret evolutionary patterns at nine markers with putatively introgressed alleles. CONCLUSION: Of the 47 genetic markers surveyed in this study, four were involved in linkage drag on chromosome 9 during introgression breeding, while alleles at five markers apparently originated from natural hybridization with S. pimpinellifolium and were associated with primitive genotypes of S. lycopersicum. The positive identification of introgressed genes within crop species such as S. lycopersicum will help inform conservation and utilization of crop germplasm diversity, for example, facilitating the purging of undesirable linkage drag or the exploitation of novel, favorable alleles.     

The identification of a gene (Cwp1), silenced during Solanum evolution, which causes cuticle microfissuring and dehydration when expressed in tomato fruit

One of the most intriguing phenomena of fleshy fruit is the ability to maintain high water content at maturity, even following harvest. This is accomplished by a fruit cuticle that is highly impermeable to water diffusion. In this paper, we report on a novel genotype of tomato, developed via introgression from the wild species Solanum habrochaites, which is characterized by microfissuring of the fruit cuticle and dehydration of the mature fruit. The microfissure/dehydration phenotype is inherited as a single gene, termed Cwp1 (cuticular water permeability). The gene was fine mapped, and its identity was determined by map-based cloning and differential expression analysis in near-isogenic lines. Causality of the Cwp1 gene was shown by the heterologous transgenic expression of the gene in the cultivated tomato, which caused a microfissured fruit cuticle leading to dehydrated fruit. Cwp1 encodes for a protein of unidentified function in the DUF833 domain family. The gene is expressed in the fruit epidermis of the dehydrating genotype harbouring the wild-species introgression, but not in the cultivated tomato. It is expressed only in the primitive green-fruited wild tomato species, but is not expressed in the cultivated Solanum lycopersicum and the closely related Solanum cheesmaniae and Solanum pimpinellifolium, indicating a pre-adaptive role for Cwp1 silencing in the evolution and domestication of the cultivated tomato.     

Investigation of tomato (Solanum lycopersicum) aminotransferases involved in biosynthesis of branched-chain-amino-acids

This study presents evidence for the role of BCAT3 and BCAT4 proteins in the synthesis of branched-chain-amino-acids in tomato Solanum lycopersicum. BCAT3 and BCAT4 genes were located on tomato chromosomal map by RFLP method (restriction fragment length polymorphism). Using confocal microscopy it was shown that BCAT3-GFP and BCAT4-GFP fusion proteins were localised in chloroplasts. It was observed that these aminotransferase isoforms exhibited distinct kinetic properties and a differential expression pattern of mRNA levels in various tomato tissues.     

醋栗番茄Solanum pimpinellifolium遗传多样性分析

野生种醋栗番茄包含丰富的变异,具有众多优良性状。本研究对从世界不同地区收集的433份醋栗番茄遗传资源进行了表型鉴定及遗传多样性分析。表型鉴定结果表明,在收集的433份醋栗番茄中约有14%为樱桃番茄,370份典型醋栗番茄中约22%的材料存在不同程度的分离。群体变异分析表明,不同性状间变异系数存在较大差异,其中柱头变异系数最大,为56.716%;花瓣数遗传稳定,变异系数最小,为2.082%;相关分析表明,多个性状间存在显著相关性;利用表型和基因型数据聚类均将醋栗番茄群体划分为两大类群;主成分分析表明,坐果率、单果重、可溶性固形物对变异的贡献率较大。研究结果将为利用醋栗番茄进行栽培种番茄遗传改良奠定一定的基础。     

Chromosomal rearrangements between tomato and Solanum chilense hamper mapping and breeding of the TYLCV resistance gene Ty-1

Tomato yellow leaf curl disease, a devastating disease of Solanum lycopersicum (tomato), is caused by a complex of begomoviruses generally referred to as Tomato yellow leaf curl virus (TYLCV). Almost all breeding for TYLCV resistance has been based on the introgression of the Ty-1 resistance locus derived from Solanum chilense LA1969. Knowledge about the exact location of Ty-1 on tomato chromosome 6 will help in understanding the genomic organization of the Ty-1 locus. In this study, we analyze the chromosomal rearrangement and recombination behavior of the chromosomal region where Ty-1 is introgressed. Nineteen markers on tomato chromosome 6 were used in F(2) populations obtained from two commercial hybrids, and showed the presence of a large introgression in both. Fluorescence in situ hybridization (FISH) analysis revealed two chromosomal rearrangements between S. lycopersicum and S. chilense LA1969 in the Ty-1 introgression. Furthermore, a large-scale recombinant screening in the two F(2) populations was performed, and 30 recombinants in the Ty-1 introgression were identified. All recombination events were located on the long arm beyond the inversions, showing that recombination in the inverted region was absent. Disease tests on progenies of informative recombinants with TYLCV mapped Ty-1 to the long arm between markers MSc05732-4 and MSc05732-14, an interval overlapping with the reported Ty-3 region, which led to the indication that Ty-1 and Ty-3 may be allelic. With this study we prove that FISH can be used as a diagnostic tool to aid in the accurate mapping of genes that were introgressed from wild species into cultivated tomato.