ABSTRACT
Pearl millet (Pennisetum glaucum L. R. Br.) is an important cereal crop grown mainly in the arid and semi-arid regions of India known to possess the natural ability to withstand thermal stress. To elucidate the molecular basis of high temperature response in pearl millet, 12 days old seedlings of P. glaucum cv. 841A were subjected to heat stress at 46°C for different time durations (30 min, 2, 4, 8, 12 and 24 h) and a forward subtractive cDNA library was constructed from pooled RNA of heat stressed seedlings. A total of 331 high quality Expressed Sequence Tags (ESTs) were obtained from randomly selected 1050 clones. Sequences were assembled into 103 unique sequences consisting of 37 contigs and 66 singletons. Of these, 92 unique sequences were submitted to NCBI dbEST database. Gene Ontology through RGAP data base and BLASTx analysis revealed that about 18% of the ESTs showed homology to genes for “response to abiotic and biotic stimulus”. About 2% of the ESTs showed no homology with genes in dbEST, indicating the presence of uncharacterized candidate genes involved in heat stress response in P. glaucum. Differential expression of selected genes (hsp101 and CRT) from the SSH library were validated by qRT-PCR analysis. The ESTs thus generated are a rich source of heat stress responsive genes, which can be utilized in improving thermotolerance of other food crops.
ABSTRACT
Salmonella enterica serovar Choleraesuis strain C500 is a live attenuated vaccine that has been widely used in Chi‐na for over 50 years to prevent piglet paratyphoid .However ,as C500 is obtained by chemical methods ,the genetic background of this strain remained unclear .In this study ,we compared the genomic differences between the virulent reference strain C 78‐2 and C500 by suppression subtractive hybridization combined with the mirror orientation selection method (MOS‐SSH ) .Six genes (asr ,ydgF ,ydgD ,ydgE ,rpoS ,and ptsG) were lost in C500 strain .Using real‐time PCR analysis ,we demonstrated that the genes regulated by rpoS ,a vital transcriptional regulator playing an important role in Salmonella infection ,were downregulated in C500 .Additionally ,the virulence of the rpoS mutant strain C78‐2ΔrpoS was 100 000 times lower than the parental strain in BALB/c mice .So loss of rpoS gene is the major factor leading to the attenuation of C500 strain .
ABSTRACT
Eichhornia crassipes is an aquatic plant native to the Amazon River Basin. It has become a serious weed in freshwater habitats in rivers, lakes and reservoirs both in tropical and warm temperate areas worldwide. Some research has stated that it can be used for water phytoremediation, due to its strong assimilation of nitro- gen and phosphorus, and the accumulation of heavy metals, and its growth and spread may play an important role in environmental ecology. In order to explore the molecular mechanism of E. crassipes to responses to nitrogen deficiency, we constructed forward and reversed subtracted cDNA libraries for E. crassipes roots under nitrogen deficient condition using a suppressive subtractive hybridization (SSH) method. The forward subtraction included 2 100 clones, and the reversed included 2 650 clones. One thousand clones were randomly selected from each library for sequencing. About 737 (527 unigenes) clones from the forward library and 757 (483 unigenes) clones from the reversed library were informative. Sequence BlastX analysis showed that there were more transporters and adenosylhomocysteinase-like proteins in E. crassipes cultured in nitrogen deficient medium; while, those cultured in nitrogen replete medium had more proteins such as UBR4-like e3 ubiquitin- protein ligase and fasciclin-like arabinogalactan protein 8-like, as well as more cytoskeletal proteins, including actin and tubulin. Cluster of Orthologous Group (COG) analysis also demonstrated that in the forward library, the most ESTs were involved in coenzyme transportation and metabolism. In the reversed library, cytoskeletal ESTs were the most abundant. Gene Ontology (GO) analysis categories demonstrated that unigenes involved in binding, cellular process and electron carrier were the most differentially expressed unigenes between the forward and reversed libraries. All these results suggest that E. crassipes can respond to different nitrogen status by efficiently regulating and controlling some transporter gene expressions, certain metabolism processes, specific signal transduction pathways and cytoskeletal construction.
Se ha convertido en una maleza importante en hábitats de agua dulce en ríos, lagos y embalses, tanto en zonas tropicales como templadas de todo el mundo. Algunas investigaciones han indicado que se puede utilizar para la fitorremediación de agua, debido a su fuerte asimilación de nitrógeno y fósforo, y la acumulación de metales pesados, su crecimiento y propagación puede desempeñar un papel importante en la ecología ambiental. Con el fin de explorar el mecanismo molecular de respuesta a la deficiencia de nitrógeno en E. crassipes, se construyeron bibliotecas de cDNA mediante síntesis adelantada y retrasada para raíces de E. crassipes en condiciones de deficiencia de nitrógeno mediante el método de hibridación supresiva sustractiva (SSH). Para este estudio se utilizaron 2 100 clones de síntesis adelantada y 2 650 de síntesis retrasada. De la biblioteca se escogieron al azar mil clones, 737 (527 unigenes) de síntesis adelanta- da y 757 (483 unigenes) de síntesis retrasada que fueron informativos. El análisis BLASTX mostró que había más transportadores y proteínas adenosilhomocisteinasa en E. crassipes cultivadas en un medio deficiente de nitrógeno; mientras que las cultivadas en un medio repleto de nitróge- no tenían más proteínas como UBR4 e3 ubiquitina-proteína ligasa y la proteína arabinogalactano 8 tipo fasciclina, así como otras proteínas del citoesqueleto, incluyendo la actina y la tubulina. Clúster del Grupo Ortológico (COG) también demostró que en la biblioteca de síntesis adelan- tada, la mayoría de los marcadores de secuencia expresada (ESTs) estaban involucrados en el transporte de coenzimas y el metabolismo.
