Abstract:Jute (Corchorus capsularis), as a natural fibre producing plant species, ranks next to cotton only. Today, biotechnological approach has been considered as most accepted means for any genetic improvement of plant species. However, genetic control of the fibre development in jute has not yet been explored sufficiently for desired genetic improvement. One of the major impediments in exploring the genetic architecture in this crop at molecular level is the availability of good quality RNA from field-grown plant t… Show more
“…It is difficult to isolate RNA from jute tissues because of the nature that much mucilage and phenolics are present in jute. The important steps taken into consideration in this study were the application of CsCl isopycnic centrifugation to remove insoluble polysaccharides, and using polyvinylpyrrolidone (PVPP) to prevent oxidation of phenolics, as reported by Samanta et al (Samanta et al, 2011). The ratios of the optical density A260/230 and A260/280 were suggesting little contamination of polysaccharides and phenolics in isolated RNA, which was found to be appropriate for further downstream applications.…”
Section: Discussionmentioning
confidence: 82%
“…Quality of mRNA plays an important role in the construction of a full-length cDNA library, and high-quality mRNA is critical to the creation of full-length cDNA (Chen et al, 2012). Jute is a fairly primitive plant species somewhat different from those of other plant species (Samanta et al, 2011). It is difficult to isolate RNA from jute tissues because of the nature that much mucilage and phenolics are present in jute.…”
White jute (Corchorus capsularis L.) is recognized as an important industrial raw material fibre crop owing to its elite characters. However, little information is known about its molecular basis and genomics. In this study, a complementary DNA library of white jute was constructed and expressed sequence tags (ESTs) were characterized. The titers of original and amplified libraries were 2.32 × 10 7 and 1.07 × 10 9 pfu/mL, respectively. The recombinant frequency was 98.3% in the library. Most of the sequences ranged from 500 to 1500 bp with an average length of 750 bp. Results show 203 (73%) ESTs exhibited significant similarity with known or putative functional nucleotide sequences in the GenBank databases. Cluster analysis allowed the identification of 61 unique sequences. These genes were classified into six types by Gene Ontology (GO) annotation. The results also indicated that unigenes of C. capsularis have higher homology to Populus trichocarpa, Ricinus communis and Corchorus olitorius. This report will provide a valuable resource for the further investigations in the gene cloning, transcription or expression for white jute.
“…It is difficult to isolate RNA from jute tissues because of the nature that much mucilage and phenolics are present in jute. The important steps taken into consideration in this study were the application of CsCl isopycnic centrifugation to remove insoluble polysaccharides, and using polyvinylpyrrolidone (PVPP) to prevent oxidation of phenolics, as reported by Samanta et al (Samanta et al, 2011). The ratios of the optical density A260/230 and A260/280 were suggesting little contamination of polysaccharides and phenolics in isolated RNA, which was found to be appropriate for further downstream applications.…”
Section: Discussionmentioning
confidence: 82%
“…Quality of mRNA plays an important role in the construction of a full-length cDNA library, and high-quality mRNA is critical to the creation of full-length cDNA (Chen et al, 2012). Jute is a fairly primitive plant species somewhat different from those of other plant species (Samanta et al, 2011). It is difficult to isolate RNA from jute tissues because of the nature that much mucilage and phenolics are present in jute.…”
White jute (Corchorus capsularis L.) is recognized as an important industrial raw material fibre crop owing to its elite characters. However, little information is known about its molecular basis and genomics. In this study, a complementary DNA library of white jute was constructed and expressed sequence tags (ESTs) were characterized. The titers of original and amplified libraries were 2.32 × 10 7 and 1.07 × 10 9 pfu/mL, respectively. The recombinant frequency was 98.3% in the library. Most of the sequences ranged from 500 to 1500 bp with an average length of 750 bp. Results show 203 (73%) ESTs exhibited significant similarity with known or putative functional nucleotide sequences in the GenBank databases. Cluster analysis allowed the identification of 61 unique sequences. These genes were classified into six types by Gene Ontology (GO) annotation. The results also indicated that unigenes of C. capsularis have higher homology to Populus trichocarpa, Ricinus communis and Corchorus olitorius. This report will provide a valuable resource for the further investigations in the gene cloning, transcription or expression for white jute.
