用基因芯片技术分析铝胁迫下小麦的基因表达谱

目的:采用基因表达谱分析方法,探讨小麦耐铝的分子机理。方法:利用抑制消减杂交(SSH)技术,以小麦的铝敏感品种Chisholm及其耐铝近等基因系Chisholm-T(其耐铝性来自小麦品种Atlas66)的根尖为材料,构建了2个铝胁迫后的SSHcDNA文库,共含有1628个表达序列标签(EST),利用这些EST制作了小麦根系的cDNA基因芯片。以cDNA基因芯片为平台,在铝胁迫后6h、1d、3d和7d,分别比较Chisholm和Chisholm-T之间的基因表达谱差异。结果:在各个时间点,耐铝和不耐铝小麦材料之间约有5%的EST表现出差异表达。对所有差异表达的EST进行测序分析,序列数据经Pipe-Online2.0进行毗连序列群(contig)拼接,发现只有8.3%的重复序列。结论:SSH是一种非常有效的差减和均一化的建库方法。对有功能注释的差异表达基因进行功能分类分析,表明这些基因参与了植物体内的电子传递、信号传导、植物保护和次生物质的代谢活动。     

Nylon Filter Arrays Reveal Differential Expression of Expressed Sequence Tags in Wheat Roots Under Aluminum Stress

To enrich differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance, cDNA subtraction libraries were generated from Al-stressed roots of two wheat (Triticum aestivum L.) nearisogenic lines (NILs) contrasting in Al-tolerance gene(s) from the Al-tolerant cultivar Atlas 66, using suppression subtractive hybridization (SSH). Expression patterns of the ESTs were investigated with nylon filter arrays containing 614 cDNA clones from the subtraction library. Gene expression profiles from macroarray analysis indicated that 25 ESTs were upregulated in the tolerant NIL in response to Al stress. The result from Northern analysis of selected upregulated ESTs was similar to that from macroarray analysis. These highly expressed ESTs showed high homology with genes involved in signal transduction, oxidative stress alleviation, membrane structure, Mg2 transportation, and other functions. Under Al stress, the Al-tolerant NIL may possess altered structure or function of the cell wall, plasma membrane, and mitochondrion. The wheat response to Al stress may involve complicated defense-related signaling and metabolic pathways.The present experiment did not detect any induced or activated genes involved in the synthesis of malate and other organic acids in wheat under Al-stress.     

Nylon Filter Arrays Reveal Differential Expression of Expressed Sequence Tags in Wheat Roots Under Aluminum Stress

To enrich differentially expressed sequence tags (ESTs) for aluminum (A1) tolerance, cDNA subtraction libraries were generated from Al-stressed roots of two wheat (Triticum aestivum L.) nearisogenic lines (NILs) contrasting in Al-tolerance gene(s) from the Al-tolerant cultivar Atlas 66, using suppression subtractive hybridization (SSH). Expression patterns of the ESTs were investigated with nylon filter arrays containing 614 cDNA clones from the subtraction library. Gene expression profiles from macroarray analysis indicated that 25 ESTs were upregulated in the tolerant NIL in response to A1 stress. The result from Northern analysis of selected upregulated ESTs was similar to that from macroarray analysis. These highly expressed ESTs showed high homology with genes involved in signal transduction, oxidative stress alleviation, membrane structure, Mg^2 transportation, and other functions. Under A1 stress, the Al-tolerant NIL may possess altered structure or function of the cell wall, plasma membrane, and mitochondrion. The wheat response to A1 stress may involve complicated defense-related signaling and metabolic pathways. The present experiment did not detect any induced or activated genes involved in the synthesis of malate and other organic acids in wheat under Al-stress.     

Gene expression profiling in maize roots under aluminum stress

To investigate the molecular mechanisms of Al toxicity, cross-species cDNA array approach was employed to identify expressed sequence tags (ESTs) regulated by Al stress in root tips of Al-tolerant maize (Zea mays) genotype Cat100-6 and Al-sensitive genotype S1587-17. Due to the high degree of conservation observed between sugarcane and maize, we have analyzed the expression profiling of maize genes using 2 304 sugarcane (ESTs) obtained from different libraries. We have identified 85 ESTs in Al stressed maize root tips with significantly altered expression. Among the up-regulated ESTs, we have found genes encoding previously identified proteins induced by Al stress, such as phenyl ammonia-lyase, chitinase, Bowman-Birk proteinase inhibitor, and wali7. In addition, several novel genes up-and downregulated by Al stress were identified in both genotypes.     

