Document Type : Original Article
Authors
- Masoud Fakhrfeshani 1
- Farajollah Shahriari Ahmadi 2
- Mohammad Zare Mehrjerdi 3
- Fatemeh Keykha Akhar 1
1 Department of Genetic and plant production, Collage of Agriculture, Jahrom University, Jahrom, Iran
2 Department of Biotechnology and Plant Breeding, Ferdowsi University of Mashhad, Mashhad, Iran
3 Agriculture Faculty of Shirvan, University of Bojnord, North Khorassan, Iran
Abstract
Introduction
Among abiotic stresses, Salinity has been increasing over the time for many reasons like using of chemical fertilizers, global warming and rising sea levels. In deed salinity stress and high concentration of ions are of the most determinative factors that simultaneously affect genetical, biochemical and physiological processes of a plant. Some of plants known as halophytes have developed mechanisms that help them to avoid or tolerate the saline conditions. With the aim of understanding the probable mechanisms of saline tolerance in Aeluropus littoralis (as a halophyte) and the saline susceptibility of Oriza sativa var. IR64 (as a glycophyte), In the present study, the similarity of Cu/Zn Superoxide Dismutase (Cu/Zn SOD) gene sequences, the differential biochemical indexes such as aromatic and instability indices of them were evaluated in silico, based on amino acid composition of their Cu/Zn SOD. the gene expression pattern and fluctuation of Cu/Zn SOD as a ROS scavenger (in root and shoot) was also assessed and compared in a span of exposer time to stress and different salt concentratio
Materials and methods
The seeds of A. littoralis and O. sativa var IR64 were prepared from Center for Research of Agricultural Science and Natural Resources (CRASN) which were sown in sand as a primary culture medium, after surface sterilization. After 21 days, the seedlings that had grown uniformly were transferred to continuously aerated hydroponic pots containing Yoshida nutrient solution. we established a stress span containing short term (6 hours), mid-term (24 and 48 hours) and long term (6 and 11 days) exposure to salinity stress. the gene expression pattern and fluctuation of Cu/Zn SOD as a ROS scavenger in Aeluropus, was evaluated at the sampling points of 6 h/100 mM, 6 h/200 mM, 6 h/300 mM, 48 h/300 mM, 144 h/300 mM and 264 h/300 mM. the elite sampling timepoint of rice, were also 6 h/30 mM, 6 h/60 mM, 6 h/100 mM, 48 hours/100mM, 144 hours/100 mM and 264 hours/100 mM. To study the structure of genes, genomic DNAs of A. littoralis and Oryza sativa L. Var IR64 were isolated from their leaf. Amplification of Cu/Zn SOD genes was performed with specific primers designed based on NCBI sequences. Amplification of their the maximum length fragments was carried out using a hot start, high fidelity DNA polymerase. The extracted fragments were cloned using pTZ57R vector in Escherichia coli (DH5α strain) and sequenced paired-end. The results were analyzed using NCBI database, and the biochemical features related to their proteins, such as isoelectric points, calculated weight, instability and aromatic indices, were analyzed Insilco using CLC Genomics Workbench 12 and Geneious Prime 2019.
Results and discussion
Comparison of the real time quantitative PCR of Cu/Zn SOD transcriptome in roots showed that Aeluropus increase the expression of these genes faster and keeps their expression in long-term while rice loses them significantly. The increased expression of Cu/Zn SOD in the roots of Aeluropus maintained but its expression in rice down regulated. rice did not show any statistically significant changes in compare to control samples and persisted in this manner until the end of investigation. But in Aeluropus, the expression showed another increase after 264 hours of being in 300mM. In the shoots of Aeluropus, the level of Cu/Zn SOD expression increased gradually and reached its highest level after 48 hours commencement of 300 mM salinity stress. While shoots of IR64 Rice did not show any statistically different expression except a very late response at 144th hour of being in 100 mM salinity stress that was only about 40 percent higher than its counterpart control samples. Analysis of amino acid sequences' indices showed that Cu/Zn SOD of Aeluropus would have a superior stability and higher aromatic value in contrast to IR64 counterpart. These indices originate from the arrangement of amino acids which they themselves are the result of nucleotide arrangement in genome.
Conclusion
The early and highly expression of Aeluropus Cu/Zn SOD gene studied in our experiment and their permanency and lasting of their activity in high concentration of salinity makes the sequence of it and its regulatory elements as a prospective candidate for future applied studies and transforming and improving salinity tolerance in many Poacea crops.
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