Rapid RNA Extraction from Eucalyptus tree and its down processing for cloning of dehydrin genes

Background: RNA extraction from tree species like Eucalyptus is very tedious and difficult task. In this study a very short and efficient method of RNA extraction from Eucalyptus has been described.Methods: A very short and efficient protocol of two steps RNA extraction has been optimized for obtaining high quality and pure RNA from different tissue types of Eucalyptus tree. In first step whole nucleic acid was extracted from plant tissues and in second step RNA was purified from nucleic acid mixture. The newly optimized rapid CTAB RNA extraction method was compared with trizol extraction method for efficiency and quality of extracted RNA.Results: The newly optimized rapid CTAB RNA extraction method was found highly efficient and suitable over the trizol method. Three Eucalyptus dehydrin genes the dehydrin-10 (DHN-10), dehydrin-1 (DHN-1), and dehydrin-2 (DHN-2) were successfully amplified, TA cloned into pTZ57/RT vector, and transformed into Top10F’ strain of E.coli. These three Eucalyptus dehydrin genes have been reported for conferring abiotic stress tolerance to the Eucalyptus plant yet have not been reported to be cloned. These cloned genes would be further manipulated for developing abiotic stress tolerance in plants of interest.Conclusion: Rapid CTAB RNA extraction method is a brief and reproducible methodology for a hard job of RNA extraction from tree plants with high phenolic contents. Keywords: Dehydrin proteins, Eucalyptus abiotic stress tolerance proteins, cloning of dehydrin genes, DHN-1, DHN-2, DHN-1

Thyroid hormone dependent gene expression

The presented work is destined to review the advances that had been made to study the role of thyroid hormone and thyroid hormone nuclear receptors in regulating the gene expression. Triiodothyronine (T3) and tetraiodothyronine (thyroxine or T4) are most important thyroid hormones. The thyroid hormones bind to their specific nuclear hormone receptors, as ligand, and play important role in gene expression and transcriptional gene regulation in human and higher animals. Thyroid hormone receptors form heterodimers by making combination with retinoid X receptors. The capability of heterodimerization of thyroid hormones generates novel complexes which allow altered specificity and higher affinity for DNA-receptor binding. Thyroid hormone receptors work as ligand activated transcription factor and play with transcriptional gene expression process. The consensus structural features of thyroid hormone receptors are N-terminal regulatory domain that contains activation function, the domain for strong gene expression and the domain for binding to DNA. The structures for individual domains have been extensively and reviewed through several latest and successful techniques.

Disease free and rapid mass production of sugarcane cultivars

Background: Sacchrum officinarum is acknowledged as a basic source for the production of sugar in Pakistan and worldwide, one of the major constituents of human diet. The presented study optimizes a convenient and successful protocol for in-vitro mass production of sugarcane comprising sixteen cultivars from various sugarcane growing areas all over Pakistan.Methods: The source plants were sampled randomly from cane growing areas all over the country. Apical region from stalks of germinated plants was taken as explant source. The growth medium used for direct regeneration and multiple shoot formation was same for all constituents but the hormonal supplementations; it comprised of MS basal medium 4.43g/l (MS SIGMA, M 5519), 3% w/v sucrose, and phytagel was added in 0.3% w/v for gelling to support the explant, 0.01mg/l activated charcoal as the carbon source, pH 5.5 to 5.8.Results: Among various concentrations of BAP used 1.0, 1.5 and 2.0mg/l in growth medium supported efficient regeneration and plenty of lateral shoots in a minimum time span almost in all cultivars. For rhizogenesis, 5.0mg/l of IAA was found to be most efficient among four different concentrations of auxin. Some cultivars have a sufficient endoauxin level and do not need any supplementation for rooting i.e., basal medium supports root induction. For long term maintenance of plant stock, MS broth with 1.0mg/l of BAP was found to be most suitable.Conclusion: Cytokinin concentrations and plant potential play an equal role in direct regeneration from meristematic tissue

Rapid RNA Extraction from Eucalyptus tree and its down processing for cloning of dehydrin genes

Background: RNA extraction from tree species like Eucalyptus is very tedious and difficult task. In this study a very short and efficient method of RNA extraction from Eucalyptus has been described. Methods: A very short and efficient protocol of two steps RNA extraction has been optimized for obtaining high quality and pure RNA from different tissue types of Eucalyptus tree. In first step whole nucleic acid was extracted from plant tissues and in second step RNA was purified from nucleic acid mixture. The newly optimized rapid CTAB RNA extraction method was compared with trizol extraction method for efficiency and quality of extracted RNA. Results: The newly optimized rapid CTAB RNA extraction method was found highly efficient and suitable over the trizol method. Three Eucalyptus dehydrin genes the dehydrin-10 (DHN-10), dehydrin-1 (DHN-1), and dehydrin-2 (DHN-2) were successfully amplified, TA cloned into pTZ57/RT vector, and transformed into Top10F’ strain of E.coli. These three Eucalyptus dehydrin genes have been reported for conferring abiotic stress tolerance to the Eucalyptus plant yet have not been reported to be cloned. These cloned genes would be further manipulated for developing abiotic stress tolerance in plants of interest. Conclusion: Rapid CTAB RNA extraction method is a brief and reproducible methodology for a hard job of RNA extraction from tree plants with high phenolic contents