scholarly journals Functional Identification of Salt-Stress-Related Genes Using the FOX Hunting System from Ipomoea pes-caprae

2018 ◽  
Vol 19 (11) ◽  
pp. 3446 ◽  
Author(s):  
Mei Zhang ◽  
Hui Zhang ◽  
Jie-Xuan Zheng ◽  
Hui Mo ◽  
Kuai-Fei Xia ◽  
...  
Keyword(s):  
Salt Stress ◽  
Salt Tolerance ◽  
Cdna Library ◽  
Molecular Mechanisms ◽  
Full Length ◽  
Cdna Clones ◽  
Functional Screening ◽  
Yeast Mutant ◽  
Full Length Cdna ◽  
Stress Related Genes

Ipomoea pes-caprae is a seashore halophytic plant and is therefore a good model for studying the molecular mechanisms underlying salt and stress tolerance in plant research. Here, we performed Full-length cDNA Over-eXpressor (FOX) gene hunting with a functional screening of a cDNA library using a salt-sensitive yeast mutant strain to isolate the salt-stress-related genes of I. pes-caprae (IpSR genes). The library was screened for genes that complemented the salt defect of yeast mutant AXT3 and could grow in the presence of 75 mM NaCl. We obtained 38 candidate salt-stress-related full-length cDNA clones from the I. pes-caprae cDNA library. The genes are predicted to encode proteins involved in water deficit, reactive oxygen species (ROS) scavenging, cellular vesicle trafficking, metabolic enzymes, and signal transduction factors. When combined with the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, several potential functional salt-tolerance-related genes were emphasized. This approach provides a rapid assay system for the large-scale screening of I. pes-caprae genes involved in the salt stress response and supports the identification of genes responsible for the molecular mechanisms of salt tolerance.

scholarly journals A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

Viruses ◽  
2018 ◽  
Vol 10 (7) ◽  
pp. 368 ◽  
Author(s):  
Maximilian Münster ◽  
Anna Płaszczyca ◽  
Mirko Cortese ◽  
Christopher Neufeldt ◽  
Sarah Goellner ◽  
...  
Keyword(s):  
Zika Virus ◽  
In Silico ◽  
Reverse Genetics ◽  
Molecular Mechanisms ◽  
Full Length ◽  
Cdna Clones ◽  
In Silico Prediction ◽  
Fluorescent Reporter ◽  
Full Length Cdna ◽  
Spurious Transcription

The Zika virus (ZIKV) has recently attracted major research interest as infection was unexpectedly associated with neurological manifestations in developing foetuses and with Guillain-Barré syndrome in infected adults. Understanding the underlying molecular mechanisms requires reverse genetic systems, which allow manipulation of infectious cDNA clones at will. In the case of flaviviruses, to which ZIKV belongs, several reports have indicated that the construction of full-length cDNA clones is difficult due to toxicity during plasmid amplification in Escherichia coli. Toxicity of flaviviral cDNAs has been linked to the activity of cryptic prokaryotic promoters within the region encoding the structural proteins leading to spurious transcription and expression of toxic viral proteins. Here, we employ an approach based on in silico prediction and mutational silencing of putative promoters to generate full-length cDNA clones of the historical MR766 strain and the contemporary French Polynesian strain H/PF/2013 of ZIKV. While for both strains construction of full-length cDNA clones has failed in the past, we show that our approach generates cDNA clones that are stable on single bacterial plasmids and give rise to infectious viruses with properties similar to those generated by other more complex assembly strategies. Further, we generate luciferase and fluorescent reporter viruses as well as sub-genomic replicons that are fully functional and suitable for various research and drug screening applications. Taken together, this study confirms that in silico prediction and silencing of cryptic prokaryotic promoters is an efficient strategy to generate full-length cDNA clones of flaviviruses and reports novel tools that will facilitate research on ZIKV biology and development of antiviral strategies.

scholarly journals Isolation of Salt Stress-Related Genes fromAspergillus glaucusCCHA by Random Overexpression inEscherichia coli

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Jie Fang ◽  
Xiaojiao Han ◽  
Lihua Xie ◽  
Mingying Liu ◽  
Guirong Qiao ◽  
...  
Keyword(s):  
Salt Stress ◽  
Salt Tolerance ◽  
Cdna Library ◽  
Chlorophyll Biosynthesis ◽  
Salinity Range ◽  
Regulation Of Transcription ◽  
Control Line ◽  
E Coli ◽  
Halotolerant Fungus ◽  
Stress Related Genes

The halotolerant fungusAspergillus glaucusCCHA was isolated from the surface of wild vegetation around a saltern with the salinity range being 0–31%. Here, a full-length cDNA library ofA. glaucusunder salt stress was constructed to identify genes related to salt tolerance, and one hundred clones were randomly selected for sequencing and bioinformatics analysis. Among these, 82 putative sequences were functionally annotated as being involved in signal transduction, osmolyte synthesis and transport, or regulation of transcription. Subsequently, the cDNA library was transformed intoE. colicells to screen for putative salt stress-related clones. Five putative positive clones were obtained fromE. colicells grown on LB agar containing 1 M NaCl, on which they showed rapid growth compared to the empty vector control line. Analysis of transgenicArabidopsis thalianalines overexpressingCCHA-2142demonstrated that the gene conferred increased salt tolerance to plants as well by protecting the cellular membranes, suppressing the inhibition of chlorophyll biosynthesis. These results highlight the utility of thisA. glaucuscDNA library as a tool for isolating and characterizing genes related to salt tolerance. Furthermore, the identified genes can be used for the study of the underlying biology of halotolerance.

