Transformation and expressional studies of GaZnF gene to improve drought tolerance in Gossypium hirsutum

Abstract

Drought stress is the major limiting factor in plant growth and production. Cotton is a significant crop as textile fiber and oilseed, but its production is generally affected by drought stress, mainly in dry regions. This study aimed to investigate the expression of Zinc finger transcription factor's gene (GaZnF) to enhance the drought tolerance in Gossypium hirsutum. Sequence features of the GaZnF protein were recognized through different bioinformatics tools like multiple sequence alignment analysis, phylogenetic tree for evolutionary relationships, Protein motifs, a transmembrane domain, secondary structure and physio-chemical properties indicating that GaZnF is a stable protein. CIM-482, a local Gossypium hirsutum variety was transformed with GaZnF through Agrobacterium-mediated transformation method with 2.57% transformation efficiency. The integration of GaZnF was confirmed through Southern blot showing 531 bp, and Western blot indicated a 95 kDa transgene-GUS fusion band in transgenic plants. The normalized real-time expression analysis revealed the highest relative fold spatial expression of cDNA of GaZnF within leaf tissues at vegetative and flowering stages under drought stress. Morphological, physiological and biochemical parameters of transgenic cotton plants at 05- and 10-day drought stress was higher than those of non-transgenic control plants. The values of fresh and dry biomass, chlorophyll content, photosynthesis, transpiration rate, and stomatal conductance reduced in GaZnF transgenic cotton plants at 05- and 10-day drought stress, but their values were less low in transgenic plants than those of non-transgenic control plants. These findings showed that GaZnF gene expression in transgenic plants could be a valuable source for the development of drought-tolerant homozygous lines through breeding.

Figure 1

Structural and functional analysis of GaZnF gene. (a) Evolution analysis of HD-ZF family of Gossypium arboreum, (b) Clustering map of six specie’s protein (c) Analysis of GaZnF motif. The letters in the figure represent amino acids, (d,e) Secondary structure of GaZnF protein.

Figure 2

Cloning of GaZnF in TA & pCAMBIA-1301 vector. (a) Total RNA extracted from stressed Gossypium arboreum, (b) EcoRI Restriction Digestion of GaZnF cloned in TA vector, lane 1 & 2 fragment of GaZnF gene, lane 3 is negative control, Lane M is 50 bp marker of Thermo Fisher Scientific Cat# SM0613, (c) PCR Amplified fragment 531 bp of GaZnF cloned in pCAMBIA-1301 vector; lane 1 &2 no amplification of GaZnF gene in negative control , lane M is 50 bp marker of Thermo Fisher Scientific Cat# SM0613, (d) NcoI and Bgl II Restriction Digestion of GaZnF cloned in pCAMBIA-1301, lane 1 & 3 with negative control with empty vector pCAMBIA-1301, lane M is 1 Kb marker of Thermo Fisher Scientific Cat# SM1331, (e) Schematic representation of GaZnF clone in pCAMBIA-1301; R Right border, L Left Border, NOS Poly A nopaline synthase terminator, 35S promoter 35S cauliflower mosaic virus (CaMV).

Figure 3

(a) Transformation of pGaZnF in Agrobacterium. (a) Colony PCR amplification of GaZnF gene, Lane M. 100 bp marker of Thermo Fisher Scientific Cat# SM1331, Lane 1. + ve control of pCAMBIA-1301 with GaZnF gene, Lane 2–8. plasmid of colonies of Agrobacterium cells containing GaZnF gene, Lane 9–10. − ve control PCR amplification of empty pCAMBIA-1301. (b,c) Transient expression in Tobacco plants. (b,c) Agroinfiltration and histo-chemically GUS Assay in Tobacco species for transient expression of GaZnF.

