Construction and Characterization of a Goat Mammary Gland cDNA Library

Han, Xue; Luo, Jun; Wu, Ning; Matand, Kanyand; Yang, Bao Jin; Wu, Hui; Zhang, Li Juan; Wang, Hai Bin

doi:10.1007/s12033-007-9020-9

Cited by 6 publications

(3 citation statements)

References 20 publications

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“…A high-quality expression cDNA library can provide molecular resources for analyzing the genes involved in biology and studying their protein functions and interactions facilitate the constructing of the protein network stemming from a known protein. cDNA libraries have been constructed for many plant species ( Lee et al, 2012 ; Ma et al, 2009 ), animals ( Han et al, 2008 ; Ma et al, 2011 ) and microorganisms ( Wang and Chen, 2011 ; Zhao et al, 2009 ) and genes related to biological processes have been determined. Also, genes related to the biology of fungal pathogens, such as pathogen invasion, development, survival, pathogenicity and virulence can be discovered with a cDNA library.…”

Section: Discussionmentioning

confidence: 99%

Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata

Gao

Jing

et al. 2015

The Plant Pathology Journal

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Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5′ end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about 6.39 ×105 transformants/3 μg pGADT7-Rec. The titer of the primary cDNA library was 2.5×108 cfu/mL. The numbers for the cDNA library was 2.46×105. Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a “bait” to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.

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Section: Discussionmentioning

confidence: 99%

Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata

Gao

Jing

et al. 2015

The Plant Pathology Journal

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“…Total cellular RNA was isolated from mouse C57BL/6J brain (male) by modification of a standard procedure [4,5]. Briefly, 1 g tissue was ground in liquid nitrogen and homogenized in 5 mL solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate pH 7.0, 0.5% sarkosyl, 0.1 M 2-mercaptoethanol) using a Polytron PT3000 homogenizer (Brinkmann) followed by addition of 500 μL 2 M sodium acetate pH 4.0.…”

Section: Total Rna Isolationmentioning

confidence: 99%

“…The mouse brain standard cDNA library and fulllength cDNA library were constructed using our previously reported method [4][5][6]. Purified regular mRNAs (1 μg) and unknown amount of purified full-length mRNAs were primed by adding 200 ng biotinylated oligo-dT primer with an integrated…”

Section: Full-length and Standard Cdna Library Constructionmentioning

confidence: 99%

Mouse brain full‐length cDNA library construction by negative selection of intact mRNAs

Li³

et al. 2010

Biotechnology Journal

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Synthesis of full-length cDNA libraries is an essential step for the study of gene function. The method for selecting the intact mRNA directly affects the number of full-length transcripts. We have developed a novel method for intact mRNA selection based on the elimination of uncapped mRNAs. A negative-selection strategy that removes both uncapped mRNA and other non-mRNA molecules that present a phosphate at the 5'-end has been applied in the mRNA purification procedures. Briefly, after performing a standard mRNA purification, a biotinylated oligoribonucleotide is ligated to the 5-end phosphate of uncapped mRNAs. Streptavidin extraction is then performed to remove truncated and non-mRNAs from the intact mRNAs. By comparing random sequencing results of mouse brain full-length and standard cDNA libraries, there was a significant increase of full-length clones with the modified procedure. The results showed that the full-length library contained more than 68% full-length clones with the 5'-end positions ranging between -485 to +100 compared to the standard library with 33% of full-length clones and 5'-end positions ranging between -233 to +100. The data were analyzed using the t-test with the significance level set at p<0.05. The statistical results showed that there were significant differences between two libraries in both 5'-end position and mRNA size (p<0.05).

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Screening and structural and functional investigation of a novel ferritin from Phascolosoma esculenta

et al. 2017

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Ferritins are primary iron storage proteins and play a crucial role in iron storage and detoxification. Yeast two-hybrid method was employed to screen the cDNA library of Phascolosoma esculenta. Sequence of positive colony FER147 was analyzed. The higher similarity and conserved motifs for ferritin indicated that it belonged to a new member of ferritin family. The interaction between Ferritin and Fer147 was further confirmed through co-immunoprecipitation. The pET-28a-FER147 prokaryotic expression vector was constructed. The expressed recombinant Fer147 was then isolated, purified, and refolded. When ferritins were treated by different heavy metals, several detection methods, including scanning electron microscopy (SEM), circular dichroism (CD), and inductively coupled plasma-mass spectrometry (ICP-MS) were applied to examine the structures and functions of the new protein Fer147, recombinant P. esculenta ferritin (Rferritin), and natural horse-spleen ferritin (Hferritin). SEM revealed that the three ferritin aggregates changed obviously after different heavy metals treatment, meanwhile, a little different in aggregates were detected when the ferritins were trapped by the same heavy metal. Hence, changes in aggregation structure of the three proteins are related to the nature of the different heavy metals and the interaction between the heavy metals and the three ferritins. CD data suggested that the secondary structure of the three ferritins hardly changed after different heavy metals were trapped. ICP-MS revealed that the ferritins exhibit different enrichment capacities for various heavy metals. In particular, the enrichment capacity of the recombinant Fer147 and Rferritin is much higher than that of hferritin.

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Construction and Characterization of a Goat Mammary Gland cDNA Library

Cited by 6 publications

References 20 publications

Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata

Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata

Mouse brain full‐length cDNA library construction by negative selection of intact mRNAs

Screening and structural and functional investigation of a novel ferritin from Phascolosoma esculenta

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