Chalcone isomerase cDNA cloning and mRNA induction by fungal elicitor, wounding and infection

Se estudio la sintesis regulada por el ambiente de productos naturales de fenilpropanoide mediante examen de la expresion de la isomerasa de chalcona (CHI) que codifica el gen. Esta enzima cataliza un paso comun a la sintesis de los pigmentos flavonoides y de las fitoalexinas isoflavonoides. Se construyo un banco genetico gt11 lambda utilizando ARNm de cultivos de celulas de frijol tratadas con un desencadenante fungico. Se obtuvieron 2 clones positivos mediante seleccion de recombinantes 10(5) con un antisuero para purificar la CHI del frijol. La identidad de las secuencias clonadas se confirmo mediante translacion selecta de hibridos y mediante la produccion de polipeptidos antigenicos de trasuntos sintetizados in vitro. La adicion del desencadenante a los cultivos celulares dio lugar a la acumulacion rapida de ARNm de CHI, con niveles max. a las 3-4 h despues de la adicion del desencadenante. Tambien se acumulo ARNm de CHI durante la infeccion natural de hipocotilos con el organismo patogeno fungico Colletotrichum lindemuthianum y en hipocotilos lesionados mecanicamente. La cinetica de la acumulacion del ARNm de CHI en respuesta a estas senales ambientales fue sorprendentemente similar a la del ARNs que codifica otras 2 enzimas de recorrido de fenilpropanoide, como son la amonialiasa de fenilalanina y la sintasa de chalcona. A diferencia de las familias multigenicas que codifican estas 2 enzimas, la isomerasa de chalcona es codificada por un solo gen que es regulado por varios estimulos ambientales. (RA-CIAT) spa

The environmentally regulated synthesis of phenylpropanoid natural products was studied by examining the expression of the gene enconding chalcone isomerase (CHI). This enzyme catalyzes a step common to the synthesis of flavonoid pigments and isoflavonoid phytoalexins. A lambda gt11 library was constructed using mRNA from cell cultures of beans treated with a fungal elicitor. Two positive clones were obtained by screening 10(5) recombinants with an antiserum to purify bean CHI. The identity of the cloned sequences was confirmed by hybrid-select translation and the production of antigenic polypeptides from transcripts synthesized in vitro. The addition of the elicitor to the cell cultures resulted in the rapid accumulation of CHI mRNA, with max. levels achieved 3-4 h after elicitation. CHI mRNA also accumulated during the natural infection of hypocotyls with the fungal pathogen Colletotrichum lindemuthianum, and in mechanically wounded hypocotyls. The kinetics of accumulation of CHI mRNA in response to these environmental signals was strikingly similar to that of mRNAs enconding 2 other phenylpropanoid pathway enzymes, phenylalanine ammonialyase and chalcone synthase. In contrast to the multi-gene families enconding these 2 enzymes, chalcone isomerase is enconded by a single gene which is regulated by several environmental stimuli. (AS) eng