Subject(s)
Environmental Restoration and Remediation , Expressed Sequence Tags , Eichhornia/genetics , Genes, Plant , Nitrogen/metabolism , Phosphorus/metabolism , Eichhornia/classification , Eichhornia/metabolism , Gene Library , Nitrogen/deficiency , Polymerase Chain ReactionABSTRACT
OBJECTIVE: To investigate differential gene expression profiling of symbiotic germinated seeds of Dendrobium officinale. METHODS: cDNAs from 5-week symbiotic germinated seeds and 5-week aseptic cultivated seeds, taken as the tester and driver respectively, were used to construct a suppressive subtractive hybridization (SSH) cDNA library. RESULTS: By sequencing positive clones and BLASTx analysis against GenBank database, 100 expressed sequence tags (EST) homologous to plant known genes were obtained. Functional annotation revealed that they were grouped into serials of cellular processes including cell and chromosome structure, RNA synthesis, signal transduction, energy metabolism, protein synthesis and degradation, and cell defense, etc. Real-time quantitative RT-PCR analyses showed that the five randomly selected genes were all up-regulated in symbiotic germinated seeds. CONCLUSION: The symbiotic seed germination of D. officinale is involved in multiple pathways, and the results of this study will lay a foundation for further molecular elucidation of seed germination in Orchidaceae.
ABSTRACT
OBJECTIVE: To screen differentially expressed genes on the roots of Dendrobium candidum induced by a symbiotic mycorrhizal fungus, the differential cDNA library was constructed. METHODS: The ds cDNAs were synthesized and enriched by SMARTer PCR cDNA synthesis kit using total RNA of D. candidum roots symbiotic with the fungus as templates. The differential cDNA library was constructed by using suppressive subtraction hybridization (SSH). RESULTS: The differential cDNA library, containing 1975 clones in the storage capacity, was constructed successfully. It was detected that above 90% clones could be amplified effective products. The lengths of the differential cDNA fragments cloned were 150 bp to 1 kb by electrophoresis detection to 20 randomly selected clones. Then, the 20 ESTs similarity analysis based on BLASTx software was carried on and most of the cloned genes were the defensive genes of plant responding to environmental stresses. CONCLUSION: Using this technology system could construct a fine differential cDNA library and be operated easily, especially suitable to the rare species.
ABSTRACT
Objective To study the effects of the recipe for activating blood circulation and nourishing qi on expression of mitochondrial ribosomal protein L51 (MRPL51) in CVB3 infection model in rat cardiac myocytes, to reveal the pathogenesis of CVB3 myocarditis in the genetic level, to explore the therapeutic mechanism of the recipe for activating blood circulation and nourishing qi on CVB3 myocarditis, and to confirm the validity of the recipe for activating blood circulation and nourishing qi on CVB3 myocarditis. Methods After establishing CVB3 infection model and treatment model with recipe for activating blood circulation and nourishing qi by culturing neonatal rat myocardial cells, a modified suppression subtractive hybridization (SSH) was used to isolate differentially expressed genes between two model groups. These results were further verified by fluorescence RT-PCR. Results The results of SSH showed that gene expression of the treatment group was lower than that of the CVB3 infection group. The results of fluorescent RT-PCR which agreed with that of SSH displayed the threshold cycle number (Ct) in the treatment group was higher than the virus group. Conclusion Up-regulation of MRPL51 might be one of the pathogenesis of CVB3 myocarditis. The recipe for activating blood circulation and nourishing qi could treat viral myocarditis by regulating the expression of MRPL51.
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Suppression subtractive hybridization (SSH) libraries between cDNA in stages I (previtellogenic) and III (cortical rod) ovaries of the giant tiger shrimp (Penaeus monodon) were established. In all, 452 ESTs were unidirectionally sequenced. Sequence assembly generated 28 contigs and 201 singletons, 109 of which (48.0 percent) corresponding to known sequences previously deposited in GenBank. Several reproduction-related transcripts were identified. The full-length cDNA of anaphase promoting complex subunit 11 (PmAPC11; 600 bp with an ORF of 255 bp corresponding to a polypeptide of 84 amino acids) and selenoprotein M precursor (PmSePM; 904 bp with an ORF of 396 bp corresponding to a polypeptide of 131 amino acids) were characterized and reported for the first time in penaeid shrimp. Semiquantitative RT-PCR revealed that the expression levels of PmSePM and keratinocyte-associated protein 2 significantly diminished throughout ovarian development, whereas Ser/Thr checkpoint kinase 1 (Chk1), DNA replication licensing factor mcm2 and egalitarian were down-regulated in mature ovaries of wild P. monodon (p < 0.05). Accordingly, the expression profiles of PmSePM and keratinocyte-associated protein 2 could be used as biomarkers for evaluating the degree of reproductive maturation in domesticated P. monodon.
ABSTRACT
Human bone marrow mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tenden, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells could be isolated from marrow aspirates of human and animals. This study was designed to identify and characterize genes specifically expressed by osteogenic supplements-treated cells by suppression subtractive hybridization(SSH) method. The results were as follows: 1. 2 genes were upregulated genes in osteogenic diffeentiation of hMSCs, which is further proved by Northern blot analysis. 2. IGFBP-2 has been identified playing an important role in bone formation. 3. HF1 was also upregulated during osteogenic differentiation, but its role in bone formation is not clear yet.