Estudo do número de cópias do gene da sintase do casbeno em Jatropha curcas L.

Abstract

Jatropha curcas L., best known in Brasil as pinhão manso and purgueira, it’s nowadays an object of many studies given the large amount of it’s different uses. The most important reason for many of these studies it is the fact that the oil extracted from the seeds can be used to produce a biofuel in engines run by diesel. Besides, there are an increasing interest for the kernel meal obtained after the oil extraction because of its high value in proteins, which could be used as a supplement in livestock feed, once eliminated all of its toxic constituents. The most harmful ones are phorbol esters, diterpenoids whose biosynthetic pathway was not completely elucidated, even though it is known the crucial participation of the casbene synthase enzyme. It has been demonstrated by studies the existence of many copies for the casbene synthase gene in the genome of J. curcas, but the number of copies is uncertain, thus this particular study has as a goal to inquire how many copies there is, through Southern Blot method. First, it was performed several DNA extractions of young leaves so that we could obtain two differents probes (using two sets, forward and reverse, of primers) marked with digoxigenin, acquire from PCR. Part of this extracted DNA were digested with restriction enzymes such as Pst I, Hind III, NcoI, NdeI e EcoR V. To verified if the probes were marked correctly with digoxigenin, tests were performed using 1.5% agarose gel and Dot Blots. At last, the Southern Blot experiment was carried out, followed immediately for the detection of the hybridization between probes and DNA attached to the nylon membrane. The results showed that at least the positives controls were successfully transferred from the gel to the membrane. However, was not possible to see any reaction on the columns where is was applied digested DNA. One of the reasons may be the lack of recognition from the probe to the DNA, causing the non hybridization between both of them. The amount of DNA digested put on the wells of the gel might have been insufficient for the hybridization with the probe since there was no hint of a band on the membranes.