Muhammad Ashraf

Functional Characterization of a Bidirectional Plant Promoter from Cotton Leaf Curl Burewala Virus Using an Agrobacterium-Mediated Transient Assay

The C1 promoter expressing the AC1 gene, and V1 promoter expressing the AV1 gene are located in opposite orientations in the large intergenic region of the Cotton leaf curl Burewala virus (CLCuBuV) genome. Agro-infiltration was used to transiently express putative promoter constructs in Nicotiana tabacum and Gossypium hirsutum leaves, which was monitored by a GUS reporter gene, and revealed that the bidirectional promoter of CLCuBuV transcriptionally regulates both the AC1 and AV1 genes. The CLCuBuV C1 gene promoter showed a strong, consistent transient expression of the reporter gene (GUS) in N. tabacum and G. hirsutum leaves and exhibited GUS activity two- to three-fold higher than the CaMV 35S promoter. The CLCuBuV bidirectional genepromoter is a nearly constitutive promoter that contains basic conserved elements. Many cis-regulatory elements (CREs) were also analyzed within the bidirectional plant promoters of CLCuBuV and closely related geminiviruses, which may be helpful in understanding the transcriptional regulation of both the virus and host plant.

Role of epicuticular waxes in the susceptibility of cotton leaf curl virus (CLCuV)

Cotton leaf curl virus (CLCuV) is the causal agent of the damaging disease of cotton that is caused by number of begomaviruses and vectored by silver leaf whitefly. In the present study, an attempt was made by infecting Gossypium arboreum variety 786, its wax mutant GaWM3 along with Gossypium hirsutum MNH-93 with viruliferous whiteflies. The presence of symptoms on leaves and amplification by PCR for virus in G. hirsutum MNH-93 and wax mutant GaWM3 but not in G. arboreum variety 786 clearly determined the presence of virus in G. hirsutum MNH-93 and wax mutant GaWM3 but not in G. arboreum variety 786. The results indicate that wax may act as physical barrier and provide hindrance in transfer of virus by whitefly.

Herbicide-resistant cotton (Gossypium hirsutum) plants: an alternative way of manual weed removal

BackgroundCotton yield has been badly affected by different insects and weed competition. In Past Application of multiple chemicals is required to manage insects and weed control was achieved by different conventional means, such as hand weeding, crop rotation and polyculture, because no synthetic chemicals were available. The control methods shifted towards high input and target-oriented methods after the discovery of synthetic herbicide in the 1930s. To utilise the transgenic approach, cotton plants expressing the codon-optimised CEMB GTGene were produced in the present study.ResultsLocal cotton variety CEMB-02 containing Cry1Ac and Cry2A in single cassette was transformed by synthetic codon-optimised 5-enolpyruvylshikimate-3-phosphate synthase gene cloned into pCAMBIA 1301 vector under 35S promoter with Agrobacterium tumifaciens. Putative transgenic plants were screened in MS medium containing 120xa0µmol/L glyphosate. Integration and expression of the gene were evaluated by PCR from genomic DNA and ELISA from protein. A 1.4-kb PCR product for Glyphosate and 167-bp product for Cry2A were obtained by amplification through gene specific primers. Expression level of Glyphosate and Bt proteins in two transgenic lines were recorded to be 0.362, 0.325xa0µg/g leaf and 0.390, 0.300xa0µg/g leaf respectively. FISH analysis of transgenic lines demonstrates the presence of one and two copy no. of Cp4 EPSPS transgene respectively. Efficacy of the transgene Cp4 EPSPS was further evaluated by Glyphosate spray (41xa0%) assay at 1900xa0ml/acre and insect bioassay which shows 100xa0%mortality of insect feeding on transgenic lines as compared to control.ConclusionThe present study shows that the transgenic lines produced in this study were resistant not only to insects but also equally good against 1900xa0ml/acre field spray concentration of glyphosate.

Dynamic Proline Metabolism: Importance and Regulation in Water-Limited Environments

Abstract Proline is an important imino acid that accumulates in plants in response to different environmental constraints including water-limited conditions. The literature shows a positive association between proline accumulation and drought tolerance in plants. However, some recent findings highlighted that induction of drought tolerance in plants is not only due to proline accumulation and turnover, but also due to metabolism of proline that is responsible for maintaining plant growth during drought conditions. Plants mutant for enzymes involved in the biosynthesis Δ1-pyrroli [pyrroline-5-carboxylate synthetase 1 (P5CS1)] and catabolism [proline dehydrogenase 1 (ProDH1)] of proline exhibited significant growth reduction under drought conditions. Plants exposed to drought show accumulation of proline that in turn maintains the redox status. A variety of cellular processes mediate the biosynthesis and catabolism of proline. Presently, only a few mechanisms dealing with this imino acid are fully elucidated. For instance, highly abscisic acid (ABA)-induced protein phosphatase 2Cs differentially influenced the endogenous levels of PDH1 and P5CS1 proteins apart from the same increase in the cellular levels of proline. Proline biosynthetic (P5CS1) and catabolic (PDH1) genes are regulated via some posttranslational changes that are not fully known. In this chapter, we have discussed proline metabolism in plants under limited water regimes. We have also provided the details of proline association with other key physiochemical attributes under water deficit conditions.

Identifying salt stress-responsive transcripts from Roselle ( Hibiscus sabdariffa L.) roots by differential display

No previous study has been reported on the salt-modulated gene(s) of roselle ( Hibiscus sabdariffa L.). Identifying the potentially novel transcripts responsible for salt stress tolerance in roselle will increase knowledge of the molecular mechanism underlying salt stress responses. In this study, differential display reverse transcriptase PCR (DDRT-PCR) was used to compare the overall differences in gene expression between salt-stressed and control plants. A total of 81 primer combinations were used and false positive clones were rejected during a screening and quality control assay. The remaining nine cDNA transcript fragments were extracted from the gel, reamplified, cloned and sequenced. A homology search revealed that four transcripts showed significant homology with known genes. Out of five transcripts, real-time PCR demonstrated that four exhibited high expression in salt-stressed root tissues relative to the control and one transcript was down-regulated. These transcripts may be useful for improving tolerance in salt stress-sensitive plants. Keywords: Roselle, Hibiscus Sabdariffa L., differential display, salt-stress, differentially expressed transcripts, signal transduction. African Journal of Biotechnology , Vol 13(53) 4775-4781