Figure 4

(a-f) Agrobacterium mediated Transformation of GaZnF in Gossypium hirsutum and Molecular analysis of transgenic plants. (a) Seed Germination, (b) A cut was given at apical meristem of isolated embryos from seeds and co-cultivation with Agrobacterium containing GaZnF, (c) Embryos on media containing plates, (d) Plants with developed shoots and roots, (e) Shifting of plants in soil containing pots, (f)shifting of pots containing plants in Green house for acclimatization. (g,h). Genomic DNA extraction and PCR amplification T0 generation. (g) Genomic DNA of transgenic and control plants, M. λ DNA/Hind III, Lane 1–6. Genomic DNA of GaZnF gene, (h) PCR amplification of genomic DNA of T0 generation’s transgenic and control cotton plants, Lane M was 100 bp Marker (Thermo scientific Cat. No.SM0241), Line 1,2,3,4,5 and 6 were Lane PF0022, PF0027, PF0031, PF0035, PF0039 and PF0054 respectively, Lane 7 was positive control and Lane 8 was negative control.

Figure 5

Confirmation of transgene (GaZnF) through southern and Western blot analysis. (a,b) Lane M. 1 kb DNA Ladder, Lane 1. Negative Control (DNA of untransformed plant), Lane 5 is positive control (plasmid DNA of pCAMBIA-1301 with GaZnF gene), Lane 2, 3, 4 & 6, 7 (PF0027, PF0035, PF0039, PF0048 and PF0054 respectively). Selected Transgenic Cotton Plant Samples (GaZnF) were digested with Nco I and Bgl II probed with biotin-labeled GaZnF DNA to assess copy number of the transgene in the transformed plants. The presence of bands at size of positive control confirms the transgene’s integration in transgenic plants as compare to un-transformed plants that is used as negative control. Lane M contains 1 Kb ladder, lane 1 represent negative controls, lane 2, 4 and 6, 7 indicate the integration of GaZnF gene in selected transgenic cotton plant samples, (c) Lane –ve. total soluble protein of the non-transformed cotton plant, Lane + ve, purified GaZnF protein; Lanes 1–4, total soluble proteins of the transgenic cotton plants (PF0035, PF0039, PF0048 and PF0054).

Figure 6

Quantitative real time PCR to determine the expression of transgenic cotton plants (GaZnF). mRNA samples of Line PF0054 exhibit the different expression levels at both vegetative and flower stages in leaf and stem tissues of transgenic and control plants, GAPDH was used as an internal control for normalization. The means ± SD of three biological replicates are presented. Error bars represent the standard deviation of the mean. Above the bars **** represent significant expression level of transcript in transgenic plants as compare to non- transgenic plants at the P < 0.05 level according to LSD multiple range test.

Figure 7

Effect of drought stress on growth indicators of transgenic cotton plants with GaZnF gene. (a) Comparison of plant height of transgenic and control plants, (b) Comparison of root shoot ratio of transgenic and control plants, (c) Comparison of root to total plant weight ratio of transgenic and control plants, (d) Comparison of stem to total plant weight ratio of transgenic and control plants, (e) Comparison of leaf to total plant weight ratio of transgenic and control plants, (f) Comparison of percentage of reduction in biomass of transgenic and control plants. The means ± SD of three biological replicates are presented. Error bars represent the standard deviation of the mean. Above the bars ****, ***, **represent significant and ns represent non-significant differences between transgenic and non-transgenic plants respectively at the P < 0.05 level according to LSD multiple range test.

Figure 8

Effect of drought stress on physiological parameters of transgenic cotton plants with GaZnF gene. (a) Comparison of Leaf relative water content of transgenic and control plants, (b) Comparison of chlorophyll of transgenic and control plants, (c) Comparison of photosynthesis rate of transgenic and control plants, (d) Comparison of transpiration rate of transgenic and control plants, (e) Comparison of stomatal conductance of transgenic and control plants, (f) Comparison of proline content of transgenic and control plants, (g) Comparison of soluble sugar of transgenic and control plants. The means ± SD of three biological replicates are presented. Error bars represent the standard deviation of the mean. Above the bars ****, ***, ** represent significant and ns represent non-significant differences between transgenic and non-transgenic plants respectively at the P < 0.05 level according to LSD multiple range test.