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MutantGossypium universal stress protein-2 (GUSP-2) gene confers resistance to various abiotic stresses in E. coli BL-21 and CIM-496-Gossypium hirsutum

ABSTRACT: Gossypium arboreum is considered a rich source of stress-responsive genes and the EST database revealed that most of its genes are uncharacterized. The full-length Gossypium universal stress protein-2 (GUSP-2) gene (510 bp) was cloned in E. coli and Gossypium hirsutum, characterized and point mutated at three positions, 352–354, Lysine to proline (M1-usp-2) & 214–216, aspartic acid to serine (M2-usp-2) & 145–147, Lysine to Threonine (M3-usp-2) to study its role in abiotic stress tolerance. It was found that heterologous expression of one mutant (M1-usp-2) provided enhanced tolerance against salt and osmotic stresses, recombinant cells have higher growth up to 10-5dilution in spot assay as compared to cells expressing W-usp-2 (wild type GUSP-2), M2-usp-2 and M3-usp-2 genes. M1-usp-2 gene transcript profiling exhibited significant expression (8.7 fold) in CIM-496-Gossypium hirsutum transgenic plants and enhance drought tolerance. However, little tolerance against heat and cold stresses in bacterial cells was observed. The results from our study concluded that the activity of GUSP-2 was enhanced in M1-usp-2 but wipe out in M2-usp-2 and M3-usp-2 response remained almost parallel to W-usp-2. Further, it was predicted through in silico analysis that M1-usp-2, W-usp-2 and M3-usp-2 may be directly involved in stress tolerance or function as a signaling molecule to activate the stress adaptive mechanism. However, further investigation will be required to ascertain its role in the adaptive mechanism of stress tolerance.

SUBMITTER: Hafeez M

PROVIDER: S-EPMC8516947 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Abiotic stress is a major environmental factor that limits cotton growth and yield, moreover, this problem has become more and more serious recently, as multiple stresses often occur simultaneously due to the global climate change and environmental pollution. In this study, we sought to identify genes involved in diverse stresses including abscisic acid (ABA), cold, drought, salinity and alkalinity by comparative microarray analysis. Our result showed that 5790, 3067, 5608, 778 and 6148 transcripts, were differentially expressed in cotton seedlings under treatment of ABA (1 μM ABA), cold (4°C), drought (200 mM mannitol), salinity (200 mM NaCl) and alkalinity (pH=11) respectively. Among the induced or suppressed genes, 126 transcripts were shared by all of the five kinds of abiotic stresses, with 64 up-regulated and 62 down-regulated. These common members are grouped as stress signal transduction, transcription factors (TFs), stress response/defense proteins, metabolism, transport facilitation, as well as cell wall/structure, according to the function annotation. We also noticed that large proportion of significant differentially expressed genes specifically regulated in response to different stress. Nine of the common transcripts of multiple stresses were selected for further validation with quantitative real time RT-PCR (qRT-PCR). Furthermore, several well characterized TF families, for example, WRKY, MYB, NAC, AP2/ERF and zinc finger were shown to be involved in different stresses. As an original report using comparative microarray to analyze transcriptome of cotton under five abiotic stresses, valuable information about functional genes and related pathways of anti-stress, and/or stress tolerance in cotton seedlings was unveiled in our result. Besides this, some important common factors were focused for detailed identification and characterization. According to our analysis, it suggested that there was crosstalk of responsive genes or pathways to multiple abiotic or even biotic stresses, in cotton. These candidate genes will be worthy of functional study under diverse stresses.

Project description:BackgroundThe sequencing and annotations of cotton genomes provide powerful theoretical support to unravel more physiological and functional information. Plant homeodomain (PHD) protein family has been reported to be involved in regulating various biological processes in plants. However, their functional studies have not yet been carried out in cotton.ResultsIn this study, 108, 55, and 52 PHD genes were identified in G. hirsutum, G. raimondii, and G. arboreum, respectively. A total of 297 PHD genes from three cotton species, Arabidopsis, and rice were divided into five groups. We performed chromosomal location, phylogenetic relationship, gene structure, and conserved domain analysis for GhPHD genes. GhPHD genes were unevenly distributed on each chromosome. However, more GhPHD genes were distributed on At_05, Dt_05, and At_07 chromosomes. GhPHD proteins depicted conserved domains, and GhPHD genes exhibiting similar gene structure were clustered together. Further, whole genome duplication (WGD) analysis indicated that purification selection greatly contributed to the functional maintenance of GhPHD gene family. Expression pattern analysis based on RNA-seq data showed that most GhPHD genes showed clear tissue-specific spatiotemporal expression patterns elucidating the multiple functions of GhPHDs in plant growth and development. Moreover, analysis of cis-acting elements revealed that GhPHDs may respond to a variety of abiotic and phytohormonal stresses. In this regard, some GhPHD genes showed good response against abiotic and phytohormonal stresses. Additionally, co-expression network analysis indicated that GhPHDs are essential for plant growth and development, while GhPHD genes response against abiotic and phytohormonal stresses may help to improve plant tolerance in adverse environmental conditions.ConclusionThis study will provide useful information to facilitate further research related to the vital roles of GhPHD gene family in plant growth and development.

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MutantGossypium universal stress protein-2 (GUSP-2) gene confers resistance to various abiotic stresses in E. coli BL-21 and CIM-496-Gossypium hirsutum

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