Expressed sequence tag-based gene expression analysis under aluminum stress in rye

To understand the mechanisms responsible for aluminum (Al) toxicity and tolerance in plants, an expressed sequence tag (EST) approach was used to analyze changes in gene expression in roots of rye (Secale cereale L. cv Blanco) under Al stress. Two cDNA libraries were constructed (Al stressed and unstressed), and a total of 1,194 and 774 ESTs were generated, respectively. The putative proteins encoded by these cDNAs were uncovered by Basic Local Alignment Search Tool searches, and those ESTs showing similarity to proteins of known function were classified according to 13 different functional categories. A total of 671 known function genes were used to analyze the gene expression patterns in rye cv Blanco root tips under Al stress. Many of the previously identified Al-responsive genes showed expression differences between the libraries within 6 h of Al stress. Certain genes were selected, and their expression profiles were studied during a 48-h period using northern analysis. A total of 13 novel genes involved in cell elongation and division (tonoplast aquaporin and ubiquitin-like protein SMT3), oxidative stress (glutathione peroxidase, glucose-6-phosphate-dehydrogenase, and ascorbate peroxidase), iron metabolism (iron deficiency-specific proteins IDS3a, IDS3b, and IDS1; S-adenosyl methionine synthase; and methionine synthase), and other cellular mechanisms (pathogenesis-related protein 1.2, heme oxygenase, and epoxide hydrolase) were demonstrated to be regulated by Al stress. These genes provide new insights about the response of Al-tolerant plants to toxic levels of Al.     

Transcriptional analysis of normal human fibroblast responses to microgravity stress

To understand the molecular mechanism（s） of how spaceflight affects cellular signaling pathways, quiescent normal human WI-38 fibroblasts were flown on the STS-93 space shuttle mission. Subsequently, RNA samples from the spaceflown and ground-control cells were used to construct two cDNA libraries, which were then processed for suppression subtractive hybridization （SSH） to identify spaceflight-specific gene expression. The SSH data show that key genes related to oxidative stress, DNA repair, and fatty acid oxidation are activated by spaceflight, suggesting the induction of cellular oxidative stress. This is further substantiated by the up-regulation of neuregulin 1 and the calcium-binding protein calmodulin 2. Another obvious stress sign is that spaceflight evokes the Ras/mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling pathways, along with up-regulating several Gl-phase cell cycle traverse genes. Other genes showing upregulation of expression are involved in protein synthesis and pro-apoptosis, as well as pro-survival. Interactome analysis of functionally related genes shows that c-Myc is the ＂hub＂ for those genes showing significant changes. Hence, our results suggest that microgravity travel may impact changes in gene expression mostly associated with cellular stress signaling, directing cells to either apoptotic death or premature senescence.     

Identification of early salt stress response genes in tomato root by suppression subtractive hybridization and microarray analysis

High salinity is one of the most serious threats to crop production. To understand the molecular basis of plant responses to salt stress better, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes involved in the early stage of tomato responses to severe salt stress. First, SSH libraries were constructed for the root tissue of two cultivated tomato (Solanum lycopersicum) genotypes: LA2711, a salt-tolerant cultivar, and ZS-5, a salt-sensitive cultivar, to compare salt treatment and non-treatment plants. Then a subset of clones from these SSH libraries were used to construct a tomato cDNA array and microarray analysis was carried out to verify the expression changes of this set of clones upon a high concentration of salt treatment at various time points compared to the corresponding non-treatment controls. A total of 201 non-redundant genes that were differentially expressed upon 30 min of severe salt stress either in LA2711 or ZS-5 were identified from microarray analysis; most of these genes have not previously been reported to be associated with salt stress. The diversity of the putative functions of these genes indicated that salt stress resulted in a complex response in tomato plants.     

