Análise bioquímica e proteômica parcial das proteínas do látex da planta medicinal Himatanthus drasticus e suas ações em defesa vegetal

Abstract

Latex is a fluid which contains many biomolecules. It is stored in cells called laticifers and it is exuded when the plant is damaged mechanically. Its components and its applications in some fields, such as the agricultural one, have been the subject of many scientific works. Himatanthus drasticus is a latex plant whose latex is used by the population of the Ceará State as a phytotherapic to treat and prevent some diseases. The aim of this work was to study biochemical and proteomics aspects of proteins from this latex besides to prospect biotechnological activities from these biomolecules against agricultural crops. The HdLP-90 fraction was obtained through ammonium sulphate precipitation (90% saturation) of the latex, post remotion of the rubber through centrifugation. HdLP-90 makes up approximately 5.6% of the total latex mass. The soluble protein content was estimated as 0,66 ± 0,01 mg/mL through the Bradford protein assay. Unidimensional electrophoresis analysis revealed the presence of at least eight protein bands in between 97 kDa and 14.4 kDa. The chromatographic processes carried out in DEAE-Sepharose Fast Flow column, either in pH 8,0 (Tris-HCl 0,05 M) and pH 5,0 (Sodium Acetate 0,05 M), were not capable of separating the proteins from HdLP-90 efficiently. When Sodium Acetate 0,05 M pH 5,0 buffer was used, however, more contaminants were removed once the major chromatographic peak (PII HdLP-90 DEAE-Shepharose ff pH 5,0) lacks the brown pigments present in HdLP-90. The bidimensional electrophoresis of this peak revealed that most of the proteins were focalized in the pH interval of 4,0 to 4,6. MS analysis, post chromatography carried out in AKTA-FPLC, identified the proteins from this peak as osmotin-like or thaumatin-like proteins. The Dot Blot using antiosmotin antibodies corroborated this result. None of the protein samples from H. drasticus latex were able to degrade azocasein substrate. They were not also recognized by Dot Blot using antiprotease antibodies, which indicates the absence of proteases in those samples. The presence of chitinases was confirmed through chitinolytic assay using colloidal chitin as substrate. The biological activities against phytopathogens showed that HdLP-90 did not cause deleterious effect on the mycelial growth of Colletotrichum gloeosporioides. Despite this, it caused large toxicity on larval and adult insect development of Callosobruchus maculatus. The methodology performed in this study allowed us to obtain a concentrated protein fraction from the H. drasticus latex, which contains ostomins and chitinases, and, these may be related to the strong insecticidal activity against C. maculatus.