“… Fig. 2 Integrity and separation of RNA samples isolated from tossa jute stem tissue (3 days old seedling stage) on agarose gel: a using modified SDS/Phenol method; b using SDS/Phenol method (Mahmood et al 2011 ); and c using HBIC method (Samanta et al 2011 ) Fig. 3 Integrity and separation of RNA samples isolated from tossa jute bark tissue (at 65 days after sowing) on agarose gel: a using SDS/Phenol method (Mahmood et al 2011 ); b using HBIC method (Samanta et al 2011 ); and c using modified SDS/Phenol method …”
Section: Methodsmentioning
confidence: 99%
“… Integrity and separation of RNA samples isolated from tossa jute stem tissue (3 days old seedling stage) on agarose gel: a using modified SDS/Phenol method; b using SDS/Phenol method (Mahmood et al 2011 ); and c using HBIC method (Samanta et al 2011 ) …”
Section: Methodsmentioning
confidence: 99%
“…This problem is particularly acute in case of tossa jute bark rich in mucilage; a highly acidic and proteinaceous compound (Stephen et al 2006 ). Mucilage often binds to other secondary metabolites, co-precipitates with nucleic acids during extraction (Samanta et al 2011 ) and thereby adversely affect downstream operations like gene expression analysis (Mahmood et al 2011 ). Jute plants are also rich in phenolic compounds (Oboh et al 2012 ) that produce quinones upon oxidization and hinder RNA isolation and/or downstream applications by binding with RNA (Loomis 1974 ).…”
Tossa jute is an important natural fiber crop of Southeast Asian countries including India, Bangladesh, China, Thailand, Myanmar etc. Traditional industrial application of jute fiber is limited to the packaging products like hessians, sacks, etc. and the fiber found unsuitable for textile industries largely due to significantly high lignin content. Therefore, understanding genetic factors underlying lignin biosynthesis in tossa jute holds promise for jute based product diversification. The major limiting factor in undertaking such study is unavailability of efficient protocol for RNA extraction at secondary growth active stage of tossa jute. Here we report a simplified, swift and cost effective protocol for isolating fairly good quality RNA from bark tissue of 65-days-old field grown tossa jute plant with active secondary growth. The purity, concentration and integrity of extracted RNA ascertained. To confirm downstream amenability, isolated RNA samples were reverse transcribed and PCR analysis done by using CCoAMT1 primer. Results established that method of RNA extraction presented here is an improvement over the other methods, particularly for bark tissue of field grown tossa jute at advance developmental stage. Therefore, present study will enhance our ability to understand expression pattern of fiber formation and maturation related genes in mature bark tissue that holds key for much talked genetic manipulation of fiber quality via lignin optimisation in the crop.
The present study documented the predominant role of WRKY transcription factor in controlling genes of different pathways related to fibre formation in jute and could be a candidate gene for the improvement of jute fiber. Understanding of molecular mechanism associated with bast fibre development is of immense significance to achieve desired improvement in jute (Corchorus sp.). Therefore, suppression subtractive hybridization was successfully applied to identify genes involved in fibre developmental process in jute. The subtracted library of normal Corchorus capsularis as tester with respect to its fibre-deficient mutant as driver resulted in 2,685 expressed sequence tags which were assumed to represent the differentially expressed genes between two genotypes. The identified expressed sequence tags were assembled and clustered into 225 contigs and 231 singletons. Among these 456 unigenes, 377 were classified into 15 different functional categories while others were of unknown functional category. Reverse Northern analysis of the unigenes showed distinct variation in hybridization intensity of 11 transcripts between two genotypes tested. The findings were also documented by Northern and real-time PCR analysis. Varied expression level of these transcripts suggested their crucial involvement in fibre development in this species. Among these transcripts, WRKY transcription factor was documented to be a most important transcript which was in agreement with its known role in other plant species in possible regulation related to cell wall biosynthesis, expansion and lignification. This report constitutes first systematic analysis of genes involved in fibre development process in jute. It may be suggested that the information generated in this study would be useful for genetic improvement of fibre traits in this plant species.
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