Aluminium-responsive genes in sugarcane: identification and analysis of expression under oxidative stress

Suppression subtractive hybridization (SSH) technology was used to gain preliminary insights into gene expression induced by the phytotoxic aluminium species, Al(3+), in sugarcane roots. Roots of hydroponically-grown Saccharum spp. hybrid cv. N19 were exposed to 221 microM Al(3+) at pH 4.1 for 24 h, a regime shown to inhibit root elongation by 43%, relative to unchallenged roots. Database comparisons revealed that, of a subset of 50 cDNAs ostensibly up-regulated by the metal in the root tips, 14 possessed putative identities indicative of involvement in signalling events and the regulation of gene expression, while the majority (28) were of unknown function. All of the 50 cDNAs sequenced displayed significant similarity to uncharacterized plant expressed sequence tags (ESTs), approximately half (23) of which had been derived from other graminaceous crop species that had been subject to a variety of stresses. Analysis of the expression of 288 putative Al(3+)-inducible genic fragments indicated higher levels of expression under oxidative (1 mM diamide for 4 h) rather than Al(3+) stress. By deploying SSH, this study has provided an indication of the nature of genes expressed in sugarcane roots under Al(3+) stress. It is anticipated that the information obtained will guide further exploration of the potential for manipulation of the Al tolerance characteristics of the crop.     

Differential expression of expressed sequence tags in alfalfa roots under aluminum stress

To gain a better understanding of differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance and to investigate the molecular mechanisms of Al toxicity, cDNA subtraction libraries were generated from Al-stressed roots of alfalfa (Medicago sativa L.) compared with no Al-stressed ones, employing suppression subtractive hybridization. Using differential screening technique in which the probes were labeled with DIG, we identified 45 non-redundant ESTs in Al-stressed alfalfa root tips with significantly altered expression. Among the up-regulated ESTs, we have found genes encoding identified proteins, including malate dehydrogenase, 6-phosphogluconate dehydrogenase, peroxidase, and an ABC transporter, while the down-regulate genes included ATPase, secretory carrier membrane protein 2, pectinesterase inhibitor. In addition, two novel ESTs, EW678752 and EY976957, up- and down-regulated by Al stress were sequenced. Analyzed by real-time PCR, the expressions of EST EW678718, EW678739, EY976969 and EW678728, which encode for ABC transporter, malate dehydrogenase, peroxidase and 6-phosphogluconate dehydrogenase correspondingly, increased 1.64-, 2.75-, 3.27- and 6.54-folds, respectively, and the expression of EY976957 encoding for ATPase decreased 3.27 folds. The expression of EST EW678752 increased 34.54-fold, while that of EY976957 decreased 16.68 folds. It suggested that the two novel ESTs maybe play a significant role in the aluminum tolerance of alfalfa.     

Identification and comparative analysis of aluminum-induced microRNAs conferring plant tolerance to aluminum stress in soybean

Aluminum (Al) toxicity in acidic soils is a major factor restricting crop production. Although the molecular mechanisms of Al responses have been extensively investigated, microRNA (miRNA) mediated differential Al tolerance in different soybean genotypes remains largely unknown. In this study, two soybean [Glycine max (L.) Merr.] genotypes, Al-tolerant BX10 and Al-sensitive BD2, were treated with 0 and 50 μM AlCl3 and then used to construct the miRNA libraries for deep sequencing. Results revealed 453 miRNAs, whose expression patterns were affected by Al stress. We also identified 32 differentially expressed miRNAs: 19 in BX10, 7 in BD2, and 6 in both genotypes. The gene ontology analysis of their putative target genes indicated that stress-responsive genes and amino-acid-metabolism-related processes preferentially existed in BX10. Comprehensive analysis demonstrated that conserved miRNAs, such as gma-miR166k/o, gma-miR390g, and gma-miR396c/k, mediated root elongation in BX10, whereas gma-miR169r triggered oxidative stress in BD2. These processes could be regarded as important mechanisms conferring differential Al tolerance in BX10 and BD2. This study provided new insights into different Al response mechanisms in various soybean genotypes.     

Mapping of genes controlling aluminum tolerance in rice: comparison of different genetic backgrounds

Aluminum toxicity is the main factor limiting the productivity of crop plants in acid soils, particularly in the tropics and subtropics. In this study, a doubled-haploid population derived from the rice ( Oryza sativa L.) breeding lines CT9993 and IR62266 was used to map genes controlling Al tolerance. A genetic linkage map consisting of 280 DNA markers (RFLP, AFLP and SSR) was constructed to determine the position and nature of quantitative trait loci (QTLs) affecting Al tolerance. Three characters - control root length (CRL), Al-stressed root length (SRL) and root length ratio (RR) - were evaluated for the DH lines and the parents at the seedling stage in nutrient solution. A total of 20 QTLs controlling root growth under Al stress and control conditions were detected and distributed over 10 of the 12 rice chromosomes, reflecting multigenic control of these traits. The two QTLs of largest effect, qALRR-1-1 and qALRR-8 for root length ratio (a measurement of Al tolerance) were localized on chromosomes 1 and 8, respectively. Three other QTLs in addition to qALRR-8 were apparently unique in the CT9993 x IR62266 mapping population, which may explain the high level of Al tolerance in CT9993. Comparative mapping identified a conserved genomic region on chromosome 1 associated with Al tolerance across three rice genetic backgrounds. This region provides an important starting point for isolating genes responsible for different mechanisms of aluminum tolerance and understanding the genetic nature of this trait in rice and other cereals.     

