Bushra Rashid

Identification of micro-RNAs in cotton.

The plant genome has conserved small non-coding microRNAs (miRNAs) genes about 20-24 nucleotides long. They play a vital role in the gene regulation at various stages of plant life. Their conserved nature among the various organisms not only suggests their early evolution in eukaryotes but also makes them a good source of new miRNA discovery by homology search using bioinformatics tools. A systematic search approach was used for interspecies orthologues of miRNA precursors, from known sequences of Gossypium in GenBank. The study resulted in 22 miRNAs belonging to 13 families. We found 7 miRNA families (miR160, 164, 827, 829, 836, 845 and 865) for the first time in cotton. All 22 miRNA precursors form stable minimum free energy (mfe) stem loop structure as their orthologues form in Arabidopsis and the mature miRNAs reside in the stem portion of the stem loop structure. Fifteen miRNAs belong to the worlds most commercial fiber producing upland cotton (Gossypium hirsutum), five are from Gossypium raimondii and one each is from Gossypium herbaceum and Gossypium arboreum. Their targets consist of transcription factors, cell division regulating proteins and virus response gene. The discovery of 22 miRNAs will be helpful in future for detection of precise function of each miRNA at a particular stage in life cycle of cotton.

Transformation and inheritance of Bt genes inGossypium hirsutum

Transgenic plants offer many unique opportunities for managing pest populations. However, the inheritance, integration, and expression of multiple transgenes are prerequisite for maintaining sustainable resistance against insects in crops. We took a gene-pyramiding approach to produce Bt cotton expressing two Bt genes,cry1Ac andcry2A. Using sonication-assistedAgrobacterium-mediated transformation (SAAT), we achieved an efficiency of 6.26%. Putative transgenic plants were confirmed via PCR, Southern hybridization, and western-blotting. Those showing mortality of 75 to 100% for the second instar ofHeliothis armigera (compared with 0% for the control) were considered Bt-positive. Transgenes were segregated according to a 3:1 Mendelian inheritance pattern in the T1 generation forHeliothis resistance. In our insect bioassay, the control plants showed >95% leaf damage, and insects reached the 4th instar stage of larval growth. In contrast, leaf damage on transgenic plants was limited to only a few bites, and insect mortality was 75 to 100%. ELISA confirmed transgene expression, and Bt protein was detected in leaf tissue. This performance was consistent with that of the parent transgenics. PCR and Southern blots verified integration of thecry1Ac andcry2A genes into the progeny. Therefore, this strategy provides a pathway toward cotton improvement and the development of durable resistance against insect damage.

Identification and expression of six drought-responsive transcripts through differential display in desi cotton (Gossypium arboreum)

There is not enough information available on drought-modulated gene(s) in Gossypium arboreum, which can be a valuable gene pool for improving modern cotton cultivars. In the present work, differential display reverse transcriptase PCR (DDRT) was used to compare overall differences in gene expression between water-stressed and control plants. By screening 93 primer-pair combinations, the DDRT technique resulted in upregulation of 30 cDNA transcripts. Through reamplification and quality control assay, ten cDNA transcripts appeared false positive. The remaining 20-cDNA transcripts were extracted from the gel, reamplified, cloned and sequenced. Homology search revealed that six transcripts showed significant homology with known genes. Real-time RT-PCR showed that, among six transcripts, five showed significant overexpression in water-stressed leaves as compared to control. This is an important finding since there are only few reports of universal stress protein, and transposable elements are available in plants but none in cotton under drought condition.

Isolation and functional analysis of cotton universal stress protein promoter in response to phytohormones and abiotic stresses.

The 949 bp promoter fragment upstream from the translation initiation site of the GUSP gene encoding a universal stress protein was isolated from the genomic DNA of Gossypium arboreum. Some putative cis-acting elements involved in stress responses including E-box, ABRE, DPBF-box, and MYB-core elements were found in the promoter region. In an Agrobacterium-mediated transient expression assay, strong activation of the GUSP full promoter region occurred in tobacco leaves following dehydration, abscisic acid, salt, heavy metal, gibberellic acid and dark treatments. Deletion analysis of the promoter revealed that the dehydration, abscisic acid and salt responses were affected by the deletion between −208 and −949 bp and showed 2–4-fold induction. However, in response to dark, gibberellic acid and heavy metals the induction was only 2-fold. These findings further our understanding of the regulation of GUSP expression. This is an important study as no report of this universal stress protein promoter is available in literature.

Herbicides and Pesticides as Potential Pollutants: A Global Problem

Herbicides and pesticides have been used to control, eliminate or destroy pests in order to protect human being’s food. This technology could be economical and effective if the selection of herbicides and pesticides is based on its mode of action, chemical nature, method and time of application and nature of crop. They have been extensively studied for their toxic potential to biological systems. Herbicides and pesticides are gradually more water soluble, polar and heat stable, therefore it is difficult to reduce their lethalness and to fade away them from the atmosphere. They are highly selective, and found to be toxic to a number of people in industry, agriculture and public health work places. They have harmful effects directly or indirectly on soil, environment, surface and ground water natural flora and fauna, aquatic life which will ultimately adversely influence the human beings and livestock. So, likely impact of herbicides and pesticides on atmosphere and community health is of great significance regardless of their noticeable benefits. It is likely to reduce the selection of pest resistance by preventing the contact between pesticide which act in a particular way and the pest population and to subsequently apply pesticides from diverse classes of compounds having dissimilar modes of accomplishment. Integrated Pest Management (IPM) is intended to protect the maximum likely risks to agriculture as well as environment by using cost-effective measures and pest management will prolong for improvements with the advent of new and improved technologies.

