Detecção de genes e expressão enzimática em cultivares de arroz (Oryza sativa L.) crescidas sob estresse salino

Abstract

In Rio Grande do Sul State, the main system for irrigation of rice cultivation is by flooding, can lead to the salinization of soils with inadequate drainage, especially at the coastal region where crops using water from the Laguna dos Patos, which is subject to the salinization by sea water. This is a major environmental problem in the rice production. This survey aimed to examine the expression of enzyme in Oryza sativa L. ssp. indica S. Kato e Oryza sativa L. ssp. japonica S. Kato, grown in different levels of salinity, in order to identify genes involved in tolerance to salinity, based on the assumption that the second subspecies show greater tolerance to salinity. In the experiment were used Oryza sativa L. ssp. japonica S. Kato (BRS Bojuru, IAS 12-9 Formosa and Goyakuman) and Oryza sativa L. ssp. indica S. Kato (BRS-7 Taim, BRS Querência and BRS Atalanta). The seedling was done in plastic trays, containing sand washed as substrate. The seedlings were transferred to greenhouse with 10 days of emergency under temperature 25 °C and humidity 85 % controlled and placed in basins of 15 L containing nutrient solution of Hoagland half strength increased of 0, 25, 50, 75 and 100 mM NaCl. Seedlings were collected at 14, 28, 42 and 56 days after the transfer and immediately stored in ultrafreezer to -70 °C to subsequent analyses. The plant tissues were macerated and placed in tubes eppendorf with extractor solution of Scandálios. The electrophoresis was performed in 7% of polyacrylamide gels placed in vertical vats. The bands were revealed for several enzymes systems: superoxide dismutase, peroxidase, catalase, esterase, xvi glutamate dehydrogenase, alcohol dehydrogenase, fosfoglucose isomerase, malate dehydrogenase, málica enzyme, alpha amylase and glucose-6-phosphate dehydrogenase. Through the search in silico, conducted with the National Center for Biotechnology Information identified the genes AY785147 - SOS and AF319481 - CK1 involved in the salinity tolerance. The detection of the gene was the extraction of DNA using the method CTAB 2%, followed by reactions of PCR thermocycler held on through the use of primers also drawn in silico. The products of amplification were detected by agarose gel electrophoresis of 1.5%. The view of the DNA stained with bromide etídio was made on ultraviolet light and scanned images. The expression of enzymes involved in the mechanisms of tolerance to salt stress is greater in O. sativa ssp. japonica. Fragment of SOS1 gene was found in all cultivars, except for BRS Atalanta. CK1 gene is present in all cultivars evaluated. It allows to conclude that enzyme systems were more expressed in cultivars O. sativa ssp. japonica, in the leaves and the 14 DAT, featuring bands more intense as the increase of salinity. The expression of enzymes involved in the mechanisms of tolerance to salt stress is greater in O. sativa ssp. japonica and the genes studied are present in both subspecies.