The role of organic acids in the short- and long-term aluminum tolerance in maize seedlings (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Aluminum (Al) affects numerous physiological processes in plants. However, Al tolerance mechanisms mediated by increased synthesis of organic acids (OAs) have been outlined recently. In this study, we examined the role of OAs in the short (1–8 h) and long-term (4 days) Al tolerance in maize seedlings. Exposure to Al stress for 4 days results in a rapid inhibition of root growth. Al induced morphological changes in the maize roots, especially at a higher solution of Al concentration (1,000 μM Al). The increase in Al accumulation in roots, including strongly elevated levels of Al accumulated in root cell walls suggests that Al tolerance in maize is mediated in part by higher accumulation of Al in the roots. The enhanced citrate exudation, which was only observed at 1,000 μM Al may lead to detoxification of Al by formation of OA–Al complexes in the root apoplast. This mechanism has been suggested to play a significant role in Al resistance response in maize. The short-term responses underlying internal detoxification via OA-chelators were also investigated. Succinate, malate, citrate and total root OA contents decreased markedly, 2 h after the Al exposure. At 4 and 8 h time points, OA contents increased or remained unchanged, except for that of malate which decreased. The level of OAs in shoots, on the other hand, showed alterations that were less pronounced in response to Al. Specifically, the citrate and total OA concentrations significantly increased at 4 h, but showed a pronounced decrease at the 8 h time point. Based on our findings, we propose that multiple responses, including Al exclusion by Al accumulation in root cells and citrate efflux, may contribute towards higher Al resistance in maize. The rapid OA changes in responses to short-term Al treatment may not be responsible for Al tolerance. However, increased OA synthesis observed in this study may be involved in diminishing the stress triggered by Al. The molecular aspects underlying Al resistance mechanism via Al-induced expression of the enzymes catalyzing OA synthesis and metabolism remain to be elucidated.     

Molecular mapping of genes conferring aluminum tolerance in rice (Oryza sativa L.)

Crop productivity on acid soil is restricted by multiple abiotic stress factors. Aluminum (Al) tolerance seems to be a key to productivity on soil with a pH below 5.0, but other factors such as Mn toxicity and the deficiency of P, Ca and Mg also play a role. The development of Al-tolerant genotypes of rice is an urgent necessity for improving crop productivity in developing countries. Inhibition of root growth is a primary and early symptom of Al toxicity. The present study was conducted to identify genetic factors controlling the aluminum tolerance of rice. Several parameters related to Al tolerance, most importantly the relative root growth under Al stress versus non-stress conditions, were scored in 188 F₃ selfed families from a cross between an Al-tolerant Vietnamese local variety, Chiembau, and an Al-susceptible improved variety, Omon269–65. The two varieties are both Oryza sativa ssp. indica, but showed a relatively high level of DNA polymorphism, permitting the assembly of an RFLP map consisting of 164 loci spanning 1,715.8 cM, and covering most of the rice genome. A total of nine different genomic regions on eight chromosomes have been implicated in the genetic control of root and shoot growth under aluminum stress. By far the greatest effects on aluminum tolerance were associated with the region near WG110 on chromosome 1. This region does not seem to correspond to most of the genes that have been mapped for aluminum tolerance in other species, nor do they correspond closely to one another. Most results, both from physiological studies and from molecular mapping studies, tend to suggest that aluminum tolerance is a complex multi-genic trait. The identification of DNA markers (such as WG110) that are diagnostic for aluminum tolerance in particular gene pools provides an important starting point for transferring and pyramiding genes that may contribute to the sustainable improvement of crop productivity in aluminum-rich soils. The isolation of genes responsible for aluminum tolerance is likely to be necessary to gain a comprehensive understanding of this complex trait. Received: 29 March 2000 / Accepted: 16 August 2000