electrophoresis, a way to identify A wheat grain variety by the migration of its protein (Cécile)26/9/2016 Poly-acrylamide gel electrophoresis (PAGE) is a technique well known in laboratory of biotechnology or biochemistry. I don’t know this technique… reminder: A mix of macromolecules, protein or nucleic acids from DNA molecules, is submitted to an electric field (fig 1). Thus, charged molecules migrate on gel. This migration occurs more or less quickly depending on several parameters such as length, conformation and charge of molecules. It allows to separate (fig 1 (B)) the different molecules in the initial mixture and to deduce information like molecular mass… Fig 1: Principle of electrophoresis A concrete application of this scientific method is the identification of wheat varieties, and you’ll discover in this article how. Proteins of wheat are spread into 4 classes: albumin, globulin but also gliadin and glutenin, wheat storage proteins. Gliadin can be separated by electrophoresis. [1] The great diversity of gliadin (around 120 alleles) [2] generate specific profiles for each seed variety. Selection 40 representative grains from billions of seeds Some wheat contracts are appropriated to bread making industry but some other are only suitable for animal feed. So let’s imagine that you are working in a company that sells flour for milling activities and you receive something like 30 trucks a day full of wheat. How to be sure that your supplier is really delivering milling wheat ? You can determine one seed variety using PAGE. But you should wonder how to do when you receive several tonnes, corresponding to several billions of seeds of wheat a day? The idea is make the analysis on a representative sample taken every 300 to 500 tonnes delivered. A representative sample initially weighs 1 kg and then an apparatus is used to remove 1 seed every 10 seed. You obtain 40 representative grains from billions of delivered grain. Prepare a mixture of macromolecule for each seed First of all you need to prepare the mixture that will be used for electrophoresis. Each seed has to be crushed and put in a solution of ethanol chlorure, saccharose and crystallised purple dye for 24 hours. The day after, the mixture is stirred for 15 minutes and centrifuged for 5 minutes. It is ready to be deposited on gel and start electrophoretic migration. However, at this end of this step, it is impossible to see the seed profile, and just like pictures needed to be developed, we use a solution of electrophoretic profile is soaked on trichloroacetic acid and coomassie blue dye to bring out the lines. Indeed, coomassie blue binds to amino acids (lysine and arginine mostly) of proteins The longer you let it soak, the strongest the colour will be. Usually, it is let 24h. From a column of blue line to the name of wheat variety The 40 profiles appear on 40 columns on the analytic paper (fig 2) from electrophoresis, each one corresponding to one seed. A profile is constituted by several blue lines numbered, corresponding to gliadin (or glutenin) fractions. Several years of observation and study of profiles from wheat of a known variety allowed to create trees or key of identification. Fig 2: analytic paper : electrophoretic profiles Thus, line by line, you follow a branch leading to the name of the seed variety (fig 3). Fig 3: Tree of identification What is the electrophoretic profile of milling wheat?
The difficulty is that a milling wheat contract can be a mix of several varieties. So you’ll not obtain 40 times the same profile, but something like 10 to 15 different profiles. Results are expressed in % of a variety. If 7 seeds or more among the 40 (15% of the contract) are of a non milling variety, the whole contract is considered as non compliant. Determining Wheat geographical provenance As all French variety has a known key of identification, this technique can also be used to determine wheat geographical provenance, an important information for company promoting “French product” for example. PAGE, a perfect technique? However, this technique pose certain problems. Proteins from damaged seeds can be not visible because they don’t bind to dye or need too much time to take a sufficient coloration. This technique is long: if you counted well, 3 days are necessary between sampling and variety identification. Any solution? A faster method, PCR (polymerase chain reaction), can also be used to amplify and study DNA from a single seed but it can be expansive and more complicated to practice and analyse. References [1] "Différenciation Des Variétés De Céréales Par Électrophorèse". Agrarforschungschweiz.ch. N.p., 2016. Web. 25 Sept. 2016. [2] KLEIJER, G. Sélection Des Variétés De Blé Pour La Qualité Boulangère. 1st ed. Station fédérale de recherches en production végétale de Changins, 2016. Web. 25 Sept. 2016.
0 Comments
Leave a Reply. |