Abstract
Cotton crop suffers shortage of irrigation water at reproductive stage which reduces the yield and fibre quality. Universal stress proteins belong to Pfam00582 which enables several plants to cope with multiple stresses via ATP binding. GUSP1 (Gossypium arboreum USP) is one of such proteins; its amino acids were mutated after in silico simulations including homology modeling and molecular docking analysis. Transgenic cotton plants were developed through Agrobacterium mediated genetic transformation by using mutated pmGP1 and non mutated pGP1 constructs under CaMV35S promoter. PCR and semi-quantitative PCR analyses confirmed the amplification and expression of transgene in transgenic plants. It was revealed that leaf relative water content, total chlorophyll content, CO2 assimilation as net photosynthesis, stomatal conductance, total soluble sugars and proline content was significantly increased at P ≤ 0.0001 and P ≤ 0.001 in both the pmGP1 and pGP1 transgenic plants as compared to non transgenic control plants. Moreover, relative membrane permeability and the transpiration rate were reduced significantly at P ≤ 0.0001 and P ≤ 0.001 respectively in transgenic plants under drought stress. Furthermore, the T1 transgenic seedlings containing pmGP1 mutated construct showed longer roots under desiccation stress imposed by 5% PEG. Transgene inheritance into the T1 progeny plants was confirmed by amplification through PCR and integration through Southern blot. Hence, our results pave the way to utilize the mutagenized known genes for increasing endurance of plants under drought stress. This will help to increase our understanding of drought tolerance/ sensitivity in cotton plants at the molecular level.
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Abbreviations
- RMP:
-
Relative membrane permeability
- LRWC:
-
Leaf relative water content
- PN:
-
Net photosynthesis
- E:
-
Transpiration rate
- gs :
-
Stomatal conductance
- USP:
-
Universal stress proteins
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12298_2021_1048_MOESM1_ESM.tif
Supplementary Fig. 1 PCR amplification of GUSP1 in total DNA of transgenic cotton plants containing mutated (pmGP1) and non mutated (pGP1) construct. a: Lanes 1-5: total DNA isolated from transgenic plants. 4 µl of DNA sample was resolved onto 0.8% agarose gel). b: PCR amplification of pmGP1with mutated region specific primers. M: 100bp ladder (Fermentas); lane1: -ve control; lane 2: positive control; lanes 3-26 PCR amplified180bp fragment of pmGP1. (9 µl of PCR product was resolved onto 0.8% agarose gel). c: PCR amplification of non mutated pGP1. M: 100bp ladder (Fermentas) lane1: +ve control; lane2-16 PCR amplified 500bp fragment of pGP1; lane 17: -ve control (9 µl of PCR product was resolved onto 0.8% agarose gel). (TIF 1562 KB)
12298_2021_1048_MOESM2_ESM.tif
Supplementary Fig. 2 PCR amplification of GUSP1 in total DNA of T1 generation of transgenic cotton plants containing pmGP1 & pGP1 construct. a): PCR amplification of pmGP1 transgene in T1 transgenic plants. 350bp fragment of pmGP1 was resolved onto 0.8% agarose gel. M: 100bp Ladder (Fermentas). Lane1: +ve control (Plasmid DNA of pmGP1). Lane 2: -ve control (Genomic DNA from non-transgenic plants). Lanes 3-19: PCR from Genomic DNA of plants containing pmGP1. b): PCR amplification of pGP1 transgene in T1 transgenic plants. 450bp fragment of pGP1 was resolved onto 0.8% agarose gel. M:100bp Ladder (Fermentas). Lanes 1-17: PCR from Genomic DNA of plants containing pGP1. Lane 18: +ve control (Plasmid DNA of pGP1). Lane 19: -ve control (Genomic DNA f (TIF 1288 KB)
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Hassan, S., Ahmad, A., Batool, F. et al. Genetic modification of Gossypium arboreum universal stress protein (GUSP1) improves drought tolerance in transgenic cotton (Gossypium hirsutum). Physiol Mol Biol Plants 27, 1779–1794 (2021). https://doi.org/10.1007/s12298-021-01048-5
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DOI: https://doi.org/10.1007/s12298-021-01048-5