Dehydrin (DHN) is known to play an important role in plant response and adaptation to abiotic stresses (drought, high salinity, cold, heat, etc.). Previous research reported the increased expression of DHN in sugarcane stems exposed to drought stress for 15 days which may be controlled by its corresponding stress inducible promoter. The DHN promoter was succesfully isolated from sugarcane variety PSJT 941 (Pr-1DHNSo) and was cloned to pBI121 expression vector fused to a β-glucuronidase (GUS) reporter gene. The aim of this research was the functional testing of the Pr-1DHNSo promoter through transformation into tobacco plant treated with in vitro drought stress. Genetic transformation of Pr-1DHNSo construct was conducted by Agrobacterium tumefaciens. The transformed tobacco was then subjected to drought stress treatment using 40% PEG 6000 for five sequential incubations (0, 12, 24, 48 and 72 hours). The GUS assay reveal that the transformed tobacco treated with drought stress showed a blue color denoting GUS activity in leaf, stem and root tissues and this expression increased along with the length of the drought treatment. The analysis of gusA gene using real time-qPCR normalized to the L25 reference gene also showed that the expression increased in line with the length of time of drought stress. The results presented in this study indicated that the Pr-1DHNSo promoter from sugarcane was expressed and induced by drought stress treatment in tobacco.
Sugarcane plantations in Indonesia have been expanded and shifted to the marginal land characterized by long drought period, therefore, an attempt has been initiated to generate drought tolerance varieties through genetic engineering. It could be conducted by inserting the gene that involve in plant adaptation response to drought stress such as dehydrin (DHN) into sugarcane genome. The promoter of sugarcane DHN gene was isolated and transformed into sugarcane in the previous research. This study aimed to demonstrate the functionality of sugarcane DHN promoter through expression analysis of DHN regulatory genes that play a role in response to drought stress. Expression analyses using RT-qPCR were also conducted on regulatory genes of sugarcane that inserted by Pr-1DHNSo construct treated with drought stress. The results showed that the expressions of SoMYB, SoWRKY, SoNAC, and SoDHN genes were escalated on sugarcane 16 days after stress treatment ranging from 353 to 4067 folds relatively to untreated samples in which SoNAC gene showed the highest expression. On the other hand, the analysis on transgenic sugarcane carrying DHNpromoter construct showed SoNAC and SoDREB expression increased after 72 hours under drought stress. The expression values of SoNAC in transgenic and non-transgenic plants under drought condition were 4.79 and 4.99, respectively. Meanwhile, the expression values of SoDREB in transgenic and non-transgenic plants under drought condition were 13.2 and 13.3, respectively. The results of these experiments showed that the promoter construct of Pr-1DHNSo was induced by drought stress treatments highlighting the regulation of several upstream genes of SoDHN.
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