RESUMEN
The tobacco ntf4 mitogen-activated protein (MAP) kinase gene (and its encoded protein p45(Ntf4)) is expressed at later stages of pollen maturation. We have found that the highly related MAP kinase SIPK is also expressed in pollen and, like p45(Ntf4), is activated upon pollen hydration. The MAP kinase kinase NtMEK2 activates SIPK, and here we show that it can also activate p45(Ntf4). In an attempt to inhibit the function of both MAP kinases simultaneously we constructed a loss-of-function mutant version of NtMEK2, which, in transient transformation assays, led to an inhibition of germination in the transformed pollen grains. These data indicate that NtMEK2, and by inference its substrates p45(Ntf4) and/or SIPK, are involved in pollen germination.
Asunto(s)
Germinación , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Nicotiana/enzimología , Polen/enzimología , Sustitución de Aminoácidos , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Fosforilación , Plantas Modificadas Genéticamente , Plantas Tóxicas , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Nicotiana/genética , Agua/metabolismoRESUMEN
We report here a new selectable marker for tobacco immature pollen transformation based on the expression of dihydrofolate reductase (dhfr) gene which confers resistance to methotrexate (Mtx). Two immature pollen transformation approaches, i.e., male germ line transformation and particle bombardment of embryogenic mid-bicellular pollen have been used for the production of stable transgenic tobacco plants. In the first method, two methotrexate-resistant plants were selected from a total of 7161 seeds recovered after transformation experiments. In the second method, four methotrexate-resistant plants were obtained from 29 bombardments using 3.7 x 10(5) pollen grains per bombardment. Southern analysis confirmed the transgenic nature of T0 and T1 candidate transgenic plants, and a genetic analysis showed that the transgenes are transmitted to subsequent generations.