Ayesha Latif

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.

Sucrose synthase genes: a way forward for cotton fiber improvement

Cotton is a premium source of natural fiber and is considered “white gold” in the textile industry. Fiber length, strength and fineness are the key considerations for the industry. Longer fibers are machine friendly because they are easily spinnable. Recent advancements in genetic engineering, including the development of DNA markers and quantitative trait loci (QTLs), together with genome sequencing and gene expression profiling, have provided new avenues for improving fiber production and quality. In plants, sucrose synthase (SUS) is the key enzyme that catalyzes the reversible cleavage of sucrose and uridine diphosphate (UDP) into fructose and UDP-glucose. Sucrose is the main mobile sugar in plants moving from source to sink. It regulates resource partitioning between active sinks, especially in cotton embryos and fibers, and therefore is directly involved in determining fiber yield and seed quality. SUS actively takes part in regulating the competition for nutrients among sink tissues through balancing osmotic potentials by providing hexoses and an efficient supply of UDP-glucose the substrate for cellulose synthase. Cotton transformation has been used to improve fiber characteristics by altering cell wall properties through the manipulation of expression of fiber genes. Overexpression of the SUS gene from natural or synthetic origins in cotton can be an excellent way to solve potential problems associated with poor fiber length and other fiber quality traits. Increased SUS activity can result in more hexoses, increasing the osmotic potential and thereby driving the water influx that creates high turgor pressure in fiber cells resulting in enhanced fiber elongation. Moreover, increased SUS gene transcript levels in vegetative tissues of the plant will elevate seedling biomass and seed number. Fiber length and seed number both contribute towards final yield and the SUS genes as key regulators of sink strength in cotton perform this dual function that is directly related to cotton productivity. Hence manipulation of the SUS gene family is considered a promising approach to improve cotton fiber yield and quality. This review focuses on the biochemical and physiological roles of the SUS genes and there value for cotton fiber improvement.

A concise review of poultry vaccination and future implementation of plant-based vaccines

Every year the growth of the poultry industry is severely threatened by a number of infectious viral, bacterial and parasitic diseases. There are a number of vaccines to control these diseases including inactivated virus vaccines, attenuated virus vaccines, live virus vaccines, and subunit vaccines, but they are often relatively expensive and require cold storage and trained people to administer them, especially in developing countries. Plant-based vaccines provide a better option to control these diseases in low profit margin poultry industry. Still there are some challenges in the field of plant-based, so called ‘green’ vaccines. Injection-based oral priming is a big challenge for commercialisation of green vaccines so, new techniques are needed in the field of plant-based vaccine to pass these barriers for commercialisation. This discusses the potential for plant-based vaccines and whether they are good option to control poultry diseases.

Escherichia coli expression of NDV fusion protein gene and determination of its antigenic epitopes

Abstract Recurrent outbreaks of Newcastle disease have questioned the usage of existing vaccines that whether they are still adequate to protect clinical diseases and inhibit virus transmission in poultry. Advancement in molecular biology has led to the production of recombinant vaccines in recent years, which can be a more useful strategy to control infections of Newcastle disease virus (NDV). Studies indicate that the pathogenic nature of NDV is mediated by its membrane associated fusion (F) protein. Here we report the cloning of the full-length F gene-pET30a and its expression in Escherichia coli BL21 DE3 cells through isopropyl β-D-1-thiogalactopyranoside induction. Transferring the protein on nitrocellulose membrane in Western blotting confirmed its specificity with histidine-tagged antibody reaction at the proper size of 67 kDa. Protein purification with nickel charged sepharose column affinity chromatography resulted in a single band of 67 kDa purified His-tag F protein on SDS-PAGE. Analysis of its immunogenicity through bioinformatics tools revealed that more than 70% of its sequence is antigenically active comprising 24 linear immunogenic peptides predicted by the Linear epitope prediction tool and 9 immunogenic peptides predicted by ElliPro. This is a key achievement of the study, which may lead towards recombinant vaccine production in future. In conclusion, our findings suggest that rather than employing live viral vaccines, using a purified immunogenic recombinant F protein as a vaccine or cloning the same gene in a suitable plant vector for production of edible vaccine will provide better protection against the NDV into chicken.