Establishment and Identification of a Normalized Full-Length cDNA Library of CCRI 36

2009 ◽  
Vol 35 (4) ◽  
pp. 602-607 ◽  
Author(s):  
Dong WU ◽  
Jun-Jie LIU ◽  
Shu-Xun YU ◽  
Shu-Li FAN ◽  
Mei-Zhen SONG
Keyword(s):  
Cdna Library ◽  
Full Length ◽  
Full Length Cdna

Construction of a Full-Length cDNA Library of Gossypium hirsutum L. and Identification of Two MADS-Box Genes

2011 ◽  
Vol 10 (1) ◽  
pp. 28-40 ◽  
Author(s):  
Li-na WANG ◽  
Dong WU ◽  
Shu-xun YU ◽  
Shu-li FAN ◽  
Mei-zhen SONG ◽  
...  
Keyword(s):  
Gossypium Hirsutum ◽  
Cdna Library ◽  
Full Length ◽  
Mads Box ◽  
Mads Box Genes ◽  
Gossypium Hirsutum L ◽  
Full Length Cdna

Cloning and characterization of full-length cDNA clones encoding chalcone synthase from the orchid Bromheadia finlaysoniana

1998 ◽  
Vol 36 (9) ◽  
pp. 647-656 ◽  
Author(s):  
Chye-Fong Liew ◽  
Chong-Jin Goh ◽  
Chiang-Shiong Loh ◽  
Saw-Hoon Lim
Keyword(s):  
Chalcone Synthase ◽  
Full Length ◽  
Cdna Clones ◽  
Full Length Cdna

scholarly journals Generation and Analysis of Expressed Sequence Tags (ESTs) from Muscle Full-Length cDNA Library of Wujin Pig

2014 ◽  
Vol 13 (2) ◽  
pp. 378-386
Author(s):  
Su-mei ZHAO ◽  
Yong-gang LIU ◽  
Hong-bing PAN ◽  
Xi ZHANG ◽  
Chang-rong GE ◽  
...  
Keyword(s):  
Cdna Library ◽  
Expressed Sequence Tags ◽  
Full Length ◽  
Full Length Cdna ◽  
Expressed Sequence

scholarly journals Physiological and transcriptomic analyses reveal the mechanisms underlying the salt tolerance of Zoysia japonica Steud.

2020 ◽  
Author(s):  
Jingjing Wang ◽  
Cong An ◽  
Hailin Guo ◽  
Xiangyang Yang ◽  
Jingbo Chen ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Salt Stress ◽  
Stress Response ◽  
Salt Tolerance ◽  
Molecular Mechanisms ◽  
Salt Tolerant ◽  
Zoysia Japonica ◽  
Salt Stress Response ◽  
Leaves And Roots ◽  
Time Point

Abstract Background: Areas with saline soils are sparsely populated and have fragile ecosystems, which severely restricts the sustainable development of local economies. Zoysia grasses are recognized as excellent warm-season turfgrasses worldwide, with high salt tolerance and superior growth in saline-alkali soils. However, the mechanism underlying the salt tolerance of Zoysia species remains unknown. Results: The phenotypic and physiological responses of two contrasting materials, Zoysia japonica Steud. Z004 (salt sensitive) and Z011 (salt tolerant) in response to salt stress were studied. The results show that Z011 was more salt tolerant than was Z004, with the former presenting greater K+/Na+ ratios in both its leaves and roots. To study the molecular mechanisms underlying salt tolerance further, we compared the transcriptomes of the two materials at different time points (0 h, 1 h, 24 h, and 72 h) and from different tissues (leaves and roots) under salt treatment. The 24-h time point and the roots might make significant contributions to the salt tolerance. Moreover, GO and KEGG analyses of different comparisons revealed that the key DEGs participating in the salt-stress response belonged to the hormone pathway, various TF families and the DUF family. Conclusions: Z011 may have improved salt tolerance by reducing Na+ transport from the roots to the leaves, increasing K+ absorption in the roots and reducing K+ secretion from the leaves to maintain a significantly greater K+/Na+ ratio. Twenty-four hours might be a relatively important time point for the salt-stress response of zoysiagrass. The auxin signal transduction family, ABA signal transduction family, WRKY TF family and bHLH TF family may be the most important families in Zoysia salt-stress regulation. This study provides fundamental information concerning the salt-stress response of Zoysia and improves the understanding of molecular mechanisms in salt-tolerant plants.

scholarly journals Infectious Transcripts from Cloned Genome-Length cDNA of Porcine Reproductive and Respiratory Syndrome Virus

1998 ◽  
Vol 72 (1) ◽  
pp. 380-387 ◽  
Author(s):  
J. J. M. Meulenberg ◽  
J. N. A. Bos-de Ruijter ◽  
R. van de Graaf ◽  
G. Wensvoort ◽  
R. J. M. Moormann
Keyword(s):  
Cdna Clone ◽  
De Novo ◽  
Infectious Clone ◽  
Full Length ◽  
Respiratory Syndrome Virus ◽  
Cdna Clones ◽  
Genome Length ◽  
Full Length Cdna ◽  
Lung Macrophages ◽  
Growth Properties

ABSTRACT The 5′-terminal end of the genomic RNA of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was determined. To construct full-length cDNA clones, the 5′-terminal sequence was ligated to cDNA clones covering the complete genome of LV. When RNA that was transcribed in vitro from these full-length cDNA clones was transfected into BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells, no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. Two nucleotide changes leading to a unique PacI restriction site directly downstream of the ORF7 gene were introduced in the genome-length cDNA clone. The virus recovered from this mutated cDNA clone retained the PacI site, which confirmed the de novo generation of infectious LV from cloned cDNA. These results indicate that the infectious clone of LV enables us to mutagenize the viral genome at specific sites and that it will therefore be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.

Sign in / Sign up

Export Citation Format

Share Document