ANÁLISIS DEL RNAm DEL GEN QUE CODIFICA PARA LA ENZIMA ESCUALENO SINTASA (sqs) EN CULTIVO DE CÉLULAS DE Kalanchoe daigremontiana

Abstract

Introduction: Kalanchoe daigremontiana is a plant that exhibit medicinal properties thanks to the occurrence of bufadienolides. These secondary metabolites presents cytotoxic activity against different carcinogenic cellular lines. Bufadienolides belong to the triterpens group. Escualene sintase enzyme (SQS) plays an important role in biosynthesis and accumulation of bufadienolides. Nevertheless, the extraction and purification of such metabolites requires great quantities of vegetal material. However, it is necessary to develop a sustainable biotechnological system to get a viable alternative for the production of these metabolites. In this way, plant cell and tissue culture could be an important tool. Objective: The aim of present study was to analyze the expression of the gene that codify to SQS enzyme as well as to detect the presence of bufadienolides in non-differentiated in vitro cells of K. daigremontiana. Methodology. An in vitro cell culture of this plant was established using stem explants growing in a Murashige and Skoog (MS) medium added with a mixture of two phytoregulators: bencylaminopurine (BA 1mg/L) and 2,4- dichlorophenoxyacetic acid (2,4-D 0.5 mg/L). Duplication time and relative growth velocity were obtained from the callus growth kinetics. The molecular study was performed with a Northern blot analysis. The mRNA was extracted and quantified from root, leaf and callus of K. daigremontiana. The plasmid that contains the SQS gene (pBI121sqs) was obtained using a maxi-preparation protocol and purified by the kit GeneClean® II Kit Bio101. The heterologous probe was no-radioactive marked and hybridized. The detection was performed by a colorimetric reaction using the Biotin DecaLabelTM DNA Labeling kit. The obtaining of blue precipitate is characteristic of mRNA presence. Finally, bufadienolides were extracted with different polarity solvents and identified using thin layer chromatography and antimony trichloride as a specific revelator. Results: Green-light colored calluses were obtained at the tenth day of culture The kinetics data showed that callus growth presented a duplication time of 5.35 days and relative growth velocity of 0.187 d-1; also, four stages of callus development were observed: adaptation, exponential growth, stationary and death. From these data, four growing days were selected with the aim to detect the presence of bufadienolides as well as the expression of the gen (sqs) that codifies to SQS enzyme. Regarding the detection of bufadienolides using thin layer chromatography, these were found in all four stages of growing callus of K.daigremontiana. The results showed that the most remarkable stage was the stationary phase followed by the death phase. During adaptation and growing stages, both, precursors and bufadienolides were found. In the other hand, the expression of sqs gen was found in all stages. The mRNA of sqs gene was clearly detectable in both, stationary and death stages, while in the growing stage was found in low levels; in the adaptation stage, non-expression of the gen was detected. It is consider that these results are originals relating to the analysis of expression of the sqs gen and the detection of bufadienolides in in-vitro non-differentiated cell cultures of this plant. Finally the experimental evidences suggest that the expression of the gen sqs is closely related with the biosynthesis of bufadienolides.