Functional genomics of maize submergence tolerance and cloning of the related gene <Emphasis Type="Italic">Sicyp51</Emphasis>

In this study, SSH (Suppression Subtractive Hybridization) and cDNA microarray were used to identify genes associated with waterlogging response of maize roots. Mo17 and Hz32 are two maize inbred lines with differential tolerance to hypoxia. Seedlings of the inbred lines with two leaves were submerged in hypoxia buffer. SSH libraries were constructed with cDNA samples from roots. Both forward and reverse subtractions were performed for each inbred line, and 105 positive clones induced by hypoxia were selected by differential screening. The treated and control message RNA were hybridized with the cDNA microarray of Mo17, sequentially, 57 of 3-fold differentially expressed clones were obtained. A total of 162 positive clones were all sequenced. Bioinformatics analysis showed these positive clones represent 85 TUGs, including genes involved in several biochemistry pathways, such as glycolysis, protection, signal transduction, cell construction and energy metabolism and 41 EST with unknown function. Comparison between Mo17 and Hz32 indicates that genes related to hypoxia tolerance have different expression patterns in submerged roots. Several positive clones' expression patterns were revealed by Northern or RT-PCR, and a new gene (Sicyp51), which may contribute to hypoxia tolerance, was identified.     

Functional genomics of maize submergence tolerance and cloning of the related gene Sicyp51

In the middle and lower Yangtze River area, the major corn-growing region of South China, seasonal rainfall greatly affects maize plantation. Maize seed-lings meet with excessive precipitation and low tem-perature in spring, and when they grow up to begin flowering, they usually encounter Mei-yu storm ac-companied by hot days. At the same time, bad irriga-tion system and a higher level of underground water cause waterlogging, which further leads to yield losses. In order to reveal the molecu…     

小麦抗病基因表达谱中的文库构建与筛选方法研究

以抗白粉病品系“百农 32 17×Mardler”BC5F4为材料 ,构建了白粉病菌诱导的普通cDNA文库和抑制消减杂交(SSH)cDNA文库。分别对两文库进行了一定规模的测序 ,获得普通cDNA文库不重复ESTs 387条和SSHcDNA文库ESTs 76 0条。将获得的ESTs与GenBank序列进行了BLASTn、BLASTx同源性分析。结果表明 :在普通文库中 ,一些参与光合作用与核糖体构成等的基因出现频率较高 ,而获得的抗病相关基因则较少。消减文库在构建方法、抗病相关基因的富集等方面具有明显的优越性 ,是目前抗病基因表达谱研究中的较好方法。利用高密度点阵膜杂交技术对两文库的筛选结果表明 ,该方法具有相对简便易操作、杂交膜可反复使用等优点 ;但也存在mRNA及同位素用量大等问题。经筛选 ,消减文库中有 5 4 1%的功能已知ESTs为抗病相关基因 ,被证明参与了小麦抗白粉病反应     

Identification of ESTs differentially expressed in green and albino mutant bamboo (Bambusa edulis) by suppressive subtractive hybridization (SSH) and microarray analysis

To gain a better understanding of gene expression in bamboo (Bambusa edulis Murno), we have used a combination of suppressive subtractive hybridization (SSH), microarray hybridization analysis, sequencing, and bioinformatics to identify bamboo genes differentially expressed in a bamboo albino mutant. Ten expressed sequence tags (ESTs) were found to be differentially expressed; these were isolated and sequenced. RT-PCR analysis of these ESTs supported the results of the microarray analysis. Six ESTs that were nucleus-encoded exhibited differential expression patterns in the green wild-type bamboo relative to the albino mutant. These genes (exception being the Rubisco small subunit) were non-photosynthesis-related genes. The development of a specific SSH cDNA library in which most of the chloroplast-encoded or photosynthesis-related genes had been subtracted proved to be useful for studying the function of non-photosynthesis-related genes in the albino bamboo mutants with aberrant chloroplast genome. The combined use of this SSH library with microarray analysis will provide a powerful analytical tool for future studies of the bamboo genome.     

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.     

Size-selection of cDNA libraries for the cloning of cDNAs after suppression subtractive hybridization

Here we describe the establishment of size-selected cDNA libraries for the cloning of full-length cDNAs that were initially identified by suppression subtractive hybridization (SSH) technology as being differentially expressed. First, the SSH-cDNA fragments were used as 32P-probes to verify their level and differential pattern of expression by virtual Northern and to establish their corresponding full-length cDNA size. Second, cDNAs were separated by size on agarose gels and used to construct size-selected cDNA plasmid libraries, which were then screened by colony hybridization with the SSH-cDNA fragments. We conclude that the described approach complements SSH technology by allowing efficient cloning and characterization of the corresponding full-length cDNA from any desired cell type or species. This approach will give researchers the ability to specifically target and study differentially expressed genes in an efficient manner for functional genomic studies.     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

Gene expression patterns in Atlantic salmon Salmo salar: gene expression during osmoregulation in intestine tissue

Ireland has the world's largest stocks of wild Atlantic salmon. A better understanding of gene expression will benefit conservation of wild stock as well as salmon aquaculture. We describe the PRTLI project designed to advance the fundamental understanding of the genome of Atlantic Salmon Salmo salar . The major objective is to create the first comprehensive database of gene expression and functional information using cDNA libraries and Microarray technology. One key area of interest to salmon biology is osmoregulation, which is critical to the ability of salmon to adapt in seawater. Tissues implicated in this process are the gills, intestine and skin. To initiate studies, SSH (suppression subtractive hybridization) libraries were constructed from intestine RNA extracted from smolts sampled in January and May. A number of potentially interesting clones have been identified, among those a heat shock protein, hsp90 in the reverse library. Others SSH libraries from various tissues (pituitary, hypothalamus, brain, gill, intestine, head kidney and spleen) have also been constructed and will be used to construct a 5000 clone microarray slide. This slide will then be used to elucidate gene expression profiles in various tissues. Further sample collection has been carried out to answer questions regarding biologicaldifferences between one‐ year and two‐year old parr and wild and hatchery smolt.     

Identification and characterization of differential gene expression in the mesocarp and kernel of oil palm nuts using suppression subtractive hybridization

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.     

Expression patterns in soybean resistant to Phakopsora pachyrhizi reveal the importance of peroxidases and lipoxygenases

Soybean rust caused by Phakopsora pachyrhizi Sydow is a devastating foliar disease that has spread to most soybean growing regions throughout the world, including the USA. Four independent rust resistance genes, Rpp1–Rpp4, have been identified in soybean that recognize specific isolates of P. pachyrhizi. A suppressive subtraction hybridization (SSH) complementary DNA (cDNA) library was constructed from the soybean accession PI200492, which contains Rpp1, after inoculation with two different isolates of P. pachyrhizi that result in susceptible or immune reactions. Both forward and reverse SSH were performed using cDNA from messenger RNA pooled from 1, 6, 12, 24, and 48 h post-inoculation. A total of 1,728 SSH clones were sequenced and compared to sequences in GenBank for similarity. Microarray analyses were conducted on a custom 7883 soybean-cDNA clone array encompassing all of the soybean-rust SSH clones and expressed sequence tags from four other soybean cDNA libraries. Results of the microarray revealed 558 cDNA clones differentially expressed in the immune reaction. The majority of the upregulated cDNA clones fell into the functional category of defense. In particular, cDNA clones with similarity to peroxidases and lipoxygenases were prevalent. Downregulated cDNA clones included those with similarity to cell-wall-associated protein, such as extensins, proline-rich proteins, and xyloglucan endotransglycosylases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.