Overexpression of a Gossypium hirsutum Stress-Associated Protein Gene (GhSAP1) Improves Salt Stress Tolerance in Transgenic Tobacco
Objective]Stress associated proteins (SAPs) is a type of novel stress response-inducing zinc finger proteins with A20/AN1 zinc-finger domain. The objectives of this study are to isolate a member of stress associated protein (SAP) gene family, analyze its responses to abiotic stress, and provide a potential candidate gene for improving abiotic stress tolerance in cotton by biotechnological approaches.[Method]A cotton zinc protein gene GhSAP1 was isolated based on in silico cloning and confirmed by RT-PCR analysis, further, recombinant plasmid vector with GhSAP1 was transformed into purified cotton mesophyll protoplast mediated with PEG for elucidating the characteristics of encoded proteins and subcellular location by a transient expression system. The expression patterns of GhSAP1 in different tissues and under salt stress were analyzed through real-time RT-PCR. By leaf disc method, Agrobacterium tumefaciens-mediated transformation, and tissue culture, different transgenic tobacco lines with high ectopic overexpression of GhSAP1 were obtained, further, their determination of physiologic index related to abiotic stress was finished and the potential of improving abiotic stress tolerance in plant was elucidated. [Result]GhSAP1 contained ORF length of 543 bp and encoded a polypeptide containing 180 amino acids with typical A20/AN1 zinc finger structural domain. The theoretical iso-electric point was 8.97 with a calculated molecular weight of 19.3 kD. Subcellular location showed that GhSAP1 was localized in cell nucleus. The expression patterns in different tissues and organs showed that GhSAP1 expressed preferentially in root, stem, and leaf tissues, but the expression level was the lowest in young ovules at five days post anthesis. GhSAP1 was accumulated significantly when induced after 2 h under salt stress treatment. Ectopic overexpression of GhSAP1 in tobacco plants was performed via leaf disc transformation mediated by Agrobacterium tumefaciens, and was confirmed in both genomic and transcriptional level by PCR and RT-PCR in transgenic tobacco seedlings. Ten tobacco transgenic lines were preliminarily obtained through resistance selection with 100μg·mL-1 Kan culture medium for transgenic progeny, and confirmed by PCR and RT-PCR of GhSAP1. The result showed that GhSAP1 was integrated into tobacco genome validity with high ectopic overexpression. Further, three transgenic tobacco positive lines were selected by random and analyzed under salt stress. Treating the seedlings of germination for seven days using 200 mmol·L-1 NaCl, 12 days later, the average survival rate of transgenic tobacco lines was 81.7%, which was 3 times higher than those of wild-type plants, and the relative root length in transgenic plants was 3.05 cm in average, significantly higher than wild type plants. Further, L2 pure lines with higher GhSAP1 expression was selected to elucidate physiologic index related to abiotic stress. In detail, when treating the plants with 9 to 11 leaves using 200 mmol·L-1 NaCl, 30 days later, the transgenic lines overexpressing GhSAP1 showed better SOD activity and increased total soluble sugar content, with net increase value of 23.89 U·g-1 FW and 8.37 %, respectively, which was significantly higher than non-transgenic plants. Compared with non-stress treatment, the reduction of the chlorophyll content of transgenic plants was only 0.17 mg·g-1 FW, in contrast to the 0.39 mg·g-1 FW of the non-transgenic plants. After treatment under salt stress condition, the MDA content of transgenic plants only increased by 7.42 nmol·g-1 FW, however, the MAD content of the non-transgenic plants increased by 20.85 nmol·g-1 FW. Meanwhile, the transgenic plants had the better transport ability of K+from plant root to green tissue, the K+/Na+ratio of above-ground part was 1.23, with higher transport ability compared with wild type plants.[Conclusion]It was concluded that GhSAP1 plays an important role in response to salinity stress in plants, overexpression of GhSAP1 can improve significantly tolerance to salt stress in transgenic tobacco plants.
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