Identification, sequencing and characterization of a stress induced homologue of fructose bisphosphate aldolase from cotton

A drought-induced homologue of fructose-1, 6 bisphosphate aldolase full length cDNA (GaAldp) was isolated and sequenced by gene homology and rapid amplification of cDNA ends (RACE) from cotton variety FDH-786 (Gossypium arboretum). Quantitative polymerase chain reaction (PCR) analysis showed that drought stress, salinity and exogenous treatment with abscisic acid (ABA) enhanced the accumulation of GaAldp transcripts in the leaf and stem tissues of the plant. An alignment of the 1413 bp cDNA and 1937 bp genomic DNA sequences revealed that GaAldp has an open reading frame (ORF) encoding 394 amino acids and contains five introns. The predicted amino acid sequence is homologous to a heat-induced isoform of oat chlorplastic fructose bisphosphate aldolase (FBP) (AF216582) and NPALDP1 from Nicotiana paniculata. The GaAldp sequence includes a novel stroma targeting N-terminal transit peptide (TP) of 45 amino acids, which is 63-76% similar to other chloroplastic TPs.Key words: Fructose-1, 6 bisphosphate aldolase, ...

Transformation and Evaluation of Cry1Ac+Cry2A and GTGene in Gossypium hirsutum L.

More than 50 countries around the globe cultivate cotton on a large scale. It is a major cash crop of Pakistan and is considered “white gold” because it is highly important to the economy of Pakistan. In addition to its importance, cotton cultivation faces several problems, such as insect pests, weeds, and viruses. In the past, insects have been controlled by insecticides, but this method caused a severe loss to the economy. However, conventional breeding methods have provided considerable breakthroughs in the improvement of cotton, but it also has several limitations. In comparison with conventional methods, biotechnology has the potential to create genetically modified plants that are environmentally safe and economically viable. In this study, a local cotton variety VH 289 was transformed with two Bt genes (Cry1Ac and Cry2A) and a herbicide resistant gene (cp4 EPSPS) using the Agrobacterium mediated transformation method. The constitutive CaMV 35S promoter was attached to the genes taken from Bacillus thuringiensis (Bt) and to an herbicide resistant gene during cloning, and this promoter was used for the expression of the genes in cotton plants. This construct was used to develop the Glyphosate Tolerance Gene (GTGene) for herbicide tolerance and insecticidal gene (Cry1Ac and Cry2A) for insect tolerance in the cotton variety VH 289. The transgenic cotton variety performed 85% better compared with the non-transgenic variety. The study results suggest that farmers should use the transgenic cotton variety for general cultivation to improve the production of cotton.

Prediction of Host-Derived miRNAs with the Potential to Target PVY in Potato Plants

Potato virus Y has emerged as a threatening problem in all potato growing areas around the globe. PVY reduces the yield and quality of potato cultivars. During the last 30 years, significant genetic changes in PVY strains have been observed with an increased incidence associated with crop damage. In the current study, computational approaches were applied to predict Potato derived miRNA targets in the PVY genome. The PVY genome is approximately 9 thousand nucleotides, which transcribes the following 6 genes:CI, NIa, NIb-Pro, HC-Pro, CP, and VPg. A total of 343 mature miRNAs were retrieved from the miRBase database and were examined for their target sequences in PVY genes using the minimum free energy (mfe), minimum folding energy, sequence complementarity and mRNA-miRNA hybridization approaches. The identified potato miRNAs against viral mRNA targets have antiviral activities, leading to translational inhibition by mRNA cleavage and/or mRNA blockage. We found 86 miRNAs targeting the PVY genome at 151 different sites. Moreover, only 36 miRNAs potentially targeted the PVY genome at 101 loci. The CI gene of the PVY genome was targeted by 32 miRNAs followed by the complementarity of 26, 19, 18, 16, and 13 miRNAs. Most importantly, we found 5 miRNAs (miR160a-5p, miR7997b, miR166c-3p, miR399h, and miR5303d) that could target the CI, NIa, NIb-Pro, HC-Pro, CP, and VPg genes of PVY. The predicted miRNAs can be used for the development of PVY-resistant potato crops in the future.

Microarray: gateway to unravel the mystery of abiotic stresses in plants

Environmental factors, such as drought, salinity, extreme temperature, ozone poisoning, metal toxicity etc., significantly affect crops. To study these factors and to design a possible remedy, biological experimental data concerning these crops requires the quantification of gene expression and comparative analyses at high throughput level. Development of microarrays is the platform to study the differential expression profiling of the targeted genes. This technology can be applied to gene expression studies, ranging from individual genes to whole genome level. It is now possible to perform the quantification of the differential expression of genes on a glass slide in a single experiment. This review documents recently published reports on the use of microarrays for the identification of genes in different plant species playing their role in different cellular networks under abiotic stresses. The regulation pattern of differentially-expressed genes, individually or in group form, may help us to study different pathways and functions at the cellular and molecular level. These studies can provide us with a lot of useful information to unravel the mystery of abiotic stresses in important crop plants.

Plant Genetic Engineering: Problems and Applications

Introduction of Biotechnology has lead us to a stage where agricultural progress seems to be much different as before. It has introduced farmers to improve agricultural products while reducing the use of pesticides, and holds increased potential for pharmaceuticals and biomedical requirements. Technology has been improved to use the methods of transformation of agriculturally important crops by using different methods such as Agrobacterium and biolistic gun. Genetically engineered/modified crops, by Agrobacterium-mediated transformation system are becoming common in many countries. The health concerns on plants, animal and human life due to GM foods is unpredictable. However, plant genetic engineering can be considered either a progress or a threat to mankind depending on the way it is used.